Nghiên cứu tách chất có hoạt tính sinh học từ nọc rắn hổ mang naja

DAI HOC QLOC GIA HA NOI TRirClNG DAI HOC KHOA HOC TliNHIEN m TEN DE TAI: NGHIEN CUU TACH CHAT CO HOAT TINH SINH HOC TlTNOC RAN HO MANG NAJA • • Ma so: QT-06-46 Chii tri de tai: TS Tran Thi Hong Tham gia: CN Nguyen Thanh Hanh OAI HOC Q U O C GIA HA ^ ^ , •RUNGTAM I HONG TIN THU Vl£N Di/IM HA NOI - 2006 BAO CAO TOM TAT Ten de tai: Nghien ci'Cu tdch chat co hoai tinh sink hoc lie noc ran ho nian^^ •'^(•{h^ Ma so: QT-06-46 Chu tri de tai: TS Tran Thi Hong Cac can bo tham gia: - CN Nguyen Thanh Hgnh Muc tieu va npi dung nghien cuu: * Muc tieu: Tach chat c6 hoat tinh sinh hoc tir noc ran h6 mang Naja * Npi dung nghien curu: - Phan tach cac phan co hoal tinh sinh hoc noc ran ho mang - Xac dinh luang phan lu ciia cac chat tach tir noc ran ho mang - Khao sat hoat tinh sinh hoc cua cac chat tach tij noc ran ho cnang Cac ket qua dat duoc: - Thuc hicn da\ du cac nuic lieu da dc - Cong bo 01 bai bao tren Tap chi Duac Heu; 01 bai tren Tap chi phan tfcl Hoa, Ly va Sinh hoc CHU TRI DE TAI DON VI QUAN LY PGS TS, Pham Quoc Trieu TS, Trdn Thi Hong TRLONG DAI HOC KHOA HOC TUNHIEN CS.IS I/i4i/w cj{(n^A4- SUMMARY L Title: Isolation of biological active substance from Naja snake venom Code: QT-06-46 Director: Dr Tran Thi Hong Members: Bachelor Nguyen Thanh Hanh Researching Objects and Contents Researching Objects: Extracted of biological active substance from snake venom Researching Contents: - Isolation of biological active substance from snake venom - Determinate molecular weights of biological active substance from snake venom - Test antiviral and antimicrobiol properties of biological active substance from snake venom Results - Carry out outlined plan - Published two articles, one is on Journal of Medicinal Materials and other on Journal of Analytical Sciences MUC LUC M6 DAU ^ ChUdng 1: Tong quan 1.1 Cac chat co hoat tinh sinh hoc lij noc ran 1.1.1 - Toxin tu noc ran 1.1.2 - Enzyme 1.2 Kha nang ling dung cac chat co hoat tinh sinh hoc tach tir noc rdn Chuong 2: Phuang phap nghien cihi 10 2.1 Hoa chat 10 2.2-Thietbi: ' 10 2.3 - Phuang phap nghien curu 10 2.3.1 - Phan tach cac phan co hoat tmh sinh hoc cua noc ran 10 2.3.2 - Cac phuang phap xac dinh protein va h(xil en/yme 10 Chuang 3: Ket qua va ban luan 12 3.1 Phan tach cac phan co hoat ti'nh sinh hoc (HTSH) noc rdn 12 Xac dinh luang phan tir cua cac chat noc ran ho mang 17 3.3 - Khao sat hoat tinh sinh hoc cua cac chat tach lis noc ran ho mang 19 KET LUAN 19 TAI LIEU THAM KHAO 20 PHU LUC 22 CAC CONG TRINH DA CONG BO 23 x^ MO DAU Viet Nam n^m vung nhiet doi gio mua nong.alm, da dang sinh hoc, CO nhieu ngu6n gen quy hiem dac trung cua vung Dong Nam A Mot nhung nguon gen quy hiem la cac loai ran co noc doc [3] Ngu6n r^n doc (thuoc ho Elapidae va Viperidae) a Viet Nam rat phong phii, CO cac loai nhu: Ho mang chua Ophiophagus harnah Ho mang banh Naja naja Ran luc Vipera Ubetina, Bungarus fasciatus, Gomxosoma pvasma [3] Co nhung trai ran moi nam co the thu duoc khoang 20-30 kg noc ran doc tho (tirang duang 3-5 kg noc dong kho) nhir trai ran VTnh San, Vlnh Tuang, Vinh phiic, trai xkx\ Dong Nai, trai r^n Quan khu Nhung viec nghien cijru sir dijng nguon nguyen lieu cho cong nghe sinh hoc va che tao thuoc chua benh chua dugc lien trien mot each thoa dang O Viet Nam, noc ran doc da duoc sir dung nhu nguon duac lieu quy Viec nghien cuu noc r^n doc nhu noc ran ho mang, noc bo cap da duac b^t d^u i\i nhung nam 90 [1-2] Song song vai cac ket qua nghien cihi phong thi nghiem, da co nhung ket qua ling diing nhu su dung noc tho lam duac pham chong dau xuang khap thit ran duac sii dung lam nguon thiJc an dac san cao cap Noc ran la hon hap ciia rat nhieu chat co hoat tinh sinh hoc (HTSH), phan Ian so la cac protein Vc ban chat cau true, cac protein chia lam loai: enzyme va toxin Cac toxin noc ran la cac polypeptide va theo tinh chat tac dong, cc the chia lam ba loai chfnh: neurotoxin (doc to than kinh), cardiotoxin hoac cytotoxin (doc to vai tim), hematoxin (doc to vai mau) [1 3] Cac enzyme tim thay noc ran deu la cac enzyme thuoc lap thu> phan Hydrolase [2] Trong so cac enzyme thi Ribonuclease, Phospholipase va Protease duac quan tam nhieu han ca Cac ket qua phong thi nghiem mai chi diing a cho tach rieng / loai phan noc rdn la toxin va enzyme Vi vay hieu qua sir dun^ cua tung phan rieng biet noc r^n ban che do chua tan diin^ bet duac gia tri quy hiem ciia noc ran [2] Vai muc dfch nang cao gia tri cua noc ran mot nguon duac lieu co gi; tri, dac trung cho nguon gen quy hiem ciia hau nhiet dai nong aim Vie Nam, chiing toi de xuat thuc hien dc tai: "Nghien cuu uk h c hat co hoat tim sinh hoc iU noc ran ho mang Naja " CHUONG 1: TONG QUAN 1.1 Cac chat co hoat tinh sinh hoc tu noc rdn Noc rdn liic m6i tiet la mot chat long, mau hai vang luang nuac co the chiem tai 70 - 80% Noc lin la hon hap ciia rat nhieu chat co hoat tinh sinh hoc, ph^n Ian so la cac enzyme Tuy nhien, doc tinh ciia noc chu yeu cac toxin co ban chat peptit va protein phan tu luang thap gay Cac phSn co hoat tinh sinh hoc noc rdn, \'^ ban chat cau true, CO the chia lam loai: protein va khong protein Loai phan protein lai chia lam loai: cac enzyme va cac toxin Loai phan khong protein lik cac chat chong dong mau va cac chat gay di ung Noc r4n ihuang lac dong len cac he ca, than kinh, tim mach va he ho hap Noc ran gay cac hien tuang nguy hiem a viing mo nhiem doc cue bo, lam giam kha nang dong mau, lam ton thuang mach mau va d^n den giam kha nang bao ve ciia mach mau Luang te bao mau l6ng so co the se giam rat nhanh va cac tieu c^u bi tan Nhung trieu chiing cua phoi rat thuang gap a nhung nan nhan bi ran can, hien tuang mat mau co the xuat hien a phoi, mang bung, than va tim Hien tuang thuang xuat hien cung vai nhung thay doi v6 hoat dong cua tim va chuc nang thi giac Noc ran rat doc nhu vay co the khai quat tac dong ho tra giSa cac enzyme va toxin ciia noc ran nhu sau: ran can gay vet thuang thi dau tien, cac enzyme ihuy phan pha va cac mang te bao vat chu ma duang cho cac toxin va protein doc xam nhap vao te bao chu, phat buy tac dung gay doc, lam te liet moi va co the gay tu vong 1.1.1 - Toxin tir noc r^n Cac toxin noc ran la cac polypeptide va theo tinh chat lac dong, CO the chia lam ba loai chinh: neurotoxin (doc to thdn kinh), cardiotoxin hoac cytotoxin (doc to vai tim) hematoxin (doc to vai mau) [9-12] Hematoxin Hematoxin la cac ph^n protein chinh noc cac loai rdn Agki, cac phan co tai 26 protein co cac chiic nang di^u chinh mau nhu cac serine protease, chiing la cac toxin gay xuat huyet (haemorrhaaic toxins) [8] Neurotoxin Neurotoxin co tai 11 loai duac tim thay noc ran Naju Cat Neurotoxin co dac trung cobra Mot so Naja neurotoxin rat giong \x trinh tu amino axit Vi du: cobrotoxin III va cobrotoxin IV sions ta 93% Neurotoxin va Neurotoxin giong tai 71% Chi co acutolysin neurotoxin tu noc Deinagkislrodon acutus co trinh lu amino axit khong gion; Irinh tu amino axil cua bat cii neurotoxin nao noc ran Naia Cardiotoxin Nguai la da tim thay 14 ho (families) Cardiotoxin noc ran Trong CO ho tim thay noc rdn Agki Hai ho vixd tim thay noc ran Agki va noc r4n Naja Con lai bon ho tim thay noc ran Niper Trong noc rin Naja, Cardiotoxin co rat nhidu chiing c6 luang phan tu tu 6,7 den lOkDa Cac toxin tijr noc r^n v6 ciing doc, co the so sanh ngang vai toxin ciia mot so dong thuc vat khac cung nhu mot so loai lao [4] Ve mat cau true, cac doc to tiJf cac loai dong vat hoac mot loai thuc vat thuang CO cau triic la protein Vi du: Rixin tir cay thdu dau tia Ricinus communis la glycoprotein a-Cobraloxin lir noc ran ho mang cobra Naja naja atra Trung quoc hoac Dai loan co 62 amino acid vai cau disulfide, co khoi luang khan tu 6,949 Da, LD50 chuol tr^ng la 0,09 i^g/g [13] Cac doc to tu cac loai thuc vat thuang la cac alcaloid [4] a-Cobratoxin tan nuac Hoat tinh bi mat co mat cac chat khu , rat b^n a nhiet 20°C a-Cobraloxin lac dong nhu mot chat doi khang (antagonist) chong chat nhan (receptors) nicotinic acetylcholine chiic nang liep hap than kinh ca (neuromuscular junctions) Nguai la tach duac protein Atropor;in and Kaotree co tinh chat chong ung thu (Anticancer) tu noc ran Mai gam Crotalus atrox (Western diamondback rattlesnake) va ran Ho mang Naja kaouthia (Thailand cobra) co luang phan tu 35.000 va 6.000 dalton tuang ling Hai protein co lac dong phoi hap vai lam tang boat tinh lieu diet te bao he in vitro va in vivo, 0,5 meg lieu diet hoan loan 10'' le bao ung thu cac loai khac Cung luang ay khong lam hu hai tai cac te bao than, la lach va gan In vivo, Atroporin va Kaotree bao ve su tan cong ciia u co Iruang lao nen bai cac te bao ung thu myeloma (u tuy) cua chuol [14-15] 1.1.2 - Enzyme Cac enzyme tim thay noc ran deu la cac enzyme thuoc nhom thuy phan Hydrolase (EC 3) nhu: phospholipase L-arginine-ester hvdrolase, phosphatase kiem, phosphotase axil, transaminase, DNase ribonuclease, ATPase, endonuclease, phosphomonoeslerase, cholineslerase hvaluronidase, va 5'-nuc]eolidase [6] Trong do, cac enzyme chu yeu gom ribonuclease, phospholipase va proteinase Trong so cac enzyme noc rdn ke tren, phospholipase ribonuclease duac nghien cuu nhieu han ca Cac enzyme d6u thuoc lap phu cac enzyme tac dong tai lien ket Ester (EC 3.1) Proteinase Cac proteinase lij noc ran cung chia lam loai: proteinase co kim loai (Metalloproteinase) va proteinase khong co kim loai (no metailoproieinasc) Trong noc cac ran Naja thuang chiia cac proteinase khong c2,5 mM) lai hoal hoa enzyme EDTA lai co tac dung uc che thuan nghich enzyme So sanh Ribonuclease iii noc ran An Do va viing Trung A Lien Xo cho thay r^ng: Ribonuclease lijr noc r^n An Dp dac hieu v6i axil cytidylic, khong thuy phan ADN [23], Ribonuclease tu noc rkn ho mang vung Trung A Lien Xo la endoribonuclease, rat giong deoxyribonuclease tu tuyen ciia dong vat, khong CO tinh dac hieu vcd base niia, chi khac biet a cho enzyme cung khong thuy phan ADN ma thuy phan ARN [22] 1.2 Kha nang umg dung cac chat co hoat tinh sinh hoc tach tu noc r^n Cho tai nay, noc r^n doc da duac su dung nhu mot duac lieu qiiy hiem Thong dung han ca, noc r^n doc duac sii dung de san xuat mot so loai thuoc xoa bop ngoai da co tac dung dieu tri dau day than kinh, viem ca, dau khap kinh nien Vi du nhu mot so loai ma xoa bop ngoai da ciia nuac ngoai dyac san xuat tir nguyen lieu la noc r^n nhu: Vipratox, Vipraxal, Nizozal, Scobral Dendroloxin la Protein toxin duac tim thay Ian dau lien noc ran doc Chau Phi (Dendroaspis), chiing chiia 57-60 goc axil amin Cac toxin tac dung len kenh th^n kinh kali Nhirng phat hien hua hen se che lao nhOng thuoc mdi chOa cac benh than kinh nhu benh Alzheimer, benh dong kinh Mot vai ket qua khac viec lam sang to moi lien he giua cau Iriic va boat tinh cua cac dendroloxin dang phat trien, va no hiia hen cac thuoc giong nhu cac toxin lu nhien se duac thiel ke va long hap, dieu da thu but su quan lam ciia cac hang nhu hang Wyelh Ayerst Trong ca the l6n tai enzyme bien doi angiolesin (ACE) Enzyme gay nen su co mach mau din den tang huyet ap Tir noc r^n Brazil Bothrops jaracusa nguai ta tach duac chat lic che enzyme (ACE) la nhiing peptide nho de su dung lam thuoc de dieu tri benh huyel ap cao nhu thuoc captoril enapril, lisinopril Mot vai protein nho (khoi luang phan tu khoang 5000-9000 Da) da duac tach iCr noc ran luc co tac dung lie che lien ket tieu cau Tren ca sa nghien cuu cac peptide nay, nhung cong tac vien cua hang Genenlech da thiel ke cac phan tu peptide nho de lam thuoc kiem tra su dong cue mau khong binh thuang Trong so cac enzyme duac phan tach tu noc r4n ho mang nhu Ribonuclease, Phospholipase A2 Lipase thi Ribonuclease (RNase) da duac lim thay nhi^u ung dung ban ca \ hoc Tu thap ky 70 cua the ky iruoc cac che pham Ribonuclease da duac ung dung a Lien Xo de: dieu tri dac hieu nhieu loai benh virus khong chi a nguai, ma ca a vat nuoi [25] Cac chat hoal hoa Ribonuclease co the duac su dung de dieu tri benh nhidm IrCing virus Chat boat boa Ribonuclease L lie che, ngan chan phat trien sinh san cua nbidu virus te bao [26] Cac ch^' pham boat boa Ribonuclease L nhu oragen co kha nang tie che qua trinh nhan doi ADN virus cac dai thuc bao mang bung in vitro va gan chuol nhiem virus (MHV)-3in'vivo[7, 24] Hien nay, nguai ta dang tap trung vao cac Ribonuclease co boat tinh cytotoxin hay boat tinh chong ung thu Cac Ribonuclease da ma mc)l huang nghien cuu mai nham tao cac che pham duac cao cap de san xuai thuoc chiJa ung thu Theo thong bao 11 thang 10 nam 2005 ciia Cong ty duac Alfacell (Uc) ONCONASE (ranpirnase) la enzyme dang duac thu nghiem lam sang giai doan III doi vai 850 benh nhan boa tri lieu chong ung thu tuyen va ung thu trung bieu mo ac tinh (Malignant Mesothelioma) va thii nghiem lam sang giai doan I/II doi vai ung thu phoi (Non-Small Cell Lung Cancer NSCLC) [27] Cung theo tin ciia Cong ty duac Alfacell, thi Hoi ung thu My (American Cancer Society) cho rang ung thu phoi (NSCLC) lai My la benh gay tu vong cao nhal, chiem tai 80% Nam 2004, co gan 160.000 chci tai My benh ung thu Nguai ta tinh cho tai nam 2012 thi thuoc chiia NSCLC phai len tai ly USD Nhu vay, neu Ribonuclease ihuc su la duac pham cao cap de san xuat thuoc chCra ung thu, thi loai ngudi lai co them mot phuang lien hxxu hieu chong lai can benh nguy hiem MUC LUC TAP 11, S6 3/2006 (Trang 97-142) Trang NGHlfiN CtlU KHOA HOC CONTENTS VOL 11 (3), 2006 (Pages Pa^e 97-142) SCIENTTHC RESE.\RCH 97 Danh luc Do cay thuoc Viet Nam nam 2006Nguyen Van Tap T The Red List of Medicinal Plants of Vietnam 2006 -Nguyen Van Tap 106 Su phan bo cua ngu gia bi ht/ong va ngu gia bi gai Viet Nam - Pham Thanh Huyen yguyen Tap, Ngo Van Trai, U Thanh San .\'gd Due Phacmg, Cu Hai Long, Sguyen Ba Hoat !06 Distribution of Acanihopanax graciiisiyius U'.W.Smilh and A irifoliaius ( D Merr in Vietnam - Pham Thanh Huyen, Nguyen Tap Ngo Van Trai, Le Thanh Son, Ngo Dac Phuong, Cu Hat Long, \guyen Ba Hoal Nghien cOru kha nang nhan giong va bao ion ngu gia bi huong va ngu gia bi gai a Viet Nam - Pham Thanh Huyen, Nguyen Tap, U Thanh Son, Ngo Due Phurnig, Cu Hai lj)ng, Dinh Van My, Nguyen Bd Hoat, Nguyen N"hia Thin 108 Propagation and conservation of Acanihopanax graciiisiyius \\\ ^V Smith and thfoliatus var setosus Li in Vietnam Pham Thanh Huyen Nguyen Tap Ngo Van Trai, Le Thanh Son Ngo Due Phuong, Cu Hai Long, Dinh Van My Nguyen Da Hoat Nguyen Nghia Thin Nghien cuu phan flavonoid cay ban - Nguyen Quoc Thuc, "Nguyen Thuang Dong Bui Thi Bung Trdn Van Thuy, Dang \ ij Luang ' ' Study on Flavonoids m Ban [Hypericum -' japonicum Thunb e\ Murr.j - Vj^uytn ^uoc Thuc, Nguven Thuang Dong Bui Thi Bang Tran \ an Thuy Dang \ u Luong Chemical Study on I Sirnbilanthes acrucephitlus T Anders • T T Thuy, T V Sung G Adam ' ':~ Chemical Study on Strohilanthes acrocephaius T Vndcrs • T T Thiiv T V Sung, G .\d(un Khao Siil Ihiinh phan hoa hix cua than re cay gimg gio - Nguyen The Dung, Pham Khtic Tiep, Nguyen Kim San, Mai Kim Nhung, Pham Minh Diep '.ZZ Study on the Chemical Composiliun o( Zingiber zerumbei Sm - Nguyen The Dung, Pham Khac Tiep, Nguyen Kim Son .Mai Kim Nhung, Pham Minh Diep TJc dung lang cucmg the luc cua re cii cay sam ffiio - Dao Thi Vai Mai Tat To, Nguyen Thuang Dong, VH Thi Tram ll-i The Forced Effects of Extract of Radix Hibisci sagittifolii - Dao Tlii Vui, Mai Tat To Nguyen Tliuong Dong, Vu Thi Tram Tac dung cua che phjim silymarin chiet xuat tir qua cue gai di thuc tren mo hinh gay viem gan man thiic nghiem a chuot cong trang - Trinh Thi Diep, Nguyen Thuang Dong Biii Thi Bang, Nguyen Kim Phuang, Dd Thi Phuang 12S Effects of Silymarin Extracted from Introduced i\Clk Thistle on Experimental Chronic Hepatitis Model in Rats - Tnnh Thi Diep, Nguyen Thuong Dong, Bm Thi Bang, Nguyen Kim Phuong, Do Thi Phuong Phuong phap len men vdi chiing vi khuan sinh proteinase tach chiet chitin tir v6 torn D6 Ngoe Tu, Nguyen Van Thiel 132 Method of Fermentation with Proteinase Producing Bacteria for Extraction of Chitin from Shrimp Shells - Do Ngoc Tu, Nguyen \'an Thiel Nghien cuu tach, chiet va su dung toxin co hoat tinh sinh hoc lis noc ran ho mang Viet Nam - Trdn Dinh Toai, Nguyen Van Thiet, Le Mai Huang, Nguyen Thanh Hanh, D6 Trung Sy, Nguyen Kim Phatmg, Tran ihi H6ng 13" Study of Extraction and Application of Biologically Active Toxins from Vietnamese Naja naja Venom - Tran Dinh Toai, Nguyen Van Thiet, Le Mai Huong, Nguyen Thanh Hanh, Do Trung Sy, Nguyen Kim Phuong, Tran thi Hong Giai ihucmg Sach Viet Nam va ngiy Hoi ton vinh van boa doc Ian thijr nhat 28 ttiang nam 2006 141 The National Prize of Vietnamese Book and the First Holiday of Cultural Reading May 25*" 2006 1J2 14, Tap chi Duac lieu., tap IIj so 3/2006 95 Tap chiDuac lieu, tap 11^ ,6312006 (Trang 137-140) NGHIEN CCr TACH, CHIET VA SUDLISG TOXI>' CO HO.AT TINH SINH HOC TUNOC RAN H MANG VIET NAM Trdn Dinh Toai 'Nguyen Van ThieY, Le Mai Huang', Nguyen Thanh Do Trung Sy', Nguyen Kim Phuang", Tran thi Hong' I- I len KHdiCN \'ict Sam, 2- Vien Duac lieu 3d Y ie 3- Tmcmg DHKHTN' DKQG Ha Noi Ilanh' Summary Study of Exrracrion anci Application of Biologically Active Toxins from Vietnamese Aaya naja Venom Biologically active components have been extracted from snake venom us:ni^ column chromatography with superdex - 75, enzyme and toxin prepara:u)ns have been ooiained The puot production of massage cream wiih punned loxm extracted from \aja naja venom and Abater - soLuhie excipients has been earned out: 'Watox'* cream •Wiitox" cream, in addition to antihacierial acUvity against Escnericnui coli and P^eudomonas aeruginosa, is effective m the treatment of neuraig'ui myaigia myosins and jnronic arihrai'^ia In comparison wuh massage cream made from crude snal " d u n i r u u y trinh tach n e n g biet timg - h a n c:; hoa^ linh s^j^h hoc lii no'c ran voi m u c dich ;hu duoc nieu q u a cao ahai Do dat d u r e m u c JIL." n a \ ^huniz '^^' ^^ ^^^ ^"^^^ SuperGex-:-^ lam lat mang n o cot sac kv x l C m m duoc can baniz boi d u n " d;ch Tach cac phan dc:an prciem duoc ticn hann j ^ ^ ^,^f^^_ ^^^ ^^ ^uacW^n hanh k-n m a v ' : ? L C boi cac pnuong phap sac ky nhu sac ky ;rao doi fpi^h Periormance L:ou:d Chrcmaic^rarnv Lon ircn cot v6i C \ [ - va DEAiZ-c^iluiosc sac kv saniz loc phan tu tren coi voi Seonoex G-T5 , ^^'- " ^ " ^ P " ^ ^-^ ^''^ ^ ^ '^^^ ^ ^ ^ ' ^ ^^' superdcx-75 va sac kv hao phu tren 2ei Ca-P '°r^'"' '™"§ '""^ ^"= " S " - ^ ^^'" ^^'^^ - " - ' ^ •' ^ ' ' tjcu duac kicm ira ham luang proicji c ITJnh I j n - Phuang phap thu hoai tinh smh hoc Dua vao thi proiem cho phep ihu cac phan TT • i_ uu • , ^.; - tk'.r.^ nh-.n T-^,-^ n doan chiia cac chan co hoai ::r.n iinh hoc Hoat tmh sinh hoc cua cac tnann pnan tacn ^ ^ tir npc ran hO mang duoc tien hinh theo phuang *^^^^ ^ ^ ^ • phap Vanden Bergher va Vlietmck ("; tai phong Sinh hoc thuc nghiem, Vien Hoa hoc cdc o,4 htTp chat Tliien nhien, Vien Khoa hoc va Cong nshe Viet Nam I c - Phuong phap thu duac ly cua cac che pnam Đ J: ,|^ ô^ *ã ^ xoa bop tir toxm Phuons phao bao che va thu duoc ly caa cac che phiim'xoa bop tu toxm duoc tien hann tai Khoa duac ly, Vien Duac lieu Bo y te - Cong thuc b a o c h e k e m xoa bop: Dd so sanh tac d u n g giam dau cua toxm, chiing toi bao c h e Icai kem Mot loai tir toxin tmh sach, mot loai tir n o c ran va tinh toan "•^ T^ „ ^,^r r-in"' Tuong cho luane toxm t r o n e K e m noc ran luuuu • , ^ ••k'^rn Tnvn" d u a n g voi luang t o x m trcng K e m toxm K e m noc rdn: N o c ran d o n e kho 0.02g; m o • ^^^f^(^r nen luuug -jr^ ,J -•— iO 10 :o •iC 50 "0 •:0 •u ^_^^ ^^ ^^^^^ ^^^ ^ ^ ^^^^^ ^^^^^^^ ,^^^^ ^^^ ^^^^ ^^^^^ ^j^- ^y i.^.^oauciease; 2-?hosphoiipase; 3-Proieaie; -UTjxm , ^ , ; / - - Xuc dinh trorvj luan'' pnan tu cua cac ilianii ^ ^ ^ ^ ^K pnan iron''noc ran no man^ K ^ Ki'ch thuoc phan tir c i a cac 'iiann m a n HOC ran ho m a n e duoc xac dinh dua vao cac Tap chi Duac lieu, tap IL so 312006 Marker chu^n: Ribonuclease A (dimer, 27,4 kDa), Trypsin (24 kDa), Trypsin Inhibitor (20kDa) va Insulin (5,75 kDa) Ket qua sic ky cac Marker chuin duoc trinh hav o hinh luong phan tir chimg 6-7 kDa Ket qua nav phu hap voi ket qua cua mot so tac gia da cong bo tnxac day ve toxin tir noc ran ho mang Naja naja Viet Nam [9] Ket qua khao sat hoat tinh smh hoc cac che phim noc r ^ h6 mang cho ihay cung nhu phospholipase, toxm co hoat tinh khang vi smh vat kiem dinh (bang 1) Bang Hoal linh khang vi smn vat ka^m dmh ciia cdc che phaim tir noc ran hO mang Nong (iz cne toi thieu MIC, ugymj No Vi khuan Qr{-: Mau Hinh D6 ihi chuan de xac dmh luong phan tir cac che phdm Markers: 1- Ribonuclease A 2- Trypsir^ 3- Trypsm Inhibitor, 4- Insulin £ coil ' 1 PhosDHoiiDase Tir cac gia tn luang phan tir cua cac Marker chuan xay dung thi chuan de xac dinh luang phan tir cac che pham noc ran ho mane Ket aua cho thav toxin co Nam men P ' Berjc:rass j S ' ' C albicansi cer^vtsiae 50 [-1 L', Toxin - Sudung toxin tinh sach tic noc rdn de san xuat kem xoa bop Bang St) sanh tac dung duac ly ciia che pham xoa bop tu loxm VCTI che pham \ca hop \u noc ran (T= Thai ;:ian chiu dau ciia chuol ) Tj I giay) Lo thi nghiem % Uing so voi To 23.1 r2.1 Doi chirng uang Q = 18 : i , r 1.: Doi chung aen D= 19 Mo noc ran Mo I ox in ,3-1 = 1-5 13.8 = 1.: % uing so voi To 23.0 = 2,6 Tang 8,4'^c Tang 4.:^c Tang S.Q^c P-.*-> 0,05 ?*.**-> 0.0: '.*•-"> 0.05 21.5 = 2.0 23.1 =2.2 23.3 = 2.6 Giam 5.3'^c Tang l.S'~c Tan 2,6^ P-,">0,05 >*,"""> 0.0: 25-4=1.7 29.6=3.0 28.5=3.0 Tang 9.8^0 Tang 28.4f-c Tano 23.5^ ?*.•"*> 0.05 P*.-""*=0.05 ?*.•""•> 0.05 37.5=59 45.2 = 49.5=9,9 Tang 51,6% Tang 90.2% Tang 108,1^^0 PV"'6i: •Oncjr CJ;! ;nnici[>-:rs jnd Me:hod" claims prcducrs for treatmu cancer usme Kaoirce .Atropor.n ir J combinaiicn: 'i^ccr.\z'.z\ \ :)S(;\ Pracncai Hanubook on Enzvmeology "' Hign shccoi", Moscow, 2"2 p; Vanden 3er;^cr ''^".•x'cj'.c:< A J ;'^J! ; .Method 01 Plant 3iochcmistrv, -17-/^9; S Turner R.\ (1965) Scrceninti mc:hcds :n pnamiacoici;y .i,cacemic Press Nc'.v York ang London, 100 - 108; Hoiing Ngoc Ann; Dang Trln Koang; Tmong Nam Hai -Ciao sat va :ach :o.\in xis noc ran hd manu Viet Nam (-V Vaja) TuvCn tap bao cao !"l6i nghi khoa hoc Vien Hoa hoc Vicn K H i O V N , Ha Noi 7:ianizl2 nam 2C05 Tr: '9-83 (Tiep theo trang 142) Ve hinh thtic hoa sy Nguyen Tnanh Dam uy vien Thu ky Hoi dong cham Sacr dec r.njn ;' ::"joc X Cac r:e: qua r ii r 11 IT c." c u r r c c^ c t a c r r s n c L ::c:"u:otcx::: ncac rvtotc:::;: c-^.i r;e.T.a'.ox:r u6c L '.'C^ rr.uu _ - Cac en::-:ne :i:r ma-/ :;-an- r L.3' C enz".':y-e ".r.uoc ]Cr tr.u' ::r.c;" : a r: cua r.o': rar — :>: r.::uor cuoc :ieu tn uac trunp cnc r.Tccr ger quy nitr :r.: r.au r.r.iet dc: nor.g i.rr '/:e: Xa.T : Phosonoiinase va Protease nnieu non zn cue ".,.- ' IP* •.'-' naja I m U ~ ~L ]'2 C tu t,' li, : t:er nanr ngr.ien ^ u rr.a.te (Naja t:r.r s;r.h hoc - N g u y e n ly: ARN h a p p h u cue dai d buoc •enzyme, toxmj n h i m t i m h i e u n h i i n g gia t n song 260 n m Khi ARN bi t h u y p h a n m a t JUoc heu cua no p h u c vu t r o n g y hoc e quang n PHUONG PHAP NGHIEN G i n ; ciia dung dich tang (hieu ung h y p e r c h r o m ) , D u a vao su t h a y doi OD^on ta H o a c h a t dupe h e a t t i n h cua R i b o n u c l e a s e [7], • N'oc r i n ho m a n g (Naja naja) dong kho (ijang a m p u i g) duOc m u a cii i a n g nghe nuoi - Don VI h o a t dp: Mot don \q h o a t dp ciia Ribonuclease la luong e n z y m e xuc tac thiiy ran Vinh Son h u y e n V i n h T u d n g , t i n h V m h p h a n ARN t r o n g cac dieu kien toi Uu (nong Phuc co c h a t bao hoa t"C p h o n g pH to'i Uu) tam - Hoa cnat u n h khiet ciia h a n g Sigma: Curat t a n g m a t q u a n e cua hon hop p h a n u n g (1 Gtvcme (Glvv Superdex-75 Auenm nbonucieotide mi) tai bade song h a p t h u 260 nm len 0.01 don vi sau 60 giav p h a n ung, Hoat dp e n z y m e uuoc u-uc v.ep tren :na\- T h i e : b i : - FPLC I Float Performance L;qu:d quang Diode Aimav SP ^452 Hewlett P a c k a r d hoac tren inay Gene Quar.L AKX/AD.V t."i"iroinatoei*aphy) calculator • HPLC (Hie:h Performance Liquid - Phuang phap xac dinh hoct C'lromaLo^.-apnv - Sac ky long hieu n a n s c a o X g u v e n tac cua pnuong pnap; p n o s p n o h p a s e - Quant^ tu ngoai :-;uc tac phan u n g chuyen hoa le-rirhm : h a n h \\[W '.].- ".am ' a n h m.ay khu;i\' :u ivsoiec'.tnin va 'Ziai ohop.g ix:: cc; ",u •>! Do :] P h u o n ' p h a p n^hicMi c u u a Phan , tinh sinh tdch cdc hoc cua noc phan \:.:cc co hoat WA P h a n tacn cac t h a n n pna:-; co aoat -cr.r ^\r.r r.cx cua ;ioc can duoc u e n ':\ur.:' oa;::; p^uonc^ phap sac ky voi cot S u p e r d e \ X :i:0 :^ -Cm.m; !em ' n t r a t pH = 2.51 id hoat phap xac dinh protein enzyme JO'; ;u :•• ' u- I:-T.'Z '•'".ihm m.iu dan 'jui r.oa: t m n \x:t oeo '-u lO la:n mat cua aiau '.vsoiecuhm :ao tiiar.r p.oac cdjr.'j !i rdn lUjr-z cho pi"! en piKjsorici'-'pase b Cdc phuang phospholipase cua cresoi Cresoi co ':uc :'.;: h a p :hu o buoc song 57S nm - Phuang phap xac dinh hoat protea.'ie Xgu'cei"; tac cua phUOng pi:ap p i ' o t e a s e xuc rac p h a n Ung t h u y p h a n protein t n a n h p e p t i a e va ammo ax:t Tien h a n h p h a n ung t h u y phan • Phaang phap xac dinh protein casein- protein du trU b a n g protease tach H a m luong p r o t e i n t r o n g cac d a n g mdu noc noc r a n Do s a n p h a m tao t h a n h b a n g p h u o n g ran va d u n g dich d a o c xac d m h b a n g p h u d n g p h a p q u a n g d buoc song 2S0n.m- T u xac oi-.ap q u a n g pho: h a p t h u cue da: cua c.Mr.% aicn dinh hoat cua p r o t e a s e arotein o cac bucc song ^SOnm iu'Oc :ren c Phuang mav q u a n g pno tu n g o a : LX 1601 i n i m a a z u Doc :inh ciia cac che pham, toxm :ach tu noc phdp thii doc tinh cua toxin sau ham lucng prote-.n aucc xac uiru: :neo can ducc : h u hoat tinh tren ':nu6t aacn Co7Bl/6 o;eu [61 VOI ca hai gtong c6 t r o n g lucng l X g Che - Phuong phap xac dinii noa: dd cua Ribonuclease A d e n i n r b o n u c i e o t : d e iARX'^ ^a cc cr.a: dac liieu ciia R i b o n u c l e a s e m p h a m toxm d a n g d u n g dich duoc t:em vao bap :hit chuot vci lieu lucng Xmii/ccn Im.; che pham c h u a 0Xi2 m.g toxm Do doc ch:a lam .3 muc do: - K h o n e doc - Doc va - Doc chet [51 d Phuang phdp khao sdt hoat tin/: sinii hoc Lac ;:;ir, ixaat >;i' noa; t m n sn-,:: ;i(ic j u o c tXc .tan: ta: priop.t: Sir.n hnr thUc ]"igiii('':r V-.'cr Hca Inn: cac Ho;- c n a : Thitm niiidn \'icr Khoa a^>' -,-; ::i - o n 1" r pna:.1 ci^a a.oc a ca• c enz y m t \-a t u n g c a r a.a:aiL- :a '-au :;unc sat: sac ^ aeu a u n r ;:ie:-a :aa a a: r ! t:a a a a '" a i '^ •"r ^Hma •- Dua vao tn.: pro;:e:; : t •VC: -:e: cua :iiu cioc n a cu: uox :n cu:~ a ::a - a: J C.T c:: c r "•;] ::" a CnC •j ;_'->_;- :ana -^ :-.ar "' aoa ' :;•'''• ::.:iX :"i(-:";c MIC a;' r „ enz\"m.f' :\r c aua c^ir ' • • " • ' - - - .-., ^ ' r , - •.-•• :i ;:ur Hi i-:i:TQL"A\'A BAX LXAX I^han tacdi cue ; r IT -mi": h o c cun ihanh p h d n oo n o a ' n o c :-an h a n e vol ^j'.• k' o, ,-'u nci dcN-7r) H / n / j :: Ale; c:uc va li X j c dmii ironci l u o n a n n a n lu cua c a c c h a i u o n d noc run ho n i a n a r\ ^ ; A' V ' V" tr-ao ca- ^•e :'•:•: \ \e• Xam _- • r e a co 5C ao • Hinh I Ac: (7i^c Ji'ac aa^.a -""orcac ,"''.': aep de ;';ac uma T.c.a.a Ti-t* cua aac cia^a piidn doan sac rv: "-, ^-'rotease: 4- Toxm ' u a It r a a a a t u cac c a e ran no Ribonuclease co toi p h a n d e a n t u o n g u n g 40 VOI cac oaa t n lupng p h a n :u khac n h a u la 37.5; 19 Gia t n t r o n g lucng p.".an tu cao 3C n h a t cua e n z y m e t h u y p h a n nav a 37.5 kDa ohu hop VOI ket qua xac dinh ucng p h a n tu b ^ n g sac ky sang loc p h a n :u vci chat m a n g la Sepitauex G-7o P h o s p h o l i p a s e c6 lucng ohdr tu 14-15 kDa, X h u vav, :rong luong a h a a tii cua P h c s o h o i i p a s e noc can ho m.aag-V.a:c -laic \':et Xam gar g:6ng vc: ket CiUa -cr.z "' "::h:1; va: E !ui via P aeruginosa -• v.a x:c:n '.a U'uc'u I 5'"' Lia^ani Phosnl";oim;iso a:vj :a: l:'. .; :' ag hd ban '.•hai: la :n6t toxm aoi la Heracx:: a 'ac dung uc cae v^rus Heaa-^- :fu"aalex ar ca tach :ox: ho mang Viet Nam N S'a:a T '•ao Hoi aghi knoa ace \ :tlm; ,1 aoc KHdiCNXdet Xam Ha Xoi T: 2(:X5 Tr: 79-S3 (2005i Xg^a',-en Van Thiec Sgiiien cuu hoac :: ribonucleolytic ciia noc ran ho mang V Nam Naja najaj Tap chi Ducc leu T "• tr; 151-135 (2002) Brockerhoff H Jensen R '7 Lipolytic cnz^m ^:XB-XIir' Mcscc^v : " - ; l - " Vanuen Bergher D A Vaetmck \ "Screening methods Xr zn::bcc:cr:ai z zniicirai agents f^om nighe^ alant' \Ie':lacd :n Pla.nt Bicca.ema.stc" • •^ Claaae; i'-d9 ::991j XS aatent??: 5.565.431 Cancer Ccd Inhibi:< :rznd Method", claims products 'or treat: ns jancer using Kaotree Airoponn or a combination (1996) ^^ '^>^ann o h ^ n hoa hoc va hoat tinh chong oxi hoa c u a t m h d a u q u a tnia la S^^i^eclens L.jViet N a m ^*"^^:cai composition aiid anuoxidative activity oi dill 'anethum c\ne:hum 50 graveciens L -essent;ai oil '•^ ^':einam Trdn Thu Huang, Ngu:en Dac V:nh 11 Nghien cuu p h a n u n g tao phuc c h a t g:Ua CodlO va N i ( n ) vdi thuoc t h u PAN :rang :n6i t r u o n g T.axen d a n h gia k h a n a n g ap d u n g vao p h a n tich d o n g thdi CoiII.' -/a X:XI 00 Study of com.plex f o r m a t i o n r e a c t i o n s of Co(II) N i d i ) a n d PAN r e a g e n t m miceilar m.edia Chu Dinn Bin'n P':c:n: Luan 12 Phan v.cn xac d i n h d u luong ':::e:hv:si:lbestroi mot so loai t h u va r a u a a De:eraa:aa.t:on of t h e ct^ntents -f c:etn',-:st:lbestroi :n :neats and e s ^ s Nguyen XX Thanh Binh P c a a j T': -: XX.v, -Vg:^_VLa'7 Tai Luang Nguven Thanh Minh Ngucen Vzn Huns 13,Xg.^.iea cuu cniet t a c h va t m n cnat cua :oxm va m.ot so enzvm t h u v p h a a :a ac:;: :;ah smn a:c :u ape r a n no mamz V:.:^: Xam NAJA NAJA :ir :;:ucv jii rne :s(jjai:on oaa a r a p e r t i e s of luxin anu -oine Ircuclv'Sr •a^c-ca-r; :c-::n \ e : a, a X U \ X U A m a K e -•mom Tran Din/j T'O: T-"::: To.: Hois I Xac u a : : a a m Xronc: ;n'X -''- aju"*-d: vl •,a iuaai: va ia; a:ea"; ":-a;";a :,a 'cd^a - u " ' l-^aa,' D''ua''n:a.i:;u!": o[ -^om'" :n:c;-' a'/:ne:::s Hucai: [\h'^ - Ha Tin:: m a cac^^ ••;:•':": ;:: -au )i l-'ac.c Z: i-c ,,a: : Lam \'s> T'r: X \::r L or '.'' \oc :::ia uanc: •.'e' X^r^'r: 1!I' v,::a \:r.v.cr.'j: phap loc^ aoc - "rac a.:.;:":-: :u.: "c-a :::a auiimz uc a:o OOAC u::a j : u a X a a a a m a : va KaJii)romua 'rone: moi :auanc: la:: -u:::a:a': Determa.nation oi traci? imoun.cs oi AsiIII) based on :ts mdiibition •^i:eci }n "a.c c u a x ••"actaj:: :i^'cx*^i"0 !-;,ihua: a r a a a ; : r m a p;ai!i:m aromiae :n suitua:c ic:a •:""