Nghiên cứu tách chiết, tinh chế và tạo chế phẩm chymopapain dược dụng từ nhựa quả đu đủ Việt Nam

• N g h i e n CUMJ - Ky thuat<br /> <br /> <br /> N g h i e n c u u t a c h c h i e t , t i n h c h e v a<br /> <br /> <br /> t a o c h § p h a m c h y m o p a p a i n d i m e d u n g<br /> <br /> <br /> t u n h u a q u a d u d u V i e t N a m<br /> <br /> Nguyen Van Rur, Nguyen Thj Loan 1<br /> <br /> <br /> <br /> <br /> Nguyen Thj Thanh Nhai 2<br /> <br /> <br /> <br /> <br /> 'Truong DH Duqc Ha Noi<br /> 2<br /> Truong Cao dang Duqc TV Hdi Duong<br /> <br /> Dat van de Thiet bi: May do quang pho hip thu U-1800,<br /> Cay 6*u du la mpt trong nhQ'ng loai cay trong may ly tam lanh Hereus 15R, may ly tam<br /> pho bien d nude ta cung nhu a nhieu nude thudng, may dilu nhiet, may ddng khd, kinh<br /> nhiet ddi khac vdi nhieu cong dung trong ddi hiln vi, noi dun each thuy, dung cu thuy tinh khd<br /> song va y hpc. Trong mu du du co ba enzym va sach, tu sly.<br /> chinh, bao gom papain, chymopapain va PhiFong phap nghien cu>u<br /> peptidase ' . Trong do chymopapain (EC<br /> |4 5,141<br /> Phuong phap xac dinh ham lupng<br /> 3.4.22.6) chiem ti le cao. Gan day, enzym nay protein: theo phuong phap Gornal, tinh theo<br /> dang dupe quan tam nhieu va dupe su dung dudng chuin xay dung bing casein.<br /> vao viec chua tri cho cac benh nhan bi kho Phuong phap xac dinh hoat dp protease:<br /> cung cac khdp xuong, dac biet la kho khdp cot Nguyen tic: Dudi tac dung cua enzym,<br /> song lung va co bing each tiem hoSc dip che protein bj thuy phan giai phdng cac acid amin<br /> pham chymopapain ma khong can phai giai tan trong acid tricloacetic. Phin protein cdn lai<br /> phiu sau mpt thdi gian djch nhay d khdp xuong tua trong acid tricloacetic. Tua nay phan ung vdi<br /> dupe phuc hdi '. De gop phin nghien cuu<br /> 1<br /> thuoc thu Gornal tao phuc mau tim hong. Do<br /> v l chymopapain trong cay du du d Viet Nam quang d 550 nm, cuvet 1cm. Dua vao biiu dd<br /> chung toi thuc hien d i tai nay vdi muc tieu la mlu tinh ra hoat tinh cua enzym.<br /> chilt tach va tinh chi dupe chymopapain co dp Tiin hanh: Pha loang enzym din ndng dp thich<br /> tinh sach khac nhau tu nhua qua du du, chuin hpp bing dung djch dem pH 7,0. Su dung co chit<br /> bi tieu chuin hoa d i tao dupe cac chi phim la casein 1%.Cho vao 3 ong nghiem nhu sau:<br /> chymopapain dung lam thuoc va thuc phim Ong co 6ng thtr dng enzym<br /> chuc nang. Hda chat<br /> chat (ml) (ml) (ml)<br /> Nguyen lieu va p h u ' e n g phap Casein 1% 2 2 0<br /> n g h i e n CUPU Oem phosphat<br /> 0,1 0 2<br /> Nguyen lieu: Nhua qua cdn non cua cay du pH 7.2<br /> du (Carica Papaya L, Caricaceae ). Cystein 0.04 M 0,5 0,5 0,5<br /> Hda chat va thuoc thu : Carboxymethyl<br /> 1<br /> Dung dich enzym 0 0,1 0,1<br /> cellulose (CMC), dung dich casein 1%, cystein Mat 66 quang D1 D2 D3<br /> 0.04M, acid tricloacetic 5%, thuoc thu Gornal, dem De 1h u am 37°C, sau dd cho vao mdi dng<br /> phosphate pH=7,0, amonisulphat 45% va 65% 5ml acid tricloacetic 5%. Lie diu d l d nhiet dp<br /> bao hda (bh), dem acetate 0,02 M pH=5 cd EDTA phdng 10 phut, ly tam lay tua. Thuc hien phan<br /> (0 0001 M), dem acetate 0,7 M pH=5, dung dich vdi 5 ml thudc thu Gornal. De on dinh d nhiet dp<br /> HCI d cac nong dp, Na Benzoyl-L-Arginine Ethyl phdng 30 phut, do quang d 550 nm, cuvet 1cm.<br /> Ester (BAEE), Ethylen diamin tetra acetic Acid Dua trdn bieu do mlu tinh ra lupng enzym bj<br /> (EDTA), L-Cystein, Hydrochlorid HCI, Sigma. thuy phan:<br /> <br /> TAP CHI DUQC HQC - 10/2010 (SO 414 NAM 50) 37<br /> • N g h i e n CLPU - K y thuat<br /> <br /> P = P1 + P2 - P3 E. Dung djch chuin (Solution-Standardized)<br /> Trong do: Sodium Hydroxide (NaOH) 100 mM<br /> P1: lugng protein trong ong co chat. (Pha thanh 50 ml trong nude da loai ion lanh,<br /> P2: lugng protein trong Ing Enzym. su dung Sodium Hydroxide, Anhydrous, Sigma<br /> P3: lugng protein trong ong thu. Stock No. 505-8. Standardize according to the<br /> Lugng protein trong m5i 6ng tinh theo ACS Reagent Procedure. 1)<br /> dudng chuan n6ng 66 protein - mat dp quang F. Dung djch enzym chymopapain<br /> Hoat dp protease (HdP) cua chymopapain (Pha trudc khi dung, pha dung dich<br /> dugc tinh theo cong thuc: chymopapain co chua 0,75 - 1,5 units/ml trong<br /> P.B.L.10 6<br /> nude da loai ion)<br /> HdP (nanokatal) = - Tiin hanh:<br /> M.m.T Dung pipett (loai 1ml), cho cac thuic thu<br /> (reagent) sau vao mpt Ing nghiem thich hpp:<br /> Trong do:<br /> P: lugng protein bi thuy phan (mg). Ong thu:<br /> B: dp pha loang cua dung djch che pham + Thuic thu A (BAEE) 7,00 ml<br /> enzym. + Thuoc thu C (Cys) 1,00 ml<br /> L: so lien ket peptid cua protein (cua casein + Thuoc thu D (NaCI) 1,00 ml<br /> Ia 209.10 ). 3<br /> Trpn bing may khuiy giu d 25°C. Diiu chinh<br /> M: trqng lugng phan tu gam cua protein (cua pH din 6,3 d 25°C bing NaOH 0,1 M.<br /> casein la 236.10 ). 5<br /> Sau do them:<br /> T: thdi gian thuy phan (giay). Thuic thu F (Dung dich) 1,00 ml<br /> m: lugng miu thu cua enzym (ml). Diiu chinh pH din 6,2; bit diu tinh thdi gian<br /> Phwong phap xac dinh hoat dp dac hieu phan ung. Tiin hanh phan ung tu 1 - 5 phut.<br /> chymopapain (EC 3.4.22.6) f4A,4J<br /> Duy tri pH hin hgp phan ung d pH 6,2 bing<br /> - Nguyen tic: each them thuoc thu E tung lugng nho (0,025 -<br /> BAEE + H 0 2 > Na-Benzoyl-L-<br /> C h Y m o p a p a i n<br /> 0,050 ml). Ghi the tich thuic thu E da dung de<br /> Arginine + Ethanol duy tri pH d 6,2 va thdi gian tiin hanh.<br /> (BAEE = Na-Benzoyl-L-Arginine Ethyl Ester, - Tinh toan hoat dp chymopapain (HdCP):<br /> Dieu kien: T = 25°C, pH = 6,2) (M).(V aOH).(1000).(df)<br /> N<br /> <br /> <br /> <br /> - Phirong phap: Dinh lugng the tich HdCP (U) /ml enzym =<br /> - Cac thudc thu (reagents) (T).(L)<br /> A. Na-Benzoyl-L-Arginine Ethyl Ester (BAEE) M = Ning dp moi cua NaOH<br /> 80 mM V oH = The tich thuic thu E (ml) dung de<br /> Na<br /> <br /> <br /> (Pha thanh 100 ml trong nude da loai ion, su duy tri pH d 6,2<br /> dung Na-Benzoyl-L-Arginine Ethyl Ester, 1000 = He s i doi tu millimol thanh micromol<br /> Hydrochlorid, Sigma Prod. No. B-4500.) theo dinh nghTa don vj enzym<br /> B. Ethylen diamin tetra acetic Acid (EDTA) df = He s i pha loang<br /> 20mM T = Thdi gian phan ung duy tri pH d 6,2 (phut)<br /> (Pha thanh 100 ml trong nude da loai ion, su L = The tich enzym da su dung (ml)<br /> dung Ethylen diamin tetra acetic Acid, Disodium units/ml enzym<br /> Salt, Dihydrate, Sigma Stock No. ED2SS.) HdCP (U) /mg bot che pham =<br /> C. Dung dich L-Cystein 50 mM, pH 6,2 va mg bot che pham/ml enzym<br /> 25°C (Cys) units/ml enzym<br /> (Pha thanh 50 ml trong thuoc thu B, su dung L- HdCP (U) /mg protein =<br /> Cystein, Hydrochlorid, Monohydrate Sigma Prod. mg protein/ml enzym<br /> No. C-7880. Diiu chinh pH 6,2 bing 1 M NaOH.) Oinh nghTa don vj chymop- .in (U):<br /> D. Dung dich Sodium Chloride (NaCI) 3,0 M Mpt don vj chymopapain I) xuc tac thuy<br /> v<br /> <br /> <br /> (Pha thanh 50 ml trong nude da loai ion, su phan 1,0 umol BAEE thanh Na-benzoyl-L-<br /> dung Sodium Chloride, Sigma Prod. No. S-9625.) arginin trong 1 phut d pH 6,2 va d 25°C.<br /> <br /> 38 TAP CHI Dl/OC HOC - 10/2010 (SO 414 NAM 50)<br />  Nghien ciru - Ky thuat<br /> <br /> - Ket qua dinh lupng miu chuin hoat dp ester, 5 mM L-Cystein, 2 mM acid<br /> enzym chymopapain: Ethylendiamintetraacetic, 300 mM sodium<br /> Su dung M Na-benzoyl-L-arginin ethyl chlorid la 0,75 - 1,5 U chymopapain.<br /> K i t qua t h y c n g h i e m<br /> Xay dung dudng chuan protein<br /> Du'6-ng chuin nfing do p rote In - m At dd quang<br /> <br /> <br /> <br /> <br /> Nong Tua (bo)<br /> • Phan doan NaCI 50% bh, pH 2, qua dem<br /> - Ly tam lanh Dich ly tam<br /> - Phan doan NaCI bh hoan toan, pH 2 -•Djch (bo)<br /> - O i 1 h, ly tam lanh<br /> Tua chymopapain Ddng kho<br /> <br /> Hinh 4: So do qui trinh tinh che chymopapain tir 1b<br /> Kit qua thu dupe trong bang 3:<br /> Bang 3: Kit qua vi protein va HdP cua cac giai doan tach va tinh chi chymopapain<br /> ^~~^~^Th6ng so Khoi Protein Protein HdP tong HdP rieng HdP/ml Dp Hieu<br /> Chat li$u~~~^-~-^^ luong tong (mg) (mg/ml) (nk) (nk/mg) (nk/ml) tinh sach suit<br /> Phandoan 65%<br /> 6,06 (g) 226,6 37,4 260200,0 1267,8 42937,3 1,99 24,0<br /> (NH )2S0 bh<br /> 4 4<br /> <br /> <br /> Pd sau hip phu 130 (ml) 212,3 1,6 241867,3 1139,2 1860,5 1,79 22,3<br /> Phan doan NaCI<br /> 100 (ml) 197,1 1,9 222835,0 1130,4 2228,3 1,78 20,5<br /> 50% bh<br /> Phan doan NaCI bh 2.12(g) 14,9 7,0 25594,7 1717,8 12072,9 2,70 2,4<br /> <br /> <br /> <br /> <br /> Hinh 5: Hinh anh bot dong khd chymopapain<br /> <br /> 40 TAP CHI DUQC HQC - 10/2010 (SO 414 NAM 50)<br /> • N g h i e n CCPU - Ky thuat<br /> <br /> Thir tieu chuan chat luang che phim<br /> <br /> Chymopapain Tieu chuan<br /> TT Chi tieu Phuangphap nghien ciru Che pham chuan<br /> Bpt dong kho mau trang due USP 30 Oat<br /> 1 Tinh chat cam quan Oat<br /> (DO My 30)<br /> 2 pH 6,5-7,5 USP 30 Oat (6,9) Oat (6,8)<br /> Phan img xuc tac cua chymopapain<br /> 3 Oinh tinh: (+) Duong tinh (+)<br /> USP 30<br /> 4 Oo nhiem khuan Oat yeu cau USP 30 / DDVN 3 Oat Oat<br /> Oat hoat lyc chymopapain<br /> 5 Oinh lirong 1717,8 nK/mg 1500,0 nK/mg<br /> USP 30<br /> Oat 0,65- 1,5 U Oat 0,75- 1,5 U<br /> 6 HdCP Xac dinh hoat do dac hieu chymopapain<br /> chymopapain chymopapain<br /> <br /> Ban luan tuong duong vdi cac tieu chuan cua che phnhu<br /> SCr dung phuang phap ket tua phan doan cam quan, dp pH, dp nhiim khuan, hoat dp<br /> [2,4,5,8,9] ^ g o, j ^fljjp, chymopapain tu protease va hoat dp dac hieu 0,65 - 1,50 U<br /> g c c<br /> <br /> <br /> nhua qua du dd Viet Nam. Tiep do su dung cac chymopapain.<br /> phuong phap hap phu tren CMC, tham tach, Summary<br /> phan doan bang NaCI... ' ' da dat ket qua<br /> |4,8 9<br /> Chymopapain (EC. 3.4.22.6 ) is a thiol<br /> budc dau, tao dupe che pham chymopapain cd protease, it was isolated from the latex of the<br /> dp tinh khiet khac nhau la 1a va 1b. Che pham unripe fruit of the papaya (Carica Papaya L,<br /> chymopapain tinh che tu che pham 1b co dp Caricaceae). We was extracted and refined to<br /> tinh sach cao vdi HdP rieng tu 1300 - higher purificatory level of chymopapain for the<br /> 1700nk/mg protein va dp tinh sach la tu 2,0 den pharmaceutical preparations. This study shows<br /> 2,7 lan so vdi che pham 1b ban dau la rat co y that the proteolytic activity of the crude preparation<br /> nghTa ung dung thuc te. Budc dau cung da is 636.26 nk/mg protein. The use some of the<br /> danh gia dupe mpt so tieu chuan ca ban cua precipated technics as the isolated and purified<br /> mpt che pham enzym dung de chi thu6c va with NaCI and (NH ) S0 and adsorbent reaction<br /> 4 2 4<br /> <br /> chung tdi da tien hanh so sanh vdi tieu chuan with carboxymethyl cellulose made from adsorbent<br /> cua che pham chymopapain chuan, ket qua la cotons, the proteolytic activities of refined<br /> kha quan. Tuy v$y, che pham nay vln dang chymopapain is 1717.80 nk/mg protein (hat was<br /> dupe tiep tuc hoan chinh de cd the tieu chuan increased 2.7 fold. The specific activity of<br /> hda dung pha che thanh thudc tiem cung nhu chymopapain (0.65 - 1.5U) was compared with<br /> mdt sd dang thuoc dac biet khac de sdm dupe level of standard preparation (0.75- 1.5U).<br /> su dung trong diiu tri benh cd hieu qua. Tai lieu t h a m k h a o<br /> Ket luan 1. Oo Tit Loi, Nhung cay thuoc va vi thuoc Viet<br /> Tu nhua qua du du tuoi da chiet tach va tinh Nam, NXB Y hoc, (2005).<br /> che bang NaCI tao dupe chymopapain 1a cd 2. Nguyln Due Lugng va cong su Cong nghe<br /> HdP la 1078,4 nk/mg protein vdi dp tinh sach la Enzym, NXB DHQG TP. Ho Chi Minh, 216-227,<br /> 1,57 lan, bang (NH ) S0 thu dupe (2004).<br /> 4 2 4<br /> <br /> <br /> chymopapain 1b cd HdP la 1267,8 nk/mg 3. Nguyln Trong Can, Nguyln Thi Hien, 06 Thi<br /> Giang, Trin Thj Luyln, Cong nghe enzym, NXB Ndng<br /> protein vdi dp tinh sach la 1,99 lan.<br /> Nghiep TP. Ho Chi Minh, (1998).<br /> Dac biet su dung ky thuat hap phu tren 4. Le Thi Phu, Nguyln Thi Thu Sang, Khao sat<br /> carboxymethyl cellulose va tiip tuc kit tua vdi tinh sach enzym chymopapain trong mu trai du du<br /> mudi NaCI. Tinh chi dupe chymopapain cd hoat Viet Nam, Tap chi Phat then KH&CN , tap 9, si 5 -<br /> dp rieng la 1717,8 nk/mg protein vdi dp tinh 2006, 59-65.<br /> sach la 2,70 lin. Chi phim nghien cuu budc 5. Mitsuo Ebata, Kerry T. Yasunoba, Isolation,<br /> diu da dat dupe mdt so tieu chuin chit lupng Chrystallization, and preliminary characterization, the<br /> <br /> TAP CHI DUQC HQC - 10/2010 (SO 414 NAM 50) 41<br /> • N g h i e n CLFU - K y thuat<br /> <br /> journal of biologial chemistry, vol 237, N 4, April 1962,<br /> 2<br /> 10. Mala B. Rao, Aparna M. Tanksale, Mohini S.<br /> USA, (1961). Ghatge, and Vasanti V. Deshpande, " Sources of<br /> 6. Barrett, Alan J; Buttle, David J.; Rich, Daniel H, proteases ", Microbiology and Molecular Biology<br /> Pharmaceutical composition of purified chymopapain, Reviews, (1998).<br /> Patent No. 5468480, (1995). 11. Americal Chemical Society, Washington, DC,<br /> 7. Dando PM et al., unoglobin E antibodies to Reagent Chemicals ACS Specification, Volume II, 2-<br /> papaya proteinases and their relevance to 26, (1993).<br /> chemonucleolysis, Spine. Department of 12. Kapsalis AA, Stern IJ, Bornstein I. The fate of<br /> Biochemistry, Strangeways Research Laboratory, chymopapain injected for therapy of intervertebral<br /> Cambridge, UK, 1;20(9):981-5, (1995). disc disease. J Lab Clin Med, 1974 Apr; 83(4):532-<br /> 8. Rubens Monti et al. Biological Faculty, Estadual 540.<br /> Paulista university, Brasil, Purification of papain from 13. Bradford DS, Cooper KM, Oegema TR., Jr<br /> fresh latex of Carica papaya, Brazilian Archives of Chymopapain, chemonucleolysis, and nucleus<br /> Biology and Technology, v.43, n.5, p. 501-507, (2000). pulposus regeneration, J Bone Joint Surg Am, 1983,<br /> 9. Sidney P. Colowick, Nathan O.Kaplan, Dec; 65(9): 1220-1231.<br /> Methods in Ezymology, vol II, vol XIX, McCollum-Pratt 14. Staley Zucker et al, The proteolytic activities<br /> Institute, the Jonh Hopkins university, Baltimore, of chymopapain, papain and papaya proteinase III.,<br /> Maryland, (2008). Biochim. Biophys, Acta 828,196-204, (1985).<br /> <br /> <br /> <br /> T h a m d o d i e u k i e n d j n h h r o n g d o n g t h d i<br /> <br /> <br /> p a r a c e t a m o l v a i b u p r o f e n t r o n g h u y e t t i r o n g<br /> <br /> <br /> b S n g p h i r o n g p h a p S K L H N C<br /> <br /> VTnh Djnh, Le Ngoc Thanh True*<br /> Khoa Duqc, Dgi hoc Y Duqc TP. HCM<br /> <br /> Dat v i n d i - Xay dung va thim dinh qui trinh djnh lugng<br /> Hien nay co nhieu che pham la nhQ'ng "che ding thdi paracetamol va ibuprofen trong huyit<br /> pham tuong duong dugc hpc" (pharmaceutical tuang bing SKLHNC.<br /> equivalence / pharmaceutical alternative) ' . Viec<br /> 4|<br /> V a t lieu va p h i f c n g p h a p<br /> kiem tra chat lupng chu yeu dua tren su kiem - T i t ca h6a chit va dung m6i su dung la<br /> tra cac tinh chat ly hoa theo tieu chuan djnh sin. loai tinh khiit phan tich, loai dung cho SKLHNC.<br /> Viec danh gia in vitro nay khong phan anh thuc - P va I chuin lam viec dupe cung cip tu<br /> su hieu qua tri lieu cua mpt thuic trong co thi vi Vien Kiem nghiem TP. HCM. Vien nen Y<br /> sinh kha dung cua mpt thuic phu thupc vao rlt<br /> (Pymexpharm, so lot: 010605, so d§ng ki: VNA-<br /> nhieu yiu t i . Hien nay viec nghien cuu ve tuang<br /> 1971-04) va vien nen doi chiiu R (luu hanh thj<br /> duang sinh hpc chi dua tren hudng dan thu<br /> trudng, so lot: 403671, so dang ki: VNB-0525-<br /> tuang duang sinh hpc cua ASEAN la chu yeu<br /> [2], D i gop phin chuan bj cho cong tac nghien 00) chua 325 mg P va 200 mg I. miu tring su<br /> cu'u nay, chung toi thuc hien de tai: "Thim dd dung la huyet tuong dupe cung cap tu Vien<br /> diiu kien dinh luong ddng thdi paracetamol va truyen mau va huyet hpc TP. HCM.<br /> ibuprofen trong huyit tuong bing phuong phap - Djnh lugng P & I:<br /> SKLHNC" voi muc tieu: Chuan bi dung dich chuan<br /> - Tham do dieu kien dinh lupng chi pham Dung djch chuin P: h6a tan chinh xac 22 mg<br /> chua paracetamol va ibuprofen trong huyit tuong P trong 10 ml acetonitril.<br /> nhim tao so sd du lieu cho viec thu tuong duong Dung dich chuin I: h6a tan chinh xac 13,4<br /> sinh hpc cua nhung chi phim nay. mg I trong 10 ml acetonitril.<br /> <br /> 42 TAP CHI DUOC HQC - 10/2010 (SO 414 NAM 50)<br />