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

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 cu

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 H O C Q U O C GIA HA ^ ^ ,

•RUNGTAM I HONG TIN THU Vl£N Di/IM

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 thanh phan co hoal tinh sinh hoc trong noc ran ho mang

- Xac dinh trong 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 ra

- Cong bo 01 bai bao tren Tap chi Duac Heu; 01 bai tren Tap chi phan tfcl Hoa, Ly va Sinh hoc

TRLONG DAI HOC KHOA HOC TUNHIEN

CS.IS I/i4i/w

Bachelor Nguyen Thanh Hanh

4 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

5 Results

- Carry out outlined plan

- Published two articles, one is on Journal of Medicinal Materials and other

MUC LUC

ChUdng 1: Tong quan 5

1.1 Cac chat co hoat tinh sinh hoc lij noc ran 5

1.1.1 - Toxin tu noc ran 5

1.1.2 - Enzyme 6 1.2 Kha nang ling dung cac chat co hoat tinh sinh hoc tach tir noc rdn 8

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 thanh phan co hoat tmh sinh hoc cua noc ran 10

2.3.2 - Cac phuang phap xac dinh protein va h(xil do en/yme 10

Chuang 3: Ket qua va ban luan 12

3.1 Phan tach cac thanh phan co hoat ti'nh sinh hoc (HTSH) trong noc 12

rdn

3 2 Xac dinh trong luang phan tir cua cac chat trong 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 trong 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 trong nhung nguon gen quy hiem do 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 9 Nhung viec nghien cijru

sir dijng nguon nguyen lieu do 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 trong phong

thi nghiem, da co nhung ket qua ling diing nhu su dung noc tho lam duac pham chong dau xuang khap con 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 trong so do la cac protein Vc ban chat cau true, cac protein nay chia lam 2 loai: enzyme va toxin

Cac toxin trong 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 trong noc ran deu la cac enzyme thuoc lap thu> phan Hydrolase [2] Trong so cac enzyme do thi Ribonuclease, Phospholipase

va Protease duac quan tam nhieu han ca

Cac ket qua trong phong thi nghiem mai chi diing a cho tach rieng / loai thanh phan trong noc rdn la toxin va enzyme Vi vay hieu qua sir dun^ cua tung thanh phan rieng biet trong noc r^n con ban che do 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 khi hau nhiet dai nong aim Vie

Nam, chiing toi de xuat thuc hien dc tai: "Nghien cuu uk h c hat co hoat tim

CHUONG 1: TONG QUAN

1.1 Cac chat co hoat tinh sinh hoc tu noc rdn

Noc rdn liic m6i tiet ra 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 trong so do la cac enzyme Tuy nhien, doc tinh ciia noc chu yeu do cac toxin co ban chat peptit va protein phan tu luang thap gay ra

Cac thanh phSn co hoat tinh sinh hoc trong noc rdn, \'^ ban chat cau true, CO the chia lam 2 loai: protein va khong protein Loai thanh phan protein lai chia lam 2 loai: cac enzyme va cac toxin Loai thanh 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 ra cac hien tuang nguy hiem a viing mo nhiem doc cue bo, lam giam kha nang dong mau, lam ton thuang thanh mach mau trong va d^n den giam kha nang bao ve ciia thanh 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 nay 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: khi 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 con moi va co the gay ra tu vong

1.1.1 - Toxin tir noc r^n

Cac toxin trong 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 thanh ph^n protein chinh trong noc cac loai rdn Agki,

cac thanh phan nay 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 trong noc ran Naju Cat Neurotoxin co dac trung cobra Mot so Naja neurotoxin rat giong nhau \x

trinh tu amino axit Vi du: cobrotoxin III va cobrotoxin IV sions nhau ta

93% Neurotoxin 6 va Neurotoxin 7 giong nhau 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 trong noc ran Naia

Cardiotoxin

Nguai la da tim thay 14 ho (families) Cardiotoxin trong noc ran Trong

do CO 6 ho tim thay trong noc rdn Agki Hai ho vixd tim thay trong noc ran

Agki va trong noc r4n Naja Con lai bon ho tim thay trong noc ran Niper

Trong noc rin Naja, Cardiotoxin co rat nhidu chiing c6 trong 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 4 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 trong nuac Hoat tinh bi mat khi co mat cac chat khu , rat b^n a nhiet do 20°C a-Cobraloxin lac dong nhu mot chat doi khang (antagonist) chong chat nhan (receptors) nicotinic acetylcholine trong chiic nang liep hap than kinh ca (neuromuscular junctions)

Nguai la tach duac 2 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

trong luang phan tu 35.000 va 6.000 dalton tuang ling Hai protein nay co lac dong phoi hap vai nhau lam tang boat tinh lieu diet te bao trong he in vitro va

in vivo, 0,5 meg lieu diet hoan loan 10'' le bao ung thu cac loai khac nhau 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 trong 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 nay d6u thuoc lap phu cac enzyme tac dong tai lien ket Ester (EC 3.1)

CO kim loai (metalloproteinase) [16-17] Cac metalloproteinase la cac haemoloxin Vi du: Proteinase a; Proteinase b; Proteinase c tii' noc ran Nhal

Ban Agkistrodon halys blomhojfii (Mamushi) cac proteinase co kim loai

[18-20]

Cac proteinase co kim loai (Metalloproteinase) ' tijr noc cac rdn

Agkistrodon thuang co trong lugng phan iii khoang 30-60 kDa [21]

Ngoai cac proteinase, trong noc ran con co cac protein khac Cac

protein rat dac trung cho noc r^n ho mang Naja la latisemin, ophanin va nalrin, dac trung cho noc ran Viper la saxalilin, calrocollastatin and

mamushigin, khong co protein nao dac trung cho ca hai loai noc neu tren [8]

Phospholipase

Cac phospholipase chia lam 2 nhom :

Phospholipase A (EC 3.1.1.4) va Phospholipase A^ (EC 3.1.1.32) thuoc nhom cac phospholipase thuy phan lien ket Carboxy Ester (EC 3.1.1 Carboxylic Ester Hydrolases)

Phospholipase C (EC 3.1.4.3) va Phospholipase D (EC 3.1.4.4) thuoc nhom cac phospholipase thuy phan lien kei phosphodiester (EC 3.1.4 Phosphoric Diester Hydrolases)

Trong cac phospholipase thi phospholipase A co y nghTa ban ca' Phospholipase A2 lac dong tai \i iri C-2 trong phospholipid, giai phong axil arachidonic Axil arachidonic duac giai phong sc la ca chat de tong hap prostaglandins va leukotrien

Theo cac nghien cuu \6 Ribonuclease tu noc ran ho mang \aing Trung A ciia Lien X6 cu, boat tinh Ribonuclease trong noc cua ba loai ran Vipera

lehetina, Naja oxyana va Echis corinatus co the khac biet nhau tai 10 l^n, trong

do noc ran ho mang Naja oxxana co boat tinh Ribonuclease cao nhal Ribonuclease lay tCr noc ciia ran ho mang Naja oxyana co pH 161 uu la 7,6 - 7,8,

duac boat hoa bai ion Mg"^ va khong thuy phan ADN Enzyme nay co do ben vai nhiet khong cao, mat 50% boat tinh khi dun each thuy 5 phiil a 50°C va mat hoan loan boat tinh khi xu ly a 70'G San pham ciia phan ling thuy phan ARN do enzyme nay xiic lac la hon hap cac oligonucleotide tu dimer den hexamer (chii yeu la dimer va irimer) [22]

Ribonuclease tinh khiet \ii noc ran ho mang Naja naja a vung Guntur cua

An Do duac nghien cuu rat ly m\ cac dieu kien anh huang tai hoal tinh cua

enzvmc [23] Enzvme nay hoal done tot tron^ khoane nhiet do tu 30 - 60"C \"ai

nhiet do loi uu la 37°C Mot so ion kim loai nhu Mg-"", Ni-^"" va Mn'* \ai n6ng do thap lam tang hoat tinh enzyme, nhung a nong do cao lai tie che manh Mot so

ion kim loai khac chi lai \xc che vai bat ky n6ng do nao nhu Zn""", Cu'^ Hg" dac

biet la Zn^"" lic che rat manh a nong do 2.5 mM (chi con 9% boat tinh)

Nhidu kim loai khac anh hucmg phuc tap len boat tinh: K"", Ca""" a nong do

thap (0,2 va 0,4 mM) tie che enzyme, nhung a nong do cao (>2,5 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], con Ribonuclease tu noc rkn ho mang vung Trung A Lien Xo la

endoribonuclease, rat giong deoxyribonuclease tu tuyen tuy ciia dong vat, khong

CO tinh dac hieu vcd base niia, chi khac biet a cho enzyme nay 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 trong noc ran doc Chau Phi (Dendroaspis), chiing chiia 57-60 goc axil amin Cac toxin nay tac dung len kenh th^n kinh kali Nhirng phat hien nay hua hen se che lao ra nhOng thuoc mdi chOa cac benh than kinh nhu benh Alzheimer, benh dong kinh Mot vai ket qua khac trong 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 nay da thu but su quan lam ciia cac hang nhu hang Wyelh Ayerst

Trong ca the l6n tai enzyme bien doi angiolesin (ACE) Enzyme nay 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 ra 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 ra cac phan tu peptide nho de lam thuoc kiem tra su dong cue mau khong binh thuang

Trong so cac enzyme duac phan tach ra tu noc r4n ho mang nhu Ribonuclease, Phospholipase A2 Lipase thi Ribonuclease (RNase) da duac

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 trong te bao [26] Cac ch^' pham boat boa Ribonuclease L nhu oragen co kha nang tie che qua trinh nhan doi ADN virus trong cac dai thuc bao mang bung in vitro va trong 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 nay da ma ra mc)l huang nghien cuu mai nham tao ra cac che pham duac cao cap de san xuai thuoc chiJa ung thu

Theo thong bao ngay 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 trong boa tri lieu chong ung thu tuyen tuy

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

do benh ung thu nay Nguai ta tinh cho tai nam 2012 thi thuoc chiia NSCLC phai len tai 4 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 nay

CHUONG 2: PHLONG PHAP NGHIEN ClT^

2.1 Hoa chat

- Noc rdn ho mang {Naja naja) dong kho (dang ampul 5 gj dupe mua lu"

lang nghe nuoi r^n VTnh San, huyen VTnh Tuang, tinh VTnh Phiic

- Hoa chat tinh khiet PA:

- FPLC (Float Performance Liquid Chromatography)

- HPLC (High Performance Liquid Chromatography - Sac k\ K)ng hicu nang cao)

- Quang pho tii ngoai

- May ly tam lanh, may khuay \\x

2.3 - Phuong phap nghien cuu

2.3.1 - Phan tach cac thanh phan co hoat tinh sinh hoc cua noc ran

Phan tach cac thanh phan co hoat tinh sinh hoc ciia npc ran duac tien hanh b^ng phuang phap sdc ky \'ai col Superdex-75 (60 x 20mm) dem citrat,

p H - 2 , 5 1

2.3.2 - Cac phuang phap xac dinh protein va hoat do enzyme

- Phuang phap xac dinh protein

Ham luang protein trong cac dang m^u npc ran va dung dich duac xac dinh b^ng phuang phap quang pho: hap thu cue dai cua dung dich protein a cac buac song 280nm duac do tren may quang pho tu ngoai UV16()1 Shimazu, sau do ham luc^fng protein duac xac dinh theo bicu do [29]

- Phuang phap xac dinh hoat do ciia Ribonuclease

Adenin ribonucleotide (ARN) la co' chat dac hicu cua Ribonu^lcaNC

- Nguyen ly: ARN hap phu cue dai a buac song 260 nm Khi ARN bi

thuy phan mat dp quang ciia dung dich lang (hieu ling hyperchrom) Dua vao

su thay doi OD260 ^a do duac boat tinh ciia Ribonuclease [5]

- Dan vi boat dp: Mot dan vi hoat dp cua Ribonuclease la luang enzyme xiic lac Ihiiy phan ARN trong cac dieu kien toi uu (n6ng do ca chat bao hoa

fC phong, pH toi uu), lam lang mat dp quang ciia hon hap phan ung (1 ml) tai

bu6c song hap thii 260 nm len 0,01 don vi sau 60 giay phan ung

Hoat dp enzyme duac do true liep tren may quang pho Diode Array SP

8452 Hewlett Packard hoac tren may Gene Quant ARN/ADN calculator

- Phuang phap xac dinh hoat dp phospholipase

Nguyen tdc cua phuang phap: phospholipase xiic tac phan ling chuyen boa lecithin thanh lysoleciihin va giai phong axit beo lu do [6] Do duac luang axil beo tu do hoac luang lysoleciihin tao thanh hoac luang lecithin mat di cho phep danh gia hoal tinh ciia phospholypase Axil beo tu do lam mat mdu ciia cresol do Cresol do co cue dai hap thu a buac song 578 nm

- Phuang phap xac dinh hoat do protease

Nguyen tac cua phuang phap: protease xiic tac phan ung thuy phan protein thanh peptide va amino axit Tien hanh phan ung thuy phan casein- protein du triJ bang protease tach ra noc rdn Do san pham lao thanh bang phuang phap quang pho a buac song 280nm Tu do xac dinh boat dp ciia protease

11

CHUONG 3: KET QUA \ A BAN LUAN

3.1 Phan tach cac thanh phdn co hoat tinh sinh hoc (HTSH) trong noc r^n

De' tach rieng biet timg thanh phdn co HTSH tu npc rAn chiing toi kra chpn SuperDex G 75 lam chat mang cho cot s^c ky 60xlOmm duac can bang bai dung dich dem citrat SuperDex G 75 cho phep sang Ipc phan tu co kich thuac tu 3.000 den 60.000 Da Sic ky duac tien hanh tren may FPLC (Floating Performance Liquid Chromatography)

M6i Ian tien hanh sac ky luang npc r^n tho duac su dung la 800mg The^ tich

dung dich thu duac sau skc ky la 60ml bo 15ml dau, the ti'ch con lai chia thanh 90 phan doan, m6i phan doan 0.5ml Dua vao hoat tinh enzyme thu duac va 66 thi

protein, thu cac phan doan chiia cac thanh ph^n co HTSH khac nhau: Ribonuclease, Phospholipase, Protease Toxin (bang 1)

Bang 1 - Thanh phan cac phan doan thu duac sau s^c ky

No Thanh phan cac phan doan

220mg ~ 27.5%

174.8mg~ 21.85%

176.5mg~22.06257o Cac thanh phan khac nhau cua noc rin trong tiing ong nghiem deu duac kiem tra ham luang protein va hoal tinh cac enzyme Ket qua thu duac trinh bay trong bang bang 2

Bang 2: Ham luung protein va hoat tinh enzyme trong cac ong nghiem (phan doan • 0,5 ml) thu duoc sau s^c ky

n

AE (Ribonuclease)

n 111 :^^

i

AE (Phospholipase)

• 0

i 0 ' 0 ' 0

o.o;

0.032

0.06 0.187 0.125 0.186 0.168 0.11 0.176 0.176

n

1

0,1524 0,1398 0.1318 0,1171 0.1054 ' 0.0918

i 0.0887

0.3057 0.3055 0.3053 i

1

0.3283 0.3290 0.3433 0.3470 0.3420 0.3403 0.3443 0,3475 0,3375 0.2924 0.2429 i 0.2095 0,1528 0,1471 0.1264 0.1384 0.1464 0.1542

! 0.1735

0.0346 0.0413 0,0619 1 0.069 0,0936 0,0969 0,0979 0,0989 0,0681 0,0549 0,0422 0,0535 0,0552 0,0497 0,0519 0,0465 0,0464 0,0272 0,0183 0,0182 0,0204 0.0108 0,0149

0.153 0.179 0.159 0.09 0.066 0.074 0.075 0.068

0.1450 0.1300 0.1318 0.1225 0.1274 0.1272 0.1139 0.1108 0.1046 0.1164 0.1172 0.1082 0.1006 0.1017

1 0.0937 0.0771 0.0769

0.0062 0.0048 0.0038

0

0

0,0003 0.0025

Tir cac ket qua thu duac trinh bay trong bang, ve d6 thi boat tinh enzyme

trong cac ong nghiem (phan doan - 0.5 ml) thu duac sau siic ky (Hinh 1)

10 20 30 40 50

No phan doan

60 70 80 90

Hinh 5- Tong hap cac ket qua xac dinh Protein va hoat tinh ciia cac enzyme

trong cac phan doan sac ky

3.2 Xac dinh trong lirong phan tu cua cac chat trong noc rdn ho mang

Trong luang phan tu cac thanh ph^n trong noc ran ho mang duac xac dinh

dua vao cac Marker chuan; Ribonuclease A (27,4 kDa), Trypsin (24 kDa) Trypsin Inhibitor (20kDa) va Insulin (5,75 kDa) Ket qua sac ky cac Marker chuain dugc trinh bay tren hinh Bang 4 Tir cac gia tri trong luang phan tu cua cac Marker chuan xay dung do thi chu^n de xac dinh trong luang phan tu cac che pham trong noc ran ho mang (Hinh 6)

35 V,ml

fPinh 6 : Do thi chuan de xac dinh trong luang phan tu cac che pham

Markers: l- Ribonuclease A, 2- Trypsin, 3- Trypsin Inhibitor, 4- Insulin

Tren ca sa do thi chuan (Hinh 6 Bang 2) cho phep de^ xac dinh trong luang phan tir cac che phiim trong noc ran ho mang Ket qua xac dinh trong liKmg phan tu cac che pham trong noc ran ho mang dugc trinh bay trong bang 3

'JC )r • A M A N O ' ri^jNG ^ - ^ T H Q N G TIN IHU viEN

17

Bang 3: Trong luang phan tu ciia cac Marker chuan va cac che pham

trong noc ran ho mang

7-10

6-7

Ket qua xac dinh trong luang phan tu cac che pham trong noc ran h6 mang

Naja naja Viet Nam cho thay:

Ribonuclease co tai 4 phan doan tuang ung vai cac gia tri trong luang phan

tu khac nhau la 37,5; 19; 9 va 4-5 KDa

Phospholipase co trong luang phan tii 14-15 kDa Nhu va\ trong luang phan

lU cua Phospholipase noc ran hc) mang Naja naja Viet Nam g^n giong \a\ ket qua cong bo v6 trong luang phan tu Phospholipase A2 chiet tii noc ran cobra Naja n

kaouthia (Thai Ian), goi la Hcrpoxin co trong luang phan tu 14 kDa [28]

3.3 - Khao sat hoat tinh sinh hoc ciia cac chat tach tir noc ran ho mang

Ket qua khao sat hoat tinh sinh hoc ciia RNase va cac che phaim con lai iii

npc rdn ho mang duac trinh bay trong bang 4

Bang 4: Hoat tinh sinh hoc ciia cac che pham tir noc ran ho mang

Hoat tinh khang vi sinh vat kiem dinh

Cac gia tri trong bang 4 cho thay: Phospholipase thu duac tu noc ran co boat

tinh khang vi khuan P aeruginosa va khang nam men S cerevisiae, con Toxin thu duac tir noc rdn co boat tinh khang vi khuan E coU va P aeruginosa

KET LUAN

De tai da thuc hien tot cac muc tieu va noi dung nghien cuu dat ra:

- Da nghien curu, long quan tai lieu ve cac chat co boat tinh sinh hoc trong noc r^n va kha nang ung dung chiing

- Da phan tach cac chat co hoat tinh sinh hoc tu noc ran ho mang Naja naja

bang phuang phap s^c ky cot vai chat mang la SuperDex G 75

- Da xac dinh trong luang phan tu cua cac chat trong noc ran ho mang, ket qua cho thay, Ribonuclease co lai 4 phan doan tuang ling vai cac gia tri trong luang phan lii khac nhau la 37.5; 19; 9 va 4-5 KDa Phospholipase co trong luang phan tu 14-15 KDa

- Ket qua khao sat boat tinh sinh hoc cua cac chat tach tu noc r^n ho mang cho thay Phospholipase thu duac tij noc ran co hoat tinh khang vi

khuan P aeruginosa va khang nam men S cerevisiae, con Toxin thu duac tiJ noc rin co boat tinh khang vi khuan E coli va P aeruginosa

19