@ Nghién citu - Ky thuat Nghién cifu thanh phan héa hoc va tac dung ha dung huyét cua day dau xuong ( Tinospora sinensis (Lour.) Merr) trén mé hinh chudt nhat gay dai thao duéng bang streptozotocin Dé Ngoc Lién, Lé Thj Xoan, Nguyén Thj Thanh Ngan, Nguyén Thi Thuy Quynh 4. Mé dau Hign nay, dai théo duéng (TD) la mat trong 3 bénh c6 téc dé phat trign nhanh nhat. Theo WHO, thé gidi dang cé khong 200 triu nguéi mac bénh nay va dy tinh d&n nam 2025 con sé mac bénh ién t6i 300-330 triéu nguai lll, Mac du da duge mo ta tit 1500 nim truéc céng nguyén nhung vie nghién ctu thudc diéu tri DTD van dang duge tiép tuc vi hau hét cdc thude tan dugc duac str dung phé bign hién nay c6n gay tac dung phu hay chi phi dat 46. Day dau xuong (DBX) (Tinospora sinensis (Lour.) Mert) phan bé 6 Viét Nam, Lao, Trung Quée va mét sé niféc khac & viing Béng Nam A. Dugc ligu nay da dugc biét dén véi tac dung chéng viém, chéng oxy héa, ite ché phan tng mién dich, chéng DTD B.81, Tac dung ha duéng huyét, chéng DTD lia mét s6 loai cing chi véi né cing da dugc ching minh trén mé hinh déng vat (9-13, Nghién ctu nay nham phan tich dinh tinh va dinh luong mét s6 hop chat thuc vat thir sinh trong cc dich chiét ti day dau xuang va tac dung ha dung huyét ca mét sé phan doan dich chiét cla v6 than DDX trén chudt nhat gay BTD bang strep- tozotocin (STZ). Nguyén liéu va phuong phap Nguyén ligu thyc vat Day dau xuong (DDX)(Tinospora sinensis (Lour) Mer) dugc thu hai tai Thai Nguyén vao thang 11 nam 2006 va da duge chuyén gia thuc vat dinh loai. V6 than DBX (500 g) sau khi da sdy kho 850-55°C dude ngam trong ethanol 80% & nhiét do phéng. C6 can dich chiet thé, thu dutéc cao ethanol (38,49). Sir dung 35g cao ethanol dé chiét qua n- hexan, sau d6 tip tuc chiét qua ethyl acetat, cat 8 Truimg Dai hoc Khoa hoc Tu nhitn, Dai hoc Quée Gia Ha loai dung méi, cusi cling thu dugc hai phan doan 1a phan doan nuéc (16,19) va phan doan ethyl acetat (2,899) Béng vat Chust Swiss, gidng duc, 4 tuan tudé (trong lugng 20-229) do Vién Vé sinh Dich t8 TW cung cap. Chust duge nudi 6 nhiét d phong (20-25°C), chu ky sang: tdi la 12:12. Thitc &n tong hap-ciia Vien Vé sinh Dich té TW va nue uéng duce cung cap khéng han ché. Nghién citu thanh phan héa hoc Dé nghién city thanh phan héa hoc cila cac dich chiét tir DDX, ching téi sit dung céc phuong phap nhur dinh tinh mét sé nhOm hop chat thyc vat thir sinh bang cac phan img héa hoc dac tang 5. §I, inh long hgp chat phenolic tng sé theo phuong phap Folin—Ciocalteau !"1l va sac ky lbp méng trén silicagel (Merck Alufolien 60 Fysq) 2. Phuong phap gay DTD thyc nghiém Chuét nhat cho nhin déi 16 gid truéc khi tién hanh gay PTD bang STZ (Sigma, St. Louis, MO.,USA). Chudt duoc tiém mang bung dung dich STZ pha trong dém citrate (0,01M, pH 4,5) véi liu duy nhat 150 mg/kg thé trong. Chudt déi ching chi tiém dém citrate. Sau 72 gig tiém STZ, nhimg con chust c6 néng dé glucose huyét de ai lén han 13,9 mmoiil (250 mg/dL) duge xem nhur da mac bénh DTD va tiép tuc dude sif dung trong thi nghiém U.7 10. 12} Nghién ctu anh hudng cila cae dich chiét DPX trén chuét nhat gay TD bdi STZ Chudt nhat BTD d3 nhin déi 16 gid duoc chia thanh 5 Id (4-5 confé thi nghiém), mdi 16 duge ap dung cae céch xirly khac nhau: TAP CHi DUGC HOC - 10/2007 (SO 378 NAM 47)@ Nghién cuu - Ky thuat L6 1: Khéng diéu tri (16 déi ching) L62: Diéu tri bang uéng cao ethanol (600 majkg). L6 3: Diéu tri bang uéng phan doan nuéc (600 magikg) Lé6 4: Biéu tri bang udng phan doan ethyl acetat (600 mg/kg). L6 5: Biéu tri bang tiém insulin tac dung nhanh (Polfa, Ba Lan) liéu 1 Ulkg. ‘Mau toan phan, duoc lay tir dudi chudt 6 thai Giém 0 gid (théi diém ngay truéc khi diéu tri bang cac liéu phap) va 8 2 gid, 4 gid, 8 gid, 10 gid sau khi diéu tri. Xac dinh néng dé glucose huyét bang may do duéng huyét va bé kit thir tung tmg (One Touch Ultra, Johnson & Johnson, USA). Két qua Thanh phan héa hoc Tat két qua cdc phan tng dinh tinh cho thay thanh phén hop chat thir sinh trong DBX rét phong phu: flavonoid, tanin, alcaloid, cc polyphenol va glycosid trong dé alcaloid va tanin chigm thanh phan chi yéu, Can cif vao mic dé phan img cing cho thay thanh phan chd yéu trong phan doan nutc a alcaloid va tanin, trong phan doan ethyl acetat la flavonoid (bang 1). Bang 1. Két qud cdc phan ing dinh tinh thanh phan mét sé hgp chdit ty nhién te dich chiét cia DDX , ; Mau Nhom chat Phan img dac trun m ning dic iMG “Caoethanol__PDnude PD ethyl acetat Shinoda: = - ++ Flavonoid toe = = = Catechin Vanitin/HCl = ~ Vanilin’ H.SO, = ry > Polyphenol = yeneno! Tanin ‘Acatat c rZ er + GelatinvNaCt S73 + + NaOH 10% + + = Polyphenol FeCl, 5% + ++ ++ Glycoside Keller-killiant + ___Vans-Mayer Alcaloid Bouchardat +t +++ ‘Dragendroff + 7H 7 ‘Dau (+) chi phan tng duong Diu (-) chi phn ung m tinh Két qua dinh lugng hop chat phenolic tong so cho thay thanh phan hgp chat phenolic trong phan doan ethyl acetat nhiéu nhat, chiém 15,39%, tiép. theo la trong phan doan nubc chiém 9,52% va cudi cing 18 trong cao ethanol chiém 9,05%. ‘Sée ky Idp méng trong hé dung moi TEAF 5:3:1:1 (loluene-ethy! acetat-acetone-formic acid) cho nhiéu bang vach vdi mau sc khac nhau khi nhugm bang H,SO, 10%. Trong dé, cao ethanol cho nhiéu bang vach nhat (12 bang), tiép theo la phan doan nuéc (4 bang) va phan doan ethyl acetat (6 bang). Khi so sanh hé sé Rf ta thay c6 3 bang chung cho ca 3 dich chiét trén do [a cac bang Rf = 0,505, Rf = 0,437 va Rf = 0,379. Anh huéng ctia céc dich chiét DDX trén chugt nhat gay OTD bing STZ Ti bang 2 cho thay, sau 72 gid tiam STZ chudt bi OTD, biéu thi bdi néng dé glucose huyét tang TAP CHE DUGC HOC - 10/2007 (SO 378 NAM 47) cao, trong khi chust déi chimg co néng dé dudng huy€t 8 mifc binh thuting, Biéu dé chimg t8 STZ da gay DTD trén chugt va nhing con chust da bi DTD dude si dung trong nghién cttu tac dung ha du’ing huyét clia dich chiét DBX. Hinh 1, Sdc ky d6 cae dich chiét cba DBX. (1) Cao ethanol, (2) Phan doan nude (3) Phan doan ethyl acetat@ Nghién citu - Ky thuat Anh hudng cia cao ethanol lén néng dé glucose huyét ciia chudt nhat BTD do STZ K@t qua cho thay, cao ethano} lam tang dung huyét trén chudt TD sau 2 gid udng véi ligu 600 mg/kg khi so sénh v6i chuét déi ching (P<0,05, t- test). Sau 4 gid, 8 gid va 10 gid, cao ethanol mat tac dung tang dung huyét, bigu thi bang dutng huyét cotia chudt khéng khac biét véi dung huyét cla chuét déi chtmg (bang 2). Két qua ciing cho thay, néng 46 dung huyét chudt uéng cao ethanol tai cdc thai diém 4 gid, 8 gid va 10 gid giam so vai thoi diém 2 gid (thoi diém duéng huyét tang do tac dung ca cao ethanol). Trong khi 46, voi chuét déi chimg, dudng huyét tai cdc thai diém trén lai khong giam tham chi con cao hen. Diéu nay da chimg té rang cao ethanol cé tac dung lam tang duéng huyét trong thdi gian ban dau, sau do né lai c6 tac dung ha dung huyét. Bang 2. Sv thay d6i néng dé glucose huyét ca chudt nhét DTD gay bdi STZ khi duoc diéu tri bang céc dich chiét tu OBX va insulin. Lé chust Néng dé glucose huyét (mmolil) thi nghigm “72 gid Ogi6 2918 4918 8 gio 40 915 Dem citrate 330.9 5.420,7 : - STZ khong Gidutl ——3,740,5 -28,216,6 25,744 28,6251 26,445.20 24,5845 STZ*Ca0 ethanol 2AOX 26,0860 33.4173" 303332 18.838,1 20,72106 STZ*+PB nude 3510 28,0880 18,018,1" 15,347,3* 10,423.7" 10,742,0" Siar © ethyl 3512 25,346,9 23,724,5 16.6: 8647.5" 10,213, STZ* insulin 31a14 26,83,3 10,7228" 15,9120" 147210 21,2943 -72 gid la thai dim ngay trude khi gay bénh, 0 gid la tho’ diém bat déu diéu tr trén chudt BT. Butng huyét chuét DTD diéu tri bang DDX va insulin dugc so sénh véi chudt BT khéng diéu tri, () chi P< 0,08, (*) chi P<0,01 Anh huéng cita phan doan nuéc lén néng do glucose huy@t cua chuét nhét DTD do STZ Can cit vao bang 2 ta thay, phan doan nuée 06 tac dung ha duéng huyét trén chudt OTD do STZ 6 tat c& cac thdi diém nghién cit: 2 gid (P<0,05, test), 4 gid, & gid va 10 gid (P<0,01, test) khi so Anh v6i I6 d6i chimg. Anh hudng cilia phan doan ethyl acetat lén néng dé glucose huyét cua chudt nhat DTD do STZ Phan doan ethyl acetat 06 tac dung ha duéng huyét d cdc thai dima Gid (P<0,08, t-test), 8 gid va 10 gid (P<0,01, test) sau khi udng so v6i 16 d6i ching, © thai diém 2 gid sau khi udng dich chiét, néng dé duéng huyét cé giam, tuy nhién st thay déi nay khOng co nghia théng ke. Anh huéng cua insulin lén néng dé glucose huyét cia chu6t nhat DTD do STZ Theo bang 2, insulin o6 tac dung ha duéng huyét t8t trén chuét nhdt OTD 6 cac théi diém 2 gid, 4 gid (P<0,01, t-test), va 8 gid (P<0,05, t-test) so véi chuét déi chimg. Sau 10 gid tiém insulin, néng 46 dung huyét gidm, tuy nhién lai khong én dinh nén khéng c6 ¥ nghia théng ke. 10 Thao luan Streptozotocin (2-deoxy-2-(3-methyl-3-nitro soureido)- D-glucopyranose) la chat dang duge stt dung réng rai dé gay cdc mé hinh dong vat DTD thuc nghiém. N6 cé tac dung pha hily khéng phuc hdi t8 bao B cla dao tuy, do dé lam mat hay suy gidm qua trinh tiét insulin ("4 Vily do trén, ligu phap tiém insulin trén chudt DTD do STZ trong mé hinh ‘tvén c6 hiéu qua rat tét, tac dung giam nhanh duéng huyét ngay sau 2 gid tiém va odn tac dung sau 8 id. Tuy nhién, insulin tai khong 6 hiéu qua lau dai, diéu nay da dugc ching minh d mé hinh trén bai sur giam néng 46 glucose huyét khéng o6 y nghia théng ké sau 10 gid tiém. Ngoai tac dung ha dung huyét, cao ethanol con gay tang éuéng huyét, tac dung nay kha nhanh va {6n tai trong théi gian ngan ban dau fic thir nghiém. Do do, rat 06 thé trong cao ethanol con chifa mot hap chat nao dé gay tng duéng huyét Trong khi cao ethanol chua thyc sy lam ha dudng huyét higu qua thi qua qua trinh tach chiét, tir né lai thu dude hai ché pham la phan doan nuéc va phan doan ethyl acetat o6 tao dung ha duéng huyét tt, khéng odn phat hién thy téc dung tng SO 378 NAM 47)@ Nghién citu - KY thuat dung huyét 6 bat ky thai diém nghién citu nao va 6 thé coi la tudng ty véi tac dung cilia insulin 6 thé diém sau 4 gid va sau 8 gid, hon han insulin 6 thai diém sau 10 gio diéu tri, Rat c6 thé chinh n-hexan lay di thanh phn lam t8ng dung huyat va toai no ra kh@i cao ethanol. Biéu nay gai y ching t6i can phai phan tich thanh phan héa hoc ca phan doan nay trong cac nghién citu tiép theo. Phan dean nuéc va phan doan ethyl acetat déu 6 tac dung ha dung huyét trong khi thanh phan h6a hoc olla chéing khéng ging nhau. Phan doan nuGc co nhiéu alcaloid, tanin, cn mot sé polyphe- nol va c6 rat it flavonoid. Phan doan ethyicetate cha yéu chifa flavonoid, ngoai ra cén mét sé tanin, polyphenol va khéng cé alcaloid (bang 1). Nhu vay, alcaloid khéng phai la thanh phan ¢6 tac dung dung huyét trong DDX. Cac flavonoid chiém ti lé khac nhau trong phan doan nude va phan doan ethyl acetat, trong khi tac dung ha duéng huyét cia chiing khéng khac biét. Do 6, hoat tinh ha duéng huyét cia DBX rat c6 thé la do thanh phan cac hgp chat phenolic néi chung. Theo ching téi, cdc tanin va polyphenol tré thanh ting cit vién sé 1 la nhém chat cé tac dung ha duéng huyét ciia DEX. Két luan 1. Phan doan dich chiét bang ethanol 80% cia v6 than DPX c6 cdc nhém chat alealoid, tanin, flavonoid, glycosid va mét sé polyphenol _khac, trong d6 alcaloid, tanin 1a thanh phén cho yéu. Thanh phan chi yéu cia phan doan auc la alcaloid va tanin, ota phan doan ethy! acetat la flavonoid, 2. V6i liu 600 ma/kg, cao ethanol 80% cila vd than DPX, cila cac phan doan nuéc va ethylacetat cila né déu co tac dung ha dudng huyét trén chust gay DTD bai STZ. 3. Phan doan nuéc va phan doan ethyl acetat o6 tac dung ha duéng huyét tét hon cao ethanol va cd tac dung tuong ty nhu insulin vai liéu 1 Utkg & cdc. thai diém 4 gid, 8 gid sau khi diéu tri cling véi 600 mg/kg cila cac phan doan chat khé cia dich chiét T. sinensis trén chudt nhat gay DTD bdi STZ. Hon nifa, sau 10 gid diéu tri, tac dung ha duéng huyét cila hai phan doan nudc va ethyl acetat van con higu qua kéo dai han so vai insulin. Summary Chemical composition of Tinospora sinensis dried stem barks extracted by various solvents and submitted to thin layer chromatography analysis. in addition, the hypoglycemic effect of some extract fractions was tested for on STZ induced diabetic mice. TAP CHI DUOC HO 10/2007 (SO 378 NAM 47) 80%-ethanol extracts of Tinospora sinensis barks showed the presence of alcaloid, tanin, flavonoid, glycoside and polyphenol. Of these, alcaloid and tannin were the main components. Dried extracts from 80%-ethanol, aqueous and ethyl acetate fractions were demonstrated to have hypoglycemic effects on STZ-induced diabetic mice at a dose of 600mg/kg of body weight. Tai ligu tham khao 4. Ta Vin Binh, Ben dai tho dung - ting glucose mau, ‘Nab Y hoe, (2006)Ha Ni 2. Nguyen Thanh Cat, Bao Hit Vinh, Cae phuomg php sic by, Neb Khoa hoc va Kj thuél, Ha Noi (1985) 3. 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