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 trong nhieu cdng trinh nghien ciiu ciia cac nha k[.]
Trang 1Buoc 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 trong
nhieu cdng trinh nghien ciiu ciia cac nha khoa bgc tren the' gidi [7] Ngoai Igi ich udng nude che hang ngay 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 thanh 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 nay se lam tang y nghia va gia tri ciia cay che trong 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 nay
Bao cao nay trinh bay mot sd ket qua danh gia sa bd tac dung
Trang 2khang 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
2 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 2 tai Tan Cuang (Thai
Nguyen) Mau la sau khi thu hai dugc rira sach va xir ly diet enzim d 120"C, say nhe d 50°C den khd tuyet ddi, rdi tan thanh bdt, bao quan trong 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 thanh 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 trong lugng 20-25g/ con
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 2.2 Chie't xudt vd dinh lugng flavonoit bang phucfng phdp Talli [ 8,4] 2.2 3 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 do [6]
2.2.5.Ddnh gid hogt tinh chdng viem in vivo tren md hinh gdy viem loet thuc nghiem [3]
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,
Trang 3„fe ^^x ^uy vijgc 3 J Gur.g la mdi trudng Sabauraud cd pH mdi
trudng = 6,8
• Cach tie'n banh: Do len dia petri da tiet triing 1 Idp thach gdc
(10ml) va dd tiep Idp thur 2 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 thanh hinh trdn cd dudng kinb 0,6 cm va cd khd'i lugng khoang
33,5 - 34 mg, say vd triing d 120°C trong 60 phiit Cho cac khoanh
gia'y nay 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 do dung dich flavonoit tdng so la 50mg/ml va danh
gia mire do khang khuan thdng qua gia tri cua dudng kinb vdng vd
khu^n sau 24 gid ke tir khi cay vi khuan vao mdi trudng nudi ca'y Thi
nghiem dugc lap lai 3 Ian va lay gia tri dudng kinb vdng vd khuan
trung binh Ke't qua dugc trinh bay d bang 1
Bang 1: Ket qua djnh tinh khang khuan ciia F.TC
STT Ten vi khuan Ducmg kinh vong vd khuan
(mm)
1 Staphylococcus aureus 30,0
2 Escherichia coli 21,0
3 Salmonella typhi 25,0
4 Shigella flexneri 24,0
5 Pseudomonas aeruginosa 22,0
6 Klebsiela pneumoniae 21,0
7 Bacillus subtilis 18,5
8 Candida stellaloides 7,5
9 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 9 chiing vi khu^n va na'm nghien ciiu
vdi cac miic do kbac nhau, nhin cbung tac dung khang na'm to ra yd'u
nha't Tac dung manh nhat the hien ddi vdi cau triing gram (-f-)
Staphylococcus aureus, tic tac dung ddi vdi chimg nay cd the' suy ra
Trang 4tac dung vdi cac loai cau triing kbac nhu Streptococcus, Diphlococcus Ngoai ra 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
trong 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
ra tac dung vdi cac vi khuan kbac trong nhdm nay 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 khi 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 khi tinh hinh khang thud'c cua cac vi khuan ngay cang tang Han niia, cac khang sinh ngudn gdc thuc vat thudng cd do 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 do lie che' tdi thieu
ciia flavonoid ddi vdi mdi loai vi khuan bang phuang phap pha loang ndng do Cac dich flavonoid dugc pha loang dan tdi khi dat tdi ndng
do ma tai dd flavonoid khdng cdn tac dung lie che' su phat trien cua vi khuan Khoang ndng do cud'i ciing nay tie'p tuc dugc chia nhd vdi do chinh xac ± 0,001mg/ml de thu dugc gia tri MIC Ke't qua eu the dugc trinh bay d bang 2
Cac ke't qua trinh bay tren bang 2 cho thay d ndng do pha loang
Trang 5256 ian so vdi ndng do 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 tuy
nhien yeu ban Ddi vdi hai chiing nam la Candida stellaloides, Candida albicans thi ngay d ndng do pha loang 1/2 F.TC da khdng the
hien tac dung
Bang 2 Djnh lugng khang khuan ciia F.TC
T
T
1
2
3
4
5
6
7
8
9
Ten vi khuan
Nong do man (so
Idn pha loang)
Staphylococcus
aureus
Escherichia coli
Salmonella typhi
Shigella flexneri
Pseudomonas
aeruginosa
Klebsiela
pneumoniae
Bacillus subtilis
Candida stellaloides
Candida albicans
Gia tri MIC cua cac flavonoit
2 +++
+++
+++
+++
+++
+++
+++
-4
+++
+++
+++
+++
+++
+++
+++
-8
+++
+++
+++
+++
+++
+++
+++
-16
+++
+++
+++
+++
+++
+++
+++
-32
+++
+++
+++
+++
+++
+++
++
-64
+++
+++
+++
+++
+++
+++
+
-128
++
+ ++
+ +
+
-256
+
± +
±
±
±
-3.2 Khao sat hoat tinh chdng viem in vivo cua chiet pham flavonoit 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 do phii chan chudt Xac dinh su kbac biet giua Id chiing va cac Id
thir nghiem
Trang 6Sau cac budc tham dd, chiing tdi da chia Id thi nghiem lam 3 nhdm gdm nhdm 1 cho udng che pham F.TC (vdi lieu Ig/kg), nhdm 2 tiem che pham F.TC dudi da (vdi lieu 0,lg/kg) va nhdm 3 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 5 con chudt Cac Id thi nghiem neu bi giam sd lugng do bi che't trong qua trinh thi nghiem se dugc lam lap lai de' dam bao sd ca the' cua mdi thi nghiem la 5 ( 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 khi chua gay viem
T = ( V 2 - V l ) x 100%/VI
trong dd VI la the tich chan chudt trudc khi gay viem va V2 la the' tich chan chudt sau khi gay viem
- Ty le phan tram lie che':
I % = (Toe trung binh - T, trung binh) x 100%/ T^c trung binh Tren bang 3 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 do the' tich chan chudt la 150 phut sau khi gay viem
Cac tac ddng dimg thud'c theo 3 dudng udng, tiem, bdi da dugc thuc hien sau khi gay viem 5 phiit Ke't qua dugc trinh bay tren bang
4, 5, 6
Bang 3: Theo doi sir thay ddi the tich chan chudt
gay viem khdng dieu tri
Chi tieu thi So lieu thuc nghiem
nghiem
Thdi gian sau khi 30 60 90 120 150 180 210 240 gay viem (phut)
Thay doi the tich 23 29 45,7 50,5 65,5 65,3 60,5 61,4
Trang 7oaug t; rvua iiaiig uw ^ c y.^ heo dudng uong ciia chiet pham
flavonoit la che F.TC
Cac 16 thi nghiem
L6 chumg
F.TC
Do tang the tich chan chuot (gia trj T )
55,5 ±5,6
34,2 ± 3,6
% tTc che ( gia tri I % )
0
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
L6 chumg gay viem khong
diing thudc
F.TC
Dp tang the tich chan chuot (gia trj T%)
55,5 ±5,6
14,2 ±1,2
% tfc che (gia tri I %)
0
74,4
Bang 6: Kha nang lie che viem theo dudng bdi ngoai da
ciia chiet pham flavonoid che xanh F.TC
Cac 16 thi nghiem
L6 chiing gay viem
khdngdung thudc
F.TC
Do tang the tich chan chuot
(gia trj T%)
55,5 ±5,6
22,0 ± 5,5
% tTc che (gia trj I %)
0
60,4
Cac sd lieu khao sat trinh bay tren bang 4, 5, 6 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 1 ndng do cho mdi dudng dung thud'c Chiing tdi by vgng cac nghien
Trang 8ciiu vdi nhieu ndng do se cho phep lua chgn ra ngudng ndng do de phat buy hieu luc chdng viem ciia che' pham che xanh
4 Ket luan
Tir cac sd lieu thuc nghiem cho phep chiing tdi riit ra mot sd ke't luan ban dau nhu sau:
1 Che' pham flavonoit che F.TC cd tac dung lie che' sir phat trie'n
cua tat ca 9 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 ra ye'u nha't
2 Ndng do lie che' tdi thieu cua F.TC khac nhau 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)
3 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 do kbac nhau tren mo hinh gay viem thuc nghiem tren chudt Miic do lic che' theo 3 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
1 Bd mdn vi sinh vat, Dai bgc Y Ha Ndi, 2003, Vi sinh y hgc, Nha
xuat ban Y hgc
2 Pham Hoang Hd ,1993 Cdy cd Viet Nam Nha xua't ban Tre,
tap 3: 67-68
3 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
Trang 9't »^anneii K J r.,iyyo natural Products Isolation Humana
Press: Harbon J B, 1994
5 Biochemistry of phenolic compounds Academic press, London
and NewYork:115
6 Method for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved Standard - fifth edition - 2000, p 13 Vol 17, No2
7 Weisburger JH., 1997 Tea and health: a historical perspective
Cancer Lett 1997 Mar
8 (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
anti-bacterial 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%)