xay dung quy trinh ky thuat tach chiet khao sat tinh khang khuan va kha nang chong oxy hoa cua mot so hop chat thu cap tu la cay xuan hoa - Tại 123doc.vn

8/14/2019 xay dung quy trinh ky thuat tach chiet khao sat tinh khang khuan va kha nang chong oxy hoa cua mot so hop c

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BGIO DC V O TOTRNG I HC NNG LM THNHPHHCHMINH

BMN CNG NGHSINH HC************************

KHA LUN TT NGHIP

XY DNG QUY TRNH KTHUT TCH CHIT, KHO

ST TNH KHNG KHUN V KHNNG CHNG OXY

HA CA MT SHP CHT THCP TL CY

XUN HOA (Pseudranthemum palati ferum).

Ngnhhc: CNG NGHSINH HC

Nin kha: 2003 2007

Sinh vin thc hin: THTY PHNG

Thnh phHCh MinhThng9/2007


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BGIO DC V O TOTRNG I HC NNG LM THNH PHHCH MINH

BMN CNG NGHSINH HC**************************

XY DNG QUY TRNH KTHUT TCH CHIT, KHO

ST TNH KHNG KHUN V KHNNG KHNG OXY

HA CA MT SHP CHT THCP TL CY

XUN HOA (Pseudranthemum palati ferum).

Gio vin hng dn: Sinh vin thc hin:

TS. PHAN PHC HIN THTY PHNG

Thnh phHCh MinhThng 9/2007


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iii

1 LI CM TCon xin khc ghi cng n ba mv nhng ngi thn nui dng v gio dc connn ngi.

Ti xin gi lng bit n n:

- Ban GimHiu Trng i Hc Nng Lm Tp. HChMinh.- Ban Gim c Trung Tm Phn Tch V Th Nghim Ha Sinh, Trng i

Hc Nng Lm Tp. HCh Minh.

- Thy chnhim, thy c bmn Cng NghSinh Hc.htrvto iu kin thun li cho ti trong sut qutrnhhc tp vhonthnh

ti.

Ti xin chn thnhbit n:

- TS. Phan Phc Hin.- KsTrnh Phi Ly.

tn tnhhng dn, gip, ginhnhiu thi gian vcng sc truyn t nhng

kin thc, kinh nghim qugi, to mi iu kin tt cho ti honthnhkhalun ny.

Ti xin gi li cm n n:

- ThS. Hunh Kim Diu tn tnhgiptrong vic ly mu lXun Hoa vnggpnhng kin qubucho lun vn.

- TS. Nguyn Ngc Hi, PhngVi Sinh Khoa Chn Nui ThY, Trng i HcNng Lm.

- CvChinh Cng By, chcstrHonNgc 7 Nga Ty Ninh tntnhgip vcung cp slng ln lXun Hoa.

Chn thnh cm n cc bn lp Cng NghSinh Hc 29 ng vin, gip ti

trong sut thi gian lmti.


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iv

TM TT

ThTuPhng, i Hc Nng Lm Tp. HCh Minh. Thng 3/2007. Xy dng quy trnh kthut tch chit, kho st tnh khng khun v khnng

chng oxy ha ca mt shp cht thcp tl Xun Hoa (Pseudranthemum

palatiferum).

Gio vin hng dn: TS. Phan Phc Hin

tic tin hnh ti:

- Phngthnghim hal, Trung Tm Phn TchvThNghim HaSinh,Trng i Hc Nng Lm Tp. HChMinh.

- PhngVi Sinh, Khoa Chn Nui ThY, Trng i Hc Nng Lm Tp. HChMinh.

Thi gian tin hnh: t15/3/2007 n 30/7/2007.

Ni dung tin hnh:

- Tin hnh chit tchccphn on tcao khngkhun nht bng sc kct vsc klp mng, sau thnghim khnng khngkhun ca ccphn on

nyvi vi khunE.coli, Staphyloccoccus aureus,Pseudomonas aeruginosa.

- Tin hnhsc kct, sc klp mng vsc kiu chtchchit mt shp cht thcp tphn on chot tnhkhngkhun mnh nht.

- Gi mu chy ph cng hng tht nhn xc nh cu trc ca cc hpcht c tch chit phngphn tch cu trc, Vin Ho Hc,Trung tm khoa

hc TNhin, Cng NghQuc Gia H Ni.

- Thnghim khnng chng oxy ha ca l Xun hoa bng quy trnh DPPH.Kt qut c:

- Xcnh c cao chloroform chot tnhkhngkhun mnh nht vi nng c chti thiu l:

MICE. coli = 500 g/ml.


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v

MICsalmonella= 500 g/ml.

- Tchchit c 18 phn on vxcnh c phn on chot tnh khngkhun lXH9, XH10, XH11, XH12, XH13.

- Tin hnh tch chit trn phn on c hot tnh khng khun ny thu c 2hp cht XH-K11 v XH-K13.

- Sau khi chy phv tham kho mt sti liu, nhdanh c XH-K11 lapigenin 7-O-- glucoside, c cng thc phn t: C21H20O10v cht XH-K13 l

- sitosterol-3-O- -glucoside, c cng thc phn t: C35H60O5.

- Kho st tnh chng oxy ha ca l Xun Hoa cho thy hm lng cc phn tc chDPPH l kh cao (85,7%), thng qua so snh chsIC50ca mu Xun

Hoa vi Vitamin C:

IC50 xun hoa = 66,55 (g/ml).

IC50 vitamin c = 57,05 (g/ml).


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vi

SUMMARY

Do Thi Tuy Phuong, Nong Lam University Ho Chi Minh City.

Establishing the method of isolating, researching antibacterial activity and

antioxidant property of secondary metabol ite compounds extr acted from Xuan H oa

leaves.

Researching Place: Physicochemiscal department, Analysis and Biochemistry Center,

Nong Lam University Ho Chi Minh City.

Content:

- Isolating and purifying a number of fractions of this glue by columnchromatography (CC) and thin layer chromatography (TLC), after that

researching antibacterial property of these fractions forE. coli, Staphylococcus

aureus, Pseudomonas aeruginosa.

- Using CC and TLC to isolate and purify metabolite compounds of theseantibacterial fractions.

- Studying antioxidant activity of Xuan Hoa leaves by using DPPH process.Result:

- Identifying chloroform glue is the most antibacterial activity, with :MICE. coli = 500 g/ml.

MICsalmonella= 500 g/ml.

- Getting 18 fractions and identifying the most antibacterial active fraction.- Purifying and determining molecular formula of the two compounds: apigenin

7-O-- glucoside and - sitosterol-3-O- -glucoside.

- Determination of antioxidant property of Xuan Hoa extract shows that themolecular volume which inhibits oxidant activity is high (85,7% Vitamin C):

IC50 xun hoa= 66,55 (g/ml).

IC50 vitamin c = 57,05 (g/ml).


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vii

MC LC

NI DUNG TRANG

Li cm n.................................................................................................................... iii

Tm Tt ......................................................................................................................... iv

Mc lc .......................................................................................................................... vii

Danh sch cc chvit tt ............................................................................................. xi

Danh sch cc hnh v biu ...................................................................................... xii

Danh sch cc bng v s ....................................................................................... xiii

1. MU ................................................................................................................... 1

1.1. t vn ............................................................................................................. 2

1.2. Mc ch - Yu cu ................................................................................................ 2

1.2.1. Mc ch.............................................................................................................. 2

1.2.2. Yu cu ................................................................................................................ 2

2. TNG QUAN TI LIU ......................................................................................... 3

2.1. Gii thiu vcy Xun Hoa................................................................................... 3

2.1.1. c imthc vt hc ......................................................................................... 3

2.1.2. c im hnh thi .............................................................................................. 3

2.1.3. Phn b ................................................................................................................ 4

2.1.4. Nhn ging .......................................................................................................... 4

2.2. Cng dng ca cy Xun Hoa................................................................................ 4

2.2.1. Theo kinh nghim dn gian ................................................................................. 4

2.2.2. Tcdng sinh hc ............................................................................................... 5

2.3.1.1. Bo vtbo gan............................................................................................. 52.3.1.2. nh hng ln bnh tiu chy heo ............................................................... 10

2.3. Thnhphn ha hc ............................................................................................... 13

2.4. i cng vmt shp cht hu ctnhin ..................................................... 15


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viii

2.4.1. Alcaloid ............................................................................................................... 15

2.4.2. Flavonoid ............................................................................................................. 16

2.4.3. Tinh du .............................................................................................................. 16

2.4.4. Saponin ................................................................................................................ 17

2.5. i cng vvi khun ng rut ........................................................................ 17

2.5.1. nh ngha ........................................................................................................... 17

2.5.2. Hnhth............................................................................................................... 17

2.5.3. Tnhcht nui cy ............................................................................................... 18

2.5.4. c tnhsinh ha................................................................................................. 18

2.5.5. Sc khng ....................................................................................................... 19

2.5.6. c t.................................................................................................................. 19

2.5.7. Cu trckhngnguyn........................................................................................ 19

2.5.8. Khnng gy bnh .............................................................................................. 21

2.6. Mt sc tnhca vi khun thnghim .............................................................. 21

2.6.1. Staphyl ococcus aureus .......................................................................................21

2.6.2.Pseudomonas aeruginosa....................................................................................21

2.6.3. Escherichia coli ...................................................................................................21

2.6.4. Salmonella ...........................................................................................................22

2.7. Hot tnh chng oxy ha DPPH............................................................................. 23

3. VT LIU V PHNG PHP TIN HNH...................................................... 24

3.1. Vt liu ................................................................................................................... 24

3.2. a im v thi gian thc hin ............................................................................. 24

3.2.1. a im .............................................................................................................. 25

3.2.2. Thi gian ............................................................................................................. 253.3. Thit b, dng cv ha cht ................................................................................. 25

3.3.1. Thit bv dng c.............................................................................................. 25

3.3.2. Ha cht .............................................................................................................. 27


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ix

3.4. Phng php tin hnh........................................................................................... 28

3.4.1. Xl nguyn liu ................................................................................................ 28

3.4.2. iu chccloi cao ........................................................................................... 28

3.4.2.1. iu chcao ete du ha .................................................................................. 28

3.4.2.2. iu chcao chloroform .................................................................................. 29

3.4.2.3. iu chcao n-butanol..................................................................................... 29

3.4.2.4. iu chcao nc ............................................................................................ 29

3.4.3. Kho st tnh khng khun ca cc loi cao ....................................................... 31

3.4.4. Chit xut mt shp cht cao chloroform ........................................................ 32

3.4.4.1. Sc k ct ........................................................................................................ 32

3.4.4.2. Sc k lp mng .............................................................................................. 35

3.4.4.3. Sc k iu ch................................................................................................ 37

3.4.4.4. Kthut kt tinh phn on ............................................................................. 38

3.4.5. Kho st tnh khng khun ca cc phn on c tch chit tcao chloroform

....................................................................................................................................... 38

3.4.6. Thnghim hot tnh chng oxy ha DPPH...................................................... 40

3.4.6.1. Cc tnh cht ca gc tdo DPPH ................................................................... 40

3.4.6.2. Quy trnh thhot tnh chng oxy haDPPH ................................................. 40

4. KT QUV THO LUN .................................................................................. 44

4.1. Thnghim khnng khng khun ca cc loi cao chit .................................... 44

4.2. Kt qutch chit ca cao chloroform v cc phn on ca chng.................... 45

4.2.1. Kt qutch chit ca cao chloroform ................................................................ 45

4.2.2. Kho st phn on XH9, XH10, XH11, XH13. ................................................ 47

4.2.2.1. Phn on XH11 .............................................................................................. 474.2.2.2. Phn on XH9 v XH10................................................................................. 47

4.2.2.3. Phn on XH13 .............................................................................................. 48

4.3. Cu trc ha hc ca cc cht tinh khit. ............................................................... 49


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x

4.3.1. Cht XH-K13 ...................................................................................................... 49

4.3.2. Cht XH-K11 ...................................................................................................... 50

4.4. Th nghim kh nng khng khun ca cc phn on c chit t cao

chloroform. .................................................................................................................... 52

4.5. Kt quca thnghim chng oxy ha................................................................. 55

4.5.1. Kt quo OD i vi vitamin C ....................................................................... 55

4.5.2. Kt quo OD i vi dch chit l Xun Hoa.................................................. 56

5. KT LUN V NGH....................................................................................... 58

5.1. Kt lun .................................................................................................................. 58

5.2. ngh................................................................................................................... 59

TI LIU THAM KHO ............................................................................................. 60

PHLC ...................................................................................................................... 62


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xi

DANH SCH CC CHVIT TT

BHI Brain Heart Infirm

C Chloroform

Cfu Colony forming unit

COSY Correclation Spectroscopy

d Double: mi iDEPT Distortionlees Enhancement by Polarization Transfer

DMSO Dimethylsulfuoxid

DPPH 1,1 - diphenyl - 2 - picrylhydrazyl

E Ete du haHMBC Heteronuclear multiple bond correction

HSQC Heteronuclear single quantum coherence

IC50 Inhibitory Concentration 50: nng c ch50% DPPHIR Infrared Spectrometry: hng ngoiJ Coupling constant: hng sghp cpLD50 Lethal dose 50: liu gy cht 50% sc thm Multiplet: mi aMe Methanol

MIC Minimum Inhibitory Concentration: nng c chti thiuMS Mass Spectrometry: khi ph

Nt Nh trnNMR Nuclear Magnetic Resonnace: cng hng tht nhnRf Retention factor

s Single: mi nt Tripplet: mi baTSA Tryptic Soy Agar


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DANH SCH CC HNH V BIU

Hnh 1.1: Cy Xun Hoa............................................................................................. 3

Hnh 3.1: Cc pha trong bnh lc qul........................................................................ 29

Hnh 3.2: Cht phn tch c a vo c t sc k................................................... 33

Hnh 3.3: Bnh trin khai sc k lp mng ................................................................... 36

Hnh 3.4: Sc k iu ch............................................................................................. 37

Hnh 3.5: Cc nng pha long ca vitamin C v mu Xun Hoa sau khi cho DPPH

vo................................................................................................................................. 43Hnh 4.1: Thnghim cc phn on khngE.coliv Salmonella .............................44

Hnh 4.2: Cht XH-K11 trn bn mng ........................................................................ 47

Hnh 4.3: Cht XH-K13 trn bn mng ........................................................................ 48

Hnh 4.4: Kt quthnghim cc phn on khngE. Coli ...................................52

Hnh4.5 : Cc nng khng E. coli ca phn on XH9 ........................................... 53

Hnh 4.6: Cc nng khng Staphylococcus ca phn on XH9 ........................ 54

Hnh 4.7: Cc nng khng Pseudomonas aeruginosa ca XH9 .......................... 54

th4.1: Vitamin C .................................................................................................... 55

th4.2: Mu Xun Hoa........................................................................................ 56


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DANH SCH CC BNG V S

Bng 2.1: Kt ququan st vi thchut ...................................................................... 7

Bng 2.2: Kt quo hm lng MDA trong gan chut liu gy l 1ml CCl4/kg th

trng ................................................................................................................................ 9

Bng 2.3: Hm lng MDA liu gy l 0,5ml CCl4/kg thtrng chut ..................... 9

Bng 2.4: Kt quhm lng men gan ......................................................................... 10

Bng 2.5: nh hng ca l ti hay bt l Xun Hoa ln sbiu hin tng trng,

bnh tiu chy v sinh l mu ca heo .......................................................................... 11Bng 2.6: So snh hiu qucha trtiu chy .............................................................. 12

Bng 3.1 : Thtch v nng cc ng nghim ........................................................... 39

Bng 3.2: pha long v nng mu Xun Hoa..................................................... 42

Bng 3.3: pha long v nng mu vitamin C ..................................................... 43

Bng 4.1: Kt quthnghim khnng khng khun ca 4 loi cao ...................... 44

Bng 4.2: Kt qusc k ct trn Cao Chloroform.................................................. 45

Bng 4.3: Nng c chvi khun ti thiu ca cc phn on ........................... 53

Bng 4.4: Nng v kt quo OD ca vitamin C.................................................... 55

Bng 4.5: Nng v kt quo OD ca mu Xun Hoa ........................................... 56

Bng 4.6: nh hng ca mu Xun Hoa i vi hp thca DPPH ................ 57

S 3.1: Quy trnh xl nguyn liu .................................................................... 28

S 3.2: Quy trnh iu chcc loi cao ............................................................... 30

S 3.3: Quy trnh ly trch mu cho thnghim DPPH ............................................ 41


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Chng 1

MU

1.1. t vn

Cy Xun Hoa (Pseuderanthemum palatiferum) l mt loi cy c dn gian

bit n nh l mt dc liu c thcha c rt nhiu cn bnh: bnh tiu ha, tiu

ng, tr ni, chy mu, suy nhc thn kinhTuy nhin, trn thc tc rt t nghin

cu vcy ny c thlm r vn trn.

Hin nay trn thgii cha c mt nghin cu no su xa vcy ny. Cc nh

khoa hc Vit Nam cng ch mi bc u tm hiu, nghin cu v n. Trn Cng

Khnh v cng s(1999) thc tnh cp din trn chut cho thy cao ton phn l

Xun Hoakhng th hin c tnh v c tc dng c chqu trnh peroxy ha lipid

mng tbo, ngha l c xu hng bo vtbo gan. Hunh Kim Diu (2003) th

nghim l Xun Hoai vi heo cha cai sa c tc dng tng trng v cha c

bnh dch t. Vgn y, trong Hi NghKhoa Hc vCng NghHaHu CTon

Quc ln th3 HNi 112005, Phan Phc Hin vcng scng bcng trnh

phn lp Triterpenoid vSteroid tlcy Xun HoaPsenderanthemum palatiferumh

Acanthaceaebng phng phpcng hng tht nhn.

Hin nay, nhim khun ng tiu ha trnn rt phbin. Phng php cha tr

chyu hin nay l sdng khng sinh. Tuy nhin vic sdng khng sinh sdn n

ri ro do hin tng khng thuc. Do , vic tm ra mt ngun nguyn liu tnhin

c khnng khng khun scho ta mt phng php iu trmt cch hiu qui vi

i vi mt sbnh nhim khun.

Thm vo , ngy cng cnhiu mi quan tm vcccht khng oxy ha c

ngun gc tthc vt ctcdng ngn chn qu trnh oxy ha khng mong mun nh


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cc carotenoid, flavonoid, vitamin C, EV th trong cng trnhnychng ti cng

kho stkhnng chng oxy ha ca cchp cht trong l Xun Hoa.

Xut pht t yu cu trn, chng ti thc hin ti: Xy dng quy trnh k

thut tch chit, kho st tnh khng khun v khnng chng oxy ha ca mt s

hp cht thcp tl Xun Hoa (Pseudranthemum palatiferum).

1.2. Mc ch Yu cu

1.2.1. Mc ch

Xc nh c cao chloroform c hot tnh khng khun mnh nht.

Chit tch mt shp cht thcp tcao chloroform .

Xc nh phn on khng vi khun: E.coli, Staphylococcus aureus,

Pseudomonas aeruginosa.

Chit tch mt shp cht thcp tphn on khng khun.

Kho st khnng chng oxy ha ca cchp cht trong lcy Xun Hoa.

1.2.2. Yu cu

C lp 4 loi cao: Ete du ha, chloroform, n-butanol, nc. Dng phng php

nui cy vi khun trn mi trng thch xc nh cao c hot tnh khng

khun mnh nht.Tin hnh sc k ct v sc k lp mng tch cc phn on v mt shp

cht tl Xun Hoa.

Sdng phng php vng khng khun v pha long lin tc xc nh nng

c chti thiu ca cc phn on c ly trch tcao chloroform.

Tin hnh ly trch l cy Xun Hoav o hp thquang (OD) bc sng

517 nm [3] xc nh khnng chng oxy ha.


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Chng 2

TNG QUAN TI LIU

2.1. Gii thiu vcy Xun Hoa

2.1.1. c im thc vt hc [2]

Tn khoa hc:Pseudranthemum palatiferum.

H: r (Acanthaceae)

Tn thng thng: Xun Hoa, Hon Ngc, T Linh, Nht Nguyt, Thn Tng

Linh, Lan in,

2.1.2. c im hnh thi [2, 6]

Cy c thcao 1-2 m (nu trng trn t vn nh), sng lu nm. Thn cy xanh hoc

tm lc, khi gi chuyn thnh mu nu, phn ra nhiu mnh. L mc i hnh mi mc,

di 12-17 cm, rng 3,5-5 cm, gc l hi men xung.

Hoa mc kl hoc u cnh. Cm hoa di 10-16 cm. Hoa lng tnh, khng u, 5

l i ri tn ti n khi trng hp, mu trng, ng trng hp v di khong 2,5 cm, c

Hnh1.1: Cy Xun Hoa


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4

5 thychia lm hai mi, mi trn gm hai thynhdnh lin nhau n na chiu di

ca thy, mi di gm 3 thy to, thugia ca mi di c cc chm mu tm. Qu

nang 2 , mi cha 2 ht. Ma hoa tthng 4 n thng 5 m lch. Cy mc hoang ra

hoa gn nh quanh nm.

2.1.3. Phn b[6, 14]

Hin nay cy Xun Hoac trng nhiu ni nh mt loi cy thuc gia nh. Cy

mc hoang ra hoa gn nh quanh nm. Gn y c pht hin mc hoang vn

quc gia Cc Phng (Ninh Bnh), rng Bnh Nguyn (Phm Hong L, 1999).

H Ni tnm 1998 rln vic trng cy Xun Hoacha mt sbnh vtiu

ha. Thnh phHCh Minh, mt snh trng cy Xun Hoacha to bn.

c bit gn y nht chng tic bit c mt sdoanh nghip Ty Ninh dng

l v rcy Xun Hoany lm tr v c bn rng ri ra th trng vi tn gi tr

Hon Ngc.

2.1.4. Nhn ging [6]

Cy Xun Hoac th nhn ging ddng bng cch gim cnh. Ly on cnh b,

cch ngn khong 20 cm, bbt vi l pha di, cm vo t m, chmt, hng

ngy ti u (khong 2-3 ln/ngy). Cy chu t xp v m trung bnh. Chsau 1

tun cnh gim ra r. Cy pht trin nhanh. Trng khong trn 2 thng l c th

dng lm thuc cha bnh.

2.2. Cng dng ca cy Xun Hoa

2.2.1. Theo kinh nghimdn gian

Cy Xun Hoac dng trong dn gian cha nhiu bnh nh:

- Nhim khun ng tiu ho, au ddy, au bng khng r nguyn nhn.- Lot hnh t trng, chy mu ng rut.- Khi phc phc sc khocho ngi m yu, cha suy nhc thn kinh.- Chn thng chy mu, dp gy c th, chn thng sno.- Vim thn cp v mn, suy thn, i ra mu, i but, i rt.


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- Cha cc bnh u phi, u x tuyn tin lit, au gan, x gan c trng, lmgim au khi bung th gan giai on pht bnh.

- iu chnh huyt p.- Trung Quc, ngi ta dng rcy Xun Hoacha n ng tn thng.

c bit H Ni tnm 1998 rln vic trng Xun Hoacha nhng

bnh thuc ng tiu ha. Thi gian gn y, mt sVit kiu Mcng

dng cy Xun Hoacha bnh i tho ng. Bnh nhn dng lin tc cy

Xun Hoagic n nh tlng trong mu.

L Xun Hoakhng c mi, khng v, hi nht. Ngi ta thng dng l ti

ra sch, nhai vi my ht mui ri ung vi nc, gi nt ly nc ung hoc

nu n. Liu lng phu thuc vo ngi bnh, tng bnh, thng thng n 2-8

l/ngy, chia lm 2 ln trc ba n.

2.3.1. Tc dng sinh hc

2.3.1.1. Bo vtbo gan [9]

gp phn xc minh tc dng c lu truyn trong dn gian v chng minh tc

dng ca cy thuc Xun Hoa, Trn Cng Khnh v cng s ca ng tin hnh

nghin cu c tnh cp din v tc dng bo vtbo gan ca l cy Xun Hoatrnin vivo.

Thc tnh cp- Chut nht trng, chia lm 6 l, mi l 7 con.- Thuc th: Cao c tonphn c cn vhatan bng nc ct, thu c cc

dung dch c hm lng khc nhau: 33,2 mg/ml; 66,8 mg/ml; 125,2 mg/ml;

222,4 mg/ml; 367,6 mg/ml; 460 mg/ml.

- Tin hnh: Cho mi chut ung 0,5 ml dung dch thuc Xun Hoacc l khcnhau: 0,83 g/kg; 1,67 g/kg; 3,13 g/kg; 5,56 g/kg; 9,19 g/kg; 11,5g/kg thtrng

chut. Quan stdin bin hnhvi, sau git ngu nhin 3 chut 3 lkhc


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nhau: quan st i th ph tng v lm vi th cc c quan chu tc dng ca

thuc nhtim - gan - thn - ddy- rut.

- Kt qu:o cc liu: 0,83 g/kg; 1,67 g/kg; 3,13 g/kg th trng chut, sau khi ung

thuc vtrong sut qutrnhthnghim thuc khng gy thay i trng thi

hot ng ca chut: Chut vn blui ti, leo tro, chiru, lim ui

o Cc liu cao hn: 5,56 g/kg; 9,19 g/kh; 11,5 g/kg th trng chut, sau khiung thuc chut cgim hot ng, nhng sau 1githtrli bnhthng,

chut rcvonhau, mt lim dim, thnhnhng.

o Tt c cc chut th nghim ( liu trn) u khng cht. Chut sng khemnh sau 48 gi.

o Quan sti th: Ccphtng u bnhthng.o Lmvi th cc cquan: Ct thnh ming mng vnhum tbo, quan st

di knhhin vi (Bng 2.1).

Cquan

LiuTim Gan Thn Ddy Rut nh gi


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Bng 2.1: Kt ququan st vi thchut

Kt lun: Cao ton phn Xun Hoakhng gy cht chut tt ccc liu. Nh vy,

trn c tnh cp cao Xun Hoakhng thhin c tnh, khng c LD50. Thtc dng bo vtbo gan- Chut nht trng, chi lm6 l, mi lt812 con.- Cao tonphn lXun Hoa loi bchlorophyl, hatan vi nc ct.

0,83g/kg C tim

bnh

thng.

Cc tnh

mch xoang

v tnh mch

trung tm

trn thu

xung huyt.

Qun cu v

cc ng thn

bnh thng.

Nim

mc d

dy v

tuyn

bnh

thng.

Nim

mc rut

v cc

tuyn

bnh

thng.

Khng

c tn

thng

thc th

cc

tng.

9,19g/kg C tim

bnh

thng.

Tbo gan

hi to,

nguyn sinh

cht c thoi

ho nh.

Qun cu

xung huyt,

cc ng thn

bnh thng.

Nim

mc d

dy v

tuyn

bnh

thng.

Nim

mc rut

v cc

tuyn

bnh

thng.

C thoi

ho nh

gan.

11,5g/kg C tim

bnh

thng.

Tbo gan

hi to,

nguyn sinh

cht c thoi

ho nh, tnhmch trung

tm trn thu

xung huyt.

Nang

Bowmann

rng ra, t

bo thnh

ng teo nh.

Nim

mc d

dy v

tuyn

bnhthng.

Nim mc

rut v

cc tuyn

bnh

thng.

C tn

thng

nh

gan v

thn.


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8

- M hnh gy ng c gan: Gy tn thng gan chut nht bng carbontetrachloride (CCl4) theo phng php c dung thng thng (Shibayama,

1989; Yoshitake v cng s, 1991) vi liu 1ml CCl4/kg th trng (liu cao)

hoc 0,5 ml CCl4/kg thtrng (liu thp).

- Chia chut thnh6 l, mi l2 nhm:o L 1 (i chng sinh hc): Chut bnh thng, khng gy m hnh vim

gan, khng dung thuc Xun Hoa.

o L2: Gy mhnhvim gan i chng, khng sdng thuc Xun Hoa.o L3: Gy thnghim vim gan, cho ung thuc Xun Hoa trc, sau 1 gi

thtim CCl4vo.

- Nguyn tc ca phng php l xc nh hm lng malonic dialdehyde(MDA), mt trong nhng sn phm to ra bi qu trnh peroxy ho lipide mng

tbo. Sn phm MDA c kh nng phn ng vi acid thiobarbituric to

thnh phc hp trimethine cmuhng vcnh hp thu cc i 530532

nm [9]. Cng muca dung dch tlthun vi nng MDA. Hmlng

MDA tnhtheo cng thc:

X = E x 30,8

Trong : X: Hmlng MDA (mM)

E: hp th532 nm.

30,8: Hstt phn t.

Bng 2.2: Hm lng MDA trong gan chut liu gy l 1ml CCl4/kg thtrng.L n (schut) ETB MDATB

1 8 0,163 5,0296

2 12 0,458 14,1156


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9

3 8 0,348 10,7091

Ch thch:

MDA: Malonic dialdehyde.

ETB: E trung bnh.

MDATB: MDA trung bnh.

Nhn xt:

- l c sdng cao Xun Hoahm lng MDA c gim so vi l gy m hnh(gim t14,1156 xung cn 10,7091).

- Hmlng men gan (AST, ALT) qucao, khng o c trn my.- Xt nghim vi th: Cha thy du hiu bnh phc ca tbo gan do thuc iu

tr.

Bng 2.3: Hm lng MDA liu gy l 0,5ml CCl4/kg thtrng chut.

L n (schut) ETB MDATB

1 8 0,1915 5,8982

2 12 0,385 11,547

3 8 0,203 6,2463

Nhn xt:

- l c sdng cao Xun Hoahm lng MDA c gim so vi l gy m hnh(gim t11,547 xung cn 6,2463).

- Xc nh hm lng men gan trn my Cobas Mira Plus, hng Roche (Thy S).

Bng 2.4: Kt quhm lng men gan.

L n (schut) AST(U/l) ALT(U/l)1 8 270 432

2 12 9668 10870

3 8 8740 10533


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10

Nhn xt:

- Hm lng men gan gim nhng cha ng k.- Xt nghim vi th: l chut c sdng cao Xun Hoav l gy m hnh nhn

thy rng cc tbo vng thoi ho bt u thu nhv c xu hng hi

phc.

Kt lun:

Cao c ton phn Xun Hoa c khnng c chqu trnh peroxy ha lipid mng t

bo, ngha l c xu hng tc dng bo vtbo gan trn thc nghim.

2.3.1.2. nh hng ln bnh tiu chy heo [14]

Bnh tiu chy chyu xy ra heo cha cai sa. Nhiu khng sinh nh avinamycine,

colistin, oxytetracyclin, spectinomycin v trimethoprim l nhng phng php ph

bin c p dng iu trbnh. Tuy nhin, lng tn d khng sinh trong cc sn

phm ng vt c xem nh l mt nguy c tim n i vi sc khocon ngi bi

v tnh c v tnh dng ca n. Thm vo , spht trin ca cc vi khun khng

thuc dn n sdng liu lng qu cao trthnh mt vi vn trn thgii. V

vy, cn phi gim vic sdng v thay thvai tr ca khng sinh trong thc n gia

sc bng cch gia tng vic sdng probiotics v nhng sn phm tnhin khc, c

bit y ta sdng l cy Xun Hoaiu trbnh tiu chy heo.

Bng 2.5: nh hng ca l ti hay bt l Xun Hoa ln sbiu hin tng trng,

bnh tiu chy v sinh l mu ca heo.

Phng php n

i

tng

Xun

Hoa

Liu lng

(g/kg thtrng/ngy)

Qua

ng

Thi

gian

TN1C1 52 Heo

cha

- - - -

T1-1 52 L ti 1 ming 1-7


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11

T1-2 52 cai sa 1 1-30

TN2

C2 60Heo

chacai sa

- - - -

T2-1 60 L ti 0,5

ming 1-30T2-2 60Bt

0,1

T2-3 60 0,2

TN3

C3 62

Heo

cai sa

- - - -

T3-1 62 L ti 0,5

thc n 1-30T3-2 62Bt

0,1

T3-3 62 0,2

Sbiu hin tng trng c o lng da vo vic tng cn v tc tng trng

chm li. Heo tng t hn 4kg/28ngy c xem l tng trng chm li. S truyn

bnh tiu chy c o lng hng ngy da trn tlheo mc bnh trong nhng con

heo th nghim, strng hp v thi gian mc bnh.

Ngoi ra, Hunh Kim Diu v Trn vn Ha [14] tin hnh thnghim khnng tr

bnh tiu chy trn heo con v heo m, so snh vi hai loi khng sinh ang c s

dng tr bnh tiu chy heo con rt hiu qu: Coli-norgen v Cotrimxazol. Sau 3ngy iu tr t l khi bnh ca heo ln lt: Bt Xun Hoa 92,86%; Coli-norgen

90,48%; Cotrimxazol 83,33%. Tlti pht theo thtl: 7,14%; 9,52%; 14,29%.

Bng 2.6: So snh hiu qucha trtiu chy.

Loi thuc

Sheo

th

nghim

Slng heo khi bnh sau khi iu tr

Ngy thnht Ngy thhai Ngy thba

Scon % Scon % Scon %

Bt Xun

Hoa42 10 23,81 29 69,05 39 92,86

Coli-norgen 42 10 23,81 25 59,52 38 90,48


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12

Cotrimxazol 42 15 35,71 28 66,67 35 83,33

Nhn xt:

Khng c tc dng phca l Xun Hoa i vi heo trong th nghim ny. Nng c

chnh nht ca cy c tnh ton l 0,75-1,5 g l ti hoc 0,15-0,3 g bt/kg th

trng cho phng php ny. Liu lng c sdng cho vic ngn chn bnh, nng

c sdng trong th nghim nhhn nng gy c chthp nht trnh gy

tc dng ph.Sdng l hay bt Xun Hoac thci thin nhng thng so lng

ni trn v kt quc thc gii thch nh sau: Trc tin, l Xun Hoacha mt t

lcao protein th (30,8% DM: dry matter), so snh vi cvoi (18,6% DM) v cy sn

dy (18,4% DM). nh hng ca liu sdng srt nhbi v tlnhca n trong

khu phn. Thhai, l Xun Hoac hm lng cao mt sloi khong nh: Ca (6,5%

DM), K (4,4% DM), Mg (6,3% DM), Fe (0,3% DM). Hm lng Fe, c bit trong cc

cht khong cao gp 4 ln u nnh v 3 ln gan heo. Fe trong cy sgp phn ci

thin tnh trng sinh l mu bi v Fe cn thit cho heo thc y hng cu, c bit

hemoglobin c chc nng nh mt thmang oxy v cacbonic, v thn c bit nh

mt sc th hp v gii thch tnh trng ca hng cu. Thba, trong l c cha mtprotein n nh nhit c gi l pseuderantin (0,4% trong protein th) c hot tnh

mnh trong vic tiu ho casein v do vic tiu ha sa sc ci thin nu b

sung l. Tht, mt shp cht ho hc trong l v mt vi tc nhn khng khun c

thnh hng ti spht trin v min dch ca heo.

Kt lun:

Sdng bt l Xun Hoavi 0,2 g/kg thtrng cho 30 ngy i vi heo cha cai sa

v cai sa ci thin stng trng v ngn nga bnh dch t, cho nn cy trngnycnhiu trin vng thay thhoc gim sdng khng sinh trong tng lai.

2.3. Thnh phn ho hc [4, 5]


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Hin nay trn thgii cha c cng trnh no nghin cu v thnh phn ha hc ca

cy Xun Hoa, chc mt scng trnh nghin cu bc u vcy ny Vit Nam

nhTrn Cng Khnh(1998), Phan Phc Hin (2005), Hunh Kim Diu (2003).

Cc nguyn ta lng v vi lng trong l cy Xun Hoakh cao so vi cc loi cy

khc. Khng pht hin cc nguyn tkim loi nng nh Cd, Pd, As, Cr. ng ch l

hm lng Vanadi kh cao (3,75 mg/100 g l ti).

Ngoi ra, l Xun Hoacn cha y cc acid amin thit yu vi hm lng tng s

kh cao (751-1365 mg%). l cc acid amin givai tr quan trng trong sinh tng

hp protein c bp v chng mi c th. Thiu chng c thst cn nhanh. L Xun

Hoa cn giu Valin (29-1001 mg%). Thiu acid amin ny, s phi hp cc chuyn

ng ca bp tht bri lon v yu i. Valin cn nh hng n hot ng ca tuyn

ty, mt tuyn tiu ho quan trng.

Trong cc cng trnh nghin cu vthnh phn ha hc ca l Xun Hoangi ta

tm thy -Sitosterol, Phytol, 3-O-( -D glucopyranosyl)-Sitosterol, hn hp gm hai

ng phn epime stigmasterol v poriferasterol, n-pentacosan-1-ol v hn hp ca

kaumpferol 3-methyl ether 7-O- -glucoside v apigenin 7-O- -glucoside [4].

Nguyn Vn Hng vcng s(2004) [4], phn lp c tip theo l: 1-triacontanol,glycerol 1-hexdecanoate, acid palmitic v acid salicylic.

- Triacontanol: L cht rn mu trng, im nng chy 87-880C, l mt Ancol nobc mt, khng phn nhnh vi cng thc l : C30H62O. 1-triacontanol l mt

cht iu ha sinh trng thc vt. Cc nghin cu gn y cn cho thy 1-

triancontanol c hot tnh khng vim, c chcc qu trnh peroxy ha cc cht

bo c cng nh khng c stham gia ca cc enzyme v lm gim mtrong

mu.

- Glycerol 1-hexadecanoate: Dng tinh thmu trng, im nng chy 65-670C,l mt mono-ester ca glycerin vi acid Palmitic v cng thc phn t l

C19H38O34.


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14

- Acid palmitic: L cht rn mu trng, nng chy 48-510C, c c trng camt acid bo no mch thng.

- Acid Salicylic: Thu c di dng tinh thhnh kim, khng mu, nng chy 140-1420C v cng thc phn t l: C7H6O3. Acid salicylic c tc dng khng

nm v khng khun. Acid salicylic tng hp c sdng trong nhiu loi ch

phm da vo tnh cht ny.

Vi cc hat tnh nu trn, sc mt ca 1-triancontanol v acid salicylic trong l cy

Xun Hoac thgp phn gii thch cc hot tnh c pht hin thy trong cc

cng trnh nghin cu trc y cng nh gii thch tc dng cha bnh ca cy Xun

Hoa theo kinh nghim sdng trong dn gian.

Tm hp cht Dotriacontan, phytol, squalen, 24-metylencycloartanol, loliolide, -

sitosterol, -sitosterol 3 -O-D-glucopyranosid v stigmasterol 3 -O-D-glucopyranosid

ln u tin c c lp trong l cy Xun HoaPseuderanthemum palatiferum(Nees)

Radlk, h r (ancathaceae) ca Trn Kim Thu Liu vcng s(2006).

2.4. i cng vmt shp cht hu c tnhin [ 7 ].

2.4.1. Alcaloid

L nhng hp cht thin nhin vi nhiu dng cu trc khc nhau v c hot tnh sinh

hc r rt. a salcaloid hin din trong loi cy ht kn, cy c hoa nhng cng tm

thy trong ng vt, cn trng, sinh vt bin, vi sinh vt v thc vt hng. a s

alcaloid khng mu, dng kt tnh rn, c nhit nng chy hoc nhit phn hy

xc nh. Vi alcaloid dng keo, v nh hnh v alcaloid dng lng (nicotin,

coniin) hoc c mu (berberin mu vng; betanidin mu ).

Cc alcaloid dng baz tdo thng ho tan c trong dung mi hu c, tuy nhin

cc alcaloid dng bazo tcp th chha tan trong nc. Mui ca a salkaloid th


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15

ha tan trong nc. ha tan ca alkaloid v cc mui ca chng givai tr quang

trng trong knghdc phm; trong vic trch ly chng ra khi cy cv trong vic

bo chalcaloid thnh cc dng thuc ph hp.

a salcaloid c tnh kim, iu ny khin cho alcaloid cc knhy cm, dbthy

phn khi gp nh sng hoc nhit .

Nhiu alcaloid c sdng lm thuc trbnh. Alcalid c tc dng kch thch

thn kinh nh: strychnine, cafein, lobelinc kh nng dit k sinh trng v cc

nguyn sinh ng vt nh: emetin, quinin, conessin, berberin. Cc alcaloid c tc dng

c chhthn kinh trung ng nn c dng lm thuc gim au trong khi gii phu

hoc khi bbnh ung th giai on cui: morphin v cc dn xut ca morphin nh:

heroin, codein (tuy nhin cc hp cht ny c thgy ra tnh trng uoi, bun ng,

kh thv nghin thuc). C alcaloid c bo ch lm thuc gim hthn kinh

giao cm: reserpin, propanolol.

Mt salcaloid nh: berberin, palmatin, taspinc khnng c chcon men sao chp

ngc (reverse trancriptase) y l con men chyu ca retrovirus m cc loi thuc

khng HIV tp trung tiu dit.

2.4.2. FlavonoidFlavonoid l nhng hp cht mu phenol thc vt, to nn mu cho rt nhiu rau, qu,

hoaPhn ln c mu vng (do tflavus l mu vng); tuy vy, mt ssc tc mu

xanh, tm, , khng mu cng c xp vo nhm ny v vmt ha hc chng c

cng khung sn.

Cc flavonoid c thdng tdo hoc dng glicosid. Cc ng thng gp nht l

D-glucose; k l D-galactose; L-Rhamnose; L-Arabinose

Chalcone hin din t trong tnhin; flavanol v flavanonol cng him gp; flavone vflavonol phn b rng ri trong tnhin. Cc glicisid flavonol nh: rutin, quercitrin,

daempherol. Kaempherol rt thng xuyn hin din. Antocyanidine dng glicosid th


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d nh: pelargonidin; cyanidin; delphinidinto mu xanh, , tm cho nhng cnh

hoa v tri.

Cc flavonoid thng dkt tinh v thng c mu. Flavone c mu vng nht hoc

mu cam; flavonol mu vng nht n mu vng; chalcone mu vng n cam-. Cc

isoflavone; flavanone; flavanonol; leucoantocyanidine; catechin kt tinh khng mu.

Antocyanidine c mu , tm, xanh dng to mu cho nhiu loi hoa v tri.

Quercetin mi trng kim th c tc dng khng khun km nhng mi trng

acid th c tc dng rt r vi vi khun tcu vng, vi khun tcu trng Salmonella

oranienbur c tc dng khng nm thc vt.

Mt s hp cht thuc loi isoflavonoid nh rotenon (trch t dy thuc c Derris

elliptica Benth, hu) c tnh c i vi c v cn trng nhng khng c vi cc

ng vt hu nh. Cng c nhng hp cht c tnh dit cn trng nh hp cht

pyrethrin (trch tcy trtrng Chrysanthemum cinerariaefolium, hCc).

2.4.2. Tinh du

Ni chung, tinh du trch ly t thc vt c cu trc terpenoid. T2000 nm trc

ngi ta bit sdng turpentine trho. Mentol l alcol terpen trch t tinh du

bc h c khnng gy t cc b, nn gp phn vo c tnh trho ca n. Cc gia v

nh: htiu, gng, nhc u khu, inh hngbn cnh mi vlm chng c gi tr

trong ngnh n ung, chng cn c tc dng khng khun. Nhiu tinh du c khnng

khng khun mnh nh: tinh du rau ngEnhydra fluctuans; tinh du hi Illicium

verum; tinh du quCinhamomum; tinh du long no Cinnamonium campho; tinh du

gngZingiber officinalist; tinh du nghCurcuma longa

2.4.3. Saponin

L mt loi glycosid phn bkh rng trong thc vt, c mt s tnh cht c trng:khi ha tan vo nc sc tc dng lm gim sc cng bmt ca dung dch v to

nhiu bt; lm vhng cu cn gi l tnh ph huyt. Saponin thng dng v nh

hnh, c vng. Saponin rt kh tinh ch, c im nng chy thng cao t200oC tr


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17

ln v c thtrn 300oC. Saponin c thbta bi acetat ch, hydroxid barium, sulfat

amonium nn c thli dng tnh cht ny c lp saponin.

Nhiu saponin steroid l nguyn liu u tng hp cc cht hormone steroid c hot

tnh cao. Nhiu loi saponin c tc dng khng nm, khng khun.

2.5. i cng vvi khun ng rut [8, 12]

2.5.1. nh ngha

Hvi khun ng rut (Enterbacteriaceae) bao gm cc trc khun Gram m, hiu kh

hoc kkh tunghi, khng c men oxidase, ln men ng glucose c km theo sinh hi

hoc khng, khnitrat thnh nitrit, c thdi ng hoc khng, nhng nu di ng th c

nhiu lng xung quanh thn, khng sinh nha bo.

2.5.2. Hnh th

Tt ccc vi khun thuc hny u l trc khun Gram (-). Kch thc trung bnh 2-4

m x 0,4-0,6 m. Mt sloi hnh thkhng n nh, c thxut hin dng si. Nhng vi

khun di ng th c nhiu lng phn bkhp xung quanh tbo. Cc thnh vin ca h

vi khun ng rut khng bao gisinh nha bo. Msc v, c thquan st c bng

knh hin vi thng thng.

2.5.3. Tnh cht nui cy

Cc thnh vin ca h vi khun ng rut c th mc c trn mi trng nui cy

thng thng. Trong mi trng lng, c th lng cn hoc lm c mi trng, c th

pht trin thnh vng trn bmt, nhng cng c thva lm c mi trng va c cn

di y ng. Trn mi trng c c 3 dng khun lc:

- Dng S: Khun lc trn, bu, nhn bng.- Dng R: Mt khun lc kh, x x. Thng gp khi nui cy gichng.- Dng M: Hnh thc pht trin ny thng gp nhng vi khun c khnng hnh

thnh v. Khun lc nhy, kch thc ln hn khun lc dng S v cc khun lc

c xu hng ha vo nhau.

2.5.4. c tnh sinh ho

Nhng c tnh sau y thng c xc nh khi nghin cu vi khun ng rut:


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- Di ng hoc khng di ng.- Ln men hoc khng ln men mt sloi ng. Hai loi ng thng c xc

nh nht l glucose v lactose.

- Sinh hi hay khng sinh hi khi ln men ng.- C hay khng c mt senzym. Hai enzym thng c xc nh nht l urease v

tryptophanase.

- Khnng sinh ra sunfua hydro (H2S) khi dha protein, acid amin hoc cc dncht c lu hunh.

- Pht trin c hay khng pht trin c trn mt smi trng tng hp. V d:khnng sdng citrat l ngun cung cp cacbon duy nht c trong mi trng

Simmons.

2.5.5. Sc khng

V khng c khnng hnh thnh nha bo nn cc thnh vin ca hvi khun ng rut

khng c sc khng cao vi nhng iu kin ha l c bit ca mi trng. Chng d

dng b tiu dit nhit si 100oC v bi cc ho cht st khun thng thng. Tuy

nhin nhiu loi vi khun ng rut c khnng sng nhiu ngy n nhiu tun, thm

ch mt vi thng ngoi mi trng. y l iu kin thun li cc vi khun gy bnh

lan truyn.

2.5.6. c t

Hu ht cc vi khun ng rut u c ni c t. Bn cht ha hc ca ni c t l

lipoposaccharid (LPS) ca vch tbo. Ni c tchc gii phng khi tbo bli gii.

Ni c tc khi lng phn tt100000 n 500000 dalton. Ni c t tuy tnh c

khng cao bng ngoi c tnhng cng rt c (chcn 0,05 mg ni c tgit chtchut nht sau 24 gi). Ni c tc thgy ra tnh trng sc, nu khng c iu tr

tch cc kp thi, chuyn thnh sc khng hi phc dn n tvong. Ni c tkhng

bmt tnh c 100oC trong 30 pht. Ni c tl cht c khnng gy sc.


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Mt sthnh vin ca hvi khun ng rut c khnng sinh ngoi c tnhS.Shiga,

E.Coli loi Enterotoxigenic E.Coli (ECET). Ngoi c t ca S.Shiga lm cho bnh l

nng hn rt nhiu, ngoi c tLT (Labile Toxin) ca ETEC l yu tquyt nh c lc

ca vi khun ny.

2.5.7. Cu trc khng nguyn

Hvi khun ng rut c 3 nhm khng nguyn cbn: khng nguyn O, khng nguyn

H v khng nguyn K.

- Khng nguyn O:L khng nguyn thn ca vi khun. y l thnh phn khngnguyn ca vch tbo. L mt phc hp protein, poliozid v lipid, trong

protein lm cho phc hp c tnh khng nguyn, poliozid quyt nh tnh c hiu

khng nguyn cn lipid quyt ng tnh c. Khng bph hu100oC trong hai

gihoc trong cn 50% nhng bmt tnh khng nguyn khi x l bng formol

0,5%. nhng vi khun khng c vhoc mng bc (khng c khng nguyn K)

th khng nguyn O nm lp ngoi cng. Khi khng nguyn O gp khng th

tng ng sxy ra phn ng ngng kt, gi l hin tng ngng kt O vi cc

ht ngng kt nh, lc kh tan.nhng vi khun c khng nguyn K, hin tng

ngng kt O c thbche lp bi khng nguyn ny. Khng nguyn O c tnh c

hiu cao, n thng c dng phn loi vi khun. Da vo khng nguyn Ongi ta c thchia mt vi loi vi khun thnh nhiu typ huyt thanh.

- Khng nguyn H:L khng nguyn lng ca tbo vi khun, chc nhng vikhun c lng. C bn cht l protein, ph hu 100oC hoc trong cn 50%

nhng khng bphn hutrong formol 0,5%. Khng nguyn H khi gp khng th

tng ng sxy ra hin tng ngng kt H vi cc ht to hn trong hin tng

ngng kt O v rt dtan khi lc. Nhng vi khun c khnng di ng khi tip xc

vi khng thH tng ng strthnh khng di ng. Khng nguyn O v khngnguyn H c thc sn xut ring pht hin ring bit cc khng th tng

ng. c khng nguyn O, ngi ta cho vi khun ny vo cn 50%, khng

nguyn H sbph hu, khng nguyn O vn tn ti. c khng nguyn H ngi


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ta cho vi khun vo formol 0,5% th khng nguyn O b ph hu, khng nguyn H

vn cn nguyn vn.

- Khng nguyn K: L khng nguyn v hoc b mt, khng nguyn K nm bnngoi khng nguyn thn.N c thdi dng mt lp vdy, quan st c bng

knh hin vi quang hc thng thng (nh Klebsiella) hoc di dng mt lp rt

mng chc thquan st bng knh hin vi in t(nh S.Typhy). Khng nguyn

K nu che phhon ton khng nguyn O sngn cch khng cho khng thO gn

vi khng nguyn O lm cho phn ng ngng kt khng xy ra. Trong trng hp

ny cn phi ph hukhng nguyn K hoc vi khun phi c nui cy trong iu

kin khng sinh ra c khng nguyn ny.

2.5.8. Khnng gy bnh

Ni v kh nng gy bnh ca h vi khun ng rut, trc ht phi cp n cc

nhim khun ng tiu ha. Hvi khun ng rut ng u trong cc cn nguyn vi

khun gy tiu chy. C chgy bnh, v tr gy tn thng bmy tiu ha rt khc

nhau tutheo tng ging, tng loi.

Ngoi ng tiu ha, cc vi khun ng rut cn c khnng gy bnh nhiu cquan

khc nh tit niu, thn kinh, h hpCc thnh vin ca hny ng u trong cc vikhun gy vim ng tit niu, bxa cc vi khun khc. Chng cng ng u trong cc

vi khun gy nhim khun huyt. C thni khi qut bt kbnh phm no cng c th

gp thnh vin ca hvi khun ng rut.

2.6. Mt sc tnh ca vi khun thnghim

2.6.1. Staphyl lococcus aureus

- L vi khun gram dng, khng gip m, khng to bo t.- Sc khng: nhy cm vi Penicillin, Authromycin v Ampicilin.- Khnng gy bnh:

o Sng mtrn da, nim mc.o Gy ung nht, p xe.


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o Vim v b cu.o Gy nhim trng mu dn n vim phi, vim thn cp, vim tuyn sa

v vim ty xng.

2.6.2. Pseudomonas aeruginosa

- L vi khun gram m, c tim mao, vi khun sinh sc tmu xanh.- Khnng gy bnh:

o Gy bnh mxanh ng vt v ngi.o Vim bng quang,vim tai c mngi.

2.6.3. Escheri chia coli (E.coli )

- L vi khun gram m, khng bo t- Sc khng: Nhy cm vi chloraphenicol, Streptomycin, Sunfamid,

Trimethroprim.

- Khnng gy bnh:E.coli c sn trong rut, khi c thbsuy yu, sc khnggim thE.colimi gy bnh:

o Gy tiu chyo Bnh ph thng heo cono Vim rut

2.6.4. Salmonella

- Salmonella l trc khun di 0,6 0,8 m, c nhiu lng xung quanh thn, rt ding, khng c v, khng sinh nha bo, nhum Gram bt Gram m.

- Vi khun hiu kh hay kkh ty tin, mc ddng trn cc mi trng nui cythng thng, nhit thch hp 37oC nhng c thpht trin c t642oC,

pH thch hp l 7,6; pht trin c pH 6,9.

o Trn mi trng lng: Sau 56 ginui cy, vi khun lm c mitrng, sau 18 gimitrng c u.

o Trn mi trng thch thng: Khun lc trn li, bng, thng khngmu hoc mu trng xm.


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o Trn mi trng phn lp SS: Khun lc c mu hng; Trn mi trngIstrati khun lc c mu xanh.

- c t:o Ni c t: Ni c t ca Salmonella rt mnh, tim cho chut nht

hoc chut lang liu thch hp th sau vi ngy chut cht, mchut thy

rut non xung huyt, mng Payer bph n, i khi hoi t. Ni c t

c vai tr quyt nh trong tnh cht gy bnh ca Salmonella.

o Ngoi c t: Ngoi c tchhnh thnh trong iu kin invivo v nuicy kkh. Ngoi c tc thiu chnh thnh gii gii c t.

- Khnng gy bnh:o St thng hn ph thng hn.o Ngc thc n.

2.7. Hot tnh chng oxy ha DPPH [3]

Hin nay ngy cng c nhiu mi quan tm vcc cht chng oxy ha thcp tthc

vt nh l ngun bsung cho cc hthng bo vchng li soxy ha c hi tn ti

sn c trong c th. Trong i sng cc sinh vt kkh v hiu kh u thit lp mt s

cn bng tinh t gia ch li v nguy c trong vic s dng oxy t nng lng.

Trong qu trnh h hp chng to ra cc hp cht trung gian l cc gc tdo: anion

superoxide (O2), hydroxyl (OH), cht oxy ha nh H2O2c hot tnh phn ng rt ln,

m tcc cht ny cng nh cc sn phm phn ng ca chng l nguyn nhn ph

hy cc phn tsinh hc nh DNA, lipid, protein

Mc d bn thn ca sinh vt tpht trin cc hthng enzyme nhm iu ha cc

loi phn t oxy hot tnh cao ny nh superoxide dismutase, catalae, glutathione

peroxidase l cc enzyme iu ha nhm duy tr san ton trong gii hn cho php caanion superoxide, H2O2v cc hydroperoxide hu c tng ng, chng c xem l

cc enzyme khc chnh trong c th. i khi hthng bo vtrn bqu ti, khng

kh b nhim, khi thuc l, bc xtngoicc loi oxy hot tnh cao ny vt qu


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gii hn cho php sl ngun gy bnh, thc y nhanh qu trnh lo ha cho sinh vt.

Cc cht chng oxy ha tthc vt gp phn htrcho hthng bo vca c th,

ngn chn oxy ha khng mong mun nh cc carotenoid, flavonoid, vitamin C, Ev

cc hp cht trao i cht thcp tthc vt v ang c quan tm nghin cu.


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Chng 3

VT LIU V PHNG PHP TIN HNH

3.1. Vt liu

Vt liu:L cy Xun Hoa(Pseuderanthemum palatiferum).

Ngun gc ly mu:LXun Hoa c ly t2 ngun:

- Ti doanh nghip tnhn tr7 Nga (Ty Ninh).- Vn thc nghim trng i hc Cn Th.

3.2. a im v thi gian thc hin

a imTi phngthnghim ha l, Trung Tm Phn TchvThNghim HaSinh, Trng

i hc Nng Lm.

Thi gianti c thc hin qua 2 phn:

Phn 1: Tin hnhtchchit mt shp cht thcp tlXun Hoa vkho sttnh

khngkhun.

- Giai on 1: Tin hnh chit Soxhlet l Xun Hoathu cc loi cao. Thi giantin hnh tngy 1/4/2007 n 27/4/2007.

- Giai on 2: Tin hnh kho st khnng khng khun ca cc loi cao xcnh cao c hot tnh mnh nht. Tngy 29/4/2007 n ngy 6/5/2007.

- Giai on 3: Thc hin sc k ct v sc k lp mng trn cao chloroform tch cc phn on. Thi gian thc hin tngy 8/5/2007 n ngy 23/6/2007.


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- Giai on 4: Tin hnh kho st khnng khng khun ca cc phn on xc nh phn on khng khun. Thi gian thc hin tngy 25/6/2007 n

ngy 2/7/2007.

- Giai on 5: Thc hin sc k ct v sc k lp mng trn phn on khngkhun tch cht sch. Thi gian thc hin t ngy 5/7/2007 n ngy

19/7/2007.

- Giai on 6: Gi mu chy ph cng hng tht nhn xc nh cu trcca cc hp cht c tch chit phng phn tch cu trc, Vin Ho Hc,

Trung tm khoa hc TNhin, Cng NghQuc Gia H Ni.

Phn 2: Kho st khnng chng oxy haca dch chit lXun Hoa. Thi gian thc

hin t23/7/2007 n ngy 30/7/2007.

3.3. Thit b, dng cv ha cht

3.3.1. Thit bv dng c

Dng cho cc thnghim sinh ha:Tsy.

My xay mu.

Bsoxhlet.

My c quay.

Bnh lc.

Bnh lc qul.

Phu.

Giy lc.

ng chy sc ct.

Bnh tam gic 50ml.Bnh trin khai.

My lc.

Bn nh siu m.


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Micropipette loi 1001000 l.

u tipe loi 1000l.

Cn phn tch.

My lm .

My o nhit nng chy.

My o OD.

ng nghim.

Giy bc.

Parafirm.

Pipette Pasteur.

n cn.

Cy kp.

Dng cho thnghim vi sinh:ng nghim.

a petri.

n cn.

Tm.

Tsy.

Thp autoclave.

Bng khng thm.

Micropipette loi 100-1000 l.

Micropipette loi

u tipe loi

Que cy vng.Tm bng v trng.

3.3.2. Ha cht

Dng cho thnghim sinh ha:


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Methanol (Trung Quc, Merck).

Ethanol (Trung Quc).

Chloroform (Trung Quc).

Ete du ha.

Acetone.

H2S0498%.

Ht silicagel kch thc 0,0630,2 mm.

Bn mng silicagel (TLC) loi 25DCalufolien 20 x 20 cm, kieselgel 60 F254,

Merck.

Vitamin C.

DPPH (1, 1- diphenyl- 2-picrylhydrazyl).

Dng cho thnghim vi sinh:Cn.

Nc ct.

Mui.

Mi trng BHI.

Mi trng TSA.

Mi trng thch mu.

DMSO (dimethylsulfoxid).

3.4. Phng php tin hnh

3.4.1. Xl nguyn liu

S3.1: Quy trnhxlnguyn liu.


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3.4.2. iu chcc loi cao

Tin hnh chit soxhlet vi 670 g bt mu kh trong 120 givi EtOH 70%, thu ly

dch lc, c quay loi bdung mi.

3.4.2.1. iu chcao ete du ha

- Cho dch lc trn vo bnhlc qul, thm 200 ml nc ct vo, sau cho etedu ha vo.

- Dch lc chia lm 2 pha: Pha ete du ha v pha nc, hng ly dch trch etedu ha, sau c cn bng my c quay vi p sut thp thu c cao ete du

ha: 23 g

- Tng thtch ete du ha dng: 1,5 lt.

Pha ete du

Pha nc

Ra sch lti Xun Hoa

khtnhin

Sy khn trng lngkhng i nhit 40C

Xay mu thnhbt mn


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3.4.2.2. iu chcao chloroform

- Ly pha nc trn c quay loi bdung mi.- Cho chloroform vo lc, dch lc chia lm 2 pha, thu ly dch chloroform, sau

c cn bng my c quay loi dung mi chloroform thu c cao

chloroform: 42 g

- Tng thtch chloroformsdng: 2,5 lt.3.4.2.3. iu chcao n-butanol

- Phn cn li sau khi trch vi chloroform c tip tc tn trch vi n butanol.

- Dch trch thu c em c quay thu c cao n-butanol:16 g- Tng thtch n-butanol sdng: 1,3 lt.

3.4.2.4. iu chcao nc

Phn cn li em c quay thu c cao th nc.

S3.2: Quy trnhiu chccloi cao.

Dch trchEte

Bt kh670 g

chit soxhlet vi EtOH 70%

Dch trchEtOH B

Pha nc

c quay, chit vi ete du ha


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3.4.3. Kho st tnh khng khun ca cc loi cao.

- Chng vi khun:E.coli, Salmonellac ly tbnh xthy, Trng i HcNng Lm Tp. HChMinh.

- Pha long lin tip xc nng c ch ti thiu (MIC) ca cc loi cao ckhnng c chstng trng ca vi khun.

- Mi trng nui cy vi sinh vt tin hnh th nghim vi sinh vt l mitrng BHI vTSA.


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- Pha mi trng BHI:cho 15 g bt BHI v 300 ml nc ct vo bnh tam gic,un nhbng microwave trong 2 pht, sau lc bng giy lc loi bcn.

Hp khtrng autoclave 121C/15 pht.

- Cch pha mi trng TSA:Cao tht 2,5 g

Pepton 5 g

NaCl 2,5 g

Nc Ct 500 ml

Cho tt cvobnhtam gic, un nhbng microwave trong 2 pht, sau cho

2 g agar vo. Hp khtrngautoclave 121C/15 pht, ly ra voapetri.

- Dung mi ha tan 4 loi cao: DMSO.- Pha ng chun c Mc Farland nh sau: Thm 0,5 ml BaCl2 0,048M vo

99,5 ml H2SO40,18 M ng thi khuy lin tc to huyn dch.

- Phng php:o Cn 0,2 g cao cho vo 10 ml DMSO ha tan, nng cao lc ny l 0,02

g/ml.

o Pha long tip tc cao ny xung nng 0,002 g/ml (2000 g/ml) bng mitrng BHI.

o Tip tc pha long cao xung cn mt na nng : 1000 g/ml, 500 g/ml,250 g/ml, 125 g/ml.

o Xc nh nng vi khun da vo ng chun c Mc Farland 0,5: iuchnh c ca huyn dch vi khun sao cho ngang vi c ca ng Mc

Farland 0,5 bng cch cho thm nc mui sinh l 0,9%, lc ny nng vi

khun sl 108

cfu/ml.

o Tip tc pha long nng vi khun xung cn 107, 106bng mi trng BHI.o Btr thnghim vi sinh vt trn mi trng thch TSA xc nh khnng

khng khun ca 4 loi cao ny. Cao ccnng 1000 g/ml, 500 g/ml, 250


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g/ml, 125 g/ml c hatan vomi trng TSA.Nguyn tc xc nh da

vo sc chca cc nng cao pha long trn.Cy vi khun E.coliv

Salmonellanng 106ln trn cng mt a mi trng TSA, mi vi khun

chim na a.

- c kt quda trn vic quan stkhun lc c mc hay khng trn bmt mitrng thch sau 24 gi, t sxc nh c cao no c khnng c chvi

khun mnh nht v tip tc c s dng c lp mt s hp cht khng

khun bng phng php sc k ct, sc k lp mng vsc kiu ch.

3.4.4. Chit xut mt shp cht tcao chloroform

3.4.4.1. Sc k ct [1, 7]

Nguyn tc:- Sc k ct loi hp phc tin hnh trn mt ct thy tinh thng ng vi

cht hp phng vai tr tng tnh, dung mi ra ct ng vai tr tng ng

chy qua cht hp phdi nh hng ca trng lc.

- i vi mi cht ring bit trong hn hp cn tch ty theo khnng hp phvkhnng ha tan ca n i vi dung mi ra ct ly ra c ln lt trc

hoc sau.

- Cht hp phc sdng phbin l Silicagel 60 cht 0,0630,2 mm.- Dung mi: trin khai sc k ct, ngi ta c thdng tun ttng dung mi

ring bit c phn cc tng dn hoc dngmt hn hp dung mi thch hp

ra ct.

- Vic la chn kch thc ct rt quan trng, thng thng ct c ng knhnhv chiu di cng di th stch cng tt.

Tin hnh:Bc 1: Chun bct.

- Ra ct tht sch, trng li vi nc ct, sau sy kh.


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- Cho bng gn vo y ct (phi tht kho nu cht qu cht nghin cu sbmc kt li khng chy xung c, nu khng cht th khi dung mi vo nt

bong sbtri).

- Kp ct thng ng trn gi st.

Bc 2: Nhi ct.

- Cn 200 g Silicagel cho vo becher, cho ete du ha vo sao cho lng thtchlm cho silicagel trng phnh ti a, tt nht l qua m.

- Cho silicagel ttvo ct sao cho nt bng di y ct khng btri ln, kimtra tc chy dung mi ra khi ct (thng thng khong 30-50 git/pht l

t yu cu).

- Hng ete du ha v cho li vo ct vi ln n khi ct tht sn nh, bmtsilicagel phi nm ngang.

Bc 3: a cht phn tch vo ct.

Hnh 3.2: Ct sc k


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- Cao chloroform phi c sy tht kh nhit 40C ui ht dung mi,khi lng cao: 42 g.

- Cho 8 g silicagel vo trn u lng cao kh ny, nghin, sau tip tc sykh hon ton, mu lc ny phi dng bt mn.

- Sau cho ete du ha vo cao ny sao cho hn hp trthnh dng st, khnglong qu cng khng c qu khi mu vo to thnh mt lp trn bmt

silicagel.

Bc 4: Trin khai ct.

- trin khai ct ta c thdung tun tcc dung mi c phn cc tng dn.Sthay i tdung mi ny sang dung mi khc phi chuyn ttbng cch

pha tltng dn hoc gim dn.

- Sdng dung mi: ete du ha, chloroform , methanol v hn hp ca n cpha theo tltng dn: 1%, 2%, 5%, 10%, 25%, 50%, 100%.

Bc 5: Hng v kim tra cc phn on.

- Sdng cc bnh tam gic 50 ml hng tng phn on, nh du sthtca bnh.

- Kim tra cc phn on thu c bng sc k lp mng vi thuc thc trng.3.4.4.2. Sc k lp mng [1, 7]

Nguyn tc:- Sc k lp mng (SKLM) lkthut phn brn - lng; trong pha ng l

cht lng c cho i ngang qua mt lp cht hp tr(vdnh: silicagel hoc

oxid nhm) cht hp thnyc trngthnhmt lp mng, u, phln mt

nn phng nhtm king hoc tm nhm.

- Dung mi trin khai trong SKLM thng l mt hn hp c t2, 3 hoc 4 loidung mi khc nhau vi t l thch hp. ng vi mi loi hp cht phn tch

khc nhau sc mt shdung mi thch hp.


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- Sdng SKLM gp cc bnh hng cht chy ra thnh chung mt phn onnu chng cho kt quRf ging nhau.

Phng php:Chun b:

- ng mao qun: S dng pipette Pasteur, dng kp ko di u pipette do scnng ca ngn la n cn.

- ng nghim nh: Cha acetone ra mao qun sau mi ln sdng.- Bnh khai trin: Cho dung mi ete du ha, chloroform, methanol theo tlthch

hp vo bnh, sau cho mt tm giy thm vo bnh dung mi c hon

ton bo ha (khong 30 pht). Mt hu qud thy lnu bnhkhng c

bo ha thkhi dung mi lmt h hn hp, mc tin tuyn dung mi s c

hnh lm (hnh lng cho) do dung mi i hai bn cnh nhanh hn i gia

bng.

- Thuc thH2SO410%/EtOH: Pha 21ml H2SO495% vi 179 ml ethanol.- Bn mng: Dng bt ch kmt ng thng cch y 1,5 cm v mt ng

thng cch u trn 0,5 cm.

Tin hnh:

- Sdng mao qun chm cht cn kim tra vo bn mng, mi vt chm phinh, gn 2 3 mm vkhng lm rch bn mng. Vt chm v trcch cnh

y11,5 cm, vt ny cchvt kia 0,5 cm v cch bcnh ca bng 0,5 cm.

- Nhng bn mng vo bnh khai trin tht nhnhng, t bn mng hi nghing,y np kn. Lu trong thi gian ny khng di chuyn bnh. Mao qun trong

bnh s dn cht i ln.Theo di vch dung mi chy n u trn bn mngcch khong 0,5 cm th ly ra, dng my sy tc lm kh dung mi. Sau

nhng bn mng vo bnh thuc thH2SO410%/EtOH, hin vt 100C.

Bn mng


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Pht hin vt:

- Qua mu sc tnhin.- Pht quang di tia UV.- Phun thuc thcho mu c trng.

Tnh trsRf:Rf = ( Khong ng di chuyn ca hp cht) / (Khong ng di chuyn ca dung

mi).

Mt h dung mi gii ly ph hp l h sau khi gii ly s cho cc vt chnh c Rf

khong t0,30,7.

3.4.4.3. Sc k iu ch[7]

- Nguyn tc c bn cng ging nh SKLM, chkhc l sdng bng c kch cln hn (10 x 20cm).

- Dng bt ch vch sn mc xut pht cch y 1,5 cm. Sdng vi qun kodung dch mu thnh mt dy, c bngang 2-4 mm, di sut chiu ngang ca

bng nhng cn cha 1cm khong cch 2 u bng trnh bhiu ng b. Sy


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cho dung mi bay i, t bng vo bnh khai trin sc k, c cha 50 ml hn

hp dung mi gii ly. Sau khi gii ly, hin hnh vt trn bng sc nhiu dy;

dng li lam hay dao c u nhn co nhng dy lp hp thu c cha cht,

cha ring bit trong cc becher c nh s.

- Thu ly hp cht : Cht hp thu Silicagel c cha cht c cha trong mtbecher, rt dung mi chloroform vo hp cht tan vo dung mi. Hn hp

c yn 15 pht hp cht ha tan ht vo dung mi. Lc qua giy lc

hng vo mt becher khc. Thc hin strch 3-5 ln na bo m trch ht

mu cht ra khi cht hp thu.

3.4.4.4. Kthut kt tinh phn on

- Gischt A c ln mt t cht B v C. Ha tan mu vo dung mi nng, lcdung dch cn nng qua giy lc, yn cho dung mi ngui tt, hp cht A

skt tinh rn v ln trong tinh thca n mt t B v C. Lng B v C vn cn

ln trong nc ci.

- Lc ly ring tinh thA. Lp li kt tinh nh th thm vi ln na bng dungmi tinh khit scho cht A tinh khit.

3.4.5. Kho st tnh khng khun ca cc phn on c chit tcao chloroform

Hnh3.4: Sc kiu ch


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- Cc phn on thu c sc k ct c tin hnh thnghim khng khunvi vi khunE.coli, staphylococcus aureus, pseudomonas aeruginosa.

- Mi trng c sdng y l TSA, BHI.- Dung mi ha tan mu l DMSO.- Dng phng php vng khng khun kim tra phn on no c khnng

khng khun.

Tin hnh:- Mi trng TSA dng a petri vi dy 2/3 a, 72 gicho a kh

hon ton.

- Ha tan 0,4 g mu mi phn on bng 5 ml dung mi DMSO(lcnynng mu l0,02 g/ml) un nhtrn ngn n cn.

- Ct 1/3 u ng tipe, sy khtrng.- Dng u tipe c khtrng c la, mi a c 5 lvi khong cch

u nhau.

- Cy vi khun vo mi trng BHI, chun c ca huyn dch vi khun tngng vi c ca ng Mc Farland 0,5 bng cch thm nc mui sinh l

0,9%. Lc ny nng vi khun s tng ng 108. Pha long nng vi

khun xung cn 107, 106bng mi trng BHI.

- Dng tm bng v trng chm nhvo huyn dch vi khun, sau ray u tmbng ny ln a petri c sn cc lsao cho vi khun c thc tri u mt

a.

- Dng micropipette ht 100l mi phn on mu ln lt cho vo mi l. Quan st:Sau 24 gi, nu lno c vng khng khun xung quanh chng t

phn on c khng khun. Ta sdng phn on ny tip tc thnghimxcnh nng ti thiu c chvi khun (MIC).

Thnghim xc nh nng ti thiu c chvi khun:


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Pha long phn on khng khun nng 0,02 g/ml xung 0,002 g/ml (2000g/ml).

Chun b3 nhm ng nghim tng ng vi 3 loi vi khun, mi nhm 6 ng.

Bng 3.1 : Thtch v nng cc ng nghim.

Tn cc

ng nghim

Nng

mu lc

u

(g/ml)

Th tch

mu cho

vo (l)

Th tch

BHI cho

vo (l)

Nng

mu

(g/ml)

Thtch vi

khun cho

vo 1ml

mu (ml)

Nng

mu cui

cng

(g/ml)

E1, P1, S1 2000 100 900 200 1 100

E2, P2, S2 2000 200 800 400 1 200

E3, P3, S3 2000 300 700 600 1 300

E4, P4, S4 2000 400 600 800 1 400

E5, P5, S5 2000 500 500 1000 1 500

E6, P6, S6 2000 600 400 1200 1 600

- Chun b3 ng nghim lm i chng, mi ng cha 1 ml mi trng BHI.Cho E.coli, Staphyloccoccus aureus, Pseudomonas aeruginosa ln lt vo

tng ng.- tt ccc ng nghim trong tm 37C/24 gi.- Quan st s thay i c ca mi trng, s to thnh cn y v sto

vng bmt ng nghim.

3.4.6. Thnghim hot tnh chng oxy ha DPPH

3.4.6.1. Cc tnh cht ca gc tdo DPPH

- L gc tdo bn do c hiu ng lin hp trong phn t.- hp thcc i ti 517 nm do gc tdo trn Nit v cho mu tm trong

Methanol [3].

- Ccng thc cu to nhsau :

NO2

N NO2N


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- C chphn ng ca gc tdo DPPH v cht chng oxy ha:DPPH + AH DPPH-H + A

- Kt qu c nh gi thng qua gi tr IC50 (inhibitory concentration) lnng cht oxy ha cn c ch(trung ha) 50% gc tdo DPPH trong

khong thi gian xc nh.

- Mu Vitamin C c dng so snh thng gi trIC50.3.4.6.2. Quy trnh thhot tnh chng oxy ha DPPH [3]

Ly trch mu:S3.3: Quy trnhly trchmu cho thnghim DPPH.

Cn 10 g lti Xun Hoa

Nghin

Cho vo25 ml Methanol

Lc nhit phng 30 pht

Lc

Lp li quy trnh 5 ln


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Pha mu:- Cn 2 mg Cao + 5 ml Methanol, lc cho mu tan hon ton. Nng

mu Xun Hoa lcnyl400 g/ml.

- Pha long mu: Pha long nng mu xung 2, 4, 8, 16 ln.

Bng 3.2: pha long v nng mu Xun Hoa

pha long(ln) Nng mu Xun Hoa (g/ml)

2 100

4 50

8 25

16 12,5

Pha thuc th:- Cn 2,7 mg DPPH cho vo bnh nh mc 25 ml.


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- Thm25 ml MeOH, lc mnh cho tan ht, sau thm MeOH vo cho25 ml.

- n nh trong ti 30 pht, 4C. Pha dung dch chun:

- Cn 2 mg vitamin C vo bnh nh mc 5ml.- Thm 3ml MeOH vo, lc mnh cho tan ht, sau thm MeOH vo

cho 5 ml. Nng vitamin C lcnyl400 g/ml.

- Pha long dung dch chun xung 2, 4, 8, 16 ln bng MeOH.

Bng 3.3: pha long v nng vitamin C.

pha long(ln) Nng vitamin C (g/ml)

2 100

4 50

8 2516 12,5

o dung dch chun- Cho 1ml DPPH vo 1ml dung dch chun tng pha long.- n nh trong bng ti 30 pht.- o hp thquang (OD) bc sng 517nm

o mu- Cho 1ml DPPH vo 1ml mu Xun Hoa tng pha long.- tt cvo trong bng ti, 30 pht.- o hp thquang (OD) bc sng 517 nm.


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Hnh3.5: Ccnng pha longca vitamin C (bn tri) vmu XunHoa (bn phi) sau khi cho DPPH vo.


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Chng 4

KT QUV THO LUN

4.1. Thnghim khnng khng khun ca cc loi cao chit

Bng 4.1: Kt quthnghim khnng khng khun ca 4 loi cao.

Ccloi cao

Nng th

nghim cao nht

(g/ml)

Nng c ch(g/ml)

E.coli Salmonella

Ete du ha 1000 Cha c ch Cha c ch

n-butanol 1000 Cha c ch Cha c ch

Nc 1000 Cha c ch Cha c ch

Chloroform 500 c ch c ch

cao chloroformcao ete du ha

cao n-Butanol cao nc

Hnh4.1: Thnghim ccphn on khngE.colivSalmonella


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Cao chloroform c hot tnh khng khun mnh nht. nng 500 g/ml, cao

chloroform khng cho php khun lc vi khun mc trn b mt mi trng thch

TSA, trong khi nng 1000 g/ml vn tm thy khun lc mc cc cao cn li.

4.2. Kt qutch chit ca cao chloroform v cc phn on ca chng

4.2.1. Kt qutch chit ca cao chloroform

Thc hin sc k ct trn cao chloroform vi hdung mi: Ete du ha, chloroform,

methanol v hn hp ca chng. Kim tra bng sc k lp mng vi thuc th

H2SO410%/EtOH cho hin vt 100oC (Bng 4.2).

Bng 4.2: Kt qusc k ct trn cao chloroform.

Sth

tbnh

Tnphn

on

Dung

mi

gii ly

ct

Cao (g)

Sc k lp mng Hot

tnh

khng

khun

Ghi

ch

H

dung

mi

Thuc

thRf

1-26 XH1 E 4,83E:C

(9:1)H2SO4

nhiu

vt

m

tnh

27-49 XH2E:C

(95:5)

2,47E:C

(85:15)

nt ntm

tnh

50-64 XH3E:C

(9:1)1,74

E:C

(8:2)nt nt

m

tnh

65-83 XH4E:C

(8:2)1,61

E:C

(7:3)nt nt

m

tnh

84-96 XH5E:C

(7:3)1,34

C:E

(8:2)nt nt

m

tnh

97-108 XH6E:C

(6:4)1,98

C:E

(9:1)nt 4vt m

tnh

XH7 E:C 1,35 C:E nt 3vt m


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109-116 (6:4) (98:2) tnh

117-126 XH8

E:C

(6:4) 1,63 C nt 3vt

m

tnh

127-132 XH9E:C

(5:5)0,87 C nt 2 vt

Dng

tnh

kho

st

133-136 XH10E:C

(5:5)0,56

C:M

(99:1)nt 2 vt

Dng

tnh

kho

st

137-145 XH11E:C

(5:5)0,78

C:M

(99:1)nt 3vt

Dng

tnh

kho

st

146-155 XH12 C 2,56C:M

(95:5)nt

nhiu

vt

Dng

tnh

156-168 XH13 C 1,23C:M

(95:5)nt 4 vt

Dng

tnh

kho

st

169-172 XH14 C 0,96C:M

(95:5)nt 5 vt

m

tnh

173-192 XH15 C:Me

(99:1)1,67 C:M

(8:2)nt nhiu

vtmtnh

193-204 XH16C:Me

(98:2)0,48

C:M

(7:3)nt nt

m

tnh

205-216 XH17C:Me

(95:5)0,74

C:M

(7:3)nt nt

m

tnh

217-223 XH18 Me Xct

(Ch thch: Phn hot tnh khng khun tham kho mc 4.4).

Sc k ct v sc k bn mng trn Cao chloroform thu c 18 phn on. Da

vo tnhkhng khun ca cc phn on chng ti chchn phn on XH9, XH10,

XH11, XH13 tip tc kho st.


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4.2.2. Kho st phn on XH9, XH10, XH11, XH13.

4.2.2.1. Phn on XH11

Thc hin sc k iu ch trn phn on 11 vi h dung mi trin khai C: Me

(98:2) thu c hp cht mu vng, xung quanh c ln mt t tp. Kt tinh nhiu ln

hp cht ny v thc hin vic gn lc sau mi ln kt tinh, kim tra bng sc k lp

mng vi thuc thhin vt l H2SO410%/EtOH n khi thu c mt vt trn mu

vng c Rf=0,36. K hiu cht ny l XH-K11. Nhit nng chy: 237C, trng

lng 4,8 mg.

4.2.2.2. Phn on XH9 v XH10

Hai phn on ny lc u trn bn mng c vt khc nhau, sau nhiu ln kt tinh

trong chloroform ta loi c tp cht, chm bn mng thy Rf ging nhau nn gp

hai phn on ny li. Tip tc kt tinh nhiu ln trong Ethylacetat, chloroform v thc

hin vic gn lc sau mi ln kt tinh. Kt qucho dng tinh thmu vng. Kim trali bng sc k lp mng vi thuc thhin vt l H2SO410%/EtOH trong hdung

mi trin khai CHCl3:M (98:2) cho mt vt trn mu vng c Rf=0,36; nhit nng

Hnh4.2: Cht XH-K11 trn bn mng


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chy 237C trng vi cht XH-K11. Gp chung li vi cht XH-K11 phn on

XH11 cn c 12,6 mg.

4.2.2.3. Phn on XH13:

Thc hin sc k iu ch trn phn on 13 vi hdung mi trin khai l C:Me

(9:1) thu c mt hp cht mu tm. Kt tinh nhiu ln hp cht ny v thc hin vic

gn lc sau mi ln kt tinh, kim tra bng sc k lp mng vi thuc thhin vt l

H2SO410%/EtOH n khi thu c mt cht trn mu tm, c Rf=0,27. K hiu cht

ny l XH-K13. Nhit nng chy: 218C, trng lng 16,6 mg.

Cc cht tinh khit thu c tip tc c tin hnh chy phmt chiu, phhai

chiuNMR xc nh cu trc ca chng.

4.3. Cu trc ha hc ca cc cht tinh khit.4.3.1. Cht XH-K13

Sc k lp mngcho hin vt mutm, cRf = 0,27, nhit nngchy 218oC.

Hnh4.3: Cht XH-K13 trn bn mng


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Phhng ngoi IR(, cm-1) cho vch hp thu mnh, rng ca nhm OH ti 3400,20

cm-1; vch 2937,99 cm-1thhin sdao ng ca CH2, vch 1624,65 thuc dao ng

C=C, cc vch 1457,71 v 1372,95 thuc dao ng ca CH, cc vch 1064,91 v

1024,40 c trng cho lin kt ca nhm C-O (Phlc 1).

Ph khi lng MS (Ph lc 8) cho pick ion m l 577 [M + H]+ cho thy trng

lngphn tca XH-K13 l 576.

Phcng hng tht nhn 13C-NRMkt hp kthut Dept cho ta thy (phlc

3, 4):

- Trong phn t c 35 cacbon, trong c 3 cacbon t cp, 14 nhm CH, 12nhm CH2, 6 nhm CH3

- Cc mi 140,437 v 121,129ppm ldu hiu ca ni i v 140,437 l C=(Cacbon tcp c ni i), 121,129 l ca CH=.)

- Mt mi c trng 100,795ppm l mi ca acetal C1' ca ng gn vo C vtr s3; c chng minh nhphHMBC (phlc 6) cho thy stng tc

gia H1' v C3.

ng thi hng s tng tc spin-spin gia H1' v H2' l J=8 Hz, chng t lin kt

kiu trans-diaxial, kt hp vi cc sliu ph13C-NRM ca phn ng v phproton

hai chiu COSY (ph lc 7 ) cho thy ph hp vi D-Glucose v ni vi khung

aglycon bng lin kt beta.

Mt khc, vi k thut Dept 90 cho ta 5 mi cc v tr 70,09; 73,44; 76,94; 76,75;

76,69 kt hp phHSQC (phlc 5), HMBC cho bit l 5 nhm CH-OH ca gc

ng. Vy trong phn tc 1 n vng c 6 cacbon.

Sau da vo ph cng hng t ht nhn 1H-NRM (ph lc 2) xc nhn c mt

vch nhn ti dch chuyn ha hc l 5,321ppm, c cng tch phn l 1,000n vl ca H6, cng vi tn hiu ph13C-NRM 121,129 ( CH=); cng ph hp trn

phHSQC. Mt khc da vo phHMBC cho thy stng tc gia H6 vi C7, C8,

C10, C4 v stng tc gia H1 vi C3, C5, C10 nn CH2vtr s1 phi c dch


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chuyn l 36,80 ppm v C= vtr s5; chng tkhung sterol c mt ni i ti v tr

5-6.

Mt khc kt hp du hiu nh tnh trn bng mng khi sc k lp mng c mu tm

khi phun H2SO410%, sau h nng, cho php githit khung aglycon ca XH-K13

l mt Sterol c 29 cacbon cha ni i v tr 5-6 kt hp vi ngGlucose, ph

hp vi cng thc ca - sitosterol-3-O- -glucoside, cng thc phn tl: C35H60O6.

Cng thc cu to ca C35H60O6:

4.3.2. Cht XH-K11

Sc k lp mng cho hin vt muvng, cRf = 0,36, nhit nngchy 237oC.

Ph1H-NMR (DMSO, ppm, 500MHz) (phlc 9) cho:

- 2 mi i ca proton nhn thm ti = 6,45 ppm v 6,83 ppm (d, J = 2 Hz),chng tchng vtr meta.

- 2 mi i ca proton nhn thm ti = 6,94 ppm (d, J=8,5) v 7,95 ppp (d,J= 9 Hz), chng tchng vtr ortho.

Ph13

C-NMR (DMSO, ppm, 125 MHz) (ph lc 11) kt hp phDEPT (ph lc10) cho thy XH-K11 c 21 cacbon trong c 1 nhm >C=O, 1 nhm CH 214 nhm

CH, 5 nhm cacbon tcp.


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ng thi kt hp phcng hng tht nhn 2 chiu HSQC (phlc 12) v HMBC

(phlc 13) cho thy XH-K11 thuc nhm flavon v l dn xut ca apigenin.

Ph13C-NMRcho mt mi c trng 99,90 ppm l mi ca acetal C1 ca ng.

Da vo phHSQC(phlc 12) c c gi tr H1 = 5,06 ppm. Kt hp ph1H-

NMR(ph lc 9), ta c c H1 (1H, d, J=7,5 Hz), chng tng ni vi khung

aglycon bng linkt . Mt khc, phDEPT 90(phlc 10) cho ta 5 mi cc vtr

73,09; 77,16; 69,56; 76,42 v 60,60ppm. ng thi kt hp vi phHSQC, HMBC

(ph lc 13) v COSY (ph lc 14) cho bit l 5 nhm CH-OH ca gc ng

glucose. Vy trong phn tc1 n vng glucose c 6 cacbon.

T cc d liu v ph v so snh vi cc ti liu tham kho ca Phan Minh Giang(2002) v Nguyn Vn Hng (2004) [16], chng ti nhn danh cht XH-K11 l:

apigenin 7-O-- glucoside, c cng thc phn tl:C21H20O10.

Cng thc cu to ca C 21H20O10 nhsau:

4.4. Th nghim kh nng khng khun ca cc phn on c chit t cao

chloroform.

Tin hnh phng php o vng khng khun i vi 18 phn on c chit t

cao chloroform. Kt qu

phn o

n XH9, XH10, XH11, XH12, XH13c vng khng

khun xung quanh l, iu ny chng trng cc phn on ny c khnng c chs

pht trin ca vi khun.


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(1): Phn on XH9.

(2): phn on XH10.

(3): Phn on XH11.

(4): Phn on XH12.

(5): Phn on XH13.

Sau tip tc thnghim trn cc phn on khng khun ny kho st nng

ti thiu c chvi khun (MIC).

Bng 4.3: Nng c chvi khun ti thiu ca cc phn on.

Phn onNng c chti thiu vi khun thnghim (g/ml)- MIC

E.coli S. aureus Pseudomonas

XH9 400 500 500

XH10 400 500 400

XH11 300 400 300

XH12 300 400 300

XH13 300 400 400

Hnh 4.4: Kt quthnghim cc phn on khngE. coli.

(1)

(2)

(3)

(4)

(5)


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Nhn xt:

Sdng kt qutrn lm c scho vic la chn phn on no c tip tc kho

st tch chit cc hp cht bng phng php sc k ct v sc k lp mng.

Hnh4.5: Cc nng khngE.colica phn on XH9.

Hnh 4.6: Cc nng khng Staphylococcus aureusca phn on XH9.

E1 E2 E3 E4 E5 E6 Ec

S1

S2 S3 S4 S5 S6 Sc


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Hnh 4.7: Cc nng khngPseudomonas aeruginosaca XH9.

Chthch:

E1, E2, E3, E4, E5, E6: E. coli tnng t100600 g/ml.

P1, P2, P3, P4, P5, P6: Pseudomonas aeruginosa t100600 g/ml.

S1, S2, S3, S4, S5, S6: Staphylococcus aureus t100600 g/ml.

c: i chng

4.5. Kt quca thnghim chng oxy ha

% c chDPPH = (Ao- A)/Aox 100

o hp thu ca DPPH : A0= 1,67458.

A: hp thu ca dung dch phn ng.

IC50: Nng c ch50% gc DPPH.

Khnng c chgc tdo c nhgibng phng phpgc tdo bn DPPH.

4.5.1. Kt quo OD i vi vitamin C

Vitamin C c o 3 ln vly gitrtrung bnh.

Bng 4.4: Nng v kt quo OD ca vitamin C

Nng vitamin C (g/ml) A (OD o 517 nm)

100 1,361421

50 0,766121

P1 P2

P3 P4 P5 P6

Pc


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25 0,43277

12,5 0,28491

Kt quo OD v nng vitamin C c thhin di dng biu :

th4.1: Khnng c chDPPH ccnng khcnhau ca vitamin C.

Cc nng vitamin C v phn trm c chc biu thdi dng ng thng

vi phng trnh y = -1,355x + 124,8, vi hstng quan R=0,999.

Tthngoi suy gi trIC50ca vitamin C l 57,05(g/ml).

Vitamin C c dng lm cht chun so snh vi mu cn i chiu l Xun

Hoa.

4.5.2. Kt quo OD i vi dch chit l Xun Hoa

Dch chit mu Xun Hoa c o 3 ln v ly gi trtrung bnh.

Bng 4.5: Nng v kt quoOD ca dch chit l Xun Hoa

Nng dch chit lXun Hoa (g/ml) A (OD o 517nm)

100 1,12256

50 0,70756

25 0,48307

12,5 0,34129


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Kt quo OD v nng mu c thhin di dng biu :

th4.2: Khnng c chDPPH ccnng khc ca dch chit lXun

Hoa.

Ccnng mu Xun Hoa v phn trm c chc biu thdi dng ng

thng y = -1,897x + 161,4, vi hstng quan R=0.998.

Tthngoi suy gi trIC50ca mu Xun Hoal 66,55 (g/ml).

Bng 4.6: So snh khnng khng oxy ha ca vitamin C v dch chit lXun Hoa:

hp thca DPPHhp thca (DPPH +

vitamin C)

hp thca (DPPH +

mu Xun Hoa)

1,67458 1,361421 1,12256

Nhn xt:

- hp thquang ca DPPH gim t1,67458 xung 1,361421 i vi trnghp cvitamin C v gim xung 1,12256 i vi trng hp cdch chit l

Xun Hoa. iu nycngha ltrong lXun Hoa cmt shp cht chng

oxy hatcdng vi phn tDPPH lmcho hp thgim xung :


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C chphn ng ca gc tdo DPPH v cht chng oxy ha:

DPPH + AH DPPH-H + A

- So snh IC50 ca mu Xun Hoa (66,55 g/ml) vi IC50 ca vitamin C (57,05g/ml): Nng c ch50% DPPH ca mu Xun Hoa l 66,55g/ml, trong

khi nng c ch50% DPPH ca vitamin C l 57,05 g/ml, kt lun rng

hm lng cc phn t chng oxy ha ca mu Xun Hoa l kh cao (bng

85,7% vitamin C).


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Chng 5

KT LUN V NGH

5.1. Kt lun

- L Xun Hoac chit soxhlet vi dung mi ethanol, sau ln lt chit viete du ha, chloroform, n-butanol ln lt cho 4 loi cao: Cao ete du ha, cao

chloroform, cao n-butanol v cao nc. Thnghim khng khun 4 loi cao ny

i viE.coliv Salmonella, kt quxc nh c cao chloroform c hot tnh

khng khun nht, vi nng c chti thiu l 500g/ml.

- Chy sc k ct v sc k lp mng trn cao chloroform, kt qu thu c 18phn on.

- Thnghim khnng khng khun ca 18 phn on, kt quxc nh cphn on c hot tnh khng khun l:XH9, XH10, XH11, XH12, XH13.

- Kt hp kt quchy sc k ct v thkhng khun cc phn on, cui cngchn ra 4 phn on tip tc kho st l: XH9, XH10, XH11, XH13. Tip tc

kt tinh xc nh c 2 cht: XH-K11 vi nhit nng chy 237C v XH-

K13 vi nhit nng chy 218C.

- Chy phcng hng tht nhn mt chiu v hai chiu xc nh c chtXH-K11 lapigenin 7-O--glucoside, c cng thc phn t: C21H20O10v cht

XH-K13 l-sitosterol-3-O--glucoside, ccng thc phn t: C35H60O5.

- Kho st hot tnh chng oxy ha ca l Xun Hoa da vo phng php DPPH xc nh c trong l Xun Hoa c cht chng oxy havi hm lng l

kh cao (85,7% vitamin C).

5.2. ngh


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Vi shiu bit v kinh nghim c gii hn ca mt sinh vin nn kt qusc

nhng thiu xt. V thi gian v kinh ph lm ti cn hn hp nn khng thtip tc

chit

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