Buoc OAU KHAO SAT TAC D U N G K H A N G KHUAN VA CHONG VIEM TREN MO HINH O O N G VAT CUA CHIET PHAM FLAVONOIT TD LA CHE - CAMELLIA SINENSIS LINDL O KUNTZE HA THI THANH BINH, NGUYEN QUOC KHANG Khoa Sinh hoc, Trudng Dai hoc KHTN TRAN QUYNH HOA Vien Hod hoc cdc hap chat thien nhien, Vien Khoa hoc vd Cong nghe Viet Nam LMcldau Vice phat hien nhting tac dung sinh dugc bgc gia tri ciia cac bgp chat polyphenol d la che Camellia sinensis da dugc de cap tdi nhieu cdng trinh nghien ciiu ciia cac nha khoa bgc tren the' gidi [7] Ngoai Igi ich udng nude che hang ddi vdi sire khoe [1], chiing ta cdn cd the' quan tam khai thac cac bgp chat polyphenol va nghien ciiu sir dung chiing nhu nhiing che' pham thud'c dac tri cd ngudn gdc thien nhien Hang nam, ngoai viec thu hai che blip de che' bien che huong, che den v.v cdn cd mot khd'i lugng che xanh (che tuai, la banh te) khdng Id cd the' tan dung de' khai thac cac bgp cha't thien nhien cd boat tinh sinh bgc cao phuc vu cho llnh vuc y - dugc, trudc het la bgp chat flavonoit Tan dung dugc ngudn nguyen lieu se lam tang y nghia va gia tri ciia cay che nen kinb te cbung ciia dat nude va dem lai Igi ich khdng nhd cho nganh dugc De' thuc hien muc dich dd nha't thiet phai cd nhiing ngbidn ciiii ca ban tren chinh cay che Viet Nam de cd ca sd hieu bie't hudng tdi khai thac va sir dung ngudn Igi thien nhien Bao cao trinh bay mot sd ket qua danh gia sa bd tac dung 775 khang khuan, chdng viem ciia chie't pham flavonoit la che tren mot sd chiing vi khuan gay benh d ngudi va tren md binh ddng vat gay viem Doi tuTdng va phUdng phap nghien ciiru 2.1 Doi tugng nghien cihi: • Che' pham flavonoit tdng sd tach chiet tir la cay che (Camellia sinensis): La che banh te thu hai vao thang tai Tan Cuang (Thai Nguyen) Mau la sau thu hai dugc rira sach va xir ly diet enzim d 120"C, say nhe d 50°C den khd tuyet ddi, rdi tan bdt, bao quan hai Idp tiii polyetylen kin d noi khd rao thoang mat dung lam nguyen lieu cho cac nghien ciiu Tach chiet va dinh lugng flavonoit toan phan bang phuang phap Talli Hinh che' pham flavonoit toan phan tir la che (ky hieu la F.TC) • Cac cbung vi sinh vat va nam gay benh: Staphylococcus aureus, Escherichia coli, Salmonella typhi, Shigella flexneri, Pseudomonas aeruginosa, Klebsiela pneumoniae Bacillus subtilis, Candida stellaloides, Candida albicans [I] • Chudt nhat khoe manh BAL b/c lugng 20-25g/ 2.2 Phuang phap nghien cuti: 2.2.1 Chie't rut cdc chat tif nguyen lieu /5J: cdc chat dugc chie't riit bang etanol 96% tren mdy soxhlet 2.2 Chie't xudt vd dinh lugng flavonoit bang phucfng phdp Talli [ 8,4] 2.2 Xdc dinh hogt tinh khdng khuan cua che pham flavonoit theo phuang phdp khdng sinh gid'y thd'm [6] 2.2.4 Dinh lugng khdng khuan theo phuang phdp he ndng [6] 2.2.5.Ddnh gid hogt tinh chdng viem in vivo tren md hinh gdy viem loet thuc nghiem [3] Ket qua nghien cCiru 3.1 Khdo sdt hogt tinh khdng khuan cua chie't pham flavonoit toan phan FTC: • Mdi trudng nudi cay.- Mdi trudng Mueller HiUon dugc sir dung de nudi cay vi khudn Chinh pH mdi trudng = 7,4±0,2 Ddi vdi n^m, 776 „fe ^^x ^uy vijgc J Gur.g la mdi trudng Sabauraud cd pH mdi trudng = 6,8 • Cach tie'n banh: Do len dia petri da tiet triing Idp thach gdc (10ml) va dd tiep Idp thur la Idp thach dinh dudng bao gdm mdi trudng nudi cay va vi khu^n (8ml) De cho thach ddng lai Cat giay tha'm hinh trdn cd dudng kinb 0,6 cm va cd khd'i lugng khoang 33,5 - 34 mg, say vd triing d 120°C 60 phiit Cho cac khoanh gia'y tha'm dung dich cd cbiia dung dich flavonoit nghien ciiu rdi sa'y khd Dat cac khoanh gia'y da cd mlu len mat mdi trudng thach da ca'y vi khuan Dat vao tu am 37°C ddi vdi vi khuan va 28°C ddi vdi vi nam, sau 24 gid dgc ket qua ddi vdi vi khuan va 48 gid ddi vdi nam Bang phuang phap gia'y tha'm, chiing tdi tie'n banh dinh tinh khang khuan vdi ndng dung dich flavonoit tdng so la 50mg/ml va danh gia mire khang khuan thdng qua gia tri cua dudng kinb vdng vd khu^n sau 24 gid ke tir cay vi khuan vao mdi trudng nudi ca'y Thi nghiem dugc lap lai Ian va lay gia tri dudng kinb vdng vd khuan trung binh Ke't qua dugc trinh bay d bang Bang 1: Ket qua djnh tinh khang khuan ciia F.TC STT Ten vi khuan Ducmg kinh vong vd khuan (mm) Staphylococcus aureus 30,0 Escherichia coli 21,0 Salmonella typhi 25,0 Shigella flexneri 24,0 Pseudomonas aeruginosa 22,0 Klebsiela pneumoniae 21,0 Bacillus subtilis 18,5 Candida stellaloides 7,5 Candida albicans 9,0 Ket qua thu dugc cho thay flavonoit toan phan F.TC cd tac dung lie che su phat trien cua ta't ca chiing vi khu^n va na'm nghien ciiu vdi cac miic kbac nhau, nhin cbung tac dung khang na'm to yd'u nha't Tac dung manh nhat the hien ddi vdi cau triing gram (-f-) Staphylococcus aureus, tic tac dung ddi vdi chimg cd the' suy 777 tac dung vdi cac loai cau triing kbac nhu Streptococcus, Diphlococcus Ngoai cac ket qua dinh tinh cdn cho tha'y che' pham F.TC cd tac dung td't len cac chiing: • Vi khuan gay benh dudng rudt dien binh nhu Escherichia coli, Shigella flexneri • Salmonella typhi: true khuan gram (-) gay benh thuong ban da khang cac loai khang sinh nhu bactrim, tetracylin, ampicillin, cloramphenicol (nhiing thud'c dac hieu diing de' dieu tri benh thuong ban lau nay) • Pseudomonas aeruginosa va Klebsiela pneumoniae: vi sinh val gay benh ca hdi, ky sinh d ngudi, thudng gay nhiem triing ndi sinh benh vien Trong dd, Pseudomonas aeruginosa la loai ndi tieng nguy biem, khang lai nhieu khang sinh cdn Klebsiela pneumoniae gay viem phoi thudng gap d tre so sinh, ty le tir vong rat cao neu khdng dugc dieu tri sdm • Bacillus subtilis: dai dien cho nhdm true khuan gram (-I-) va sinh nha bao Tir tac dung td't vdi Bacillus subtilis thi cung cd the suy tac dung vdi cac vi khuan kbac nhdm nhu: Bacillus anthracis - gay benh nhiet than hoac vdi vi khuan gay benh udn van Tir cac ke't qua dinh tinh tren da cho thay kha nang khang khuan cua flavonoit che xanh sir dung nhu mot chat khang sinh thuc vat Viec phat hien them cac khang sinh cd ngudn gdc thuc vat la mot van de cd y nghia he't sue dac biet tinh hinh khang thud'c cua cac vi khuan cang tang Han niia, cac khang sinh ngudn gdc thuc vat thudng cd an toan cao ban va it gay cac phan iing phu Chiing tdi tiep tuc tie'n banh tim cac gia tri MIC (minimum inhibiting concentration) dac trung cho cac ndng lie che' tdi thieu ciia flavonoid ddi vdi mdi loai vi khuan bang phuang phap pha loang ndng Cac dich flavonoid dugc pha loang dan tdi dat tdi ndng ma tai dd flavonoid khdng cdn tac dung lie che' su phat trien cua vi khuan Khoang ndng cud'i ciing tie'p tuc dugc chia nhd vdi chinh xac ± 0,001mg/ml de thu dugc gia tri MIC Ke't qua eu the dugc trinh bay d bang Cac ke't qua trinh bay tren bang cho thay d ndng pha loang 778 256 ian so vdi ndng ban dau, che pham F.TC the hien tac dung rd nha't len cac cbung Staphylococcus aureus, Salmonella typhi, ddi vdi cac cbung Escherichia coli, Salmonella typhi Shigella flexneri, Pseudomonas aeruginosa, Klebsiela pneumoniae vln cd tac dung nhien yeu ban Ddi vdi hai chiing nam la Candida stellaloides, Candida albicans thi d ndng pha loang 1/2 F.TC da khdng the hien tac dung Bang Djnh lugng khang khuan ciia F.TC T T Gia tri MIC cua cac flavonoit Ten vi khuan Nong man Idn pha loang) (so 16 32 64 128 256 Staphylococcus aureus +++ +++ +++ +++ +++ +++ ++ + Escherichia coli +++ +++ +++ +++ +++ +++ + ± Salmonella typhi +++ +++ +++ +++ +++ +++ ++ + Shigella flexneri +++ +++ +++ +++ +++ +++ + ± Pseudomonas aeruginosa +++ +++ +++ +++ +++ +++ + ± Klebsiela pneumoniae +++ +++ +++ +++ +++ +++ + ± Bacillus subtilis +++ +++ +++ +++ ++ + - - Candida - - - - - - - - Candida albicans - - - - - - - - stellaloides 3.2 Khao sat hoat tinh chdng viem in vivo cua chiet pham flavon toan phan la che F.TC tren mo hinh gay viem loet thi/c nghiem Chiing tdi da tien banh thir tac dung ciia F.TC len chudt gay viem theo md hinh Winter: Tiem 0,1 ml dung dich dextran 5% vao gan ban chan chudt de tao viem tay Danh gia tac dung chdng vidm bang each do phii chan chudt Xac dinh su kbac biet giua Id chiing va cac Id thir nghiem 779 Sau cac budc tham dd, chiing tdi da chia Id thi nghiem lam nhdm gdm nhdm cho udng che pham F.TC (vdi lieu Ig/kg), nhdm tiem che pham F.TC dudi da (vdi lieu 0,lg/kg) va nhdm cho tiep xiic che pham F.TC tren da (vdi lieu 0,lg/ml) Bd tri mot Id ddi chiJ-ng Mdi Id thi nghiem va Id ddi chiing deu gdm chudt Cac Id thi nghiem neu bi giam sd lugng bi che't qua trinh thi nghiem se dugc lam lap lai de' dam bao sd ca the' cua mdi thi nghiem la ( n = 5) Ket qua danh gia tac dung khang viem cua che pham thdng qua cac chi sd: - Ty le phan tram thay ddi the' tich chan chudt so vdi chua gay viem T = ( V - V l ) x 100%/VI dd VI la the tich chan chudt trudc gay viem va V2 la the' tich chan chudt sau gay viem - Ty le phan tram lie che': I % = (Toe trung binh - T, trung binh) x 100%/ T^c trung binh Tren bang bie'u thi sir thay ddi the tich chan chudt theo thai gian gay viem Sau 150 phut the tich chan chudt dat tdi cue dai vdi Id doi chiing Vi vay chiing tdi chgn thai diem the' tich chan chudt la 150 phut sau gay viem Cac tac ddng dimg thud'c theo dudng udng, tiem, bdi da dugc thuc hien sau gay viem phiit Ke't qua dugc trinh bay tren bang 4, 5, Bang 3: Theo doi sir thay ddi the tich chan chudt gay viem khdng dieu tri Chi tieu thi nghiem So lieu thuc nghiem Thdi gian sau 30 gay viem (phut) 60 90 120 150 180 210 240 Thay doi the tich 29 45,7 50,5 65,5 65,3 60,5 61,4 780 23 oaug t; rvua iiaiig uw ^ c y.^ heo dudng uong ciia chiet pham flavonoit la che F.TC Do tang the tich chan chuot (gia trj T ) Cac 16 thi nghiem % tTc che ( gia tri I % ) L6 chumg 55,5 ±5,6 F.TC 34,2 ± 3,6 38,4 Bang 5: Kha nang itc che viem theo dudng tiem dudi da ciia chie't pham flavonoid che xanh F.TC Cac 16 thi nghiem Dp tang the tich chan chuot (gia trj T%) L6 chumg gay viem khong diing thudc 55,5 ±5,6 F.TC 14,2 ±1,2 74,4 % tfc che (gia tri I %) Bang 6: Kha nang lie che viem theo dudng bdi ngoai da ciia chiet pham flavonoid che xanh F.TC Cac 16 thi nghiem Do tang the tich chan chuot (gia trj T%) % tTc che (gia trj I %) L6 chiing gay viem khdngdung thudc 55,5 ±5,6 F.TC 22,0 ± 5,5 60,4 Cac sd lieu khao sat trinh bay tren bang 4, 5, cho thay tac dung khang viem kha rd cua cac chie't pham flavonoid che xanh F.TC tren md hinh gay viem thuc nghiem Trong dd, tac dung theo dudng tiem dudi da cd hieu luc nhanh va manh nha't (gia tri lie che dat 74,4%), tie'p de'n la dudng bdi ngoai da (gia tri lic che' dat 60,4%), dudng udng thi hien tac dung ye'u nha't (gia tri lie che' dat 38,4%) Do dieu kien thi nghiem khdng cho phep, cac thi nghiem chi diing lai d khao sat vdi ndng cho mdi dudng dung thud'c Chiing tdi by vgng cac nghien 781 ciiu vdi nhieu ndng se cho phep lua chgn ngudng ndng de phat buy hieu luc chdng viem ciia che' pham che xanh Ket luan Tir cac sd lieu thuc nghiem cho phep chiing tdi riit mot sd ke't luan ban dau nhu sau: Che' pham flavonoit che F.TC cd tac dung lie che' sir phat trie'n cua tat ca chiing vi khuan va nam nghien ciiu: Staphylococcus aureus, Escherichia coli, Salmonella typhi, Shigella flexneri, Pseudomonas aeruginosa, Klebsiela pneumoniae Bacillus subtilis, Candida stellaloides, Candida albicans Trong dd tac dung manh nhat the hien ddi vdi cau trung gram (+) Staphylococcus aureus va tac dung khang nam to ye'u nha't Ndng lie che' tdi thieu cua F.TC khac ddi vdi cac chiing vi khuan kbac nhau, cu the' la ddi vdi: - Staphylococcus aureus vd Salmonella typhi < (11256 x 50 mg/ml) - Eschericliia coli Salmonella typhi, Shigella flexneri, Pseudomonas aeruginosa, Klebsiela pneumoniae = (1/256 x 50 mg/ml) Vdi lieu Ig/kg theo dudng udng; 0,lg/kg theo dudng tiem dudi da va 0,lg/ml theo dudng tiep xiic tren da, che' pham flavonoit che xanh F.TC da the hien tac dung d nhiing miifc kbac tren mo hinh gay viem thuc nghiem tren chudt Miic lic che' theo dudng su dung theo thii tu nhu sau: dudng tiem dudi da (gia tri lie che' dat 74,4%) > dudng bdi ngoai da (gia tri lie che' dat 60,4%) > dudng udng (gia tri lie che dat 38,4%) Tai lieu tham khao Bd mdn vi sinh vat, Dai bgc Y Ha Ndi, 2003, Vi sinh y hgc, Nha xuat ban Y hgc Pham Hoang Hd ,1993 Cdy cd Viet Nam Nha xua't ban Tre, tap 3: 67-68 Balows A., Hausler W.J.Jr, Ohashi M and Turano A., 1988 Laboratory diagnosis of infectious diseases principles and practice vol.1 Springer Verlag, New York Inc., New York 782 't »^anneii K J r.,iyyo natural Products Isolation Humana Press: Harbon J B, 1994 Biochemistry of phenolic compounds Academic press, London and NewYork:115 Method for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved Standard - fifth edition - 2000, p 13 Vol 17, No2 Weisburger JH., 1997 Tea and health: a historical perspective Cancer Lett 1997 Mar (Tieng Nga) Ep^akob A U u gp, 1972, Cdc phuang phdp hod sinh thi/c vat, Nxb Khoa hgc - Leningrat tr 117 -123; 336 - 374 Abstract Investigating the anti- bacterial and anti - inflamation activity of total flavonoid extracted from tea leave Camellia sinensis Lindl O Kuntze Ha Till Thanh Binli', Nguyen Quoc Khang', Tran Quynh Hoa^ ' Hanoi University of Science; ^ Institute of Natural Product Chemistry A flavonoid preparation was extracted from TanCuong tea leave to supply for the experiment in vitro and in vivo on the bacteria species and inflammatory animal model Flavonoid from TanCuong tea leave (F.TC) exhibited antibacterial activity in vitro for the following species: Staphylococcus aureus, Eschericliia coli Salmonella typhi Shigella flexneri, Pseudomonas aeruginosa, Klebsiela pneumoniae Bacillus subtilis, Candida stellaloides, Candida albicans The best effect is on Staphylococcus aureus and the worst are for Candida stellaloides, Candida albicans In vivo, the experiments on inflammatory animal model with whhe mice BAL/bc showed that F.TC could inhibit inflammatory process By comparison three using manners of FTC for experimental animals, the best result was obtained in the subcutaneous injection manner (inhibitive rate was 74,4%), the second result was in the applying FTC on the skin (inhibitive rate was 60,4%) and the last was oral manner (inhibitive rate was 38,4%) 783 ... r.,iyyo natural Products Isolation Humana Press: Harbon J B, 1994 Biochemistry of phenolic compounds Academic press, London and NewYork:115 Method for dilution antimicrobial susceptibility tests for... bao quan hai Idp tiii polyetylen kin d noi khd rao thoang mat dung lam nguyen lieu cho cac nghien ciiu Tach chiet va dinh lugng flavonoit toan phan bang phuang phap Talli Hinh che' pham flavonoit. .. Camellia sinensis Lindl O Kuntze Ha Till Thanh Binli', Nguyen Quoc Khang', Tran Quynh Hoa^ ' Hanoi University of Science; ^ Institute of Natural Product Chemistry A flavonoid preparation was extracted