Nghiên cứu ảnh hưởng của axit asiatic tách chiết từ cây sắn thuyền syzygium resinosum g lên vi khuẩn streptococcus mutans

DAI HOC QUdC 6IA HA Ndi TRirdNG DAI HOC KHOA HOC XlT NHlfiN NGHifiN ctru ANH HU6NG CUA AXIT ASIATIC TACH CHI^T Ttr CAY SAN THUY^N Syzygium resinosum G L£N VI /t KHUAN Streptococcus mutans Ma so QT 08-30 Chu tri de tai: ThS Nguyen Quang Huy DAI HOC QUOC GIA HA NOI TRUNG TAM THONG TIN THU VIEN 000^00000 A4 Ha Ndi, 2008 Bao cao torn tat a Tdn 66 tki: Nghien curu anh hirong cua axit asiatic tach chiet tur cay S^n thuy^n (Syzygium resinosum Gapnep) len vi khuan Streptococcus mutans, Mas6: QT 08-30 b Chii tri 66 tai: ThS Nguyen Quang Huy c Cdc can bd tham gia: ThS Nguyen Quang Huy, CN Pham Anh Thuy Duomg, CN Phung Thi Thu Hucmg, CN D6 Thi Hue va SV Bui Thanh Duyen d Muc ti6u va n6i dung nghi6n cuu: - Muc tifiu Tdch chie't va nghifin cihi anh hucmg ciia axit asiatic tijr dich chiet la cay SSn thuy^n [Syzygium resinosum Gapnep) Idn vi khudn gay siu ring Streptococcus mutansphSin lap tiT nguod Viet Nam - N6i dung • Tach chiet, xac dinh c6ng thiic hoa hoc ciia axit asiatic tijr la cay San thuyen • Nghien cihi anh hucmg ciia axit asiatic ien su sinh axit va tieu diet vi khuan gay sau rang mutans H2 phan lap tiir ngucri Viet Nam • Budrc 6iu nghien cuu co che' tac dung ciia axit asiatic len vi khuii mutans H2 th6ng qua viee irc che' hoat 66 mot sd enzym co lien quan e Cac ke't qua dat duac: • Da tach chie't, tinh sach va xac dinh ca'u true hoa hoc m6t ch^t thuc vat thii ca'p thu6c nhom terpene la axit asiatic (C30H48O5) tit dich chie't la San thuyen • Axit asiatic co tac dung iic che' sir sinh axit, su tieu thu oxy va giet ciia vi khuan mutans H2 a pH 4,0 Kha nang iJc che su sinh axit tang len phoi hop voi NaF hay H2O2 Axit asiatic co tac dung giet m6t sd vi khuan duomg mieng khac la S sanguis va rattus • Axit asiatic tiir la San thuyen fc che' hoat 66 cac enzyme phosphoryl hoa ducmg-PTS (IC50 = 1,0 mM), lactate dehydrogenase (IC50 = 0,75 mM), pyruvate kinase (IC50 = , mM) lien quan de'n cac qua trinh sinh axit, ATPase (IC50 =1,5 mM) lien quan de'n qua trinh chiu axit, NADH oxidase (ICjf, = mM) lien quan de'n qua trinh tieu thu oxy ciia Streptococcus mutans H2 phin lap tiir ngucri Viet Nam f Tinh hinh kinh phi: Tdng kinh phi d^ tai la: 20.000.000 VND XAC NHAN CUA BAN CHU NHI$M KHOA CHU TRI Bi TAI (ky va ghi ro ho ten) (ky va ghi ro ho ten) PHO CHU NHIEM KHOA POSTS, ^/(a/i ^tiak ThS Nguyen Quang Huy J f ^ XAC NHJ^N CUA TRUONG »M6 MIEU TRUONG zJiiA^t/oVxO Summary a) The title of subject: Investigation of anticaries effects of asiatic acid extracted from Syzygium resinosum Gapnep on Streptococcus mutans Numerical code: QT 08-30 b) The grant holder: MSc Nguyen Quang Huy c) The Participants: MSc Nguyen Quang Huy, B.Sc Pham Anh Thuy Duong, B.Sc Phung Thi Thu Huong, B.Sc Do Thi Hue and Student Bui Thanh Duyen d) Objectives: Extraction and isolation of asiatic acid from Syzygium resinosum G leave and investigation of anticaries effects of asiatic acid to Streptococcus mutans H2 which isolated from Vietnamese patient e) The obtained results: • One compound belong to terpene group from the extracts of Syzygium resinosum was isolated, purified and identified as asiatic acid (C30H48O5) • Asiatic acid from Syzygium resinosum was inhibitory for acid production by S mutans H2 Combination of asiatic acid with NaF or H2O2 exhibited a remarkably enhanced inhibitory effect on acid production of mutans H2 In addition, asiatic acid from plants were highly lethal for mutans H2 at pH 4.0 and was shown to kill other oral bacteria such as S sanguis and S rattus • Asiatic acid from Syzygium resinosum leaves inhibited activity of glucosephosphotransferase system (IC50 was mM), lactate dehydrogenase (IC50 was 0.75 mM), pyruvate kinase (IC50 was 1.2 mM) related to acid production, ATPase (IC50 was 1.5 mM) related to acid adaptation, NADH oxidase (IC50 was I mM) related to oxygen consumption of mutans • Publications: 01 paper DANH MUC CAC K t HIEU, CAC C H VIET TAT ADP Adenosine di phosphate ATP Adenosine tri phosphate DMFT Chi sd rang sau, rang mS^t hay tram rang sau rang (Decayed Missing Filled Teeth) ICst, N6ng d6 urc che' 50% hoat 66 enzyme (Inhibitory concentration) kDA Kilo dalton Kb Kilo base LDH Lactate dehydrogenase MS Ph6 khdi (Mass spectrophotometry) NAD"^ Nicotinamide adenine dinucleotide NMR C6ng hucmg tiir hat nhan (Nuclear magnetic resonance) NOX NADH oxidase PEP Phosphoenolpyruvate PTS He thdng enzyme chuyen phosphate (Phosphotransferase system) RFLP Da hinh d6 dai cac doan phan cat gi6i han (Restriction Fragment Length Polymorphism) SDE Sao den {Hopea odorata Roxb.) SOD Superoxide dismutase STH San thuyen {Syzygium resinosum Gagnep.) TSA M6i trucmg thach dau tuomg phan giai (Tryptic Soy Agar) TY Tryptone cao na'm men (Tryptone yeast extract) MUC LUC Trang M DAU C H U O N G T O N G Q U A N TAI LIEU 1.1 Benh sau rdng va cac yeu to lien quan 1.1.1 Benh sau rang 1.1.2 Vi khuin Streptococcus mutans va kha nang gay sau rang 1.2 Cac bien phap ngdn ngura phong chong sau rang 1.2.1 Sir dung chat khang khudn 1.2.2 Sir dung cac bien phap khac 1.3 Cac chat thuc v$t thur c^'p 1.3.1 Nhom cac hap chSit terpene 10 1.3.2 Tac dung khang khudn cua cac hap chat nhom terpen 10 1.3.3 Vai net ve cay San thuyen {Syzygium resinosum G) 11 CHUONG NGUYfeN L l £ u VA PHUONG PHAP NGHlfiN CtTU 13 2.1 Nguyen lieu 13 2.1.1 Chiang vi sinh vat va mau la Sdn thuuyen 13 2.1.2 Hoa chat 13 2T.3 Thiet bi thi nghiem 13 2.2 Phuang phap nghien curu 13 2.2.1 Nu6i ca'y vi khudn va chudn bi miu te' bao 13 2.2.2 Xac dinh mu'c sinh axit ciia vi khuan bang Ihi nghiem giam pH 14 2.2.3 Xac dinh miJc d6 sdng sot ciia ciia vi khuan 14 2.2.4 Do mirc 66 tieu thu oxy ciia te bao 14 2.2.5 Xac dinh hoat 66 m6t sd enzyme lien quan ciia S mutans 15 2.2.5.7 Phan tick hoat ATPase 15 2.2.5.2 Phan tieh hoat phosphotransferase (PTS) 15 2.2.5.3 16 Phan tick hoat NADH oxidase Summary a) The title of subject: Investigation of anticaries effects of asiatic acid extracted from Syzygium resinosum Gapnep on Streptococcus mutans Numerical code: QT 08-30 b) The grant holder: MSc Nguyen Quang Huy c) The Participants: MSc Nguyen Quang Huy, B.Sc Pham Anh Thuy Duong, B.Sc Phung Thi Thu Huong, B.Sc Do Thi Hue and Student Bui Thanh Duyen d) Objectives: Extraction and isolation of asiatic acid from Syzygium resinosum G leave and investigation of anticaries effects of asiatic acid to Streptococcus mutans H2 which isolated from Vietnamese patient e) The obtained results: • One compound belong to terpene group from the extracts of Syzygium resinosum was isolated, purified and identified as asiatic acid (C30H48O5) • Asiatic acid from Syzygium resinosum was inhibitory for acid production by S mutans H2 Combination of asiatic acid with NaF or H2O2 exhibited a remarkably enhanced inhibitory effect on acid production of S mutans H2 In addition, asiatic acid from plants were highly lethal for 5* mutans H2 at pH 4.0 and was shown to kill other oral bacteria such as S sanguis and S rattus • Asiatic acid from Syzygium resinosum leaves inhibited activity of glucosephosphotransferase system (IC50 was I mM), lactate dehydrogenase (IC50 was 0.75 mM), pyruvate kinase (IC50 was 1.2 mM) related to acid production, ATPase (IC50 was 1.5 mM) related to acid adaptation, NADH oxidase (IC50 was I mM) related to oxygen consumption of mutans • Publications: 01 paper DANH MUC CAC KY HIEU, CAC CHlJ VIET T A T ADP Adenosine di phosphate ATP Adenosine tri phosphate DMFT Chi sd rang sau, rang mSit hay tram rang sau rang (Decayed Missing Filled Teeth) ICso Ndng 66 iJc che 50% hoat 66 enzyme (Inhibitory concentration) kDA Kilo dalton Kb Kilo base LDH Lactate dehydrogenase MS Ph6 khdi (Mass spectrophotometry) NAD* Nicotinamide adenine dinucleotide NMR C6ng hucmg tijr hat nhan (Nuclear magnetic resonance) NOX NADH oxidase PEP Phosphoenolpyruvate PTS He thdng enzyme chuyen phosphate (Phosphotransferase system) RFLP Da hinh dai cac doan phan cat gidi han (Restriction Fragment Length Polymorphism) SDE Sao den {Hopea odorata Roxb.) SOD Superoxide dismutase STH San thuyen {Syzygium resinosum Gagnep.) TSA Moi trucmg thach dau tuang phan giai (Tryptic Soy Agar) TY Tryptone cao nam men (Tryptone yeast extract) MUC LUC Trang M DAU C H U O N G T O N G Q U A N TAI LIEU 1.1 Benh sau rdng va cac yen to lien quan 1.1.1 Benh sau rang LI.2 Vi khudn Streptococcus mutans va kha nang gay sau rang 1.2 Cac bien phap ngdn ngura phong chong sau rang 1.2.1 Sir dung chat khang khudn 1.2.2 Sir dung cac bien phap khac 1.3 Cac chat thuc vat thur cap 1.3.1 Nhom cdc hop chSft terpene 10 1.3.2 Tdc dung khang khudn ciia cac hap chat nhom terpen 10 1.3.3 Vai net \6 cay Sdn thuyen {Syzygium resinosum G) 11 CHUONG NGUYfiN LI$U VA PHUONG PHAP NGHIEN CClU 13 2.1 Nguyen lieu 13 2.1.1 Chiing vi sinh vat va mau la Sdn thuuyen 13 2.1.2 Hoa chat 13 2.1.3 Thiet bi thi nghiem 13 2.2 Phuang phap nghien curu 13 2.2.1 Nu6i ciy vi khudn va chudn bi miu te' bao 13 2.2.2 Xac dinh mu'c d6 sinh axit ciia vi khudn bang thi nghiem giam pH 14 2.2.3 Xac dinh mu"c 66 sdng sot ciia ciia vi khudn 14 2.2.4 Do miJc d6 tieu thu oxy cua te'bao 14 2.2.5 Xac dinh hoat d6 m6t sd enzyme lien quan cua S mutans 15 2.2.5.7 Phan tick hoat dp ATPase 15 2.2.5.2 Phan tick hoat dp phosphotransferase (PTS) 15 2.2.5.3 16 Phan tick hoat NADH oxidase 2.2.5.4 Phan tick hoat dp lactate dehydrogenase 76 2.2.5.5 Phan tick hoat dp pyruvate kinase 17 2.2.5.6 Phucmg phap xac dinh cdu triic hoa hoc cdc chat thi/c vat thir ca'p 18 2.2.5.7 Phuang phap xu ly sd lieu 18 CHUONG K^T QUA 15> 3.1 NGHI£N COU Tach, tinh sach, nghien curu tinh chat cua mot sd chat thur cap co 19 hoat tinh khang khuan s^u rdng tiir la cay Sdn thuyen 3.1.1 Tim hi^u cac nhom chat thiJcSfp co la Sdn thuyen 19 3.1.2 Tach chie't tinh sach m6t sd ch^t thir dp iix Id cay Sdn thuyen 20 3.2 Tac dung cua axit asiatic tach tur la Sdn thuyen len sir sinh axit 23 cua mutans H2 3.3 Tac dung gay chet \6i S mutans H2 bai axit asiatic 25 3.3.1 Tac dung ciia axit asiatic len su giet S mutans H2 a pH va pH7 25 3.3.2 Tac dung ciia axit asiatic phdi hop voi NaF va H2O2 ddi v6i viee giet vi khudn S mutans H2 26 3.3.3 Tac dung ciia axit asitatic ddi vdi m6t sd vi khudn ducmg mieng 27 3.4 Tac dung cua axit asiatic len sir tieu thu oxy cua S mutans H2 28 3.5 Tac dung ciia axit asiatic len hoat mot sd enzyme ciia mutans 28 3.5.1 Tac dung len hoat 66 F-ATPase 28 3.5.2 Tac dung len hoat d6 phosphotransferase 29 3.5.3 Tac dung len hoat 66 pyruvate kmase 30 3.5.4 Tac dung len hoat lactate dehydrogenase 31 3.5.5 Tac dung len hoat d6 NADH oxidase 31 K£T LUAN 33 D^NGHI 33 TAI LifiU THAM KHAO 34 PHU LUC 37 ST3A-MeOD-C13CPD&OEPT DEPT90 I MMMNMBMMMMWWNBHOP^^ -"^^ 190 r ——r 180 170 ISO 1" 150 -r 140 —i 130 120 —i—-^ 110 100 90 BO r70 -T 60 50 i 40 UmAiX i 30 T" 30 DEFT135 L CH&CH3 mmmmm ikMMNP f""""V'' CH2 ""T 190 F 180 — — , 170 160 T ISO , —""-[ -• 50 40 -I 30 | 20 F' | ST3A-MeOD-Cl3CPD&DEPT DEPT90 MX k^ ,,.^ 45 —ras 30 40 V-4L >VM I « ^ l ^ « DEPT135 i 'H&CH3 i^^UoiJ 'f'lyj T 'ai »L»r> *-•»"•• I iiilJftm^iniiri [•••rnr* — rg-»i""*i T - ^ i'~iii~ i^fT^ • - ,• — aJI J •• •'F^am a n * »MB ^ w CH2 45 Jj- ^ 35 25 30 I Aw W ^ ^J^AsKWlằ ^ N IAI K^^^#ằW^ai Viet Nam Effects of Syzygium resinosum Gapnep asiatic acid on Streptococcus mutans H2 isolated from Vietnamese subjects 06 Thj Hu6, Bui Thanh Duy6n, Nguyin Quang Huy, Phan Tuan Nghia Khoa Sinh hpc, Truang D^i hpc Khoa hpc Tf/nhien DHQGHN 20 The Use of 6-Carboxynaphthofluorescein Phosphoramldite in the Automated Synthesis of Quencher-Dye Oligonucleotide Probes (QDOPs) Vu Huynh, Mehrdad Majlessi, Dennis Adelpour, and James Russell Gen-Probe Incorporation 10210 Genetic Center Drive San Diego California 92121-4362, USA 21 Xac djnh nhanh gioi tinh phoi bo bing phu'ong phap LAMP (Loop Mediated Isothermal Amplification) Rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification D^ng Hodng Lam, Phgm Van Phue, Trin Thi Thanh Khu-ang, Tru-ang Hai Nhung, Phan Kim Ngpc Tnjxmg D^i hpc Khoa hpc Tt/ nhien, DHQG TP HCM 22 Nghien ci>u anh hu'O'ng cua lutein len SIP sinh tru'ong cua vl sinh vat bj chilu tia ti> ngtKR Study on the effect cf !:itefr! or rrricroG^ganism radiated UV Phung Xuan Le T - ^ Thi f-fjye- ^ r Nguyir Vsr Wui Khoa Sinh hpc, Truxmg Dai hpc Khoa hpc Ti/nhien DHQGHN 23 Comparison and verrScstior of the phylogenetic trees constructed by different approaches Piotr Wiiczak jacek LeiLfk ~ 'Faculty of3iat::g7C2: Sc:Br:zB3, Jrnver^ r^Zlei—a 36rz ^oJar:! " Center of Excellence BicExpioratorium Umvers^ry cf'/v'ar^3w Paand 24 The sequence multiple alignment approaches and reliability Jacek Leiuk Faculty of Biological Sciences, University of Zielona Gdra, Poland Center of Excellence BioExploratorium University of Warsaw, Poland t 25 Virtual reverse translation based on the genetic relationship within homologous positions - application and accuracy Anna Lutowska\ Jacek Leluk^^ r Tac dung cua acid asiatic tip cay s i n thuy§n len vi khuIn Streptococcus mutans H2 phan lap tiF ngu'O'i Viet Nam '-• ^ o Thi Hue, Bui Thanh Duyen, Nguyfen Quang Huy, Phan Tudn NghTa Khoa Sinh hoc, Tru-ang Oai hpc Khoa hpc Tu- nhien, DHQGHN Streptococcus mutans du-ac coi la nguyen nhan chinh gay sau rang kha nang chiu acid, sinh acid dan den an mon men rang [4] Cac nghien CLHU tren the gioi da cho thay viee si> dung cac chat khang khu^n la bien phap hOu hi#u de phong ngi>a sau rang va cac benh ve rang, mi^ng Cho den fluo v i n du-ac xem la mpt tac nhan ch6ng sau rang co hi#u qua cao vS du-p-c su- dung rpng rai cac san pham v? sinh va bao v# rang Tuy nhien tinh trang nhiem Huo (fluorosis) du'O'C phat hi#n tron^ cac cpng ddng dan cu- da khien cac nha nghien ci>u va san xuit phai tim kt^m nhu'ng hp-p chat rn&'\ co the thay^ the mpt ph^n fluo hay ket hpp vp-i fluo a n6ng dp th^p ma vSn co hieu qua ch6ng sdu rang [11] Hang logt cac chat tong hp-p nhu- chlorohexidlne, triclosan, cac acid yeu va nhieu ch4t co nguon goc tM- nhiSn ti> th^-c vat da du-pc nghien cu-u v^ thi> nghicm [6, 10 12 13, 16] Yamamoto vd cpng su* (15] cho thfly cac hpp chat polyphenol thp-c vgt, dgc biet che co kha nang i>c che sy phat trien va hinh mang bam rang cua S mutans Tu*ang ty, Murata va cpng sy [6] cung da tach chiet du-pc hpp chat 7-epiclusianone ti> cay Rheedia gardneriana co kha nang u-c che enzyme glucosyltransferase cua S mutans vai vai tro tong hp-p glucan, ca sa de hinh mang bam rang K6t qua nghien cu-u tryp-c day cua ehung toi [8] cho thay dich chi^t ethanol va acid asiatic t y la cay san thuyen co tac dung giet te bao vi khuan S mutans GS-5 [8] Trong bai bao chiing toi trinh bdy nhyng ket qua nghien cyu ve anh hu-ang cua acid asiatic cua s^n thuyen len mpt so qua trinh sinh ly, hoa sinh cua Chung S mutans H2 d y p c phan \^p t y ngu-oi Vi?t Nam [9] NGUYEN LIEU VA P H U a N G PHAP NGHIEN Cl>U Nguyen li$u Chung S mutans Marquis, Tru-ang Dgi hpc Cac ehung dyp-c giii' Te bao d y p c nuoi a 37°C GS-5 S sanguis NCTC-10904 va S rattus FA-1 la qua tgng cua Giao su Robert E Rochester, Hoa Ky Chung S mutans H2 du-pc ehung toi phan lap ti> ngu-oi Vl^t Nam [9] va cay chuyen hang tuan tren moi tru-ang tryotic soy agar (TSA) cua hang Difco (Hoa ky), moi tryang ehya 3% trypton, 0,5% dich chi^t n^m men va % glucose (TYG) Hp'p chat acid asiatic tach t y sSn thuyen ta san pham eua mpt eong trinh nghien cu'u tru-oc day eua ehung toi [8] Hpp chat d y o c hoa tan ethanol de dung cho eac thi nghiem cua nghien ei>u Fhu'ang phap nghien ci>u 2.1 Chuan bi djeh ti bao tham: Dich nuoi cay te bao S mutans dyp-c rya bSng dung dich muPi khoang 1x (50 mM KCI, mM MgCb) va d u ^ c hoa lai d^m Tris- HCl 50 mM, pH 7,5 eo bo sung mM VgC:: 50 mM KCI cho dp hap thy a b y a c song 700 nm (A700) eua huy^n djeh t§ bao = 14, toluen du-pc bo sung 6k dat n6ng cuoi eung la 10%, hon hpp du-pc u a 37°C phut va r6i chuyen sang lam dong da b^ng nita long phut, va sau du-pc chuyen tra Igi u 37°C de lam tan da Bu-ae lam dong da - lam tan da du-pc lap lai l^n HPn hpp sau du-pc ly tam loai bo dich noi va hoa tra lai d f m Tris- HCl pH 7.5 vai the tich b i n g 1/10 the tich ban 6ku Dieh t6 bao chuan bj du-pe sy dgng cac thi nghiem vai enzyme, 2.2 Do mu'c cfp sinh acid cua te bao: Kha nang sinh acid cua te bao du-pc danh gia thong qua viee lam giam pH eua moi tryang ben ngoai theo phu-ang phap du-pc mo ta [13] Dich nuoi c i y te bac S r-rj:ans ij-:yz rya bo moi tru-an b^ng ly tam sau du-pe hoa tan dung djeh KCI 50 mM co chua McCi: ' -^'v' aat -f 5c khoang 5x10® CFU/ml Dp pH cua djeh t^ bao d y p c di^u ehinh d^n 7.2 b^ng KOH I M GUccse i-j-r- ot SL:'^g f a ! nong dp % , dam bao moi tru-ang du- thua ca ehat cho qua trinh duang phan, Sy giarr c ^ "^c ^ ^ J T - C ZC SJ s i - r acid qua trinh d y a n g phan du-pc b i n g may pH Horiba (Nhat Ban) 2.3 Xac djnh dg song sdt cua vi khuan: Tae dgng gay eh§t doi voi S rputans dupe tien hann thee phuang phap d y p c mo ta'trong [12], Tac dung gay chet cua dich chidt du-ae danh gia bang gia tn :: = uogN'^^c• vc?: No la so vi khuan ban dau, N la so vi khuan tgi thai diem nghien eu-u, Gia tn D tL^crg ung voi LogN/Nc = -': •= t-^r zar cathi^t de 90% q u l n the vi khuan bi g i l t chet 2.4 Do mCrc tieu thu oxy cua te bao: Ho hap cua t l bao vi knuin dypc dsn- z:a r:z-~ z-i3 - ec dc TTIJ: dp tieu thM oxy moi tryang theo phyong phap cua CacSveii va cpng 3_- ;3; s u ^ d - - ; zzs •- ^ar '^z^-.e^ ^r: a= CO toe dp tieu thu oxy eao Cac t l bao nghien cuu du'pc n j o cay sau f c ^ r -Ja Ian - ::ung a^^ - _• "w rnM co chya MgCb mM Cac dung dieh t l bao sau du'pc bo sung glucose den " : n g c^^O' c j - g 'a 0.4',, cj-^g dich te bao d y p c lac manh tren may vertex de dat dp bao hoa oxy sau sy tieu thu oxy Q-u-pc o nhiet phong, su dgng may oxy WTW (Du-e) 2.5 Xac dinh hoat dp mdt so enzyme: Hoat dp enzyme F-ATPase d y p c xac dinh theo phu'ong phap cua Sturr va Marquis [15]; Hoat enzyme PTS du-ae xac dinh theo phyang phap mo ta [1], Hoat lactate dehydrogenase (LD) du-pe xac dinh theo phyang phap cua Iwami va cpng sy [5], hoat pyruvate kinase (PK) dya tren phu-ang phap lu-png pyruvate tao K t T QUA vA T H A O LUAN A n h h i ^ a n g cua acid asiatic len qua trinh sinh acid, hd hap va g i l t cua vi k h u i n S mutans H2 dy.ng.SS^S:^SJ.S^^ nghicm nghien cu-u tac dung yc c h i d6i vai kha nang sinh acid cua vi khuan S mutans luon d y p c coi la thi nghiem khai diiu x a K i t qua nghien cu-u thu du-p-c a hinh cho thay acid asiatic^co tac dgng y c c h i s y san xuat acid eua S mutans H2 M l u d i i chu-ng nu-ac va ethanol co gia trj pH c u i i cung 3C 60 90 thi nghicm d y a i 4,0 acid asiatic vai nong dp Thai gian (phut) mM 6a Ore che s y sinh acid cua S mutans vai gia tri pH Hinh Tac dung cua acid asiatic len sy sinh acid Cling thai diem sau 90 phiit la 6,2, eao han so vol dich chilt -•-Odicbu-ngHiO; - - D6I chyng ethanol ling s6 a nong dp 0,005% Ket qua cd y nghia dac biet -A- acid asiatic; - • - Dich chiet tho vdi Chung vi khuan S mutans H2 nghien cu-u tryac day ctja ehung toi la ehung vi khuan co kha nang chiu acid mgnh [9] Acid asiatic cung i>c e h i qua trinh ho h i p tieu thg oxy eua vi khuIn S mutans H2 m l u t l bao d i i chu-ng nyac hay ethanol chi s a u j h a i gian phut lypng oxy da d y p c tieu thg h i t , a m l u co bo sung acid asiatic vai ndng dp mM sau 20 phut S mutans H2 da bi u-c e h i va khong tieu thg hit oxy hoa tan moi tru-dng (hinh 2) Nghien cu-u tru-ac day cua ehung toi [8] cho thay dieh chiet s i n thuyen va acid asiatic co tac dung g i l t vi khan S mutans GS5 d^c biet la tgi gia tri pH acid K i t qua nghien cu-u cua Chung toi cung k h l n g dinh acid asiatic eo tac dgng g i l t eac t l bao vi khuan S mutans H2 d pH (hinh 3) Ket qua tren eho thiy tgi pH 4, dich c h i l t tong so va acid asiatic deu ed tac dgng gilt vi khuan rd r#t, eg the pia trj D a mau cd acid asiatic mM la 10 phut Djeh chilt tong sd tu- la s i n thuyin d ndng dp 0,005% cung cd tae dgng g i l t S mutans H2 vdi D tu- d i n phut Cac kit qua phu hp-p vdi eac nghien cu-u tru-dc day [8, 9], vay acid asiatic cd t h i la hpp chat ddng vai trd ehu y l u sy hogt tinh khang S mutans eua s i n thuyin, Kha nang g i l t vi khuan S mutan H2 cua acid asiatic mdi trydng acid la thu vi, vi nghien cyu trydc day cua ehung tdi S mutans H2 phan l|p t y lU 1.5 20 Thdj gjan (phut) 25 Hinh Tac dung cua acid asiatic \&n vi$c tieu th^ oxy cira S mutans H2 - • - D6i chu-ng H2O: -A- acid asiatic; -•-Djchchtetthd ngydi Viet Nam khdng nhi>ng la ehung vi khuan sinh acid tdt ma cdn la ehung vi khuan chju acid manh so vdi ehung S mutans GS-5 [9] \^—""— "I 't •' -1 \ r 1- ,., „ -2 i -3 _] -4 Hinh Tac dung g i l t cua acid asiatic vdi t l bao mutans H2 mdi tru-dng pH 4,0 - • - Mlu d i i chij'ng; -A- acid asiatic - a - Dich chilt tho \5 30 45 Tlioi gian (phiil) 60 (I LS ^(1 45 T^cfi gian (phijl) fin Hinh Tac dung cua acid asiatic ddi vdi vl khu^n S sanguis (A) va S rattus (B) mdi tru-dng pH - • - Mdu d6i chdng; - • - acid asiatic ChiJng tdi cung tien hanh thi nghiem giel vi khuan tren ddi typng la eac vi khuan dydng mieng khae nhu- S, sanguis va S rattus su- dung hap chat acid asiatic Day cung la hai loat vi khuan bien dupe tim thay mang bam rang, Trong S sanguis cd kha nang sinh k i l m thi S, rattus cd kha nang gay benh nha chu [4] Gia tri doi vdi Chung S sanguis NCTC-10904 va S rattus FA-1 d ndng dp acid asiatic mM tu-ang dng la 10 phut va phut d pH (hinh 4) Tae dgng diet k h u I n eua acid asiatic khdng chi vdi S, mutans H2 ma eon vdi hai ehung vi khuan khac khoang mieng d i l u khang djnh them tiem nang chdng sau rang cua hpp chit Trong cae c h i t khang khuan bao ve rang mieng fluo du-pc To chde Y t l T h i gidi khuyen cao si> dgng vu-a cd kha nang khang khuan vu-a cd kha nang tai tgo men rang, Fluor natn (NaF) la p h i n chinh dupe su- dgnq thuoc danh rang hay nu-dc sue mieng Hydro peroxide (H2O2) la c h i t khang khuan du-pc su- dung mpt so san pham bao ve rang mieng Do vay ehung tdi da t i l n hanh nghien cdu anh hu-dng cua viec phdi hpp acid asiatie 57 vdi NaF hogc H2O2 ddi vdi S mutans H2 K i t qua cho t h I y phdi hp'p vdi NaF ndng dp 0.5 mM (hinh 5A) hay H2O2 0,6 mM (hinh 5B) thi kha nang gilt t l bao vi khuan cua acid asiatic tang len (gia tri D vdi ca hai tru-dng hap la khoang phut) Hieu qua tac dgng cua hap c h i t acid asiatic phdi hap vdi cac chat bao ve rang mieng du-dng nhu- cd tinh cpng tyang t y n h y tac dung cua s y phdi hpp cua fluo vdi cac acid y l u nghien cu-u cua Belli va cOngsy[1] n 15 W 45 60 Thdi gian (phut) 15 M) 45 fi() Thdi gian (phut) Hinh Tac dung gifet vi khuin S mutans H2 cua acid asiatic ph6l hp'p vdi NaF (A) va H1O2 (B) A n h h u a n g cua acid asiatic len mot s d enzyme cua mutans H2 • • - Mlu d6( chu-ng, -A-acid asiatic: - • - N a F hay HjOj o l di sau tim hieu tac dgng cua acid asiatie len S mutans H2, ehung tdi da t i l n hanh nghien cu-u anh h y d n g cua acid asiatie len enzyme F-ATPase va PTS eua ehung vi khuan F-ATPase va PTS ta nhyng enzyme ddng vai trd quan trpng viee chdng chju acid ciia vi khuan S mutans mdi tru-dng acid A R 100 100 so \ 60 40 • ^~~~^ xo 60 \ 40 ^ 20 20 () \ ^ 05 I 15 Nnng(!(mM) Niing ill) (mM) Hinh Tac dung cua acid asiatic len hoat F-ATPase (A) vd PTS (B) cua S mutans H2 - ^ - Mlu dii chu-ng: - • - acid asiatic K i t qua d hinh cho t h l y acid asiatic 6- ndng dp mM da yc c h i 50% hogt dp cua F-ATPase, Murata va cpng sy [7] cho thay hap chat 7-epiclusianone tach ty R gardnerriana ed tac dgng dc che sy sinh acid, tao biofilm cue S mutans Hpp c h i t vdi ndng dp 100 MQ^HII dc e h i tren 60% hogt dp cua enzyme F-ATPase a S mutans Ket qua eho thay a a d asiatic cd the la mpt chat dc che manh cua F-ATPase Doi vdi enyzme PTS nong acid asiatic dc che 50% hogt dp cua enzyme la 1,5 mM (hinh 5B) Tac dgng dc che cac enzyme F-ATPase va PTS giai thich dupe mpt phan tac dgng dc c h i cua acid asiatie len sy sinh acid cua vi khuan S mutans H2C 100 100 lUO su fiO CO 40 \ E^ ^ S 20 I •ru: N.n 0.5 I 1.5 Nnn'j ill) ( m M l Hinr ~sz di-Tc cua acid ssisHz ie" neat enzyme LD (A) PK (B) va NADH oxidase (C) cua S, mutans H2 - • - Vii- 50' z''-jng -fc- acid asiatic K i t qua nghien ojiu tac duna cua acid asiatic len pyruvate kinase, lactate denydrogenase va NADH oxidase ciJa S mu/a/7S H2 (hinh 7) cho thly'ndng dp acid asiatic dc che 50% hoat cua cac enzyme luang i^ng la 0,7 mM, 1,2 mM va 0.8 mM Viee nghien eu-u tac dgng cua acid asiatic len hoat dd mpt so enzyme tren mang va enzyme tham gia qua trinh d y d n g phan eua S mutans H2 gdp p h l n giai thich co c h i tac dung cua acid asiatic doi vdi viec uc c h i smh acid, g i l t vi khuan, Tuy nhien day mdi chi la cac k i t qua nghien cuu ban d k , c i n co them nghien cuu sau hon de co the danh gia d l y du ve tac dung chdng sau rang cua acid asiatic t d la s i n thuyin TAI LI$U THAM K H A O Belli W.A Buckley D H Marquis R.E (1995), "Week acid effects and fluoride inhibition of glycolysis by Streptococcus mutans GS-5", Can J Microbiol 41: 785-791 F" J ' Bender G.R Sutton S.V.W., Marquis R,E (1986), "Acid tolerance, proton permeabilities and membrane ATPase of oral streptococci", tntect Immun 53(2) 331-338 Caldwell CE and Marquis RE (1999) "Oxygen metabolism by Treponema denticota" Oral Microbiol Imumunol 14 66-72 Hamada S and Slade H.D (1980) Biology, immunology, and canogemcity of Streptococcus mutans Microbiol Rev 44 331384 Iwami Y Abble K Takahash A.S Yamada T, (1992) Acid production by streptococci growing at low pH in a chemostat under anaerobic conditions Oral Microbiol Immunol 7: 304-308 Ms Y , Rutherford GO Curran T M,, R&dmiller j S , Marqi^is RE (1399), Membrane locus and pH sensitivity of paraben inhibition ctalka'i orodLicticrr by oral stratGcocci' Oral Micrct-.Qi 'mmura/., 14: 244-249 fgiuiara R M ^imetiis L £ ^'irrjcia P Sa^^s M, Nag^n TJ ^z%a\er PL Koo H (2008) Inhibitory effects of 7«:;a;-5.=.- c jfzl^izsr ^-.-r-ss .^^nz^^-cz-y sr.c b.cf rr *c~a::cn b- Streptococcus mutans' FEMS Microbiol Lett t-8 NgL;yer Cuang Hay P^^an J-jzi Ngnia Hzt Van Quang Pr.an Van Kiem (2007) "Acid asiatic phan lap tu* cay sin thuyĐn ãã.S/Tf-gii::^ su;..ZIL.- Is^re:;; v.2:3c z^ng c;la no len v; trusn Streptococcus mutans" Tap chi Duoc hoc 47(375) 19-22 Nguyen Ckiang Huy P^ung Th; Thb HuT3'n_g Phan Tuan NghTa (2007) "Phan lap va nhan dgng mpt s6 Chung vi khuin Slr&JtDCOCCuS mut3'^s tjj ^ j - d i V'st fsar^.' ao chi Cong ngne S>nhhoc 5(3) 291-297 10 Nguyen Thi Mai Phu-ong, Pnan Tuln tsignia Nguyen Th| Ngpc Dao, DO Ngpc Lien, (2004), 'Thanh phSn polyphenol ciia vd qud mSng cut iGarcinia mangostana L) va tac dung i>c ch^ su- sinh acid cua vi khuan Streptococcus mutans GS-5", Tap chi Dupe hoc 2ZB ''5-2^ i : F^isn T N Kvifscn A VI V.arz:-3 R E IJCC; "Selective sensiilzation of bactena to peroside damage associatd with flouhde inniciticn of c s t a ; ^ srra pssazzzzia^ass' Oral Microbiol Imumunol 16: 28-33 12 Phan T.N Nguyen P T M Acrancnes J , Marquis RE (20G2), "Flouride and organic weak acids as respiration inhibition for oral rLreptocGCCi ;:i ac;a erv-i^ments" Oral Microbiol Imumunol., Mi2) ^^9-^24 13 Phan T.N and Marquis RE, (2006) "Triclosan inhibition of membrane enzymes of mutans streptococci", Can J Microbiol 52: 977-98314 Song J,H Yang T,C Chang K W Han S K , Yi H K , Jeon J G (2007), In vivo effects of a fraction separated from Polygonum cuspidatum root on the viability In suspension and biofilm formation of mutans streptococci, J Ethnopharmacol 112 419-425 15 SturrMG and Marquis R E (1992) 'Comparative acid tolerances and inhibitor sensitivities of isolated F-ATPases of oral lactic acid bacteria" Appl Environ Microbiol 5Q- 22Q7-229-\ 16 Yamamoto K and Ggawa T •2GCi > •Antimicrobial effects of phenolic compounds derived from plant", J Dent Res 80 724728 ' Cdng trinh dup'C thu'c hi^n co su ho trp- kmh phi cua de tai cap Dai hoc Qu6c gia Ha NOi ma s6 QT 08 30 SUMMARY Effects of Syzygium resinosum Gapnep asiatic acid on Streptococcus mutans H2 isolated from Vietnamese subjects Do Thi Hue, Bui Thanh Duyen, Nguyen Quang Huy Phan Tuan Nghia Faculty of Biology Hanoi University of Science The effects of asiatic acid extracted from Syzygium resinosum Gapnep Streptococcus mutans H2 which had been isolated from Vietnamese subjects were examined It was found that asiatic acid at a concentration of mM reduced the rale of acid production by S mutans H2 in a pH-drop assay with excess glucose Combination of asiatic acid with sodium fluoride or hydro peroxide sensitized the bacterial celts to acid killing al pH Further studies indicated that asiatic acid was inhibitory for F-ATPase Phosphotransferase system, pyruvate kinase, lactate dehydrogenase and NADH oxidase of S mutans H2 with a 50% inhibitory concentration of in the range of and mM The results suggested that asiatic acid could be a potential anticaries -jgent 59 ... Trong sd nhiitig vi khudn S mutans ludn dugc phdt hien nude bot, mang bam rang ciia ngudi va cd sd lugng cao d nhihig ngudi bi sdu rang [19] Chng vdi mutans, S sobrinus cung la loai vi khudn gay... each de phdng chdng sdu rang Id sii dung nhihig chdt lam ngpt khdc ma vi khudn khdng the tieu thu dugc 6i thay the' dudng Nhihig chd't khdng gay su sinh axit nen khdng gay sau rang Cdc hgp chd't... cdc chiing vi khudn ddi vdi ngudi sir dung chua dugc nghien cuu ky Theo lieu phdp nay, nhihig ehung vi khudn cd lgi hodc khdng gay hai dugc cdy vao mang bdm rang trudc cdc ehung khdng mong mudn