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

Cdc chat khdng khudn tu nhien nay to ra co hieu qua trong viee phong isc che' su phdt trien ciia vi khudn duofng mieng, tac dung \sc che hinh thanh mang bam rang va co kha nang irng dun

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 5 mutans H2 phan lap tiir ngucri Viet Nam

• Budrc 6iu nghien cuu co che' tac dung ciia axit asiatic len vi khuii 5 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 5 mutans H2 a pH 4,0 Kha nang iJc che su sinh axit tang len khi phoi

hop voi NaF hay H2O2 Axit asiatic con co tac dung giet m6t sd vi khuan

duomg mieng khac la S sanguis va 5 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 = 1 , 2 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, = 1 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

(ky va ghi ro ho ten)

PHO CHU NHIEM KHOA

POSTS, ^/(a/i ^tiak J f ^

CHU TRI Bi TAI

(ky va ghi ro ho ten)

ThS Nguyen Quang Huy

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 5 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

glucose-phosphotransferase system (IC50 was 1 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 5 mutans

• Publications: 01 paper

DANH MUC CAC K t HIEU, CAC C H 0 VIET TAT

ADP Adenosine di phosphate

ATP Adenosine tri phosphate

DMFT Chi sd rang sau, rang mS^t hay tram rang do 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

C H U O N G 1 T O N G Q U A N TAI L I E U 2

1.1 Benh sau rdng va cac yeu to lien quan 2

1.1.1 Benh sau rang 2

1.1.2 Vi khuin Streptococcus mutans va kha nang gay sau rang 3

1.2 Cac bien phap ngdn ngura phong chong sau rang 5

1.2.1 Sir dung chat khang khudn 5

1.2.2 Sir dung cac bien phap khac 7

1.3 Cac chat thuc v$t thur c^'p 9

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 2 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 do 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 do ATPase 15

2.2.5.2 Phan tieh hoat do phosphotransferase (PTS) 15

2.2.5.3 Phan tick hoat do NADH oxidase 16

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

glucose-phosphotransferase 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 5 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 do 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 do 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

C H U O N G 1 T O N G Q U A N TAI L I E U 2

1.1 Benh sau rdng va cac yen to lien quan 2

1.1.1 Benh sau rang 2

LI.2 Vi khudn Streptococcus mutans va kha nang gay sau rang 3

1.2 Cac bien phap ngdn ngura phong chong sau rang 5

1.2.1 Sir dung chat khang khudn 5

1.2.2 Sir dung cac bien phap khac 7

1.3 Cac chat thuc vat thur cap 9

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 2 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 Phan tick hoat do NADH oxidase 16

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 3 K^T QUA N G H I £ N C O U 1 5 >

3.1 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 trong 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 5 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 4 va pH7 25

3.3.2 Tac dung ciia axit asiatic phdi hop voi NaF va H2O2 ddi v6i viee giet vi 26

khudn S mutans H2

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 do mot sd enzyme ciia 5 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 do lactate dehydrogenase 31

3.5.5 Tac dung len hoat d6 NADH oxidase 31

K £ T L U A N 33

D^NGHI 33 TAI LifiU THAM KHAO 34

PHU LUC 37

^ M d D A U

Sau rang la m6t trong nhung benh ph6 bien hien nay Theo sd lieu dieu tra gdn day ciia Vien Rang Ham Mat Viet Nam, khoang 90% dan sd Viet Nam mac cac benh v6 rang, mieng trong do pho bie'n nhat la sau rang va viem quanh rang Benh sau rang khdng chi gay anh huong toi tinh trang su'c khoe ciia ngir5i benh ma con lam idn kem chi phi d^ cham soc va siSra chiia rang bi tdn thuang

Benh sau rang thuc chat la su tieu huy ca'u true ciia men rang va nga rang, tao

nen 16 h6ng tren bi mat rang, benh do vi khudn gay ra Trong sd han 500 loai vi sinh vat ducmg mieng Streptococcus mutans {S mutans) duac xem la nguyen nhan chinh

gay ra sau rang, do ehiing co kha nang sinh axit manh va chiu axit tdt Vi khudn nay sijr dung duomg trong thuc phdm de sinh axit lactic an mon men rang lam thanh 16 sau va gay sau rang

Cach tdt nhat d^ han che' benh sau rang la ve sinh rang mieng thucmg xuyen

vd dung each, ciing vdi viee han che' cac thiJc an, dd udng co nhi6u duofng Tuy nhien, viee dam bao ve sinh rang mieng, che' d6 an udng thucmg kho thuc hien duac

Do vay nhi^u bien phap khac nhau nham ngan ehan sau rang da duac nghien curu va ihig dung Siir dung cac chdt co kha nang khang khudn nhu fluor, triclosan,

clohecxidine la m6t bien phdp 6i phong chdng sau rang hieu qua Gdn day, nhieu

chat khdng khudn co ngudn gdc thuc vat da duac tdch chie't, nghien cuu va thir

nghiem Cdc chat khdng khudn tu nhien nay to ra co hieu qua trong viee phong isc che' su phdt trien ciia vi khudn duofng mieng, tac dung \sc che hinh thanh mang bam

rang va co kha nang irng dung trong thuc tien

O Viet Nam, cdc loai thuc vdt diing de chiJa cdc benh rat phong phii va da

dang Tuy nhien nhiitig nghien ciiu \6 ca che' diet khudn ciia cdc loai thuc vat nay

cung nhu anh hucmg ciia ehiing vdi cdc loai vi khudn gay sau rang vdn con han che Chua CO nhi^u hap cha't tinh sach duac tdch chiet nghien ciJu tdc dung len vi khudn gdy sau rang De tai nghien ciiu ciia ehung t6i nham muc dich tim hieu tdc dung ciia

axit asiatic tir la Sin thuyen len ehiing vi khudn gdy sau rang Streptococcus mutans

Ket qua nghien cihi cua de tai se la ca sa cho viee khai thdc va u'ng dung ngudn nguyen lieu nay trong cdc san phdm bao ve rdng mieng trong tuang lai

CHUONG 1: TONG QUAN TAI LIEU

1.1 Benh sau rdng va cac yeu to lien quan

1.1.1 Benh sau rang

Rang CO cin tao gdm 3 Idfp: men rang, nga rang va tuy rang (hinh 1) Men rang la Idfp ngoai cung rdn chdc nhii Men rang bao gdm chii yeu cdc tinh the

phosphate canxi dai va manh xdp canh nhau theo mot trdt tu nhat dinh Men rang rdt ben vihig, kh6ng bi v5, kh6ng bi co xdt nhung co the bi bao mon do axit a trong mieng Nga rdng la Idfp ben trong, la thanh phdn chinh ciia rang Tuy rang ndm d

chinh giiJa ciia rang, gdm n\6 lien ke't co chu'a tdn cung la than kinh va cdc mao mach lam cho rang cd cam gidc va 6i nu6i rdng Rang bi sau khi Idp men rang

khdng dugc bao ve tdt va bi axit an mon Axit hoa tan cdc tinh the' phosphate canxi

tren b6 mat rdng, tao ra cdc hd nho tren rang Cdc hd nay Idfn ddn, an sau vao cdc to

chirc ben trong ciia rdng (nga rdng, tuy rang) Vi khudn theo cdc hd sdu xam nhdp vao ben trong, gay viem tuy rang, tao ra cam gidc dau ddfn cho nguoi benh Vao giai doan cudi, cdc manh mdu va m6 thdn kinh bi che't, hinh thanh dp xe d chdn rang [5]

iMen rang (Ng^ rang Tuy Ldi

Hinh 1: Ca'u tao ciia rang [5]

Ba yeu td quan trong gay ra benh sau rang la: vi khudn, ducmg (trong thu'c an)

va thdi gian ducmg ciing vi khudn t6n tai trong khoang mieng Vi khudn gay benh

sau rang tdn tai va bam tren b6 mat rang nhd Idp mang bdm rang Chiing sir dung ducmg trong thu'c an va 66 udng de tao va phdt trie'n cdc mang bdm rdng ddng thdi

chiing tieu hod ducmg de tao axit, dn mon cac chat v6 ca d men rang va nga rang lam thanh 16 sau M6t thdi gian (khoang 20 phiit) sau khi an, lucmg axit dugc sinh ra dii Idfn de' bat ddu qua trinh men rang

Benh sau rdng chi dien ra khi ba ye'u id tren ciing tdn tai Vi the' ca sd ciia viee phong chdng benh sau rang la ngan ehan mdt hoae ca ba ye'u td xudt hien ciing mdt llie Ngoai ra, ye'u td thii tu khdc khdng kem phdn quan trong la ban thdn ngudi

benh Cdc ye'u td chii quan nhu tu6i tac, hoat ddng khdng binh thucmg cua tuyen nude bot, di tdt bdm sinh ciia rang cd the khie'n cho kha nang mdc benh tang cao va tdc dd benh tie'n tri^n nhanh

Benh sau rang la mdt trong 3 benh dugc to chiic Y te' the' gidi canh cdo ve mure

dd nguy hi^m chi sau cac benh tim mach va ung thu Trong cdc thdp nien trudc, ty le

sd ngucfi mdc benh sdu rang cao d ca nguoi Idn va tre em Trung binh mdi tre em 12 tu6i cd tur 8 de'n 10 rang sdu hoae rang da bi mdt do sau Chi sd DMFT dugc sir dung d^ phan anh sd rang sdu, sd rang mdt hay tram rang do sau Nam 1997, chi sd DMFT

d lura tudi trung nien (tur 35 de'n 44 tu6i) tai cdc nude cdng nghiep phdt trien nhu Canada, Nhdt Ban, Uc va cdc nude Bdc Au la 13,9 tuang duong mdt ngudi cd trung binh 13,9 rdng bi sdu, tram hay mdt Ty le nay d My Id cao hon 9 Nhutig ndm gdn day do cdng tdc tuyen truy6n, phdng chdng nen ty le sdu rang da giam tuy nhien cdn

d miic cao mdt phdn do di^u kien sdng dugc cai thien, lugng dudng tang lrong che'

dd dn nhu sir dung nhi6u loai bdnh keo va san phdm co dudng

6 Viet Nam theo sd lieu thdng ke 6i6u tra gdn ddy ciia Trinh Dinh Hai nam

2005, ty le sdu rang d lira tudi tren 18 la 87,5% , tre em dudi 18 tudi ty le sau rang d

mvtc 75,2% Nhin ehung d tat ea cdc nhom tudi thi ty le sdu rdng va chi sd DMFT

gia tang theo tudi Khdng cd su khdc biet v6 ty le sdu rdng giu'a nam va nil, nhung cd

su khdc biet \6 ty le sdu rang giiJa viing thanh thi va ndng thdn Ngudi sdng d thanh

thi cd ty le sdu rang cao hem ngudi ciing nhom tudi sdng d ndng thdn Khoang 10%

ddn sd Viet Nam cd rang hoan toan khde manh Cdc ket qua 6i6u tra nay cho thd'y,

viee chdm sdc va di6u tri rang sdu cho tre em, ngudi trudng thanh d nhi6u viing con ye'u, chi ddp ling dugc tir 1,49% den 6,38% nhu cdu [1]

1.1.2 Streptococcus mutans va kha ndng gdy sau rang

Nhirng vi khudn cd kha nang len men dudng thanh axit lactic tdn tai trong khoang mieng dugc xem la nguyen nhdn gdy benh sau rang Chiing chu yeu thudc 3

nhom vi sinh vat: Streptococci {S mutans Streptococcus sorbrinus Streptococcus, salivarius, Streptococcus melleri ), Lactobacilli {Lactobacillus acidophilus, Lactobacillus casein ) va Actinomyces Trong sd nhiitig vi khudn nay S mutans

ludn dugc phdt hien trong nude bot, mang bam rang ciia ngudi va cd sd lugng cao d

nhihig ngudi bi sdu rang [19] Chng vdi 5 mutans, S sobrinus cung la loai vi khudn

gay sau rang phd bie'n d ngudi Hai loai vi khudn nay ciing vdi mdt sd loai

Streptococci khdc xud't hien d mdt sd' ddng vat {Streptococcus rattus Streptococcus cricetus Streptococcus macacae, Streptococcus ferus ) dugc goi ehung la mutans

streptococci

3

p

Streptococcus mutans Idn ddu tien dugc Clack md ta nam 1924 sau khi dugc

phdn Idp tijr viing rang bi tdn thuang Tuy vay phai den nhirng nam 60 ciia the ky

XX vi khudn nay mdi dugc chii y de'n nhi^u khi cdc nha khoa hoc idp trung nghien

ciiu benh sdu rang tiir giai doan sdm 5 mutans la vi khudn Gram (+), chiu khi, tdn tai

d dang lien cdu khudn Nhieu nghien cihi da khdng dinh chiing vi khudn nay la nguyen nhdn chinh ciia benh sdu rang nhd kha nang sinh axit manh va chiu axit tdt [9, 14, 20, 23, 26]

Streptococcus mutans ien men dudng de' la'y nang lugng cho qud trinh sinh

trudng va phdt trien San phdm cudi ciing ciia qud trinh ien men dudng la axit lactic

5 mutans cd kha nang sinh axit manh bdi chiing cd kha ndng tieu thu nhieu loai carbohydrate khdc nhau Ben canh dd, S mutans cdn cd kha ndng san xud't cdc

polysaccharide ngoai bao Cdc poysaccharide cd tdc dung gdn, lien ke't cdc loai vi khudn vdi nhau hinh thanh nen mang bdm rdng, mdt ye'u td gay sau rang Ngoai ra,

5 mutans cung cd the' san xudt cdc polysaccharide ndi bao Polysaccharide ndi bao

dugc sir dung d^ ien men tao axit lactic khi khdng cd dudng tir ngoai dua vao

Dudng tiir mdi trudng ben ngoai vdo te' bao dugc phosphoryl hod chii ye'u nhd

he thdng phosphotransferase phu thudc vao phosphoenolpyruvate (PEP) Ddi vdi cdc

vi khudn Streptococci lrong mieng, ddy la enzyme ddng vai trd quan trong trong viee phosphoryl hod glucose va nhilu loai dudng khdc bao gdm mannose, fructose, sucrose, lactose va maltose [33]

Ngoai kha nang sinh axit manh, vi khudn S mutans cung nhu cdc vi khudn

khdc trong mang bdm rdng khoang mieng ludn phai ddi mat vdi stress axit sinh ra khi vi khudn tieu thu dudng Stress axit cd anh hudng bdt Igi de'n vi khudn qua viee

cd th^ lam ton thuang cdc dai phdn tijr trong dd cd ADN, protein Vi vay khi ddi

mat vdi stress axit, vi khudn S mutans cd nhihig protein va enzyme ddng vai tro bao

ve va sira chu'a cdc dai phdn tijr hay cdc he thdng sira chu'a ADN cam utig [35, 36]

Ngoai ra, Streptococcus mutans cung cd nhieu ca che' khdc nhau de chdng choi va

thich nghi vdi dieu kien mdi trudng axit [18] Mdt trong nhihig ca che'quan trong d^ thich nghi vdi di^u kien pH thdp la bam proton F-ATPase Day la enzyme lien ke't mang, cd vai trd bam proton tao ra su can bang pH cho te' bao F-ATPase gdm hai to hgp Fo va F,, td hgp FQ ndm tren mang cd hoat tinh van chuye'n proton Td hgp F, xiic tdc cho qud trinh vdn chuyen proton ke't hgp vdi su sinh tong hgp hay thuy phdn ATP [16] Khi thuy phan ATP thanh ADP va Phospho vd co, nang lugng dugc giai phdng Ndng lugng nay dugc sir dung de bam H"^ ra ben ngoai te' bao, duy tri trang thdi cdn bang pH ciia ndi bao [20]

i:

I- Mang te' bao cung ddng vai trd quan trong ddi vdi su thich nghi axil cua vi

khudn Ma va tdp ihi [28] cho rang tinh thdm proton cua mang td bao Streptococci

giam di khi te' bao thich nghi vdi mdi trudng cd linh axit cao [30] Ben canh dd cdc protein mang cung cd vai trd bao ve mang te' bao khi d dieu kien pH thdp

1.2 Cac bien phap ngdn ngura benh phong chdng benh sau rang

Benh sau rang cd th^ dugc ngan ngijra mdt cdch huu hieu bang cdeh ve sinh rang mieng thudng xuyen, ke't hgp vdi che' dd dn udng it dudng Tuy nhien, viee dam bao ve sinh rdng mieng, che' dd dn udng thudng khd thuc hien dugc Do vdy, nhieu bien phdp khdc nhau nhdm ngan ehan sdu rang da dugc nghien ciiu va litig dung 1.2.1 Sir dung cdc ehdt khdng khudn

Viee sir dung cdc chdt khdng sinh, khang khudn 6i phdng chdng benh sdu

rang, kiem sodt su hinh thanh mang bdm rang da dugc thuc hien trong nhieu nam qua Cac chdt khdng sinh nhu penicilin, ampicillin hay tetracycline cd tdc dung kim

ham su phdt tri^n ciia S mutans trong 6\iu kien in vitro Di6u thii vi la cdc dang a va

b ciia S mutans 6iu khd nhay cam vdi bacitracin va polymixin B cdn cdc dang khdc

thi khdng Tuy nhien, ke't qua kiem tra ddi vdi cdc benh nhdn lira tudi tiir 6 de'n 19 tudi cd thdi gian dai sir dung khdng sinh penicillin cho thd'y 2/3 sd benh nhdn nay benh sdu rdng vdn cdn xud't hien Mat khdc viee sir dung khdng sinh trong san pham rang mieng cd nguy ca tao ra cdc chiing vi sinh vdt khdng khdng sinh tren ddi tugng con ngudi

Nhi^u chat khdng khudn da dugc nghien ciiu sir dung trong viee ki^m soai su

hinh thanh mang bam rang va \xc che' su phdt trien ciia vi khudn S mutans nhu fluor

[9, 31], triclosan [22] Cac chd't khdng khudn cd tdc ngan ngura su gdn ke't, sir xam

nhiem ciia S mutans vao mang bdm rdng Cdc chd't khdng khudn tieu diet cdc vi

khudn do chiing tdc ddng de'n qud trinh trao ddi chd't, gdy tdn thuang mang te' bao va cdc vdt chd't di truyen nhu ADN, protein ciia vi khudn

Trong cdc chd't khang khudn sdu rang dugc nghien ciiu, viee sii dung fluor dugc coi la bien phdp phdng chdng sdu rang chii yeu trong thap ki trudc Fluor dugc

bo sung vao cdc san phdm bao ve rdng mieng dac biet la nude udng, nude siic mieng, kem ddnh rdng cd tdc dung phdng ngira sdu rang hieu qua Cac nha nghien ciiu cho tha'y bd sung fluor vao nude cd the' giam 40- 70% ty le mdc sau rang

Fluor tac ddng len nhi^u enzyme trong qud trinh trao doi chd't Enzyme

enolase d 5 mutans ddc biet nhay cam vdi fluor, su li'c che enzyme nay cd the giam

su cung cd'p phosphoenol pyruvate cho he thdng vdn chuyen dudng, do dd ij:c che su hd'p thu dudng cung nhu su thuy phdn glucose Fluor cung lic che cdc qua trinh tong hgp glycogen va cac enzyme phdn giai peroxit hydro nhu peroxidase, catalase Tdc

dung irc che' ciia fluor len cdc enzyme catalase va pseudocatalase ciia cdc vi khudn chiu H2O2 cd tdc dung lam mdt tdc dung bao ve ciia cdc vi khudn nay vdi vi khuan

khdng chiu dugc H2O2 nhu 5 mutans, tang kha ndng diet khudn cua H2O2 ddi vdi

cdc vi khudn tren mang bam rdng [30,31]

Fluor cd anh hudng de'n qud trinh vdn chuye'n H* qua mang te' bao va lam tdng tinh ddn proton ciia mang t^ bao, dac biet la d pH thap Tdc dung nay cd the dugc giai thich mdt phdn bdi tinh axit ye'u ciia fluor Ruor cd pKa la 3,15 nen cd the cho va nhdn proton theo phuang trinh sau ddy:

AH A" + H^

Khi pH mdi trudng cdng xud'ng thdp, xu the' tao dang proton hod (AH) cdng cao, ddy la dang cd th^ thdm qua hang rao ua lipit ciia mang te' bao Khi vao mdi trudng cd tinh ki^m ciia te' bao chdt, dang proton hod bi phdn ly thanh A' vd H*, lam mdi trudng ndi bao bi axit hod Tuy nhien, fluor cdn cd tdc dung irc che' ATPase-enzyme vdn chuyen proton ciia mang te' bao streptococci vd do dd lam tdng tinh thdm ciia te' bao vdi proton bdng each irc che' qua trinh vdn chuyin proton qua mang ciia te' bao dugc xiic tdc bdng enzyme

Fluor va axit ye'u la nhChig chdt li'c che' hd hdp ciia Streptococci trong mdi

trudng axit Qud trinh hd hdp ciia S mutans lien quan de'n enzyme NADH oxidase,

chii ye'u la dang san xud't HjOj Day la enzyme nhay cam cao vdi axit Do dd, trong mdi trudng axit, su hd hdp ciia te' bao nguyen ven cung nhay cam cao vdi fluor Fluor u:c che' hd hdp ciia te bao nguyen ven lien quan de'n tac dung axit ye'u dan tdi tdng tinh thdm proton ciia te' bao, axit hod te' bao chdt va ddn de'n axit li'c che' NADH

oxidase va qua trinh dudng phdn Su san xudt H2O2 d te' bao S sanguis nguyen ven,

nhdm sinh peroxit, cung bi li'c che' tuang tu bdi fluor hay cdc axit huu ca yeu khdc trong mdi trudng axit Do dd, fluor va axit ye'u li'c che qud trinh hd hdp cua cdc vi khudn dudng mieng gidn tie'p bdi su axit hod te' bao chat hem la hoat ddng nhu chdt ire che'true tie'p NADH oxidase [33]

So vdi cdc chd't khang khudn khdc fluor khdng nhiiiig cd tdc dung khdng khudn ma cdn cd tdc dung tdi tao men rdng va lam cho rang khdng tdt han vdi su an mdn ciia axit Chinh vi vdy fluor dugc xem la idc nhdn chdng sdu rang hieu qua nhdt

md de'n nay chua cd chdt nao cd the' thay the dugc Tuy nhien do su dung lau dai cdc ngudn nude hay cdc san phdm ve sinh rang mieng cd chiia fluor vdi ndng dd cao da din de'n hien tugng nhiem fluor (fluorosis) Bieu hien ciia no la lam cho mat rang

sdn sui va Idp men rang 6 vang, cd vach mau trdng trong nhihig ndm gdn day, tinh

trang nhiem flour gia tang d nhi^u nude tren the'gidi, khdng chi d nhihig nai cd ndng

dd flour cao trong nude tu nhien, ma cdn gap ca d nhihig viing cd ndng dd thdp hay 1^ tudng Da cd mdt sd cdng trinh nghien ciiu ve tinh trang rdng nhiem flour d liia tudi hoc dudng va ke't qua cho tha'y cd mdi lien quan giira ngudn nude vdi ndng dd flour Ndng do fluor trong ngudn nude Idn han 0,6 ppm thi ty le nhilm flour lrong cdng ddng tdng ro ret [9] 6 Viet Nam, tai Thdi Binh qua khao sdt thuc trang, cho thd'y ty le tre em tur 9-11 tudi d ndng thdn nhiem flour la 53,6%, trong dd viing ndi

d6ng la 75,7%, vung ven biin la 33,2% [7] Su xuat hien cua hien tugng nhiem fluor

nay ddi hdi phai cd nhihig chd't khdng khudn mdi cd kha ndng thay the' fluor hoae ke't hgp vdi fluor d nhihig ndng dd thdp ma vdn dam bao hieu qua chdng sau rang

1.2.2 Mdt sd bien phdp ngdn ngi^a, phdng benh sdu rang khdc

+ Sijr dung chdt thay the' dudng

Vi khudn sii dung dudng trong dd an thdc udng de len men, sinh axit vd ke't qua la lam mdn men rang Bdi vdy mdt trong nhihig 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 nay khdng gay ra su sinh axit nen khdng gay sau rang Cdc hgp chd't thay the' dudng nhu manitol, sorbitol la nhihig loai dudng khac nhau cd kha ndng khdng khudn nhdt dinh thdng qua viee urc che' su phdt tri^n ciia vi khudn do

chiing khdng bi S mutans tieu thu Tuy nhien sir dung thudng xuyen cae loai dudng

nay se ddn de'n hinh thanh cdc chiing vi khudn cd kha ndng ddng hod chiing Xylitol cung la mdt loai dudng cd lac dung chdng sdu rdng Ca che' tdc ddng ciia dudng nay

ien vi khudn S mutans da dugc nghien curu Khi xylitol di vao te bao se can thiep qud trinh ien men dudng ciia vi khudn, ddng thdi \xc che' canh tranh vdi

phosphofructokinase nhd chdt trao ddi trung gian xylitol- 5- phosphate Tdc dung

chdng S mutans ciia xylitol cdn the hien d mu'c do gen Xir ly S mutans bdng xylitol

cho tha'y cd su thiic ddy chon loc qudn the khdng xylitol, nhihig chiing khdng xylitol cung cd ddc tinh giam so vdi cdc chiing nhay cam vdi xylitol [40]

+ Sir dung lieu phdp thay the

Sir cd mat ciia mdt sd vi khudn cd th^ ngan ehan su dinh cu ciia vi khudn gay benh va giU sd lugng ciia chiing d mirc dd an loan, bien phdp dua tren ea sd nay de chdng nhiem khudn dugc goi la lieu phdp thay the Lgi ich cua lieu phdp thay the la

ed tac dung bao ve rdng lau dai vd it tdn kem Tuy nhien lieu phdp nay chua dugc thir nghiem nhi^u d ngudi vi tinh an loan ciia 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 khi cdc ehung khdng mong mudn xdm nhdp va dinh cu tren mang bam hoae dung mdt ehung vi khudn trung tinh canh tranh vdi ehung gay benh tren mang bam rang

+ Vacxin chdng sdu rdng

Vi khudn gdy sdu rang cd the bi tieu diet bdi cac ddp litig mien dich Hudng nghien cuoi vacxin chdng sdu rang la tao ra khdng the dac hieu vdi khdng nguyen ciia te' bao Cdc vacxin cung dugc san xuat theo hudng chdng lai enzyme

glucosyltransferase hoae protein be mat cua 5 mutans, ngan ehan vi khudn nay bdm dinh vdo b6 mat rang Tuy hudng nay ed lam giam ty le sdu rang d mdt sd ddng vat

thuc nghiem nhung khdng cd hieu qua d cdc loai linh trudng va xud't hien mot sd phan litig phu

+ Cac hgp chdt thuc vdt ih\s cdp

Cdc hgp chdt tu nhien cd trong thuc vdt dugc xem la ed nhi^u ti^m nang vtng dung trong thuc t^ Hgp chdt gallotannins tdch tijr Melaphis chinensis cd tdc dung

ire che' su hinh thanh glucan d mdt sd chiing Streptococci, catechin tur dich che Olong Nhat Ban cd hoat tinh sau rang cung da dugc biet den [37] Mdt sd nghien

ciiu phdt hien thdy cdc hgp chdt thudc nhdm polyphenol trong che xanh {Camellia sinesis L.) cd kha nang chdng sdu rdng thdng qua qud trinh dudng phdn vd li'c che'

hoat ddng ciia glucosyltransferase, ddy la enzyme cd vai tro tao glucan, ca sd hinh

thanh mang bdm rdng [22] Mdt sd hgp chat tdch tir loai Saussurea lappa, qua Cranbery hay rl ciia Polygonum cuspidatum cd kha ndng ngdn ehan sir hinh thanh

mang bam rang ciia cac vi khudn dudng mieng [41, 42]

Cdc hgp chd't polyphenol va flavonoid cd the hinh thanh lien ke't hydro vdi cdc

protein- enzyme, do 66, lam thay ddi nhieu hoat tinh ciia enzyme Nhieu flavonoid

cd tac dung kim ham cdc enzyme oxy hod- khir, qua trinh dudng phdn va hd hdp, qua trinh phdn bao, phd vd cdn bang trao ddi chdt trong cac te' bao ung thu, vi vay, kim ham sir phat trien ciia khdi u, dac biet la vao giai doan sdm

Cac tac gia Viet Nam phdi hgp cdc ngudn dugc lieu khac nhau de ndng cao hieu qua trong di^u tri cdc benh dudng mieng nhu: cao cdn la cd lao ket hgp vdi dich chie't la trdu khdng va la Id'u trong che' phdm RK 94 cd tdc dung chira viem rang [8] Nguyen Nam Hai str dung thudc sindolot (dich chie't Id trdu khdng va hat cau) lam nude xiic mieng di^u tri viem lgi, viem quanh rang, hay che' phdm BT 98

dugc bao che' tiir vd cdy Dai trdng {Plumeria acutifolia), la cd ngot {Stevia rebaudiana) va Id trdu khdng {Piper belte) cd tdc dung trong viee dieu tri benh nha

chu [7] cdc ke't qua nghien ciiu cua Nguyen Thi Mai Phuang vd tdp the cho thay cac hgp chdt polyphenol trong dich chie't mang cut cd hoat tinh lie che' kha nang

sinh axit cao, ddng thdi lie che' su hinh thanh mang bam rang ciia S mutans [6]

Nguyen Quang Huy va tdp the' [3, 4] da cd cac nghien ciiu budc ddu ve tac dung

ciia dich chi^t vd Sao den, la Sdn thuyen ddi vdi viee irc che' kha nang sinh axit tiir vi

khudn Streptococcus mutans

1.3 Cac hgp chd't thuc vat thur cap

Hgp chdt thuc vdt thu* cdp hay la cdc san phdm ciia qud trinh irao ddi chat dugc sinh ra bdi thuc vat Chiing la cdc chdt hod hoc dugc tdng hgp va chuyen hod tir cdc chdt trao ddi bdc nhdt nhu axit amin, axit nucleic, carbohydrate, lipid, peptid hodc tiir cac san phdm trung gian cua chu trinh dudng phdn, chu trinh pentose, chu trinh Kreb Khac vdi cdc chdt trao ddi bdc nhdt, giii vai trd trung tdm va tham gia true tie'p vao qua trinh trao ddi chdt ciia ca the, cdc hgp chdt thuc vdt thir sinh khdng phai la cdc yeu td dac biet cdn thiet cho qua trinh sinh trudng, phat trien, quang hgp

va sinh san, ehung dugc tao ra giu'a cdc te' bao chuyen biet vdi vai trd di^u hod mdi quan he qua lai giu'a cdc te' bao trong ca the va giira cdc cd the vdi mdi trudng sdng xung quanh

CO,

Quang hgp Chdt trao ddi bac 1

Erythrose-4- phosphate

3 phosphoglycerate (3-PGA)

Hgp chat alkaloid

Con dudng mevalonic

Hgp chait phenolic

Con dudng MEP

Hgp chat terpenes

Hinh 2 Mdi quan he giOa chdt trao ddi bdc 1 va cdc chdt thuc vdt thir cdp

Tuy thudc vao cdu triic hod hgc cdc chd't thuc vdt thii cdp dugc phdn loai thanh 3 nhdm chinh la nhdm terpene, nhdm hgp chd't phenolic va nhdm alkaloid Trong dd nhdm cd sd' lugng nhieu nhdt va dugc quan tdm nhieu la nhdm terpene

1.3.1 Nhdm cdc hgp chdt terpene

Terpene la nhdm carbohydrate thuc vdt Idn va da dang nhdt, dugc hinh thanh

iix qua trinh polymer hod cdc lieu dcm vi isoprene 5 carbon (CsHg), cd cdng thiic cau

tao ehung la (CsHx)^ Trong thuc vat terpenes dugc tdng hgp thdng qua con dudng trao ddi chdt acetate/mevanolate hoae con dudng glyceraldehyde 3-phosphale/pyruvate (hinh 2)

Hdu het cac terpene cd thd bi khir hoae bi oxy hod 6i hinh thanh nen cdc hgp

chd't terpenoids khdc nhu nhu alcohol, ketone, axit va aldehyde do vdy mdt sd tdc gia sii dung thudt ngii' "terpene" dd chi ehung mdt nhdm Idn cdc hgp chd't bao gdm ca terpene va terpenoid

Tuy thudc vao sd lugng cdc tidu dan vi isoprene cd trong phdn tii ma terpenes dugc ehia nhd thanh mdt sd phdn nhdm ehinh nhu :

+ Hemiterpene chiia 1 dan vi isoprene (5 nguyen tir C)

+ Monolerpene chiia 2 dan vi isoprene (10 nguyen tii C)

+ Sesquiterpene chiia 3 dcm vi isoprene (15 nguyen tir C) dugc coi la phdn nhdm Idn nhd't trong terpene

+ Diterpene chiia 4 dan vi isoprene (20 nguyen tir C) Trong tu nhien cd the gap cdc diterpene d ca gidi thuc vdt va ddng vat

+ Sesterpene chiia 5 dcm vi isoprene (25 nguyen tii C)

+ Triterpene chiia 6 dcm vi isoprene (30 nguyen tii C) la mdt nhdm Idn cdc hgp chdt tu nhien vdi tren 40 bd khung cdu triic khac nhau

+ Tetraterpene chiia 8 don vi isoprene (40 nguyen tii C) vd polyterpene cd chu'a sd dan vi isoprene tren 8

Trong nhdm triterpene con chiia nhdm Saponin la nhihig hgp chat thudc nhdm glycosid dac biet, dugc tieh luy d mot sd loai thuc vdt, khi dem thuy phdn se dugc dudng va sapogenin,

Tuy theo ban chdt hod hoc ciia sapogenin ma ngudi ta ehia cac saponin thanh

2 loai: cdc saponin triterpenoid va cdc saponin steroid Cac saponin cd dac tinh la tao bot khi hoa vao nude va Idc manh va ddc ddi vdi cac ddng vat cd mau lanh

1,3.2, Tdc dung khdng khudn cua cdc hgp chd't thudc nhdm terpene

Nhi^u hgp chat thuc vat thir cap trong nhdm terpene cd hoat tinh sinh hoc dugc sii dung lam thudc de dieu tri nhi^u loai benh khac nhau :

10

+ Trong nhdm triterpene cdc hgp chat glycosid trg tim cd the gap d mdt sd

loai thudc cac hg Triic dao {Apocynaceae), Hoa mom chd {Scrophulariaceae), Ddu tdm {Moraceae), Hanh tdi (Liliaceae)

+ Cdc saponin triterpenoid cung dugc ling dung nhieu trong y dugc, do chiing cd tinh khang viem, chiJa ho, long ddm Trong re cua loai Cam thao

{Glycyrrhiza uralensis Fisch) ngudi ta da tdch dugc mdt saponin triterpenoid goi la

glycyrrhizin cd tdc dung chdng viem va da dugc bao che' thanh kem da dilu tri benh nha chu

+ Cdc sesquiterpene ed hoat tinh sinh hgc manh, chiing dugc sii dung rdng rai trong y dugc (khang khudn, khang ndm, diet mu6i ) Hgp chd't artemissinin la

mdt sesquiterpene laeton tdch chie't tur loai Thanh hao hoa vang {Artemisia annua L.)

cd tdc dung diet ky sinh tr^ng sdt ret manh, hien da va dang dugc ling dung dd di^u tri benh

+ Hgp chdt triterpenoid dugc tdch chie't tiir loai Rostellularia procumbens cd

tac dung diet khudn vdi MIC trong khoang 1,56-100 ^ig/ml Trong mdt nghien ciiu

khdc, tac gia Angeh va tdp the [10] da tdch 5 triterpenoid tiir la cua loai Combretum imberbe Trong dd, mdt triterpenoid: la,3P-dihydroxy-12-oleanen-29-oic cd hoat tinh lie che' vi khudn gdy sdu rang Streptococcus mutans Vdi ndng dd tir 16-62

|ig/ml hgp chd't nay da lie che' 50% sd lugng vi khudn S mutans Cdc hgp chd't terpenoids dugc tdch tur loai Pterocarpus indicus, pentacyclic triterpene tdch tiir hg Combretaceae d Chdu Phi deu cd tdc dung khang khudn [10]

6 Viet Nam da cd mdt sd cdng trinh cdng bd cdc nghien ciiu tdch chie't, dac tinh sinh hoc, hda hgc ve nhdm hgp chdt triterpene trong mdt sd loai thuc vdt [2] Tuy nhien, cho den nay chua cd nhi^u tdc gia lim hieu ve kha nang khang khudn cua nhdm hgp chdt nay ddi vdi vi khudn gay sau rang, dac biet la kha nang khdng khudn

sdu rdng 5 mutans dugc phan lap tur chinh ngudi Viet Nam

1.3.3 Mot vdi net ve cdy Sdn thuyen ( Syzygium resinosum G)

Sdn thuyen {Syzygium resinosum Gagnep) hay cdn goi la sdn xam thuyen,

thudc hg Sim la cay cd than thing diing, hinh tru, cd the cao tdi 15 m Canh nho gay

va dai, liic ddu del sau hinh tru, mau nau nhat, nhan nheo La moc ddi, phien la hinh mac thudn nhgn d gdc, nhgn tii d dinh Cum hoa mgc d ke cac la rung hay chua rung, thanh chuy dai 2 - 3 cm, thudng hop thanh nhdm dai 20 cm, true gdy nho, tan ciing bdi 3 hoa khdng cd cud'ng Nu hoa hinh le, gdn hinh cdu Qua chin cd mau tim

dd, vi nggt, chdt, Sdn thuyen cd the mgc hoang hoae dugc trdng d hdu khdp cac tinh phia Bac

11

Sa bd thanh phdn ciia Sdn thuyin gdm tinh dau, chat nhua, chat nhay, tannin

Ld sdn thuyen non cd the diing an goi, vd than de xam thuyen La tuai gia nat ddp ien vet thucmg chay mii dai dang, bdng, vet md nhiem triing, gdy xuang hd, hoai tir

da Benh vien Hihi nghi Viet Tiep sii dung la Sdn thuyen trong viee dieu tri chiJa nhihig v^t thuang nhiem triing thdng thudng cd tdc dung lam cho vet thucmg chdng khd, bdt nhiem triing, td chiic seo cd di^u kien phat trien, lam cho vet thuang chdng len da non [3]

Trong cdc nghien ciiu trudc day cua ehung tdi dich chie't bang ethanol tir la

Sdn thuyin cd tac dung lie che' su sinh axit va giet vi khudn Streptococcus mutans

GS-5 Tuy nhien chiing tdi chua tach chie't dugc hgp chdt cu the nao cd tdc dung vdi

S mutans trong dich chie't tii la Sdn thuyen

D6 tai nghien cuu cua chiing tdi vdi muc tieu nghien ciiu tdc dung cua axil asiatic tiir Id cdy Sdn thuyen len vi khudn gdy sau rang Streptococcus mutans H2

phdn Idp tur ngudi Viet Nam la mdt hudng nghien ciiu nhdm gdp phdn ndng cao viee khai thae, sir dung cdc san phdm tu nhien trong viee bao ve rang mieng d Viet Nam

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CHUONG 2: NGUYEN LIEU vA PHUONG PHAP NGHIEN CUU

2.1 Nguyen lieu

2.1.1 Chung vi khudn va mdu la Sdn thuyen

Chung vi khudn Streptococcus mutans H2 dugc phdn Idp tiir ngudi Viet Nam,

dugc cung cdp tur Phdng thi nghiem Protein tdi td hgp thudc Phdng thi nghiem trgng didm cdng nghe Protein- Enzyme

Chung vi khudn Streptococcus sanguis NCTC-10904, Streptococcus rattus

FA-1 la qua tdng cua GS Robert E Marquis, Dai hgc Rochester, Hoa Ky

Mdu Id Sdn thuyen dugc thu thdp tir vudn Thudc Ddng y d Trung Van, Tir Liem, Ha Ndi va dugc phan loai bdi TS Trdn Dinh Nghia DHQG HN

Cdc hod chd't cdn lai d^u dat dd tinh sach phdn tieh

2.1.3 Thiet bi thi nghiem

Cac thiet bi chinh diing cho cdc thi nghiem bao gdm: pH meter cua hang Orion (My) Quang phd ke'tii ngoai- nhin thd'y (UV-Vis) ciia hang Pharmacia (Thuy Dien) Mdy li tdm lanh ciia hang Kubota (Nhdt Ban) May Idc dn nhiet (Stutart scientific, Anh) May li tdm eppendorf 5147 (Eppendorf, Diic)

2.2 Phuang phap nghien curu

2.2.1 Nudi cdy vi khudn va chudn bi mdu te' bao

Chiing vi khudn dugc nudi cdy tren mdi trudng thach TSA (tryptic soy agar)

va cdy chuyen 2 tudn mot Ian, giU d 4"C

Vi khudn dugc nudi cdy tinh hodc nudi cdy Idc d 37"C trong mdi trudng TYG gdm cd 3% tryptone, 1% glucose, 0,5% cao nam men, thu te bao vi khudn bdng ly tam Gid tri pH ciia mdi trudng khi thu te' bao la khoang 4,1

Chudn bi dich te' bao thdm: te' bao dugc nudi qua dem nhu da md ta, sau dd te' bao dugc riia 1 Idn bang dung dich mudi khoang Ix (50 mM KCI, 1 mM MgCl2) va dugc hod lai trong dem Tris- HCl 50 mM, pH 7,5 cd bd sung I mM MgCl2, 50 mM

13

KCI sao cho mdt dd te' bao tuong duang OD700 = 14, bd sung them 10% loluen, iron

manh bdng may vortex Sau 66, h6n hgp dugc ii d 37"C bdng be dn nhiet trong 5

phiit, rdi chuydn sang lam ddng da bang nita Idng trong 1 phiit, lap lai hai Idn Loai

bd toluen du bdng ly tdm va riia lai 2 Idn vdi dung dich mudi khodng Ix Hod ket tua te' bao trd lai trong dem Tris- HCl pH 7,5 vdi thd lich bdng 1/10 the lich ban dau

Viee xir 1^ i6 bao vdi toluen ke't hgp vdi viee chuyen te bao qua cdc pha dm va

pha ddng dd ed tac dung lam tdn thuang cue bd mang te' bao, lam cho te' bao cd the thdm dugc cdc chdt cd khdi lugng phdn tii Idn nhung khdng lam tdn thuang cdc enzyme tren mang Vi vdy, dich te' bao dugc chudn bi theo phuang phdp nay ggi la dich te' bao tham, dugc sir dung trong cdc thi nghiem vdi enzyme

2.2.2 Xdc dinh miic dd sinh axit eiia vi khudn bang thi nghiem giam pH

Te' bao vi khudn dugc hda tan trong dung dich mudi khodng Ix sao cho mat

dd cudi ciing tuang ducmg A700 = 10 (ddi vdi dang huy^n dich) hay dung dich mudi

khodng ngdp biofilm pH cua huyin dich t6 bao hoae biofilm dugc chinh de'n 7,2

bdng NaOH IM Glucose dugc bd sung dd dat ndng dd cudi ciing la 1%, dam bao mdi trudng du thiira ca chdt cho qud trinh dudng phdn Su giam pH ciia mdi trudng dugc thuo doi bdng pH meter tai nhihig thdi diem nhdt dinh dd giam pH ciia mdi mdi trudng phan anh kha ndng sinh axit ciia te' bao

2.2.3 Xdc dinh miic dd sdng sdt cua vi khudn

Te bao vi khudn dugc riia va hod lai trong dung dich peptone 1% sao cho mdt

dd te' bao cudi ciing tuang ducmg A70()= 1 (khoang 10^ te' bao/ml) Cdc chd't nghien ciiu dugc bd sung de dat ndng dd cudi ciing mong mudn va chinh pH de'n 4 6 nhihig thdi diem nhdt dinh, 100 ^il dich te' bao dugc Idy ra pha loang trong peptone 1% theo cdc ndng dd nhd ddn 10 Idn va dugc cdy trai tren dia thach TSA Cdc dia thach ed't d 37 "C cho de'n khi cdc khudn lac hinh thanh ro ret va cd the de'm bdng mat thudng

Tdc dung gdy che't ciia dich chie't dugc danh gid bang gia tri D, la thdi gian cdn thiet dd 90% qudn the vi khudn bi giet che't Tdc dung nay cdn dugc danh gid bdng gid tri LogN/No vdi No la sd vi khudn ban dau, N la sd vi khudn tai thdi didm nghien ciiu Gia tri D tuang ihig vdi LogN/No = -1

2.2.4 Do miic dd tieu thu oxy ciia te' bao

Hd hdp ciia td bao vi khudn dugc danh gid thdng qua viee do miic dd lieu thu oxy trong mdi trudng theo phuong phdp cua Cadwell va tap the [17] sir dung cdc te bao nudi hie'u khi dd cd tdc dd tieu thu oxy cao Cac te' bao nghien ciiu dugc nudi

14

cdy sau 66 dugc riia 2 Idn vdi dung dich mudi cd chira KCI 50 mM, MgCls 1 mM

Cac dung dich te bao sau dd dugc bd sung glucose de'n ndng do cudi ciing la 0,4%,

lac manh tren may vortex 6i dat dd bao hod oxy Mdu te' bao nay dugc su dung 6i

do lugng oxy tieu thu d nhiet dd phdng, sir dung mdy do oxy WTW (Diic)

Miic dd oxy cdn lai trong mdi trudng dugc do sau mdi phiit cho den het Ddi vdi thi nghiem cd dich chie't thi mdu te' bao dugc ii vdi dich chiet nghien ciiu va do lugng oxy cdn lai ngay Idp tire

2.2.5 Xac dinh hoat dd ciia mdt sd enzyme quan trgng lien quan tdi hoat ddng sdng

ciia Streptococcus mutans

2.2.5.1 Phdn tieh hoat dp F-ATPase

Hoat dd F-ATPase dugc xdc dinh theo phuong phap ciia Sturr va Marquis [39] thdng qua viee do lugng phosphate vd co dugc giai phdng ra

Hdn hgp phan ihig bao gdm Tris-HCl 100 mM, pH 7, MgCl2 10 mM, dung djeh te' bao thdm ATP 5 mM, nude ed't (d dng ddi chiing) hodc nude ed't va dich chie't thuc vdt (d dng thi nghiem) Enzyme dugc lam bdt hoat bdng TCA 5% Ddi vdi dng ddi chiing, TCA dugc bd sung trudc khi cho co chat ATP Ddi vdi dng thi nghiem, trudc khi cho co chdt, hdn hgp dugc ii vdi dich chie't trong 10 phiit, sau dd

bd sung ATP va ii trong 25 phiit d 25''C, cudi ciing mdi them TCA dd bdt hoat enzyme va lam ngiimg phan ling

Hdn hgp phan ling dugc dem ly tdm d 10.000 vong/phiit trong 2 phiit, thu dich

trong tren tiia De dinh lugng phosphate vd ca tao thanh, cho vao mdi dng 150|il

dich trong tren tiia va 900 |il thudc thur Bencini (gdm ZnCl2 100 mM, ammonium molibdat 15 mM), 150 |j.l H2O Hdn hgp dugc Idc deu, u trong 5 phiit va do dd hdp thu d A350 Hieu sd giiia dd hdp thu ciia dng thi nghiem va dng ddi chiing phan dnh lugng phosphate vd co tao thanh

Mdt don vi hoat dd F-ATPase la lugng enzyme can thiet de giai phdng ra 1

^mol phosphate vd co trong I phiit d didu kien phan ling

2.2.5.2 Phdn tieh hoat dp enzyme phosphoryl hod dudng (PTS)

Hoat dd cua he thdng enzyme phosphoryl hod dudng (PTS) dugc xac dinh theo phuang phap cua Belh va Marquis [12]

Hdn hgp phan ling gdm Tris-HCl 50 mM, pH 7, MgCl2 20 mM, NaF I mM, va glucose 40 mM, dung dich te' bao thd'm, PEP 5 mM, ethanol (dng ddi chiing) hoae ethanol va dich chie't (dng thi nghiem) Ddi vdi dng ddi chiing, hdn hgp dugc ly tam lO.OCX) vdng/phiit trong 2 phiit de lam Idng te bao, khdng cho enzyme tiep xiic vdi co

15

chdt trudc khi bd sung PEP Ddi vdi dng thi nghiem, dich chiet dugc u vdi hdn hgp trong 10 phiit trudc khi bd sung co chdt, sau khi bd sung PEP hdn hgp dugc u d 37'C trong 20 phut, sau dd ly tam, thu dich de dinh lugng pyruvate

Di dinh lugng ndng dd pyruvate, cho vao mdi dng 300 \x\ dich trong tren tua,

400 ul dung dich P-NADH, 300 ^il H2O vd bd sung 2 ^1 LDH Hdn hgp dugc u d nhiet dd phdng trong 10 phiit sau dd dem do hdp thu d A340 Hieu sd dd hdp thu giu'a dng ddi chiing va dng thi nghiem phan dnh lugng NADH da phan ling vdi lugng pyruvate tao thanh

Mdt don vi hoat dd cua enzyme phosphoryl hod dudng la lugng enzyme xiic tdc cho phan ling phosphoryl hod 1 jimol glucose thanh 1 jimol glucose-6-phosphate, tuong duong vdi 1 fimol NADH bi oxy hod thanh NAD* trong phan ling chuydn hod pyruvate thanh lactate trong 1 phiit d di6u kien phan ling

2.2.5.3 Phdn tieh hoat dp NADH oxidase

Hoat dd ciia NADH oxidase dugc xdc dinh theo phuong phap ciia Poole va Claiborne [34] Phan ling dua tren su oxi hod NADH bdi NADH oxidase Hdn hgp phan ling bao gdm:

a) 500 ^il dem phosphate 200 mM, EDTA 1,2 mM, pH 7,0

b) 200 yl te bao thd'm

c) 100 ^1 P-NADH 2 mM

Ddi vdi dng kidm tra, hdn hgp cdc dung dich a, b dugc trdn ddu vd ly tdm d 4"C, 10.000 vdng/phiit trong 2 phiit, sau dd dugc bd sung dung dich c Ddi vdi dng

thi nghiem, hdn hgp cac dung dich a, b, dugc trdn deu, sau 66 dugc bd sung dung

dich c va trdn deu ngay lap tiic Do dd hdp thu d budc sdng 340 nm trong vdng 3 phiit Hieu sd dd hdp thu giUa dng kiem tra vd dng thi nghiem phan dnh lugng NADH da phan ling Mdt don vi hoat dd ciia NADH oxidase la lugng enzyme xiic tac cho phan ling chuyen hod 1 jimol NADH thanh NAD"^ trong 1 phiit d didu kien phan ling

2.2.5.4 Phdn tieh hoat dp lactate dehydrogenase

Lactate dehydrogenase xiic tdc cho phan ling chuyen hod pyruvate thanh lactate Hoat dd cua enzyme nay dugc xdc dinh theo phuang phdp ciia Iwami va lap thd [23] Hdn hgp phan ling bao gdm:

a) Dem Tris-HCl 100 mM, NaCl 100 mM, FBP 2 mM, pH 7,0

b) P-NADH 2 mM

c) H2O

16

d) Te' bao thdm

e) Dung dich natri pyruvate 300 mM

Ddi vdi dng kiem tra, hdn hgp cac dung dich a, b, c, d dugc trdn ddu, dugc ly

tam d 4°C, 10.000 vdng/phiit trong 2 phut, sau dd dugc bd sung dung dich e Ddi vdi

dng thi nghiem, hdn hgp cac dung dich a, b, c, d, dugc trdn ddu, sau dd dugc bd sung dung dich e vd trdn ddu ngay lap tiic Dd hdp thu d budc sdng 340 nm (A34(,) dugc do trong vdng 3 phiit Hieu sd dd hdp thu giiia dng kidm tra va dng thi nghiem phan anh lugng NADH da phan ling

Mdt don vi hoat ddng ciia lactate dehydrogenase la lugng enzyme xiic tdc cho

phan utig chuydn hod 1 ^mol pyruvate thanh 1 \imol lactate tuong ducmg vdi I

jimol NADH bi oxy hod thanh NAD* trong 1 phut d didu kien phan ling

2.2.5.5 Phdn tieh hoat dp pyruvate kinase

Pyruvate kinase xiic tdc cho phan ling chuyen hod PEP thanh pyruvate khi cd mat ciia ADP Hdn hgp phan ling bao gdm:

a) Dem Tris-HCl 100 mM, KCI 200 mM, MgClj 20 mM EDTA 1 mM, pH 7,0 b) ADP 100 mM

c) PEP 100 mM

d) Te' bao thdm

e) H2O

Ddi vdi dng kidm tra, hdn hgp cdc dung dich a, b, d, e dugc trdn deu va dugc

ly tdm d 4"C, 10.000 vdng/phiit trong 2 phiit Sau dd, dugc bd sung dung dich c va ly tam them mdt Idn nfia va thu dich tren tiia vao mdt dng nghiem sach

Ddi vdi dng thi nghiem, hdn hgp cdc dung dich a, b, d, e dugc trdn ddu, sau dd dugc bd sung dung dich c, dugc trdn deu va giii hdn hgp phan ling trong vdng 10 phiit d nhiet dd phdng, sau dd hdn hgp dugc ly tdm va thu dich tren tua nhu dng kidm tra

Phuong phdp dinh lugng pyruvate: Hdn hgp phan ling gdm: Dich tren tua, dung dich P-NADH 2 mM va dung dich Tris-HCl 100 mM, dugc trdn ddu va dugc

bd sung 2 jil enzyme lactate dehydrogenase Do do hap thu d budc sdng 340nm (A340) Hieu sd dd hdp thu giira dng kidm tra va dng thi nghiem phan anh lugng NADH da phan ling vdi lugng pyruvate tao thanh

Mdt don vi hoat ddng ciia pyruvate kinase la lugng enzyme xiic tac cho phan ling chuyen hod 1 jimol PEP thanh 1 jimol pyruvate luong duong vdi 1 jimol NADH

DAI HOC Q U O C GtA HA NOI TRUNG TAts/l THONG TIN THU VIEN

"oooloooooZT

17

bi oxy hod thanh NAD* trong phan ling chuydn hod pyruvate thanh lactate trong I phiit d didu kien phan ling

2.2.6 Phucmg phdp xac dinh cdu triic hod hgc cdc hgp chdt

Mdu nghien ciiu dugc phan tieh dd sach va xdc dinh khdi lugng phdn tir tdng

sd tren may do khdi phd Ky thudt phd khdi lugng dugc sii dung kha phd bien dd xdc dinh cdu triic hda hoc ciia cdc hgp chdt hihi co Nguyen tdc chii ye'u ciia phuang phdp phd nay la dua vao su phdn manh ion ciia phdn tir chd't dudi su ban phd ciia chiim ion ben ngoai Ngoai ion phdn tur, phd khdi lugng phdn tiir cdn cho cac dinh ion

manh khac ma dua vao 66 ngudi ta cd thd xdc dinh dugc co che' phdn manh va dung

lai cdu triic hda hgc cdc hgp chdt

Phd cdng hudng tir hat nhdn (NMR): IH-NMR (500 MHz) va 13C-NMR (125 MHz) dugc do tren may Bruker AM500 FT-NMR Spectrometer Vdi phucmg phdp cdng hudng tiir hat nhdn, chdt nghien cdu sau khi dua vao viing tur trudng Bo se

bi tdch thanh cdc miic ndng lugng khdc nhau tur cao de'n thdp Su khdc nhau nay dugc phan dnh thdng qua tdn sd cdng hudng Tdn sd nay dugc ghi lai dudi dang cdc phd proton, carbon, HMBC, HPQC nhd mdy cdng hudng tii hat nhdn Dua vao dd dich chuydn cua cdc proton se xdc dinh dugc sd nguyen lir C, H cung nhu nhihig nhdm chii'c cd thd tham gia vao bd khung carbon Didm ndng chay (Mp) dugc do tren mdy Kofler micro-hotstage Ke't hgp cdc sd lieu se cho phep xdc dinh dugc cdu triic hod hgc ciia chdt cdn nghien cuu xdc dinh

Viee phdn tieh xdc dinh cdu triic hod hgc cdc hgp chdt cd su tham gia hgp tdc ciia Vien Hod hgc va Vien Hda hgc cdc hgp chd't thien nhien thudc Vien Khoa hgc Viet Nam

2.2.7 Phuong phdp xii ly sd lieu

Cdc sd lieu dugc sur ly bdng phuong phap thdng ke

Gid tri trung binh X= S xi/ n; Phuang sai S^ =[I (xl-x2)]/ n-1; Do sai chudn Sx= S/Vn; Ke't qua x= x ± Ax

Khoang gidi han tin cay x= +(a, 0- Sx

Trong dd x l : Gid tri sd lieu thuc nghiem x= trung binh cdng n: sd Ian thi nghiem, ±(a, 0 tra theo bang vdi a= 0,95; f= n-1

18

CHUONG 3: K^T QUA VA THAO LU^N

3.1 Tach, tinh sach, nghien curu tinh chd't cua mot sd chat thu cap co hoat tinh khang khuan sau rang tur cay Sdn thuyen

Qua cdc ke't qua nghien ciiu trudc ddy [3], chiing tdi thd'y dich chie't ethanol ciia Sdn thuydn va Sao den cd tidm ndng chdng sdu rdng cao hon so vdi cdc loai dich

chie't cdn lai, do vdy nghien eiru trong 66 tai ciia chiing tdi la xdc dinh, tdch chie't,

tinh sach chdt thuc vdt thii cdp cd tac dung khdng khudn sdu rdng cd trong la Sdn thuydn

3.1.1 Tim hidu cdc nhdm chdt thuc vdt thii cd'p cd trong la Sdn thuydn

Chiing tdi da tie'n hanh tim hidu so bd thdnh phdn cdc nhdm hgp chdt thuc vdt

thii cdp trong dich chie't ciia Sdn thuydn khi dugc chie't bang cdc dung mdi khdc nhau, ke't qua (Bang 1) cho thdy vdi cac dung mdi nude, ethanol, methanol va acetone hay hdn hgp choroform : methanol dd tach chie't thi ed thd thu dugc hdu het

cdcchdt thii cdp chinh cd trong la Sdn thuydn nhu alkaloid, flavonoid, cumarin va glucoside Tuy nhien, vdi cdc dung mdi phdn cue lugng dich thu dugc nhidu hon va cung cd phan ling manh hon vdi cdc thudc thii

Bang 1 Thanh phdn mdt sd nhdm chd't thuc vat thii sinh tir dich chie't la Sdn thuydn

trong cdc dung mdi

Khdi lugng - tdch chie't (mg/g)

+ + + + + + + + +

+

+ +

-Alcaloid Van- Mayer + - + - -H +

-H-

-phdn cac hgp Cumarin NaOH 10%

+ HQ dac +

+ - -

+

-chd't Glycoside Keller- KiUian ++

+ - -

* +

H-+

+ + + +

++

+-I-+

+ +

• ^

9 Butanol Phan cue it 118 + - ++

10 Ethylacetate Phan cue it 108 +

11 Ethanol Phan cue 214 + + +-H

12 Methanol Phan cue 196 + + ++

13 Chloroform: Hdn hgp 196 + + +

methanol (3:7)

Chi4 thich: Khdng cd phan thig (-); phan ung ye'u (+); phan u'ng manh (++)

3.1.2 Tdch chie't tinh sach mdt sd chdt thii cd'p tiir Id cdy Sdn thuydn

Dd tdch, tinh sach mdt hgp chdt thuc vdt thu* cd'p tir la Sdn thuydn chiing tdi da chie't cdc chat ra khdi mdu bang cdch ngdm mdu trong ethanol (ty le 1 g mdu: 10 ml dung mdi) trong thdi gian 5 ngay (hinh ) Dich chie't sau dd dugc cd lai dd loai bd bdt dung mdi sau dd lai dugc chie't tiep bdng cdc dung mdi khac nhau vdi dd phdn cue tdng ddn cu thd la n-hexan, chloroform, ethyl acetate va n-butanol Dung mdi trong cdc phdn doan dich chie't nay duge lam bay hoi dd thu phdn doan cac chdt chie't

ra Tir 1,2kg Id Sdn thuydn nguyen lieu ban ddu chiing tdi da thu dugc 1,5 g hdn hgp vdi phdn doan chie't bdng n-hexan, 6 g hdn hgp vdi phdn doan chie't bdng choloroform, 2,5 g hdn hgp chie't bdng ethyl acetate va 2 g hdn hgp chie't bang n-butanol Nhu vdy cd thd thdy rdng chloroform cd hieu sud't chie't cao nhdt (tuong tu nhu Sao den) Khi thii nghiem anh hudng ciia cdc phdn doan nay ien kha nang sinh

axit cua S mutans GS-5, chiing tdi thdy rdng phan doan chie't bang chloroform cung the hien kha nang lie che' su sinh axit ciia S mutans GS-5 tdt hon cdc phdn doan cdn

lai (ke't qua khdng trinh bay d ddy), chinh vi vdy phdn doan nay dugc lua chgn de cho cdc budc tinh sach tiep theo

Phdn doan dich chie't bang chloroform ciia la Sdn thuydn dugc cho qua cdt sdc

ky sephadex LH 20 va cho riia chiet bdng he dung mdi chloroform: methanol (CHCI3: MeOH) vdi ty le methanol tang ddn (su phan cue tang dan) chiing tdi thu dugc 3 phan doan hgp chdt khdc nhau ky hieu Idn lugt la STl, ST2 va ST3 qua budc sdc k^ nay Phdn doan ST3 dugc tie'p tuc sdc ky qua cot sdc ky pha dao RP-18 rira chie't bang he dung mdi methanol: acetone: nude theo ty le 2: 1:2 vd thd tieh Ket qua chiing tdi da thu dugc mdt phan doan duy nhdt 1 bang khi chay kidm tra bdng sdc ky ban mdng silicagel (hinh 4) va dugc ky hieu ST3A

20

Bdt la Sdn thuydn

Chi^l vdi ethanol Cat loai bo dung mdi Dich chie't ethanol cd dac

Chie't Idn lugt vdi n-hecxan, chloroform, ethyl acetate, n-butanol (dd phan cue tang dan)

Dich chie't

n-hecxan

Dich chiet chloroform

Dich chie't ethyl acetate

Dich chie't butanol Cat loai dung mdi bang dp sudt giam Dich chi^^t chloroform

n-Gdp cac phan doan cd sdc ky

dd Rf tuang duang nhau

Chay cdt sdc ky sephadex LH-20

he dung mdi CHCiyMeOH vdi dd phan cue tang ddn

Thu dugc 3 phan doan

Cdt pha dao RP-18 vdi he dung mdi ' MeOH/acetone/H2O'(2/1/2)

ST3A Axit asiatic

Hinh 3 Cac budc tdch chiet axit asiatic tii la Sdn thuydn Ket qua phan tieh phd cdng hudng tiir hat nhan cua ST3A cho thay hgp chat nay

ed dang phd cua mdt hgp chdt triterpene cd 5 vdng 6 canh vdi nhihig tin hieu ciia 30 carbon tren phd '^C-NMR So sanh cac ke't qua phd ciia ST3A vdi cdc ket qua tuong ling eua asiaticoside cho thdy cd su phii hgp hoan toan d ca 29 vi tri cacbon (Bang 2) Ngoai su phii hgp vd tuong tac H-C trong ed'u trdc cua ST3 A (hinh 6) cung khdng dinh them cdu triic cua phdn tur ciing nhu vi tri ciia cac nhdm the' neu tren Hgp chat

21

ST3A dugc xac dinh la axit asiatic (2a,3p,23-trihydroxy-12-ursen-28-oic axit) vdi cdng thiic phdn tur la C30H48O5 (hinh 5) Ddy la Idn dau tien hgp chd't nay dugc nhan

dang, tinh sach phdt hien d cdy Sdn thuydn {Syzygium resinosum G) tai Viet Nam

-f^-0,3

ft

ST STl STJA ST2 STJ 2I CH2OH

Hmh 4- Anh sdc ky tren ban mdng Hinh 5 Cdu triic hda hoc ciia axit asiatic

mdu ST3A tach tiir la Sdn thuvdn tdch tiir la Sdn thuven

Bang 2 Ke't qua phdn tieh phd cdng hudng tii hat nhdn cua ST3A tir la Sdn thuydn

5,^*'*'' 48.00 69,66 78,22 44,09 48.20 19,07 33,63 40,78 49,00 38,97 24.45 126,65 139,78 43.37

DEPT CH2

CH

CH

CH

CH, CH2

3 , 3 8 ( l H , d , / = 9 5 H z )

1.31 1,42/1,50 1,34/1,67 - 1,68 - 2.00 5,28(lH,t,./ = 3,5Hz)

-

-22