Nghien CIFU - Ky thuat
Tai lieu tham khao
1. Nguyin Tlln Ban, V. P. Serov (2003), Danh luc cac loai thuc vat Viet Nam, tap II, Nha xuat ban Nong nghiep.
2. Vo Van Chi (2012), Ti> dien cay thudc Viet Nam, tap II, Nha xuat ban Y hpc.
3. Pham Hoang Hp (1999), Cay cd Viet Nam, tap II, Nha xuit ban Tre.
4. Tran Van On (2012), Thuc vat va nhan biit cay thuoc, Trung tam Thong tin - Thu' vien - Topong Dal hoc Du'ge Ha Noi.
5. Nguyen Viet Than (2003), Kiim nghiem Dugc lieu bing phLFong phap hien vi, Nha xuat ban Ha Ngi.
6. Vien Du'ge lieu (2014), Bao cao tdng kit Du an {Ngay nhan bai: 03/03/2016 -
Bao ton ngudn cay thudc cd tmyen, Tai lieu lu'u hanh npi bp.
7. Flora of North America Editorrial Committee (1997), Flora of North America, Vol. 3, Anemone Genus.
8. F. Gagnepain (1929), Deux Anemones nouvelles dIndo-Chine, Bulletin de la Societe Botanique de France 76(2), 315-316.
9. Roy Upton, Alison Graff, Georgina Jolliffe, Reinhard Langer, Elizabeth Williamson (2011), "Botanical Pharmacognosy: Microscopic characterization of botanical medicines", American Herbal Phannacopoeia.
(ISBN 9781420073263)
10. Wang Wencai, Svetlana N. ZIman, Bryan E.
Dutton (2001), Flora of China ,6, pp. 307-328.
Ngay duyet dang: 02/06/2016)
r
I Nghien cmi anh huong cua dich chiet la
Gymnema inodorum (Lour.) Deuce Viet Nam den muc do bieu Men gen glucose-6-phosphatase
6* gan chuot thuc nghiem
Do Thi Anh, Dang Lan Hirong Phung Thanh Hirong, Dao Thi Mai Anh*
Truang Dgi hpc Duac Hd Npi 'E-mail: mai_anh_dao(^yahop.cpm Summary
Regarding that the plant Gymnema inodorum (Lour.) Decne enjoys wide use in Northem Thailand as valuable green vegetable not only for its nourishing value, but also hypoglycemic effect, and in continuation of the previous study on the hypoglycemic effects on the streptozocine-induced hyperglycemic mice of tiie species found growing in Vietnam that showed the only mechanism that answers for this hypoglycemic effect was the inhibition of alpha glueosidase, to reveal other mechanisms responsible for the hypoglycemic effect, was investigated the influence of leaf extracts from the Vietnamese Gymnema inodomm (Lour.) Dence on hepatic G6Pase gene expression at level mRNA. The inhibition of gene expression was obsen/ed obviously in all 4/4 tested samples. This inhibitory activity was quite different from ecotype to ecotype of the species.
The highest inhibition was obtained with the ethanolic leaf extracts from the plant collected in Hoa Binh These suggested the inhibition of hepatic G6Pase gene expression at mRNA level to be another mechanism and acting synergistically with the inhibition of aiphagiueosidase to create the hypoglycemic effect of the Vietnamese Gymnema inodomm (Lour.) Decne.
Keywords: Gymnema, diabetes, hypoglycemic effect, hepatic G6Pase, gene expression.
Dat v3n d l
Sip dung thao dugc trong dieu tri dai thae dudng (DTD), vol uu the canh tranh trong chi phi, hieu qua diiu trj va dd an toan, tir lau da la mdt hudng nghien ciru thu hut dugc sg quan tam chu y cua cac nha khea hpc d nhiiu qude gia
tren toan t h i gidi. Thee thdng ke gin day cd hon 1200 loai thae dugc duge su' dung de phdng va dieu tri benh DTD, trong sd dd, han 400 loal ciing han 700 phuong thudc da dugc cac nha khoa hpc nghien ciru va chipng minh v i co c h i cung nhu hieu qua ciia chiing ddi vdi can benh nay '°i.
14 TAP CHI DirqC HOC - 6/2016 (SO 482 NAM 56)
• Nghien
CLPU- Ky thuat
Loai Gymnema inodorum (Lour.) Decne phan bd rat rdng rai d khu vuc Ddng Nam A va dugc sif dung kha phd biin nhu mdt loal rau vira ed gia tri dinh dudng lai vira cd kha nang ha glucose mau g viing phia bac Thai Lan. NhCfng nghien cifu g i n day da chi ra rang Gymnema inodorum (Lour.) Dence cung chira nhung thanh phin hda hgc va hoat tinh ha glucose mau tuong tg nhu ngudi anh em ndi tieng ciia nd la Gymnema sylvestre (Retz.) R. Br. ex Sehul P^i. 6 Viet Nam, loai thao dugc nay cung da dugc phat hien va thu hai d mdt sd dja phuong PI. Cac kit qua nghien ciru ban d i u cho thiy t i t ca cac mau Gymnema inodorum (Lour.) Decne thu hai d cac viing khac nhau eua Viet Nam deu cd kha nang ha glucose mau tdt va la nhu'ng dugc lieu d i y tiem nang de dieu tri benh DTD i^i. Nghien eifu g i n day nhit da phat hien ra ring, uc c h i enzym aiphagiueosidase d rupt la co c h i quan trpng giup Gymnema inodorum Viet Nam lam giam gluccse mau, tuy nhien day ed the khdng phai la CO c h i duy nhit i^i. Chinh vi vay, de cd the tiip tuc lam sang td ca c h i ha gluccse mau ciia loai Gymnema inodorum (Lour.) Decne d Viet Nam, gdp phin vao viee nang cao hieu qua diiu trj cung nhu dp an toan ciia mpt loai dugc lieu tiim nang, chiing tdi tiin hanh nghien ciru anh hudng ciia loai thao dugc nay d i n mirc dp biiu hien gen ciia enzym glucose-6-phosphatase (GBPase) d gan chudt thge nghiem.
D6i tipgmg va phutyng phap nghien ciPU Nguyen lieu, hda chat, dong vat thi nghiem Hoa chit: Streptozecin (Sigma); que thip Accu - chek Active (Roche Diagnostics); Trizel reagent (Invitrogen); Agarose (Merck-Miilipor); Maxima reverse transcriptase (Thenno scientific); Random primer (Thermo scientific); dNTP (Thermo scientific);
DreamTaq PCR master mix 2X; Luminaris color HiGreen qPCR Master Mix 2X; GeneRuler 100 bp DNA Ladder (Thermo scientific).
Dgng vat fhi nghiem: Chudt nhat tring 4 tuin tudi, chung Swiss, gidng due, khde manh, nang tip 18-20 g, mua tai Vien Ve sinh Dich te Trung uong.
Ddng vat sau khi mua ve dugc nudi tai phdng thi nghiem cua Bd mdn Dugc Igc Tnfdng Dai hpc Dugc Ha Ndi trong diiu kien ehiiu sang theo chu ky 12 gid sang: 12 gid tdi, dae nguge ngay dem va dugc tiip xiic tg do vdi ngudn nudc, thirc an.
Thirc an ciia chudt la thiPC an tdng hgp do Vien Ve sinh Djch t l Trung uong cung cip.
Phuwng phap nghien CIPU B6 tri thi nghiem
Chudt dugc gay tang glucose mau thuc nghiem bang each tiem mang bung STZ vol liiu 150 mg/kg. Sau 3 ngay, tiin hanh dinh lugng glucose mau ldc ddi cua chudt va chpn ra chudt cd glucose mau > 200 mg/dl (> 11 mmel/l) de tien hanh thi nghiem. Chudt tang glucose mau dugc ehia thanh cac Id, moi Id 10 con: Id chirng benh (udng dung djch NaCMC 0,5%), Id chirng duang (tiem insulin lieu 1 Ul/kg/ngay), 4 Id thip (udng hdn dieh c i n dugc lieu vdi liiu tugng duong 10 g dugc lieu khd/kg chupt) gdm cd: Id GII (dugc lieu thu hai tai Ha Ndi); Id GI2 (dugc lieu thu hai tai VTnh Phiic; Id GI3 (dugc lieu thu hai tai Bac Kan); Id GI4 (duge lieu thu hai tai Hda Binh). Ld chirng thudng dugc nudi song song ciing vdi cac Id ehudt thip nghiem d tren va dugc che udng nudc mudl sinh ly.
Sau 7 ngay diiu trj, md chudt liy gan, tach chiit ARN.
Phirong phap danh gia muv do bleu hien cua gen G6Pase
ARN tong sd dugc tach chiit bang dung dich thudc thip Triazol rdi dugc dem do quang d i xae djnh ndng dp va chay dien di de kiim tra chit lugng.
Sir dung Random primers va enzym sao chep nguge Maxima reverse transcriptase de ehuyin ARN thanh cADN. Phan irng sao chep nguge dugc tiin hanh trong dng Eppendori" 0,2 ml thee chu ky nhiet: 50°C/60', 85°C/5', 4°C/ •».
Sip dung cADN thu dugc de nghien ciru mirc dp bieu hien eiia gen GOPase bing 2 phuang phap: PCR ed diin va realtime PCR. Mire dp bieu hien gen eiia G6Pase the hien qua ty sc REL (relative expression level) giua gen dich G6Pase va gen tham ehiiu (reference gene) GAPDH.
Doi vdi phuang phap PCR ty sd REL duge tinh toan bing each chup anh san pham PCR sau khi dien di va sii' dung phin mim chuyen dung cua he thdng chup anh gel Vilber Leurmat de phan tich va tinh ty le dam dp bang ciia 2 gen.
Ddi vdi phuang phap realtime PCR, ty sd REL giO'a gen dich G6Pase va gen nen (reference gene) GAPDH dugc tinh thee cdng thirc: 2-"==', trong dd Ct la trj sd ehu ky ngudng, la ehu ky nhiet ma d tai thdi diem nay he thdng may real-time phat hien dugc tin hieu.
Trinh tg cae cap mdi dac hieu sir dung trong nghien ciru dugc trinh bay treng bang 1:
TAP CHI DirOC HOC - 6/2016 (SO 482 NAM 56) 15
Nghien CCFU - Ky thuat
Bang 1 : Tririh tu cac cap mil diirig trorig nghien ciru
Ten gen Moi xudi Moi ngum
G6Pase 5' /y\CGCCTTCTATGTCCTCTrrC 3' 5' GnGCTGTAGTAGTCGGTGTCC 3'
GAPDH 5'AAGAAGGTGGTGAAGCAGGC 3' 5' TCCAGCACCCTGTTGCTGTA 3'
Xir li so lieu
Sd lieu dugc XLP li bang phin mem Excel 2010 va SPSS 20. Sg khae biet v i thing ke ciia cac nhdm dugc tinh toan bang one-wray ANOVA, test Turkey va non-paired student's T test.
Ket qua nghien ciru va ban luan Ket qua tach chiet ARN
Sau khi kit thiie qua trinh diiu trj, tiin hanh liy gan ehudt d l tach chiit ARN. Cac mau ARN tach chilt dugc deu dugc do quang de xac dinh ndng dp va chay dien dl de kiim tra dp tpan ven phan tip.
Kit qua dugc the hien d bang 2 va hinh 1.
Bang 2: Ndng do va do tinli saeh cua cac miu ARN tach chiit duac
thu dugc dil ve sd lugng va dam bao dp tinh sach de tien hanh cac budc nghien cipu tiep theo.
Nhom
Chiing tiiiporig CiiLfng benh Chifng difcfng
GM GI2 GI3 GI4
Nong do ARN (ng/pL) (Min-max) 2791,67-11830,00 1722,22-14040,00 2645,83-13263,89 8930,56-10236,11 3104,17-11736,11 2100,00-6416,67 2472,22-16701,39
TyleOD260/OD230 (Min-max)
2,45-6,13 3,14-8,24 2,614,12 2,73-5,23 3,34-8,18 2,33-4,31 4,21-8,13
Ket qua ciia bang 2 eho thiy tu khoang 100 mg md gan, ARN tach chilt dugc tip t i t ca cac mau diu cd ndng dp nam trpng khpang tir 1700 - 16000 ng/pl ddng thdi ty le OD260/230 eiia cac miu thu dugc diu > 2. Kit qua nay chirng td ARN
Hinh 1 : Hirih anh dien di cua dai dien eae mau ARN tach chiit dirge
(Lan 1,2,3, 4, 5, 6, 7 lan lugt la cac miu gan cua cac Id:
chOrtg thucmg, chimg benh, chimg duong, GI1, GI2, GI3, GI4) Dac biet, kit qua cua hinh 1 che thay cae miu ARN khi chay dien di d i u chi tach thanh 2 bang re net tuang irng vdi vi tri rARN 183 va 28S. Kit qua nay chirng td rang cac m i u ARN thu dugc khdng bj phan hiiy va dam bao chit lugng d i tiin hanh cac budc nghien ciPU tiep thee.
Ket qua nghien ciru mirc do bieu hien cua gen G6Pase b i n g phu'O'ng phap PCR co diin
Dung enzym sao chep nguge d l ehuyen ARN tach chilt dugc thanh cADN sau dd diing 2 cap mdi dac hieu de nhan ban 2 gen (gen dieh GOPase va gen tham chilu GAPDH) bang ky thuat PCR cd dien. Dem chay dien di san pham, chup anh va phan tich dam dd bang bang he thing chup anh gel Vilber Lourmat de tinh toan mirc dp bilu hien gen G6Pase thdng qua ty sd REL, kit qua duge trinh bay treng hinh 2 va hinh 3.
REL
" 1
Hinh 2: Hinh anh dien di san phim PCR gen G6Pase (A) va GAPDH (B) a gan cua eae id chudt nghien ciru
(Lan 1: Ladder 100bps; Lan 2, 3,4, 5, 6,1, 8 lin luot la cac mau gan ciia: to chimg thudng chimg benh, chimg duong Gil, GI2, GI3, Gi4) Kit qua cua hinh 2 va 3 cho thiy G6Pase dugc
tang cudng bleu hien manh d Id chudt bj benh
"•"" trong khi d Id chudt duge diiu trj bang insulin mirc dp bleu hien ciia gen nay giam xudng gin vdi mirc bleu hien d Id chudt binh thudng. Dac biet, sau 7
„ ' ^„ • ngay diiu tri ca 4 miu Gymnema inodorum thu hai tip 4 dia phuang khac nhau d i u t h i hien tac dung lam giam manh mipc dp biiu hien cua gen G6Pase d gan chudt. Lugng mARN G6Pase tao ra d gan
1 1
Hinh 3: Mirc dd biiu hien gen G6Paseagan cua eae Id ehudt khi danh gia bing ky thuat PCR cd diin
16 TAP CHI Dl/OC HOC - 6/2016 (SO 482 NAM 56)
• Nghien
CIPU- Ky thuat
ciia chudt d cae Id diiu tri bang duge lieu khdng dil d l cd t h i phat hien bang ky thuat PCR cc dien trpng diiu kien thf nghiem ciia chiing tdi
Ket qua thu dirge tir phu'O'ng phap real time PCR
Sau khi cd kit qua so bd v i mirc dp bieu hien gen G6Pase bang ky thuat PCR cd diln, tilp tuc sip dung ky thuat real time PCR nghien ciru mirc dp bilu hien ciia gen nay. So vdi ky thuat PCR cd diin, ky thuat real time PCR ed dp nhay eae han, do dd ky thuat nay cho phep nghien ciru rd hon v i anh hudng eiia eae mau dich chilt Gymnema inodorum din mirc dp bilu hien gen G6Pase. Kit qua ciia phuang phap real time PCR t h i hien trong hinh 4.
Hinh 4: Mire dd bieu hien gen G6Pase dgan eiia cac Id ehudt nghien ciru sir diJng ky thuat
real time PCR
Kit qua cua hinh 4 tiip tuc khang djnh kha nang lam giam manh mipc dp biiu hien gen G6Pase d gan chudt bi benh eiia t i t ca eae mau djch chiit Gymnema inodorum. Hon thi, kit qua cua hinh 4 cung cho thiy ed sg khae biet trong kha nang lam giam bleu hien gen G6Pase giO'a cac miu dugc lieu. Miu dugc lieu GI4 thu hai tai Kim Bdi, Hda Binh la mau dugc lieu ed kha nang ire c h i manh nhit ddi vdl mirc dp bieu hien gen G6Pase.
Mirc dp bleu hien cua gen nay d gan eiia Id ehudt udng djch chiit GI4 giam cd y nghTa thdng ke se vdi Id chipng thudng. Id dugc dllu tri bang insulin va Id chudt udng djch ehiit GII thu hai tai Ha Ndi (p < 0,05).
Ban luan
Tan tae dudng va thoai hda glycogen d gan vdn la hai CO c h i chuyin hda chinh ma ea t h i sir dung d l duy tri lugng glucose mau khi ddi. Tuy nhien, niu cac enzym chia khda ciia hai con dudng nay bj mit kiem seat tang boat ddng se din tdi tang san xuit glucose d gan va gay tang glucose mau.
Hien tugng nay da dugc xac dinh la nguyen nhan chinh cua trieu chirng tang glucose mau liie ddi
thudng gap treng ea DTD type 1 va type 2. (fc c h i cac enzym chia khda eiia cen dudng thoai hda glycogen va tan tae dudng, vl t h i , la mdt trpng nhu'ng hudng tiip can ciia cac nha nghien ciru de tim ra cae heat chit diiu trj DTD. Mdt trong cac enzym dich quan trpng cua chiin lugc nay la GOPase, enzym chia khda nim giii' canh cira cudi ciing ciia ca hai con dudng k i tren w.
Kit qua nghien ciru cua chiing tdi cho thiy ca 4 miu Gymnema inodorum thu hai tai 4 dia phuang khac nhau d Viet Nam d i u cd kha nang ire c h i manh mirc dp biiu hien gen ciia enzym quan trpng nay. Theo cac tai lieu da dugc edng bd, tac dung ire ehl mire dp bleu hien gen GBPase eiia nhiiu duge lieu dac biet la cae duge lieu thudc ehi Gymnema diu cd lien quan d i n insulin, hormon quan trpng nhit ciia eg t h i dam nhiem vai trd ire ehi bieu hien gen ciia G6Pase. Cu the la, cae dugc lieu nay lam tang lugng hormon Insulin trong co the va insulin sau khi gin kit vdi receptor d gan se thdng qua cac cen dudng truyin tin hieu de ire c h i sg bleu hien cua enzym dich G6Pase i^'i. Khac vdi cac leal Gymnema khac cd chipa cac thanh phin hda hpc cd kha nang kich thich trge tiip t i bac beta ciia dao tuy tang bai tiit insulin, dich chiit ethanol ciia loai Gymnema inodorum thu hai tai Thai Lan lai khdng cd kha nang nay i^'.
Cae tac gia da gia thiit rang ca c h i lam tang tilt insulin ciia Gymnema inodorum la do dugc lieu nay cd kha nang kieh thieh bai tilt eae ineretin d dudng tieu hda va ehinh cac ineretin nay mdi kich thich t l bao beta ciia dao tuy tang tilt insulin. Tuy nhien, trong nghien ciru ciia minh, chiing tdi thiy rang cac miu Gymnema inodorum thu hai tai Viet Nam lai cd kha nang ire ehi bilu hien G6Pase rit manh. O eae Id ehudt duge udng dugc lieu, dae biet la Id chudt udng miu G14 thu hai tai Hda Binh, mirc bleu hien gen G6Pase xudng thip hon ca Id chudt binh thudng va Id chudt dugc dieu trj bang insulin. Mat khae, tuy ire c h i G6Pase manh han insulin, nhung mdt thi nghiem ciia chung tdi eho thiy kha nang ha glucose mau ciia Gymnema inodorum lai thip hon ed y nghTa thdng ke vdi Insulin (sd lieu ehua cdng bd). Kit qua nay ggi y ring, loai Gymnema inodorum Viet Nam cdn ed t h i chira cac hpat ehit ed kha nang ipc ehl G6Pase theo ea ehl khdng lien quan d i n insulin.
De cd the kilm chipng gia thiit nay, rit ein cd nhu'ng nghien ciru sau ban v i ca thanh phin hda hpc va ea ehi ire ehi G6Pase eiia loai Gymnema inodorum thu hai tai Viet Nam.
(Xem tiip trang 21)
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