• N g h i e n c u » u - K y t h u a t Xac dmh do tinh khiit quang hoc
Vdi mdi chit phan tich tiin hanh xac djnh tren 3 Id sin phim da dupe tdng hop. Mdi Id do 2 miu thu. Pha miu d ndng dd 100 pg/ml. Mdi miu sic ky 2 lin vdi dieu kidn sic ky da chpn.
Bang 4: Dd tinh khiit quang hoc cua cic chit phin tich (n=12)
Chat Dg tinh khiet quang hgc theo sic ky Do tinh khiet quang hgc doi quang (ee)
HN1 96,62% 93,25%
(+)N1 94,22% 88,43%
(-)N2 97,21% 94,42%
(+)N2 94,17% 88,34%
Ket luan
Dua vao cdc kit qua thuc nghidm thu dupe, chiing tdi nhan thiy ring cd the ap dung quy trinh da thiit l$p de tien hanh xdc dinh dd tinh khiit quang hpc cua cdc din chit quang hoat c/s-A/-alkyl- phthalazinon. Quy trinh cd dd chinh xdc cao, tu dd ddnh gid dupe chit lupng cua cdc san phim ddng phan dd dupe tdng hpp, gdp phin xdc djnh ngudn nguydn lidu tinh khiet vd dat tidu chuin.
S u m m a r y
An HPLC method using dehvatized cellulose chiral stationary phase for separation of the enantiomers of the newly synthesized chiral cis-N- benzyl and cis-N-heptyl phthalazinone derivatives was established and validated. Separation
efficiency and selectivity were optimized using mobile phases containing n-hexane and either isopropanol (IPA) or ethanol in variable ratios.
Also, addition of triethylamine (TEA) to the mobile phases was investigated for improvement of peak tailing. Complete separation of the enantiomers was achieved with a cellulose tris-3,5- dimethylphenylcarbamate column (Lux cellulose-1 column: 250 x 4.6 mm; 5 um) thenmostated at 30°C, a mixture of n-hexane and IPA containing 0.2% TEA (90:10, v/v) as eluent in isocratic mode, flow rate of 0.8 ml/min, and detection wavelength of 317 nm. The method was applied to determine the enantiomeric excess (ee) of cis-N-alkyl- phthalazinone showing the enantiomeric purity of the synthesized enantiomers to be 93.25%;
88.43%; 94.42% and 88.34% (ee) for (-)N1, (+)N1, (-)N2 and (+)N2, respectively.
Tai lieu t h a m k h a o
1. Margaretha Van Der Mey, Hildegard Boss, Armin Hatzelmann, Ivonne J. Van Der Laan, Geert J.
Sterk and Hendrik Timmerman , Journal of Medical Chemistry, (2002), 45, 2520-2525.
2. Margaretha Van Der Mey, Kirsten M.Bommeld, Hildegard Boss, Armin Hatzelmann, Mike Van Slingeriand, Geert J. Sterk, Kees Goubitz, Henk Schenk and Hendrik Timmerman , Journal of Medical Chemistry, (2003), 46, 2008-2016.
3. ICH Harmonised Tripartite Guideline, Validation of analytical procedures: text and methodology, (2005), 1-5.
*?
N g h i e n c u u h o a t t i n h k h a n g k h u a n s a u r a n g r
t i r d i c h c h i e t c a y x o a i ( M a n g i f e r a i n d i c a L . )
McV dau
Sdu rdng Id mdt bdnh rit phd bien trdn the gidi cung nhu d Vidt Nam. Theo sd lidu dieu tra cua Vidn Rdng Hdm Mdt Trung Uong vdo ndm 2006, khoang 90% ddn sd Vidt Nam mdc cdc bdnh vd rdng midng, trong dd phd bien nhit Id sdu rdng vd vidm quanh rdng. Ty Id ngudi Vidt Nam mic cdc bdnh vd rdng tdp trung d tudi trung nidn trong khoang 40-45 tudi vd tre em
Nguyin Quang Huy, Nguyen Thj Mai Duyen Khoa Sinh hoc, trudng Dgi hgc Khoa hgc Tu nhien dudi 12 tudi. Nguydn nhdn bdnh sdu rdng do
| cdc vi khuin cd mdt trong mang bdm rdng sinh
a acid gdy dn mdn men rdng. Trong sd cdc nhdm
n vi khuin sinh sdng d mang bdm rdng nhdm
c Streptococcus dupe coi Id nguydn nhdn chfnh
3 do chung cd kha ndng sinh acid manh cung nhu
( t chiu acid. Kidm sodt sy phdt tridn cua cdc vi
>j khuin ndy Id mdt trong nhung gidi phdp ngdn
n ngua sdu rdng.
• N g h i e n C C F U - K y t h u a t Dieu tra tim kiem cac hgp chat mai co tac dung ac chi sg phat trien cung nha sinh acid cua cac chung vi khuin gay sau rang la phuang phap hidu qua trong vide bao vd rang khoi sau Id hudng nghidn cuu dang dugc quan tam.
Nhidu logi djch chiit thuc vat dd dugc nghien cuu vd thu nghidm kha ndng didt khuin sdu rang, uc che hinh thdnh mang bdm rdng. Chen vd tdp the 1 1 1 dd nghien cuu djch chiet tu 79 lodi cdy thuoc trong dd 3 lodi Mows australis, Ludwigia octovalvis vd Thuja orientalis cd hoat tinh uc che su phdt trien cua S. mutans vdi ndng dd trong khoang tu 2 ddn 7,8 mg/ml.
Nhieu nghidn cuu gin ddy da di sdu vdo vide nghien cuu ca che tdc dung cua cdc hgp chit tu nhidn, cung nhu tdc ddng cua chung ldn cac enzym glucosyltranferases, ATPase... ddng vai trd chu dao giup Streptococcus mutans tdn tai sinh trudng phdt trien vd hinh thdnh mang bam rang'111. 6* Viet Nam, cdc dich chiet tu cac thanh phin khdc nhau cua cdy chd [Camellia sensis L), vd mdng cut (Garcinia mangostana L), Id lot {Piper lolot DC), vd sao den (Hopea Odorata Roxb), sin thuyen (Syzygium resinosum Gapnep) [ 5"7' cung cho thiy cd tdc dung trong vide uc che sinh acid cua Streptococcus mutans.
Cdy xodi (Mangifera indica L.) tu ldu dugc nhdn ddn Vidt Nam vd m0t so khu vuc Chdu A su dung trong vide chua sdu rdng'2,31. Nghien cuu cua chung tdi nhdm tim hieu tdc dung djch chiit cua vd thdn vd Id cay xodi len vi khuin Streptococcus de bo sung thdm cdc dan lidu ve su da dang nguon dugc lieu chong sdu rdng d Viet Nam. Ode bidt nghidn cuu cua chung tdi cdn dugc thu nghidm trdn doi tugng vi khuin Streptococcus phdn lap tu bdnh nhdn ngudi Vidt Nam.
Nguyen lieu va phuang phap nghien ciru Nguyen lieu
Vi khuin Streptococcus chuan S. mutans GS-5, S. sanguis la qua tdng cua Giao su R E Marquis (Oai hoc Rochester- Hoa Ky), chung S. sp 74 dugc phdn lap tu bdnh nhan sau rang Vidt Nam. Cdc chung vi khuin dugc giu vd ciy chuyen hdng tuan ldn mdi trudng thach TSA (tryptic soy agar). Te bdo vi khuan dugc nudi ciy ldc d nhidt dd 37°C trong mdi truang TYG chua 3% tryptone, 0,5% djch chiet nim men vd 1% glucoza.
Chuan bj djch chiet cay xoai
Cac phin khdc nhau cua cdy xodi (Id, vd thdn) dugc rua sach, siy khd d 40-50°C vd tdn nhd thdnh dang bpt mjn. Tiin hdnh thu djch chiit bdng cdch ngdm bdt nghien vd thdn vd Id trong ethanol 90°.. theo ty Id 1 gam b0t 10 ml ethanol. Djch chiit sau dd dugc ly tdm d i thu phin djch trong.
Phan tich sy san xuat acid qua thi nghiem lam giam pH cua moi trifdng
Cdc budc ca ban cua thi nghidm duac tiin hdnh nhu md ta cua Ma vd tap the ( 4 ), tdm tit cac budc chinh nhu sau: te bdo vi khuin dugc thu tu pha dn djnh cua qud trinh sinh trudng, dugc ly tam, rua vd hoa trd lai trong dung djch mudi (KCI 50mM cd chua MgCI21mM) vdi mdt dp te bdo khoang 4x101 0/ml. Glucose duac bd sung de dat ndng dd cudi ciing 0,5%. pH cua djch te bdo dugc dieu chinh ve 7,2 bing KOH va bit diu do su giam pH theo thdi gian.
Tac dung gay chet cua djch chiet
Thi nghidm gdy chet te bao vi khuan dugc tien hanh theo phuang phdp dugc md td bdi Phan va tap the1101. Djch chiet bdng ethanol tu Id va vd thdn xodi dugc bd sung vdo mau nghidn cuu theo ty Id nhit djnh ve the tich, sau dd dieu chinh pH bdng KOH hay HCI de cd gid trj pH mong mudn. Tdc dyng gdy chit cua djch chiit dugc bieu thj bdng gid trj LogN/No, trong dd N Id sd te bdo sdng sdt tai thdi diem thu miu vd No Id sd te bdo ban dau. Vdi cdch biiu diin ndy, gid trj LogN/No= -1 cd nghTa Id cd 90%
quin the te bdo bj giet chet.
Phan tich thanh phdn djch chiit
Sdc ky ban mdng tu cdc thdnh phan khdc nhau cua cdy xodi dugc thuc hidn trdn ban mdng trdng sin DC-Alufolien 60 F2 5 4 (Merck) vdi hd dung mdi Toluen: ethylacetat: aceton: acid focmic (TEAF) voi ty Id 5:3:1:1; hidn hinh bdng dung djch H2SO4 10%. Quan sdt vd ghi lai nhdn xdt ve mdu sdc vd he sd Rf cua cdc bdng vach.
Thu nghidm kha ndng uc chi su phdt tridn tii cdc phdn doan sau khi chay sdc ky bang mdng bing phuang phdp khuydch tdn dTa thach.
Kdt q u a v a t h a o l u a n
Danh gid tdc dung Idm gidm kha ndng sinh acid cua djch chiit xoai
Kha ndng sinh acid Id mOt ddc trung gdy bdnh cua Streptococcus mutans. Vide ddnh gid
38 TAP CHI DUQC HQC - 4/2011 (SO 420 NAM 51)
khd nang ire che sy sinh acid tu cac djch chiet la cc so1 ban dau nhdm danh gia hoat tinh cua cac dich chiet. Sau thdi gian 60 phut ddi vdi miu thi nghidm trdn chung Streptococcus mutans GS-5 gia tri pH d doi chung ethanol giam xudng 4,95 trong khi d miu cd bd sung djch chiit ty la xoai va thdn xodi deu trdn 6 vdi muc dd Id 6,22 vdi mdu thdn xodi vd 6,15 vdi Id xodi (Hinh 1A). Vdi chung S. sp 74 phdn Idp tu
7.5 7 - 6.5 6 5.5 5 H 4.5
bdnh nhdn Vidt Nam cho thiy d mdu ddi chung nude pH tut xudng 5,09 trong khi dich chiit thdn vd Id xodi hiu nhu uc che hodn todn su giam pH cua chung vi khuin ndy vdi gid trj pH tuang ung la 7,11 vd 7,15 (Hinh 1B). Vide hoat tinh uc chd sinh acid d S. sp 74 cao han so vdi S.
mutans GS-5 cd the lidn quan kha ndng chju acid cua Streptococcus phdn Idp d ngudi Vidt Nam thip han so vdi S. mutans GS-5 [ 9 ).
7.5 7 6.5 6 5.5 5 4.5
4 R
10 20 30 40 50 60 0 10 20 30 40 50 60
thfrl gian (phut) Thfrl gian (phiit)
Hinh 1: Khi ning (re chi sy sinh acid cua chung S. mutans GS-5 (A) vi S. p74 (B) D6i chung nude (•); doi chung ethanol (•); djch chiet than xoai (•); djch chiet la xoai (A) 7.5
7 6.5 6 5.5 - 5 - 4.5
0 10 20 30 40 50 60 90 100 110 120 130 140 150 160 190 th&i gian (phiit)
Hinh 2: Khi ning (rc chi sinh acid cua S. mutans GS-5
Odi chung nude (•); doi chung ethanol (•); dich chiet than xoai (•); dich chiet Id xoai (A) Chung tdi tidn hdnh phdn tich kha ndng uc xodi. Kit qua cho thiy sau khi pH miu ddi
chi vide sinh acid cua chung S. mutans GS-5 chung xudng dudi 5 (sau 90 phut) chung tdi tiip trudc vd sau khi loai bd djch chiet vd thdn vd Id tuc bd sung glucose vd t i bdo vi khuin lgi tidp
N g h i e n C L F U - K y t h u a t tuc sir dung dudng de sinh trudng va tao ra acid. Trong khi d mau thi nghiem vide loai bd djch chiet va sau dd bo sung glucose thi cac te bao khi dd bj tac dung cua djch chiet kha ndng phat trien, sinh acid la rat yeu (hinh 2). Gia trj pH cuoi cung trong miu doi chung la 5,2 trong khi miu cd bo sung djch chiet la va thdn xoai lan lugt la 6,37 va 6,61. Ket qua nay cho thay tac dung cua djch chiet la va than xoai cdn tac dung uc che su sinh acid cua S. mutans GS-5 ngay ca
sau khi da loai bd djch chiet ra khdi mdi trudng.
Ket qua nay cOng tuong tu khi ap dung doi vdi chung S. p 74 phan Idp tu ngudi Vidt Nam.
Kha nang diet vi khuin S. mutans tip djch chiit cay xoai
Be khing dinh tac dung cua djch chiet vd than va la xoai ldn vi khuin S. mutans ngoai thi nghidm tac dung ldn kha ndng sinh acid chung tdi cdn tien hdnh nghidn cuu kha ndng tidu didt cdc vi khuin ndy d hai gid trj pH mdi trudng Id trung tinh vd acid.
30 Thm gian (phut) Hinh 3: Khd ning diit vi khuin S
Doi chung nuoc (•); d6i chung ethanol pH 4 (•) vd djch chiet la xoai pH Tai pH 4 kha ndng giit chit 90% so lugng S.
mutans GS-5 vdi ca djch chiit tu vd thdn vd Id xodi deu nim trong khoang 15 phut trong khi d miu doi chung ethanol Id trdn 35 phut. Trong khi d pH 7 kha ndng giet S. mutans GS-5 tu djch chiit vd thdn vd Id xodi deu cao hon so vdi pH 4, sau 12 phut 90 % so lugng te bdo vi khuin dd bj tidu diet trong khi d miu doi chung ethanol Id gin 55 phut vd nude Id trdn 60 phut (hinh 3A).
So sdnh vdi cdc kit qua nghidn cuu trudc ddy cua Nguydn Quang Huy vd tdp the trdn doi tugng vd sao den vd Id sdn thuyen t 6 J 1 kha ndng giet vi khuin S. mutans GS-5 cua Id vd thdn xodi cao hon d pH 7,0 tuy nhidn lai thip hon d pH 4.
o 30 Thd-i gian (phut) mutans GS-5 (A) vi S.sp 74 (B)
pH7 (O); djch chiet thdn xoai pH 4 (•) vd pH 7 (•);
4 ( • ) va pH 7 (A)
Vdi chung S.sp 74 phdn Idp tir ngudi Vidt Nam thi kha ndng giit vi khuin ndy d djch chiet ethanol vd thdn xodi cd cao-hon so vdi la xodi a ca pH4 vd pH7 vdi thdi gian tuong ung khoang 14 vd 20 phut (Hinh 3B). K i t qua so vdi S.
mutans GS-5 kha ndng didt S. sp 74 cua djch chiet xodi Id tuong dong.
Tac dung mot sd phan doan t u djch chiit la va vd than xoai
Vdi muc dich budc dau tim hieu tdc dyng tir cdc hgp chit cd mdt trong djch chiet Id vd vo thdn xodi ldn vi khuin Streptococcus chiing tdi tien hdnh sdc ky ban mdng tdch chiit cdc hgp chat cd trong djch chiit (bang 1, hinh 4).
Bang 1: Cic phin doan dich chiit tir thin vi li xoii trin sic ky bin mdng
La xoai Than xoii
Phan doan Mau sac H# so Rf Phan doan Mau sic HesoRf
1 Xanh duong nhat 0,495 1 Vdng chanh 0,36
2 Hong nhat 0,52 2 Vang xanh nhgt 0,46
3 Xanh la cay vang 0,53 3 Xanh rdu ddm 0,57
4 Xanh den nhat 0,54 4 Vang nghd 0,65
5 Xanh reu dgm 0,57
6 Vang nghe 0,62 .
7 Xanh vang 0,72
40 TAP CHI DUQC HQC - 4/2011 (S6 420 NAM 51)
N g h i e n C L F U - K y t h u a t TCr dich chiet bing ethanol qua sic ky ban mong vai he dung moi TEAF (5:3:1:1) chiing toi nhdn thiy la xoai c6 7 phan d*oan (hinh 4A) nhieu phan doan han dich chiet vo than xoai 5 phan doan (hinh 4B). Tuy nhien cac bang vach d than xoai lai cho bang sic net han la xoai.
Trong cac bang vach xuat hien trong dich chiet la va vo than xoai bang co he so Rf = 0,57 thay xuat hien d ca la va than xoai.
Ket qua danh gia kiem tra hoat tinh ire che su phat trien cua cac chung Streptococcus tir cac phan doan tach tir ban sic ky duac the hien tren hinh 5. Cac phan doan tir la va vo xoai diu co tac dung ire chi su phat trien ciia cac chung Streptococcus d cac mire do khac nhau (the hien qua vdng ire chi) trong do phan doan 5 (he so Rf = 0,57) co hoat tinh manh nhit trong viec
ire che su phat trien cua ca chung vi khuin S.
mutans GS-5, S. sanguis cung nhu chung S.
sp74 phan l$p tu ngudi Viet Nam.
r S T .i -i ..i 1 B
Hinh 4: Anh sic ky ban mdng tCt dich chiit la xoai (A) va than xoai (B)
Mi yfl. »AVT: * " " I B M —
Hinh 5: Khi ning tic che sis phit triin S. sanguis (C) fi> cic phin Chiing toi dang tiep tuc tim hieu ca che tac dung ciia cac phan doan tach duac tir djch chiet la va vo than xoai len vi khuan S. mutans.
Ket luan
Dich chiet ethanol tir than xoai va la xoai co hoat tinh uc che su sinh acid cung nhu giet vi khuan S. mutans GS-5, S. sanguis vi S. sp 74 phan lap tir ngudi Viet Nam.
Qua sic ky ban mong di xic dinh mpt so phan doan tir djch chiet la xoai va than xoai trong dd phan doan co h$ so Rf =0,57 co hoat tinh ire che phat trien cua Streptococcus cao nhat.
Ldi cam an: Cong trinh duac hoan thanh vdi su hd tra kinh phi ciia Trung tam Nghien ciru Chau A va Quy Giao due Cao hoc Han Quic, Oai hoc Quoc gia Ha Npi. Be tai cd su dung cac trang thiet bj thuoc Phdng Thi nghiem TrQng diem Cdng nghe Enzym va Protein.
cua S. mutans GS-5 (A), S. sp74 (B) vi doan chay sic ky bin mdng
Summary
Mangifera indica L. (Anacardiaceae) grown commonly in many parts of the world and widely used in some Vietnamese traditional medicines was tested for treatment of dental caries. As being considered the main pathogen of dental caries due to high acidogenicity and acidurance, oral streptococci isolated from Vietnamese was subjected to the test of ethanolic leaf and branch extracts of M. indica for antimicrobial activities by acid production-inhibiting and killing methods.
The tested extracts inhibited acid production of the oral bacteria in a pH-drop assay with glucose in excess. The final pH value of the control sample dropped down to 4.5, while the pH values of the suspensions containing the plant extracts ranged from 5.84 to 7.09. Also, the extracts produced a high lethality to the oral streptococci in both acidic (pH 4.0) and neutral (pH 7.0) media. At 10% concentration, the extracts killed 90% of oral streptococci
• N g h i e n c m j - K y t h u a t population after ca. 15 min's at pH 4.0 and 20 min's at pH 7.0. Of some silica gel chromatography fractions of the extracts tested, those with Rf of 0.57 were most active. Further investigation is neccesary for isolation, characterizaion of the active components of the M. indica and defining their anticaries mechanism.
Keywords Mangifera indica L, Streptococcus mutans, sau ring, pH drop
Tai lieu t h a m k h a o
1. Chen CP., Lin CC, Namba T. Screening of Taiwanese drugs for antibacterial activity against Streptococcus mutans J. Ethnopharmacol., (1989).
27: 285-295.
2. 06 Tat Lpi . Nhung cay thuPc va vi thuoc Viet Nam. (1986)A/X6 Khoa hoc va Ky thuat.
3. Jose.S., Beegum R JC. Invitro susceptibility of viridans streptocci to leaf extracts of Mangifera Indica.
Indian J.Microbiol. (2007) 47: 160-163.
4. Ma, Y., Curran, T.M., Marquis, R.E. . Rapid procedure for acid adaptation of oral lactic-acid bacteria and further characterization of the response.
Can. J. Microbiol. (1997) 43: 143-148.
5. Nguyen Thi Mai Phuong, Bui Tuyet Anh, . Dieu tra tac dgng khang vi khuan sau rang Streptococcus mutans cua mpt so thuc vat Viet Nam. Tap chi Dime lieu (2007)12: 19-23.
6. Nguyen Quang Huy, Pham Thanh Nga, Phan Tuan NghTa, Tim hieu tac dung ch6ng sau rang cua djch chiet vo cay Sao Den (Hopea odorata R.). Tap chi Diroo hpc 2005.45 (350): 13-19.
7. Nguyen Quang Huy, Ngo Van Quang, Pham Van Kiem, Phan Tuan NghTa, Acid asiatic phan lap tu cay san thuyen Syzygium resinosum va tac dgng cua no len vi khuan Streptococcus mutans. Tap chi DuvchQC 2007.47(375): 19-22.
8. Nguyen Quang Huy, Phung Thj Thu Huong, Phan Tuan NghTa. Phan lap va nhan dang mpt so chung vi khuan Streptococcus mutans tu nguoi Viet Nam. Tap chi Cong ngh$ Sinh hoc 2007 5(3): 291- 297.
9. Phan, T.N., Krisch, A.M. and Marquis, R.E..
Selective sensitization of bacteria to peroxide damage associated with fluoride inhibition of catalase and pseudocatalase. Oral. Microbiol. Immunol. (2001) 16:
28-33.
10. Phan T.N., Reidmiller J.S., Marquis R.E.
Sensitization of Actinomyces naeslundii and Streptococcus sanguis in biofilm and suspensions to acid damage by fluoride and other weak acids. Arch.
Microbiol. (2000). 174: 248-255.
11. Xiao J., Zuo Y., Liu Y., Li J., Hao Y., Zhou X.
Effects of Nidus Vespae extract and chemical fraction on glucosyltranferases, adherence and biofilm formation of Streptococcus mutans. Arch. Oral. Biol.
(2007) 52: 869-875.
D a n h g i a t a c d u n g c u a r e c a y b a b e n h
( E u r y c o m a l o n g i f o l i a J . ) l e n h a n h v i t i n h d u e
c h u o t t h u c n g h i e m
Dat van de
Cay ba. benh va cac che pham tu ba benh trong nhung nam gan day da duac dua vao su dung dudi dang thuc pham chuc nang de tang cudng, hd tra cdc chuc ndng sinh ly d nam gidi.
Duang Thj Ly Huang1, Pham T h j Nhir Hang1, Trjnh Thj Kim Anh', Nguyen T h j Hai Ha1, Nguyen Tran Giang Hirang2, Tran Due Phan2 'Dqi hoc Duoc Ha Noi, 2Dai hoc Y Ha Noi
Tren the gidi, cac nghidn cuu thuc nghidm
ve tac dung tdng cudng chuc nang sinh san nam cua rd ba bdnh chu yeu tap trung khai thdc cdc hoat dong tinh due cua chuot due hudng ve phia chudt cai nhu hdnh vi tiep cdn, thdm nhdp duang vdt vao am dao chudt cdi, xuat tinh hay
