B GIO DC V O TO

TRNG I HC NNG LM THNH PH H CH MINH

B MN CNG NGH SINH HC
************************

KHA LUN TT NGHIP

XY DNG QUY TRNH K THUT TCH CHIT, KHO

ST TNH KHNG KHUN V KH NNG CHNG OXY
HA CA MT S HP CHT TH CP T L CY
XUN HOA (Pseudranthemum palatiferum).

Ngnh hc: CNG NGH SINH HC

Nin kha: 2003 2007
Sinh vin thc hin: TH TY PHNG

Thnh ph H Ch Minh
Thng 9/2007

B GIO DC V O TO
TRNG I HC NNG LM THNH PH H CH MINH
B MN CNG NGH SINH HC
**************************

XY DNG QUY TRNH K THUT TCH CHIT, KHO

ST TNH KHNG KHUN V KH NNG KHNG OXY
HA CA MT S HP CHT TH CP T L CY
XUN HOA (Pseudranthemum palatiferum).

Gio vin hng dn:

Sinh vin thc hin:

TS. PHAN PHC HIN

TH TY PHNG

Thnh ph H Ch Minh
Thng 9/2007

1 LI CM T
Con xin khc ghi cng n ba m v nhng ngi thn nui dng v gio dc con
nn ngi.
Ti xin gi lng bit n n:
-

Ban Gim Hiu Trng i Hc Nng Lm Tp. H Ch Minh.

Ban Gim c Trung Tm Phn Tch V Th Nghim Ha Sinh, Trng i

Hc Nng Lm Tp. H Ch Minh.

Thy ch nhim, thy c b mn Cng Ngh Sinh Hc.

h tr v to iu kin thun li cho ti trong sut qu trnh hc tp v hon thnh

ti.
Ti xin chn thnh bit n:
-

TS. Phan Phc Hin.

K s Trnh Phi Ly.

tn tnh hng dn, gip , ginh nhiu thi gian v cng sc truyn t nhng
kin thc, kinh nghim qu gi, to mi iu kin tt cho ti hon thnh kha lun ny.
Ti xin gi li cm n n:
-

ThS. Hunh Kim Diu tn tnh gip trong vic ly mu l Xun Hoa v
ng gp nhng kin qu bu cho lun vn.

TS. Nguyn Ngc Hi, Phng Vi Sinh Khoa Chn Nui Th Y, Trng i Hc
Nng Lm.

C v Ch inh Cng By, ch c s tr Hon Ngc 7 Nga Ty Ninh tn

tnh gip v cung cp s lng ln l Xun Hoa.

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

trong sut thi gian lm ti.

iii

TM TT
Th Tu Phng, i Hc Nng Lm Tp. H Ch Minh. Thng 3/2007.
Xy dng quy trnh k thut tch chit, kho st tnh khng khun v kh nng
chng oxy ha ca mt s hp cht th cp t l Xun Hoa (Pseudranthemum
palatiferum).
Gio vin hng dn: TS. Phan Phc Hin
ti c tin hnh ti:
-

Phng th nghim ha l, Trung Tm Phn Tch v Th Nghim Ha Sinh,

Trng i Hc Nng Lm Tp. H Ch Minh.

Phng Vi Sinh, Khoa Chn Nui Th Y, Trng i Hc Nng Lm Tp. H

Ch Minh.

Thi gian tin hnh: t 15/3/2007 n 30/7/2007.

Ni dung tin hnh:
-

Tin hnh chit tch cc phn on t cao khng khun nht bng sc k ct v
sc k lp mng, sau th nghim kh nng khng khun ca cc phn on
ny vi vi khun E.coli, Staphyloccoccus aureus, Pseudomonas aeruginosa.

Tin hnh sc k ct, sc k lp mng v sc k iu ch tch chit mt s

hp cht th cp t phn on c hot tnh khng khun mnh nht.

Gi mu chy ph cng hng t ht nhn xc nh cu trc ca cc hp

cht c tch chit phng phn tch cu trc, Vin Ho Hc,Trung tm khoa
hc T Nhin, Cng Ngh Quc Gia H Ni.

Th nghim kh nng chng oxy ha ca l Xun hoa bng quy trnh DPPH.

Kt qu t c:
-

Xc nh c cao chloroform c hot tnh khng khun mnh nht vi nng

c ch ti thiu l :
MICE. coli = 500 g/ml.

iv

MICsalmonella= 500 g/ml.

-

Tch chit c 18 phn on v xc nh c phn on c hot tnh khng

khun l XH9, XH10, XH11, XH12, XH13.

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

Sau khi chy ph v tham kho mt s ti liu, nh danh c XH-K11 l

apigenin 7-O-- glucoside, c cng thc phn t: C21H20O10 v 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 t
c ch DPPH l kh cao (85,7%), thng qua so snh ch s IC50 ca mu Xun
Hoa vi Vitamin C:
IC50 xun hoa = 66,55 (g/ml).
IC50 vitamin c = 57,05 (g/ml).

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 metabolite compounds extracted from Xuan Hoa
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 column

chromatography (CC) and thin layer chromatography (TLC), after that
researching antibacterial property of these fractions for E. coli, Staphylococcus
aureus, Pseudomonas aeruginosa.

Using CC and TLC to isolate and purify metabolite compounds of these

antibacterial 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 the

molecular 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).

vi

MC LC
NI DUNG

TRANG

Li cm n .................................................................................................................... iii
Tm Tt ......................................................................................................................... iv
Mc lc .......................................................................................................................... vii
Danh sch cc ch vit tt ............................................................................................. xi
Danh sch cc hnh v biu ...................................................................................... xii
Danh sch cc bng v s ....................................................................................... xiii
1. M U ................................................................................................................... 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 v cy Xun Hoa ................................................................................... 3
2.1.1. c im thc 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. Tc dng sinh hc ............................................................................................... 5
2.3.1.1. Bo v t bo gan ............................................................................................. 5
2.3.1.2. nh hng ln bnh tiu chy heo ............................................................... 10
2.3. Thnh phn ha hc ............................................................................................... 13
2.4. i cng v mt s hp cht hu c t nhin ..................................................... 15

vii

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 v vi khun ng rut ........................................................................ 17
2.5.1. nh ngha ........................................................................................................... 17
2.5.2. Hnh th ............................................................................................................... 17
2.5.3. Tnh cht nui cy ............................................................................................... 18
2.5.4. c tnh sinh ha ................................................................................................. 18
2.5.5. Sc khng ....................................................................................................... 19
2.5.6. c t .................................................................................................................. 19
2.5.7. Cu trc khng nguyn........................................................................................ 19
2.5.8. Kh nng gy bnh .............................................................................................. 21
2.6. Mt s c tnh ca vi khun th nghim .............................................................. 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 ............................................................................................................. 25
3.3. Thit b, dng c v ha cht ................................................................................. 25
3.3.1. Thit b v dng c .............................................................................................. 25
3.3.2. Ha cht .............................................................................................................. 27

viii

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

3.4.1. X l nguyn liu ................................................................................................ 28
3.4.2. iu ch cc loi cao........................................................................................... 28
3.4.2.1. iu ch cao ete du ha .................................................................................. 28
3.4.2.2. iu ch cao chloroform .................................................................................. 29
3.4.2.3. iu ch cao n-butanol..................................................................................... 29
3.4.2.4. iu ch cao nc ............................................................................................ 29
3.4.3. Kho st tnh khng khun ca cc loi cao ....................................................... 31
3.4.4. Chit xut mt s hp 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. K thut kt tinh phn on ............................................................................. 38
3.4.5. Kho st tnh khng khun ca cc phn on c tch chit t cao chloroform
....................................................................................................................................... 38
3.4.6. Th nghim hot tnh chng oxy ha DPPH ...................................................... 40
3.4.6.1. Cc tnh cht ca gc t do DPPH ................................................................... 40
3.4.6.2. Quy trnh th hot tnh chng oxy ha DPPH ................................................. 40
4. KT QU V THO LUN .................................................................................. 44
4.1. Th nghim kh nng khng khun ca cc loi cao chit .................................... 44
4.2. Kt qu tch chit ca cao chloroform v cc phn on ca chng .................... 45
4.2.1. Kt qu tch chit ca cao chloroform................................................................ 45
4.2.2. Kho st phn on XH9, XH10, XH11, XH13. ................................................ 47
4.2.2.1. Phn on XH11 .............................................................................................. 47
4.2.2.2. 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

ix

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 qu ca th nghim chng oxy ha ................................................................. 55
4.5.1. Kt qu o OD i vi vitamin C ....................................................................... 55
4.5.2. Kt qu o 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
PH LC ...................................................................................................................... 62

DANH SCH CC CH VIT TT

BHI
C
Cfu
COSY
d
DEPT
DMSO
DPPH
E
HMBC
HSQC
IC50
IR
J
LD50
m
Me
MIC
MS
Nt
NMR
Rf
s
t
TSA

Brain Heart Infirm

Chloroform
Colony forming unit
Correclation Spectroscopy
Double: mi i
Distortionlees Enhancement by Polarization Transfer
Dimethylsulfuoxid
1,1 - diphenyl - 2 - picrylhydrazyl
Ete du ha
Heteronuclear multiple bond correction
Heteronuclear single quantum coherence
Inhibitory Concentration 50: nng c ch 50% DPPH
Infrared Spectrometry: hng ngoi
Coupling constant: hng s ghp cp
Lethal dose 50: liu gy cht 50% s c th
Multiplet: mi a
Methanol
Minimum Inhibitory Concentration: nng c ch ti thiu
Mass Spectrometry: khi ph
Nh trn
Nuclear Magnetic Resonnace: cng hng t ht nhn
Retention factor
Single: mi n
Tripplet: mi ba
Tryptic Soy Agar

xi

DANH SCH CC HNH V BIU

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

Hnh 3.1: Cc pha trong bnh lc qu l ........................................................................ 29
Hnh 3.2: Cht phn tch c a vo ct 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 ................................................................................................................................. 43
Hnh 4.1: Th nghim cc phn on khng E.coli v 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 qu th nghim cc phn on khng E. 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
th 4.1: Vitamin C .................................................................................................... 55
th 4.2: Mu Xun Hoa........................................................................................ 56

xii

DANH SCH CC BNG V S

Bng 2.1: Kt qu quan st vi th chut ...................................................................... 7

Bng 2.2: Kt qu o 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 th trng chut ..................... 9
Bng 2.4: Kt qu hm lng men gan ......................................................................... 10
Bng 2.5: nh hng ca l ti hay bt l Xun Hoa ln s biu hin tng trng,
bnh tiu chy v sinh l mu ca heo.......................................................................... 11
Bng 2.6: So snh hiu qu cha tr tiu chy .............................................................. 12
Bng 3.1 : Th tch 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 qu th nghim kh nng khng khun ca 4 loi cao ...................... 44
Bng 4.2: Kt qu sc k ct trn Cao Chloroform.................................................. 45
Bng 4.3: Nng c ch vi khun ti thiu ca cc phn on ........................... 53
Bng 4.4: Nng v kt qu o OD ca vitamin C.................................................... 55
Bng 4.5: Nng v kt qu o OD ca mu Xun Hoa ........................................... 56
Bng 4.6: nh hng ca mu Xun Hoa i vi hp th ca DPPH ................ 57
S 3.1: Quy trnh x l nguyn liu .................................................................... 28
S 3.2: Quy trnh iu ch cc loi cao ............................................................... 30
S 3.3: Quy trnh ly trch mu cho th nghim DPPH ............................................ 41

xiii

Chng 1
M U
1.1. t vn
Cy Xun Hoa (Pseuderanthemum palatiferum) l mt loi cy c dn gian
bit n nh l mt dc liu c th cha c rt nhiu cn bnh: bnh tiu ha, tiu
ng, tr ni, chy mu, suy nhc thn kinhTuy nhin, trn thc t c rt t nghin
cu v cy ny c th lm r vn trn.
Hin nay trn th gii cha c mt nghin cu no su xa v cy 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) th c tnh cp din trn chut cho thy cao ton phn l
Xun Hoa khng th hin c tnh v c tc dng c ch qu trnh peroxy ha lipid
mng t bo, ngha l c xu hng bo v t bo gan. Hunh Kim Diu (2003) th
nghim l Xun Hoa i vi heo cha cai sa c tc dng tng trng v cha c
bnh dch t. V gn y, trong Hi Ngh Khoa Hc v Cng Ngh Ha Hu C Ton
Quc ln th 3 H Ni 11 2005, Phan Phc Hin v cng s cng b cng trnh
phn lp Triterpenoid v Steroid t l cy Xun Hoa Psenderanthemum palatiferum h
Acanthaceae bng phng php cng hng t ht nhn.
Hin nay, nhim khun ng tiu ha tr nn rt ph bin. Phng php cha tr
ch yu hin nay l s dng khng sinh. Tuy nhin vic s dng khng sinh s dn n
ri ro do hin tng khng thuc. Do , vic tm ra mt ngun nguyn liu t nhin
c kh nng khng khun s cho ta mt phng php iu tr mt cch hiu qu i vi
i vi mt s bnh nhim khun.
Thm vo , ngy cng c nhiu mi quan tm v cc cht khng oxy ha c
ngun gc t thc vt c tc dng ngn chn qu trnh oxy ha khng mong mun nh

cc carotenoid, flavonoid, vitamin C, EV th trong cng trnh ny chng ti cng

kho st kh nng chng oxy ha ca cc hp 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 kh nng chng oxy ha ca mt s
hp cht th cp t l 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 s hp cht th cp t cao chloroform .
Xc nh phn on khng vi khun: E.coli, Staphylococcus aureus,
Pseudomonas aeruginosa.
Chit tch mt s hp cht th cp t phn on khng khun.
Kho st kh nng chng oxy ha ca cc hp cht trong l cy 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 s hp
cht t l Xun Hoa.
S dng phng php vng khng khun v pha long lin tc xc nh nng
c ch ti thiu ca cc phn on c ly trch t cao chloroform.
Tin hnh ly trch l cy Xun Hoa v o hp th quang (OD) bc sng
517 nm [3] xc nh kh nng chng oxy ha.

Chng 2
TNG QUAN TI LIU
2.1. Gii thiu v cy 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 th cao 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.

Hnh 1.1: Cy Xun Hoa

Hoa mc k l 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

5 thy chia lm hai mi, mi trn gm hai thy nh dnh lin nhau n na chiu di
ca thy, mi di gm 3 thy to, thu gia ca mi di c cc chm mu tm. Qu
nang 2 , mi cha 2 ht. Ma hoa t thng 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 Hoa c 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 t nm 1998 r ln vic trng cy Xun Hoa cha mt s bnh v tiu
ha. Thnh ph H Ch Minh, mt s nh trng cy Xun Hoa cha to bn.
c bit gn y nht chng ti c bit c mt s doanh nghip Ty Ninh dng
l v r cy Xun Hoa ny lm tr v c bn rng ri ra th trng vi tn gi tr
Hon Ngc.
2.1.4. Nhn ging [6 ]
Cy Xun Hoa c th nhn ging d dng bng cch gim cnh. Ly on cnh b,
cch ngn khong 20 cm, b bt vi l pha di, cm vo t m, ch mt, hng
ngy ti u (khong 2-3 ln/ngy). Cy chu t xp v m trung bnh. Ch sau 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 nghim dn gian
Cy Xun Hoa c dng trong dn gian cha nhiu bnh nh:
-

Nhim khun ng tiu ho, au d dy, au bng khng r nguyn nhn.

Lot hnh t trng, chy mu ng rut.

Khi phc phc sc kho cho ngi m yu, cha suy nhc thn kinh.

Chn thng chy mu, dp gy c th, chn thng s no.

Vim thn cp v mn, suy thn, i ra mu, i but, i rt.

Cha cc bnh u phi, u x tuyn tin lit, au gan, x gan c trng, lm

gim au khi b ung th gan giai on pht bnh.

iu chnh huyt p.

Trung Quc, ngi ta dng r cy Xun Hoa cha n ng tn thng.

c bit H Ni t nm 1998 r ln vic trng Xun Hoa cha nhng
bnh thuc ng tiu ha. Thi gian gn y, mt s Vit kiu M cng
dng cy Xun Hoa cha bnh i tho ng. Bnh nhn dng lin tc cy
Xun Hoa gi c n nh t l ng trong mu.
L Xun Hoa khng 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 v t bo 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 v t bo gan ca l cy Xun Hoa trn
in vivo.
Th c tnh cp
-

Chut nht trng, chia lm 6 l, mi l 7 con.

Thuc th: Cao c ton phn c cn v ha tan 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 Hoa cc l khc
nhau: 0,83 g/kg; 1,67 g/kg; 3,13 g/kg; 5,56 g/kg; 9,19 g/kg; 11,5g/kg th trng
chut. Quan st din bin hnh vi, sau git ngu nhin 3 chut 3 l khc

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

thuc nh tim - gan - thn - d dy - 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 v trong sut qu trnh th nghim thuc khng gy thay i trng thi
hot ng ca chut: Chut vn b lui ti, leo tro, chi ru, lim ui
o Cc liu cao hn: 5,56 g/kg; 9,19 g/kh; 11,5 g/kg th trng chut, sau khi
ung thuc chut c gim hot ng, nhng sau 1gi th tr li bnh thng,
chut rc vo nhau, mt lim dim, th nh nhng.
o Tt c cc chut th nghim ( liu trn) u khng cht. Chut sng khe
mnh sau 48 gi.
o Quan st i th: Cc ph tng u bnh thng.
o Lm vi th cc c quan: Ct thnh ming mng v nhum t bo, quan st
di knh hin vi (Bng 2.1).

Cquan
Liu

Tim

Gan

Thn

D dy

Rut

nh gi

0,83g/kg

9,19g/kg

C tim

Cc tnh

Qun cu v

Nim

Nim

Khng

bnh

mch xoang

cc ng thn

mc d

mc rut

c tn

thng.

v tnh mch

bnh thng.

dy v

v cc

thng

trung tm

tuyn

tuyn

thc th

trn thu

bnh

bnh

cc

xung huyt.

thng.

thng.

tng.

C tim

T bo gan

Qun cu

Nim

Nim

C thoi

bnh

hi to,

xung huyt,

mc d

mc rut

ho nh

thng.

nguyn sinh

cc ng thn

dy v

v cc

gan.

cht c thoi

bnh thng.

tuyn

tuyn

bnh

bnh

thng.

thng.

ho nh.

11,5g/kg

C tim

T bo gan

Nang

Nim

Nim mc

C tn

bnh

hi to,

Bowmann

mc d

rut v

thng

thng.

nguyn sinh

rng ra, t

dy v

cc tuyn

nh

cht c thoi

bo thnh

tuyn

bnh

gan v

ho nh, tnh

ng teo nh.

bnh

thng.

thn.

mch trung

thng.

tm trn thu
xung huyt.
Bng 2.1: Kt qu quan st vi th chut
Kt lun: Cao ton phn Xun Hoa khng gy cht chut tt c cc liu. Nh vy,
trn c tnh cp cao Xun Hoa khng th hin c tnh, khng c LD50.
Th tc dng bo v t bo gan
-

Chut nht trng, chi lm 6 l, mi l t 8 12 con.

Cao ton phn l Xun Hoa loi b chlorophyl, ha tan vi nc ct.

M hnh gy ng c gan: Gy tn thng gan chut nht bng carbon

tetrachloride (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 th trng (liu thp).

Chia chut thnh 6 l, mi l 2 nhm:

o L 1 (i chng sinh hc): Chut bnh thng, khng gy m hnh vim
gan, khng dung thuc Xun Hoa.
o L 2: Gy m hnh vim gan i chng, khng s dng thuc Xun Hoa.
o L 3: Gy th nghim vim gan, cho ung thuc Xun Hoa trc, sau 1 gi
th tim CCl4 vo.

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
t bo. Sn phm MDA c kh nng phn ng vi acid thiobarbituric to
thnh phc hp trimethine c mu hng v c nh hp thu cc i 530 532
nm [9]. Cng mu ca dung dch t l thun vi nng MDA. Hm lng
MDA tnh theo cng thc:
X = E x 30,8
Trong : X: Hm lng MDA (mM)
E: hp th 532 nm.
30,8: H s tt phn t.

Bng 2.2: Hm lng MDA trong gan chut liu gy l 1ml CCl4/kg th trng.
L

n (s chut)

ETB

MDATB

0,163

5,0296

12

0,458

14,1156

0,348

10,7091

Ch thch:
MDA: Malonic dialdehyde.
ETB: E trung bnh.
MDATB: MDA trung bnh.

Nhn xt:
-

l c s dng cao Xun Hoa hm lng MDA c gim so vi l gy m hnh

(gim t 14,1156 xung cn 10,7091).

Hm lng men gan (AST, ALT) qu cao, khng o c trn my.

Xt nghim vi th : Cha thy du hiu bnh phc ca t bo gan do thuc iu

tr.

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

L

n (s chut)

ETB

MDATB

0,1915

5,8982

12

0,385

11,547

0,203

6,2463

Nhn xt:
-

l c s dng cao Xun Hoa hm lng MDA c gim so vi l gy m hnh

(gim t 11,547 xung cn 6,2463).

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

Bng 2.4: Kt qu hm lng men gan.

L

n (s chut)

AST(U/l)

ALT(U/l)

270

432

12

9668

10870

8740

10533

10

Nhn xt:
-

Hm lng men gan gim nhng cha ng k.

Xt nghim vi th: l chut c s dng cao Xun Hoa v l gy m hnh nhn

thy rng cc t bo vng thoi ho bt u thu nh v c xu hng hi
phc.

Kt lun:
Cao c ton phn Xun Hoa c kh nng c ch qu trnh peroxy ha lipid mng t
bo, ngha l c xu hng tc dng bo v t bo gan trn thc nghim.
2.3.1.2. nh hng ln bnh tiu chy heo [14]
Bnh tiu chy ch yu 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 tr bnh. 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 kho con ngi bi
v tnh c v tnh d ng ca n. Thm vo , s pht trin ca cc vi khun khng
thuc dn n s dng liu lng qu cao tr thnh mt vi vn trn th gii. V
vy, cn phi gim vic s dng v thay th vai tr ca khng sinh trong thc n gia
sc bng cch gia tng vic s dng probiotics v nhng sn phm t nhin khc, c
bit y ta s dng l cy Xun Hoa iu tr bnh tiu chy heo.
Bng 2.5: nh hng ca l ti hay bt l Xun Hoa ln s biu hin tng trng,
bnh tiu chy v sinh l mu ca heo.
Phng php

TN1

Xun

tng

Hoa

Liu lng
(g/kg th
trng/ngy)

Qua

Thi

ng

gian

C1

52

Heo

T1-1

52

cha

L ti

ming

1-7

11

TN2

TN3

T1-2

52

C2

60

cai sa
Heo

T2-1

60

T2-2

60

T2-3

60

C3

62

T3-1

62

Heo

T3-2

62

cai sa

T3-3

62

cha
cai sa

1
-

L ti

0,5
0,1

Bt

1-30
-

ming

1-30

thc n

1-30

0,2

L ti

0,5
0,1

Bt

0,2

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

chm li. Heo tng t hn 4kg/28 ngy c xem l tng trng chm li. S truyn
bnh tiu chy c o lng hng ngy da trn t l heo mc bnh trong nhng con
heo th nghim, s trng hp v thi gian mc bnh.
Ngoi ra, Hunh Kim Diu v Trn vn Ha [14] tin hnh th nghim kh nng 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 3
ngy iu tr t l khi bnh ca heo ln lt: Bt Xun Hoa 92,86%; Coli-norgen
90,48%; Cotrimxazol 83,33%. T l ti pht theo th t l: 7,14%; 9,52%; 14,29%.
Bng 2.6: So snh hiu qu cha tr tiu chy.
S lng heo khi bnh sau khi iu tr

S heo
Loi thuc

th

Ngy th nht

nghim S con
Bt Xun
Hoa
Coli-norgen

Ngy th hai
S con

Ngy th ba
S con

42

10

23,81

29

69,05

39

92,86

42

10

23,81

25

59,52

38

90,48

12

Cotrimxazol

42

15

35,71

28

66,67

35

83,33

Nhn xt:
Khng c tc dng ph ca l Xun Hoa i vi heo trong th nghim ny. Nng c
ch nh 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 s dng cho vic ngn chn bnh, nng
c s dng trong th nghim nh hn nng gy c ch thp nht trnh gy
tc dng ph. S dng l hay bt Xun Hoa c th ci thin nhng thng s o lng
ni trn v kt qu c th c gii thch nh sau: Trc tin, l Xun Hoa cha mt t
l cao protein th (30,8% DM: dry matter), so snh vi c voi (18,6% DM) v cy sn
dy (18,4% DM). nh hng ca liu s dng s rt nh bi v t l nh ca n trong
khu phn. Th hai, l Xun Hoa c hm lng cao mt s loi 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 s gp 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 th mang oxy v cacbonic, v th n c bit nh
mt sc t h hp v gii thch tnh trng ca hng cu. Th ba, trong l c cha mt
protein 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 s c ci thin nu b
sung l. Th t, mt s hp cht ho hc trong l v mt vi tc nhn khng khun c
th nh hng ti s pht trin v min dch ca heo.
Kt lun:
S dng bt l Xun Hoa vi 0,2 g/kg th trng cho 30 ngy i vi heo cha cai sa
v cai sa ci thin s tng trng v ngn nga bnh dch t, cho nn cy trng
ny c nhiu trin vng thay th hoc gim s dng khng sinh trong tng lai.
2.3. Thnh phn ho hc [4, 5]

13

Hin nay trn th gii cha c cng trnh no nghin cu v thnh phn ha hc ca
cy Xun Hoa, ch c mt s cng trnh nghin cu bc u v cy ny Vit Nam
nh Trn Cng Khnh (1998), Phan Phc Hin (2005), Hunh Kim Diu (2003).
Cc nguyn t a lng v vi lng trong l cy Xun Hoa kh cao so vi cc loi cy
khc. Khng pht hin cc nguyn t kim 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 Hoa cn cha y cc acid amin thit yu vi hm lng tng s
kh cao (751-1365 mg%). l cc acid amin gi vai tr quan trng trong sinh tng
hp protein c bp v chng mi c th. Thiu chng c th st cn nhanh. L Xun
Hoa cn giu Valin (29-1001 mg%). Thiu acid amin ny, s phi hp cc chuyn
ng ca bp tht b ri 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 v thnh phn ha hc ca l Xun Hoa ngi 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 v cng 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 no

bc 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 1triancontanol c hot tnh khng vim, c ch cc qu trnh peroxy ha cc cht
bo c cng nh khng c s tham gia ca cc enzyme v lm gim m trong
mu.

Glycerol 1-hexadecanoate: Dng tinh th mu trng, im nng chy 65-670C,

l mt mono-ester ca glycerin vi acid Palmitic v cng thc phn t l
C19H38O34.

14

Acid palmitic: L cht rn mu trng, nng chy 48-510C, c c trng ca

mt acid bo no mch thng.

Acid Salicylic: Thu c di dng tinh th hnh 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 s dng trong nhiu loi ch
phm da vo tnh cht ny.

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

Xun Hoa c th gp 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 s dng 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 Hoa Pseuderanthemum palatiferum(Nees)
Radlk, h r (ancathaceae) ca Trn Kim Thu Liu v cng s (2006).

2.4. i cng v mt s hp cht hu c t nhin [ 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 s alcaloid 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 h ng. 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 t do thng ho tan c trong dung mi hu c, tuy nhin
cc alcaloid dng bazo t cp th ch ha tan trong nc. Mui ca a s alkaloid th

15

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

trng trong k ngh dc phm; trong vic trch ly chng ra khi cy c v trong vic
bo ch alcaloid thnh cc dng thuc ph hp.
a s alcaloid c tnh kim, iu ny khin cho alcaloid cc k nhy cm, d b thy
phn khi gp nh sng hoc nhit .
Nhiu alcaloid c s dng lm thuc tr bnh. 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 ch h thn kinh trung ng nn c dng lm thuc gim au trong khi gii phu
hoc khi b bnh ung th giai on cui: morphin v cc dn xut ca morphin nh:
heroin, codein (tuy nhin cc hp cht ny c th gy ra tnh trng u oi, bun ng,
kh th v nghin thuc). C alcaloid c bo ch lm thuc gim h thn kinh
giao cm: reserpin, propanolol.
Mt s alcaloid nh: berberin, palmatin, taspinc kh nng c ch con men sao chp
ngc (reverse trancriptase) y l con men ch yu ca retrovirus m cc loi thuc
khng HIV tp trung tiu dit.
2.4.2. Flavonoid
Flavonoid l nhng hp cht mu phenol thc vt, to nn mu cho rt nhiu rau, qu,
hoaPhn ln c mu vng (do t flavus l mu vng); tuy vy, mt s sc t c mu
xanh, tm, , khng mu cng c xp vo nhm ny v v mt ha hc chng c
cng khung sn.
Cc flavonoid c th dng t do hoc dng glicosid. Cc ng thng gp nht l
D-glucose; k l D-galactose; L-Rhamnose; L-Arabinose
Chalcone hin din t trong t nhin; flavanol v flavanonol cng him gp; flavone v
flavonol phn b rng ri trong t nhin. Cc glicisid flavonol nh: rutin, quercitrin,
daempherol. Kaempherol rt thng xuyn hin din. Antocyanidine dng glicosid th

16

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

hoa v tri.
Cc flavonoid thng d kt 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 t cu vng, vi khun t cu 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, h u) 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 t cy tr trng Chrysanthemum cinerariaefolium, h Cc).
2.4.2. Tinh du
Ni chung, tinh du trch ly t thc vt c cu trc terpenoid. T 2000 nm trc
ngi ta bit s dng turpentine tr ho. Mentol l alcol terpen trch t tinh du
bc h c kh nng gy t cc b, nn gp phn vo c tnh tr ho ca n. Cc gia v
nh: h tiu, gng, nhc u khu, inh hngbn cnh mi v lm chng c gi tr
trong ngnh n ung, chng cn c tc dng khng khun. Nhiu tinh du c kh nng
khng khun mnh nh: tinh du rau ng Enhydra fluctuans; tinh du hi Illicium
verum; tinh du qu Cinhamomum; tinh du long no Cinnamonium campho; tinh du
gng Zingiber officinalist; tinh du ngh Curcuma longa
2.4.3. Saponin
L mt loi glycosid phn b kh rng trong thc vt, c mt s tnh cht c trng:
khi ha tan vo nc s c tc dng lm gim sc cng b mt ca dung dch v to
nhiu bt; lm v hng cu cn gi l tnh ph huyt. Saponin thng dng v nh
hnh, c v ng. Saponin rt kh tinh ch, c im nng chy thng cao t 200oC tr

17

ln v c th trn 300oC. Saponin c th b ta bi acetat ch, hydroxid barium, sulfat

amonium nn c th li 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 v vi khun ng rut [8, 12]
2.5.1. nh ngha
H vi khun ng rut (Enterbacteriaceae) bao gm cc trc khun Gram m, hiu kh
hoc k kh tu nghi, khng c men oxidase, ln men ng glucose c km theo sinh hi
hoc khng, kh nitrat thnh nitrit, c th di ng hoc khng, nhng nu di ng th c
nhiu lng xung quanh thn, khng sinh nha bo.

2.5.2. Hnh th
Tt c cc vi khun thuc h ny u l trc khun Gram (-). Kch thc trung bnh 2-4
m x 0,4-0,6 m. Mt s loi hnh th khng n nh, c th xut hin dng si. Nhng vi
khun di ng th c nhiu lng phn b khp xung quanh t bo. Cc thnh vin ca h
vi khun ng rut khng bao gi sinh nha bo. M s c v, c th quan 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 b mt, nhng cng c th va lm c mi trng va c cn
di y ng. Trn mi trng c c 3 dng khun lc:
-

Dng S: Khun lc trn, b u, nhn bng.

- Dng R: Mt khun lc kh, x x. Thng gp khi nui cy gi chng.

- Dng M: Hnh thc pht trin ny thng gp nhng vi khun c kh nng 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:

18

Di ng hoc khng di ng.

Ln men hoc khng ln men mt s loi 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 s enzym. Hai enzym thng c xc nh nht l urease v

tryptophanase.

Kh nng sinh ra sunfua hydro (H2S) khi d ha protein, acid amin hoc cc dn
cht c lu hunh.

Pht trin c hay khng pht trin c trn mt s mi trng tng hp. V d:
kh nng s dng citrat l ngun cung cp cacbon duy nht c trong mi trng
Simmons.

2.5.5. Sc khng
V khng c kh nng hnh thnh nha bo nn cc thnh vin ca h vi 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 kh nng 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 t bo. Ni c t ch c gii phng khi t bo b li gii.
Ni c t c khi lng phn t t 100000 n 500000 dalton. Ni c t tuy tnh c
khng cao bng ngoi c t nhng cng rt c (ch cn 0,05 mg ni c t git cht
chut nht sau 24 gi). Ni c t c th gy ra tnh trng sc, nu khng c iu tr
tch cc kp thi, chuyn thnh sc khng hi phc dn n t vong. Ni c t khng
b mt tnh c 100oC trong 30 pht. Ni c t l cht c kh nng gy sc.

19

Mt s thnh vin ca h vi khun ng rut c kh nng sinh ngoi c t nh S.Shiga,

E.Coli loi Enterotoxigenic E.Coli (ECET). Ngoi c t ca S.Shiga lm cho bnh l
nng hn rt nhiu, ngoi c t LT (Labile Toxin) ca ETEC l yu t quyt nh c lc
ca vi khun ny.

2.5.7. Cu trc khng nguyn

H vi khun ng rut c 3 nhm khng nguyn c bn: khng nguyn O, khng nguyn
H v khng nguyn K.
-

Khng nguyn O: L khng nguyn thn ca vi khun. y l thnh phn khng

nguyn ca vch t bo. 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 b ph hu 100oC trong hai
gi hoc trong cn 50% nhng b mt tnh khng nguyn khi x l bng formol
0,5%. nhng vi khun khng c v hoc mng bc (khng c khng nguyn K)
th khng nguyn O nm lp ngoi cng. Khi khng nguyn O gp khng th
tng ng s xy 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 th b che 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 O
ngi ta c th chia mt vi loi vi khun thnh nhiu typ huyt thanh.

Khng nguyn H: L khng nguyn lng ca t bo vi khun, ch c nhng vi

khun c lng. C bn cht l protein, ph hu 100oC hoc trong cn 50%
nhng khng b phn hu trong formol 0,5%. Khng nguyn H khi gp khng th
tng ng s xy ra hin tng ngng kt H vi cc ht to hn trong hin tng
ngng kt O v rt d tan khi lc. Nhng vi khun c kh nng di ng khi tip xc
vi khng th H tng ng s tr thnh khng di ng. Khng nguyn O v khng
nguyn H c th c 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 s b ph hu, khng nguyn O vn tn ti. c khng nguyn H ngi

20

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 bn

ngoi khng nguyn thn. N c th di dng mt lp v dy, quan st c bng
knh hin vi quang hc thng thng (nh Klebsiella) hoc di dng mt lp rt
mng ch c th quan st bng knh hin vi in t (nh S.Typhy). Khng nguyn
K nu che ph hon ton khng nguyn O s ngn cch khng cho khng th O gn
vi khng nguyn O lm cho phn ng ngng kt khng xy ra. Trong trng hp
ny cn phi ph hu khng nguyn K hoc vi khun phi c nui cy trong iu
kin khng sinh ra c khng nguyn ny.

2.5.8. Kh nng 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. H vi khun ng rut ng u trong cc cn nguyn vi
khun gy tiu chy. C ch gy bnh, v tr gy tn thng b my tiu ha rt khc
nhau tu theo tng ging, tng loi.
Ngoi ng tiu ha, cc vi khun ng rut cn c kh nng gy bnh nhiu c quan
khc nh tit niu, thn kinh, h hpCc thnh vin ca h ny ng u trong cc vi
khun gy vim ng tit niu, b xa cc vi khun khc. Chng cng ng u trong cc
vi khun gy nhim khun huyt. C th ni khi qut bt k bnh phm no cng c th
gp thnh vin ca h vi khun ng rut.

2.6. Mt s c tnh ca vi khun th nghim

2.6.1. Staphyllococcus aureus
-

L vi khun gram dng, khng gip m, khng to bo t.

Sc khng: nhy cm vi Penicillin, Authromycin v Ampicilin.

Kh nng gy bnh:
o Sng m trn da, nim mc.
o Gy ung nht, p xe.

21

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 t mu xanh.

Kh nng gy bnh:
o

Gy bnh m xanh ng vt v ngi.

Vim bng quang, vim tai c m ngi.

2.6.3. Escherichia coli (E.coli)

-

L vi khun gram m, khng bo t

Sc khng: Nhy cm vi chloraphenicol, Streptomycin, Sunfamid,

Trimethroprim.

Kh nng gy bnh: E.coli c sn trong rut, khi c th b suy yu, sc khng

gim th E.coli mi gy bnh:
o

Gy tiu chy

Bnh ph thng heo con

o Vim rut

2.6.4. Salmonella
-

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

Vi khun hiu kh hay k kh ty tin, mc d dng trn cc mi trng nui cy

thng thng, nhit thch hp 37oC nhng c th pht trin c t 6 42oC,
pH thch hp l 7,6; pht trin c pH 6,9.
o Trn mi trng lng: Sau 5 6 gi nui cy, vi khun lm c mi
trng, sau 18 gi mi trng c u.
o Trn mi trng thch thng: Khun lc trn li, bng, thng khng
mu hoc mu trng xm.

22

o Trn mi trng phn lp SS: Khun lc c mu hng; Trn mi trng

Istrati 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, m chut thy
rut non xung huyt, mng Payer b ph 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 t ch hnh thnh trong iu kin invivo v nui
cy k kh. Ngoi c t c th iu chnh thnh gii gii c t.

Kh nng gy bnh:
o St thng hn ph thng hn.
o Ng c thc n.

2.7. Hot tnh chng oxy ha DPPH [3]

Hin nay ngy cng c nhiu mi quan tm v cc cht chng oxy ha th cp t thc
vt nh l ngun b sung cho cc h thng bo v chng li s oxy ha c hi tn ti
sn c trong c th. Trong i sng cc sinh vt k kh 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 t do: anion
superoxide (O2), hydroxyl (OH), cht oxy ha nh H2O2 c hot tnh phn ng rt ln,
m t cc cht ny cng nh cc sn phm phn ng ca chng l nguyn nhn ph
hy cc phn t sinh hc nh DNA, lipid, protein
Mc d bn thn ca sinh vt t pht trin cc h thng 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 s an ton trong gii hn cho php ca
anion superoxide, H2O2 v cc hydroperoxide hu c tng ng, chng c xem l
cc enzyme kh c chnh trong c th. i khi h thng bo v trn b qu ti, khng
kh b nhim, khi thuc l, bc x t ngoicc loi oxy hot tnh cao ny vt qu

23

gii hn cho php s l ngun gy bnh, thc y nhanh qu trnh lo ha cho sinh vt.
Cc cht chng oxy ha t thc vt gp phn h tr cho h thng bo v ca c th,
ngn chn oxy ha khng mong mun nh cc carotenoid, flavonoid, vitamin C, Ev
cc hp cht trao i cht th cp t thc vt v ang c quan tm nghin cu.

24

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:
L Xun Hoa c ly t 2 ngun:
-

Ti doanh nghip t nhn tr 7 Nga (Ty Ninh).

Vn thc nghim trng i hc Cn Th.

3.2. a im v thi gian thc hin

a im
Ti phng th nghim ha l, Trung Tm Phn Tch v Th Nghim Ha Sinh, Trng
i hc Nng Lm.
Thi gian
ti c thc hin qua 2 phn:
Phn 1: Tin hnh tch chit mt s hp cht th cp t l Xun Hoa v kho st tnh
khng khun.
-

Giai on 1: Tin hnh chit Soxhlet l Xun Hoa thu cc loi cao. Thi gian
tin hnh t ngy 1/4/2007 n 27/4/2007.

Giai on 2: Tin hnh kho st kh nng khng khun ca cc loi cao xc

nh cao c hot tnh mnh nht. T ngy 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 t ngy 8/5/2007 n ngy 23/6/2007.

25

Giai on 4: Tin hnh kho st kh nng khng khun ca cc phn on

xc nh phn on khng khun. Thi gian thc hin t ngy 25/6/2007 n
ngy 2/7/2007.

Giai on 5: Thc hin sc k ct v sc k lp mng trn phn on khng

khun 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 t ht nhn xc nh cu trc

ca cc hp cht c tch chit phng phn tch cu trc, Vin Ho Hc,
Trung tm khoa hc T Nhin, Cng Ngh Quc Gia H Ni.

Phn 2: Kho st kh nng chng oxy ha ca dch chit l Xun Hoa. Thi gian thc
hin t 23/7/2007 n ngy 30/7/2007.
3.3. Thit b, dng c v ha cht
3.3.1. Thit b v dng c
Dng cho cc th nghim sinh ha:
T sy.
My xay mu.
B soxhlet.
My c quay.
Bnh lc.
Bnh lc qu l.
Phu.
Giy lc.
ng chy sc ct.
Bnh tam gic 50ml.
Bnh trin khai.
My lc.
Bn nh siu m.

26

Micropipette loi 100 1000 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 th nghim vi sinh:
ng nghim.
a petri.
n cn.
T m.
T sy.
T hp 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 th nghim sinh ha:

27

Methanol (Trung Quc, Merck).

Ethanol (Trung Quc).
Chloroform (Trung Quc).
Ete du ha.
Acetone.
H2S04 98%.
Ht silicagel kch thc 0,063 0,2 mm.
Bn mng silicagel (TLC) loi 25DC alufolien 20 x 20 cm, kieselgel 60 F254,
Merck.
Vitamin C.
DPPH (1, 1- diphenyl- 2-picrylhydrazyl).
Dng cho th nghim 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. X l nguyn liu
S 3.1: Quy trnh x l nguyn liu.

28

Ra sch l ti Xun Hoa

kh t nhin

Sy kh n trng lng
khng i nhit 40C

Xay mu thnh bt mn

3.4.2. iu ch cc loi cao

Tin hnh chit soxhlet vi 670 g bt mu kh trong 120 gi vi EtOH 70%, thu ly
dch lc, c quay loi b dung mi.
3.4.2.1. iu ch cao ete du ha
-

Cho dch lc trn vo bnh lc qu l, thm 200 ml nc ct vo, sau cho ete
du ha vo.

Dch lc chia lm 2 pha: Pha ete du ha v pha nc, hng ly dch trch ete
du ha, sau c cn bng my c quay vi p sut thp thu c cao ete du
ha: 23 g

Tng th tch ete du ha dng: 1,5 lt.

Pha nc
Pha ete du
ha

29

3.4.2.2. iu ch cao chloroform

-

Ly pha nc trn c quay loi b dung 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 th tch chloroform s dng: 2,5 lt.

3.4.2.3. iu ch cao 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 th tch n-butanol s dng: 1,3 lt.

3.4.2.4. iu ch cao nc
Phn cn li em c quay thu c cao th nc.
S 3.2: Quy trnh iu ch cc loi cao.

Bt kh 670 g
chit soxhlet vi EtOH 70%

Dch trch EtOH

c quay, chit vi ete du ha

Dch trch Ete

Pha nc

30

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

-

Chng vi khun: E.coli, Salmonella c ly t bnh x th y, Trng i Hc

Nng Lm Tp. H Ch Minh.

Pha long lin tip xc nng c ch ti thiu (MIC) ca cc loi cao c

kh nng c ch s tng trng ca vi khun.

Mi trng nui cy vi sinh vt tin hnh th nghim vi sinh vt l mi

trng BHI v TSA.

31

Pha mi trng BHI: cho 15 g bt BHI v 300 ml nc ct vo bnh tam gic,

un nh bng microwave trong 2 pht, sau lc bng giy lc loi b cn.
Hp kh trng autoclave 121C/15 pht.

Cch pha mi trng TSA:

Cao tht

2,5 g

Pepton

5g

NaCl

2,5 g

Nc Ct

500 ml

Cho tt c vo bnh tam gic, un nh bng microwave trong 2 pht, sau cho
2 g agar vo. Hp kh trng autoclave 121C/15 pht, ly ra vo a petri.
-

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 H2SO4 0,18 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 mi
trng 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: iu
chnh 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 s l 108 cfu/ml.
o Tip tc pha long nng vi khun xung cn 107, 106 bng mi trng BHI.
o B tr th nghim vi sinh vt trn mi trng thch TSA xc nh kh nng
khng khun ca 4 loi cao ny. Cao cc nng 1000 g/ml, 500 g/ml, 250

32

g/ml, 125 g/ml c ha tan vo mi trng TSA. Nguyn tc xc nh da

vo s c ch ca cc nng cao pha long trn. Cy vi khun E.coli v
Salmonella nng 106 ln trn cng mt a mi trng TSA, mi vi khun
chim na a.
-

c kt qu da trn vic quan st khun lc c mc hay khng trn b mt mi

trng thch sau 24 gi, t s xc nh c cao no c kh nng c ch vi
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 v sc k iu ch.

3.4.4. Chit xut mt s hp cht t cao chloroform

3.4.4.1. Sc k ct [1, 7]
Nguyn tc:
-

Sc k ct loi hp ph c tin hnh trn mt ct thy tinh thng ng vi

cht hp ph ng vai tr tng tnh, dung mi ra ct ng vai tr tng ng
chy qua cht hp ph di nh hng ca trng lc.

i vi mi cht ring bit trong hn hp cn tch ty theo kh nng hp ph v

kh nng ha tan ca n i vi dung mi ra ct ly ra c ln lt trc
hoc sau.

Cht hp ph c s dng ph bin l Silicagel 60 c ht 0,063 0,2 mm.

Dung mi: trin khai sc k ct, ngi ta c th dng tun t tng dung mi
ring bit c phn cc tng dn hoc dng mt hn hp dung mi thch hp
ra ct.

Vic la chn kch thc ct rt quan trng, thng thng ct c ng knh

nh v chiu di cng di th s tch cng tt.

Tin hnh:
Bc 1: Chun b ct.
-

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

33

Cho bng gn vo y ct (phi tht kho nu cht qu cht nghin cu s b

mc kt li khng chy xung c, nu khng cht th khi dung mi vo nt
bong s b tri).

Kp ct thng ng trn gi st.

Hnh 3.2: Ct sc k

Bc 2: Nhi ct.
-

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

Cho silicagel t t vo ct sao cho nt bng di y ct khng b tri ln, kim

tra 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 s n nh, b mt

silicagel phi nm ngang.

Bc 3: a cht phn tch vo ct.

34

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 sy

kh hon ton, mu lc ny phi dng bt mn.

Sau cho ete du ha vo cao ny sao cho hn hp tr thnh dng st, khng
long qu cng khng c qu khi mu vo to thnh mt lp trn b mt
silicagel.

Bc 4: Trin khai ct.

-

trin khai ct ta c th dung tun t cc dung mi c phn cc tng dn.

S thay i t dung mi ny sang dung mi khc phi chuyn t t bng cch
pha t l tng dn hoc gim dn.

S dng dung mi: ete du ha, chloroform , methanol v hn hp ca n c

pha theo t l tng dn: 1%, 2%, 5%, 10%, 25%, 50%, 100%.

Bc 5: Hng v kim tra cc phn on.

-

S dng cc bnh tam gic 50 ml hng tng phn on, nh du s th t

ca bnh.

Kim tra cc phn on thu c bng sc k lp mng vi thuc th c trng.

3.4.4.2. Sc k lp mng [1, 7]

Nguyn tc:
-

Sc k lp mng (SKLM) l k thut phn b rn - lng; trong pha ng l

cht lng c cho i ngang qua mt lp cht hp tr (v d nh: silicagel hoc
oxid nhm) cht hp th ny c trng thnh mt lp mng, u, ph ln mt
nn phng nh tm king hoc tm nhm.

Dung mi trin khai trong SKLM thng l mt hn hp c t 2, 3 hoc 4 loi

dung mi khc nhau vi t l thch hp. ng vi mi loi hp cht phn tch
khc nhau s c mt s h dung mi thch hp.

35

S dng SKLM gp cc bnh hng cht chy ra thnh chung mt phn on

nu chng cho kt qu Rf ging nhau.

Phng php:
Chun b:
-

ng mao qun: S dng pipette Pasteur, dng kp ko di u pipette do sc

nng ca ngn la n cn.

ng nghim nh: Cha acetone ra mao qun sau mi ln s dng.

Bnh khai trin: Cho dung mi ete du ha, chloroform, methanol theo t l thch
hp vo bnh, sau cho mt tm giy thm vo bnh dung mi c hon
ton bo ha (khong 30 pht). Mt hu qu d thy l nu bnh khng c
bo ha th khi dung mi l mt 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 th H2SO4 10%/EtOH: Pha 21ml H2SO4 95% vi 179 ml ethanol.

Bn mng: Dng bt ch k mt ng thng cch y 1,5 cm v mt ng

thng cch u trn 0,5 cm.

Tin hnh:
-

S dng mao qun chm cht cn kim tra vo bn mng, mi vt chm phi
nh, gn 2 3 mm v khng lm rch bn mng. Vt chm v tr cch cnh
y 1 1,5 cm, vt ny cch vt kia 0,5 cm v cch b cnh ca bng 0,5 cm.

Nhng bn mng vo bnh khai trin tht nh nhng, 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 mng
cch khong 0,5 cm th ly ra, dng my sy tc lm kh dung mi. Sau
nhng bn mng vo bnh thuc th H2SO4 10%/EtOH, hin vt 100C.

Bn mng

36

Pht hin vt:

-

Qua mu sc t nhin.

Pht quang di tia UV.

Phun thuc th cho mu c trng.

Tnh tr s Rf:
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 t 0,3 0,7.
3.4.4.3. Sc k iu ch [7]
-

Nguyn tc c bn cng ging nh SKLM, ch khc l s dng bng c kch c

ln hn (10 x 20 cm).

Dng bt ch vch sn mc xut pht cch y 1,5 cm. S dng vi qun ko

dung dch mu thnh mt dy, c b ngang 2-4 mm, di sut chiu ngang ca
bng nhng cn cha 1cm khong cch 2 u bng trnh b hiu ng b. Sy

37

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 s c 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.

Hnh 3.4: Sc k iu ch
-

Thu ly hp cht : Cht hp thu Silicagel c cha cht c cha trong mt

becher, 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 s trch 3-5 ln na bo m trch ht
mu cht ra khi cht hp thu.
3.4.4.4. K thut kt tinh phn on

Gi s cht A c ln mt t cht B v C. Ha tan mu vo dung mi nng, lc

dung dch cn nng qua giy lc, yn cho dung mi ngui t t, hp cht A
s kt tinh rn v ln trong tinh th ca n mt t B v C. Lng B v C vn cn
ln trong nc ci.

Lc ly ring tinh th A. Lp li kt tinh nh th thm vi ln na bng dung

mi tinh khit s cho cht A tinh khit.

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

38

Cc phn on thu c sc k ct c tin hnh th nghim khng khun

vi vi khun E.coli, staphylococcus aureus, pseudomonas aeruginosa.

Mi trng c s dng y l TSA, BHI.

Dung mi ha tan mu l DMSO.

Dng phng php vng khng khun kim tra phn on no c kh nng
khng khun.

Tin hnh :
-

Mi trng TSA dng a petri vi dy 2/3 a, 72 gi cho a kh

hon ton.

Ha tan 0,4 g mu mi phn on bng 5 ml dung mi DMSO (lc ny nng

mu l 0,02 g/ml) un nh trn ngn n cn.

Ct 1/3 u ng tipe, sy kh trng.

Dng u tipe c kh trng c l a, mi a c 5 l vi khong cch

u nhau.

Cy vi khun vo mi trng BHI, chun c ca huyn dch vi khun tng

ng 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, 106 bng mi trng BHI.

Dng tm bng v trng chm nh vo huyn dch vi khun, sau ray u tm

bng ny ln a petri c sn cc l sao cho vi khun c th c tri u mt
a.

Dng micropipette ht 100 l mi phn on mu ln lt cho vo mi l.

Quan st : Sau 24 gi, nu l no c vng khng khun xung quanh chng t

phn on c khng khun. Ta s dng phn on ny tip tc th nghim
xc nh nng ti thiu c ch vi khun (MIC).
Th nghim xc nh nng ti thiu c ch vi khun:

39

Pha long phn on khng khun nng 0,02 g/ml xung 0,002 g/ml (2000g/ml).
Chun b 3 nhm ng nghim tng ng vi 3 loi vi khun, mi nhm 6 ng.
Bng 3.1 : Th tch v nng cc ng nghim.
Nng
Tn cc

mu lc

ng nghim

u
(g/ml)

Th tch

Th tch Nng

mu cho

BHI cho mu

vo (l)

vo (l)

(g/ml)

Th tch vi

Nng

khun cho

mu cui

vo 1ml

cng

mu (ml)

(g/ml)

E1, P1, S1

2000

100

900

200

E2, P2, S2

2000

200

800

400

200

E3, P3, S3

2000

300

700

600

300

E4, P4, S4

2000

400

600

800

400

E5, P5, S5

2000

500

500

1000

500

E6, P6, S6

2000

600

400

1200

600

100

Chun b 3 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 c cc ng nghim trong t m 37C/24 gi.

Quan st s thay i c ca mi trng, s to thnh cn y v s to

vng b mt ng nghim.

3.4.6. Th nghim hot tnh chng oxy ha DPPH

3.4.6.1. Cc tnh cht ca gc t do DPPH
-

L gc t do bn do c hiu ng lin hp trong phn t.

hp th cc i ti 517 nm do gc t do trn Nit v cho mu tm trong

Methanol [3].

C cng thc cu to nh sau :

NO2

O2N

40

C ch phn ng ca gc t do DPPH v cht chng oxy ha :

DPPH + AH

DPPH-H + A

Kt qu c nh gi thng qua gi tr IC50 (inhibitory concentration) l

nng cht oxy ha cn c ch (trung ha) 50% gc t do DPPH trong
khong thi gian xc nh.

Mu Vitamin C c dng so snh thng gi tr IC50.

3.4.6.2. Quy trnh th hot tnh chng oxy ha DPPH [3]

Ly trch mu:
S 3.3: Quy trnh ly trch mu cho th nghim DPPH.
Cn 10 g l ti Xun Hoa

Nghin

Cho vo 25 ml Methanol

Lc nhit phng 30 pht

Lc

Lp li quy trnh 5 ln

41

Pha mu:
-

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

mu Xun Hoa lc ny l 400 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)

100

50

25

16

12,5

Pha thuc th :
-

Cn 2,7 mg DPPH cho vo bnh nh mc 25 ml.

42

Thm 25 ml MeOH, lc mnh cho tan ht, sau thm MeOH vo cho
25 ml.

n nh trong ti 30 pht, 4C.

Pha dung dch chun:

-

Cn 2 mg vitamin C vo bnh nh mc 5ml.

Thm 3 ml MeOH vo, lc mnh cho tan ht, sau thm MeOH vo
cho 5 ml. Nng vitamin C lc ny l 400 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)

100

50

25

16

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 th quang (OD) bc sng 517nm

o mu
-

Cho 1ml DPPH vo 1ml mu Xun Hoa tng pha long.

tt c vo trong bng ti, 30 pht.

o hp th quang (OD) bc sng 517 nm.

43

Hnh 3.5: Cc nng pha long ca vitamin C (bn tri) v mu Xun

Hoa (bn phi) sau khi cho DPPH vo.

44

Chng 4
KT QU V THO LUN
4.1. Th nghim kh nng khng khun ca cc loi cao chit
Bng 4.1: Kt qu th nghim kh nng khng khun ca 4 loi cao.
Nng th
Cc loi cao

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 n-Butanol

cao ete du ha

cao nc

cao chloroform

Hnh 4.1: Th nghim cc phn on khng E.coli v

Salmonella

45

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 qu tch chit ca cao chloroform v cc phn on ca chng
4.2.1. Kt qu tch chit ca cao chloroform
Thc hin sc k ct trn cao chloroform vi h dung 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 qu sc k ct trn cao chloroform.

S th
t bnh

Tn
phn
on

1-26

XH1

27-49

XH2

50-64

XH3

65-83

XH4

84-96

XH5

97-108

XH6
XH7

Sc k lp mng

Dung
mi
gii ly

Cao (g)

E:C
(95:5)
E:C
(9:1)
E:C
(8:2)
E:C
(7:3)
E:C
(6:4)
E:C

dung
mi

ct
E

4,83

2,47

1,74

1,61

1,34

1,98
1,35

E:C
(9:1)
E:C
(85:15)
E:C
(8:2)
E:C
(7:3)
C:E
(8:2)
C:E
(9:1)
C:E

Thuc
th

H2SO4

Rf

Hot
tnh

Ghi

khng

ch

khun
nhiu

vt

tnh

nt

nt

nt

nt

nt

nt

nt

nt

nt

4vt

nt

3vt

m
tnh
m
tnh
m
tnh
m
tnh
m
tnh
m

46

109-116

(6:4)

E:C

117-126

XH8

127-132

XH9

133-136

XH10

137-145

XH11

146-155

XH12

2,56

156-168

XH13

1,23

169-172

XH14

0,96

173-192

XH15

193-204

XH16

205-216

XH17

217-223

XH18

(6:4)
E:C
(5:5)
E:C
(5:5)
E:C
(5:5)

C:Me
(99:1)
C:Me
(98:2)
C:Me
(95:5)

tnh

(98:2)

1,63

nt

3vt

0,87

nt

2 vt

nt

2 vt

nt

3vt

0,56

0,78

1,67

0,48

0,74

C:M
(99:1)
C:M
(99:1)
C:M
(95:5)
C:M
(95:5)
C:M
(95:5)
C:M
(8:2)
C:M
(7:3)
C:M
(7:3)

nt

tnh
Dng

kho

tnh

st

Dng

kho

tnh

st

Dng

kho

tnh

st

nhiu

Dng

vt

tnh

nt

4 vt

nt

5 vt

nt

Dng

kho

tnh

st

m
tnh

nhiu

vt

tnh

nt

nt

nt

nt

Me

m
tnh
m
tnh
X ct

(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 tnh khng khun ca cc phn on chng ti ch chn phn on XH9, XH10,
XH11, XH13 tip tc kho st.

47

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 th hin vt l H2SO4 10%/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.

Hnh 4.2: Cht XH-K11 trn bn mng

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 qu cho dng tinh th mu vng. Kim tra
li bng sc k lp mng vi thuc th hin vt l H2SO4 10%/EtOH trong h dung
mi trin khai CHCl3:M (98:2) cho mt vt trn mu vng c Rf=0,36; nhit nng

48

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 h dung 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 th hin vt l
H2SO4 10%/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.

Hnh 4.3: Cht XH-K13 trn bn mng

Cc cht tinh khit thu c tip tc c tin hnh chy ph mt chiu, ph hai
chiu NMR 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 mng cho hin vt mu tm, c Rf = 0,27, nhit nng chy 218oC.

49

Ph hng ngoi IR (, cm-1) cho vch hp thu mnh, rng ca nhm OH ti 3400,20
cm-1; vch 2937,99 cm-1 th hin s dao 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 (Ph lc 1).
Ph khi lng MS (Ph lc 8) cho pick ion m l 577 [M + H]+ cho thy trng
lng phn t ca XH-K13 l 576.
Ph cng hng t ht nhn 13C-NRM kt hp k thut Dept cho ta thy (ph lc
3, 4):
-

Trong phn t c 35 cacbon, trong c 3 cacbon t cp, 14 nhm CH, 12

nhm CH2, 6 nhm CH3

Cc mi 140,437 v 121,129 ppm l du hiu ca ni i v 140,437 l C=

(Cacbon t cp c ni i), 121,129 l ca CH=.)

Mt mi c trng 100,795 ppm l mi ca acetal C1' ca ng gn vo C

v tr s 3; c chng minh nh ph HMBC (ph lc 6) cho thy s tng 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 s liu ph 13C-NRM ca phn ng v ph proton
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 ph HSQC (ph lc 5), HMBC cho bit l 5 nhm CH-OH ca gc
ng. Vy trong phn t c 1 n v ng 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 5,321 ppm, c cng tch phn l 1,000
n v l ca H6, cng vi tn hiu ph 13C-NRM 121,129 ( CH=); cng ph hp trn
ph HSQC. Mt khc da vo ph HMBC cho thy s tng tc gia H6 vi C7, C8,
C10, C4 v s tng tc gia H1 vi C3, C5, C10 nn CH2 v tr s 1 phi c dch

50

chuyn l 36,80 ppm v C= v tr s 5; chng t khung 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 H2SO4 10%, sau h nng, cho php gi thit 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 t l: C35H60O6.
Cng thc cu to ca C35H60O6:

4.3.2. Cht XH-K11

Sc k lp mng cho hin vt mu vng, c Rf = 0,36, nhit nng chy 237oC.
Ph 1H-NMR (DMSO, ppm, 500MHz) (ph lc 9) cho:
2 mi i ca proton nhn thm ti

= 6,45 ppm v 6,83 ppm (d, J = 2 Hz),

chng t chng v tr 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 t chng v tr ortho.

Ph

13

C-NMR (DMSO,

ppm, 125 MHz) (ph lc 11) kt hp ph DEPT (ph lc

10) cho thy XH-K11 c 21 cacbon trong c 1 nhm >C=O, 1 nhm CH2 14 nhm
CH, 5 nhm cacbon t cp.

51

ng thi kt hp ph cng hng t ht nhn 2 chiu HSQC (ph lc 12) v HMBC

(ph lc 13) cho thy XH-K11 thuc nhm flavon v l dn xut ca apigenin.
Ph 13C-NMR cho mt mi c trng 99,90 ppm l mi ca acetal C1 ca ng.
Da vo ph HSQC (ph lc 12) c c gi tr H1 = 5,06 ppm. Kt hp ph 1HNMR (ph lc 9), ta c c H1 (1H, d, J=7,5 Hz), chng t ng ni vi khung
aglycon bng lin kt . Mt khc, ph DEPT 90 (ph lc 10) cho ta 5 mi cc v tr
73,09; 77,16; 69,56; 76,42 v 60,60 ppm. ng thi kt hp vi ph HSQC, HMBC
(ph lc 13) v COSY (ph lc 14) cho bit l 5 nhm CH-OH ca gc ng
glucose. Vy trong phn t c 1 n v ng 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 t l: C21H20O10.
Cng thc cu to ca C 21H20O10 nh sau:

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 on XH9, XH10, XH11, XH12, XH13 c vng khng
khun xung quanh l, iu ny chng t rng cc phn on ny c kh nng c ch s
pht trin ca vi khun.

52

(4)

(1)
(3)

(5)

(2)

Hnh 4.4: Kt qu th nghim cc phn on khng E. coli.

(1): Phn on XH9.
(2): phn on XH10.
(3): Phn on XH11.
(4): Phn on XH12.
(5): Phn on XH13.
Sau tip tc th nghim trn cc phn on khng khun ny kho st nng
ti thiu c ch vi khun (MIC).

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

Phn on

Nng c ch ti thiu vi khun th nghim (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

53

Nhn xt:
S dng kt qu trn lm c s cho 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.

E1

E2

E3

E4

E5

E6

Ec

Hnh 4.5: Cc nng khng E.coli ca phn on XH9.

S1

S2

S3

S4

S5

S6

Sc

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

54

P1

P2
2

P3

P4

P5

P6

Pc

Hnh 4.7: Cc nng khng Pseudomonas aeruginosa ca XH9.

Ch thch:
E1, E2, E3, E4, E5, E6: E. coli t nng t 100 600 g/ml.
P1, P2, P3, P4, P5, P6: Pseudomonas aeruginosa t 100 600 g/ml.
S1, S2, S3, S4, S5, S6: Staphylococcus aureus t 100 600 g/ml.
c: i chng
4.5. Kt qu ca th nghim chng oxy ha
% c ch DPPH = (Ao- A)/Ao x 100
o hp thu ca DPPH : A0 = 1,67458.
A: hp thu ca dung dch phn ng.
IC50: Nng c ch 50% gc DPPH.
Kh nng c ch gc t do c nh gi bng phng php gc t do bn DPPH.
4.5.1. Kt qu o OD i vi vitamin C
Vitamin C c o 3 ln v ly gi tr trung bnh.
Bng 4.4: Nng v kt qu o OD ca vitamin C
Nng vitamin C (g/ml)

A (OD o 517 nm)

100

1,361421

50

0,766121

55

25

0,43277

12,5

0,28491

Kt qu o OD v nng vitamin C c th hin di dng biu :

th 4.1: Kh nng c ch DPPH cc nng khc nhau ca vitamin C.

Cc nng vitamin C v phn trm c ch c biu th di dng ng thng
vi phng trnh y = -1,355x + 124,8, vi h s tng quan R=0,999.
T th ngoi suy gi tr IC50 ca 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 qu o OD i vi dch chit l Xun Hoa
Dch chit mu Xun Hoa c o 3 ln v ly gi tr trung bnh.
Bng 4.5: Nng v kt qu o OD ca dch chit l Xun Hoa
Nng dch chit l Xun Hoa (g/ml)

A (OD o 517nm)

100

1,12256

50

0,70756

25

0,48307

12,5

0,34129

56

Kt qu o OD v nng mu c th hin di dng biu :

th 4.2: Kh nng c ch DPPH cc nng khc ca dch chit l Xun

Hoa.
Cc nng mu Xun Hoa v phn trm c ch c biu th di dng ng
thng y = -1,897x + 161,4, vi h s tng quan R=0.998.
T th ngoi suy gi tr IC50 ca mu Xun Hoa l 66,55 (g/ml).
Bng 4.6: So snh kh nng khng oxy ha ca vitamin C v dch chit l Xun Hoa:
hp th ca DPPH
1,67458

hp th ca (DPPH +

hp th ca (DPPH +

vitamin C)

mu Xun Hoa)

1,361421

1,12256

Nhn xt:
-

hp th quang ca DPPH gim t 1,67458 xung 1,361421 i vi trng

hp c vitamin C v gim xung 1,12256 i vi trng hp c dch chit l
Xun Hoa. iu ny c ngha l trong l Xun Hoa c mt s hp cht chng
oxy ha tc dng vi phn t DPPH lm cho hp th gim xung :

57

C ch phn ng ca gc t do 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 (57,05

g/ml): Nng c ch 50% DPPH ca mu Xun Hoa l 66,55g/ml, trong
khi nng c ch 50% 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).

58

Chng 5
KT LUN V NGH
5.1. Kt lun
-

L Xun Hoa c chit soxhlet vi dung mi ethanol, sau ln lt chit vi

ete du ha, chloroform, n-butanol ln lt cho 4 loi cao: Cao ete du ha, cao
chloroform, cao n-butanol v cao nc. Th nghim khng khun 4 loi cao ny
i vi E.coli v Salmonella, kt qu xc nh c cao chloroform c hot tnh
khng khun nht, vi nng c ch ti thiu l 500 g/ml.

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

phn on.

Th nghim kh nng khng khun ca 18 phn on, kt qu xc nh c

phn on c hot tnh khng khun l: XH9, XH10, XH11, XH12, XH13.

Kt hp kt qu chy sc k ct v th khng khun cc phn on, cui cng

chn 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 XHK13 vi nhit nng chy 218C.

Chy ph cng hng t ht nhn mt chiu v hai chiu xc nh c cht

XH-K11 l apigenin 7-O--glucoside, c cng thc phn t: C21H20O10 v cht
XH-K13 l -sitosterol-3-O--glucoside, c cng 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 ha vi hm lng l
kh cao (85,7% vitamin C).

5.2. ngh

59

Vi s hiu bit v kinh nghim c gii hn ca mt sinh vin nn kt qu s c

nhng thiu xt. V thi gian v kinh ph lm ti cn hn hp nn khng th tip tc
chit tch thm mt s hp cht th cp trong l Xun Hoa c hot tnh sinh hc cao.
Nu c thi gian, ti xin c php ngh tip tc phn tch cc thnh phn v tch
chit cc hp cht th cp nhm s dng chng thay th cho khng sinh trong tng
lai. Thm vo , nghin cu su hn na c th s dng cy Xun Hoa nh mt
thc phm chc nng l mt trong nhng hng v cng quan trng.

60

TI LIU THAM KHO

TING VIT

1. Nguyn Thc Ct, 1985. Phng php sc k. Nh Xut Bn Khoa Hc v K

Thut.
2. V Vn Chi, 1997. T in cy thuc Vit Nam. Nh Xut Bn H Ni.
3. Bi Trng t, Phng Vn Trung, Phan Nht Minh, Hong Th Ngc Dung v
Phm Cao Thanh Tng, 2003. Xy dng quy trnh th hot tnh khng oxy ha
DPPH v sng lc mt s cao chit t cy c Vit Nam.Tuyn Tp Cng Trnh
Nghin Cu Khoa Hc v Cng Ngh nm 2003; tr.150-155.
4. Nguyn Vn Hng, L Anh Tun v Nguyn Quyt Chin, 2004. Nghin cu
thnh phn ha hc cy Xun Hoa (Pseuderanthemum Palatiferum Nees Radlk).
Tp Ch Khoa Hc v Cng Ngh, tp 42, s 2; tr.75-79.
5. Trn Cng Khnh, Nguyn Vn Hng, Nguyn Th Thanh Nhi, L Mai Hng
v Bi Kim Lin, 1997. Gp phn nghin cu v thc vt, thnh phn ha hc
v tc dng sinh hc ca cy Xun Hoa. Tp ch dc liu, tp 3; tr. 37-41.
6. Trn Cng Khnh, 1997. S tht v cy thuc k diu-cy Xun Hoa. Tp Ch
Thuc v Sc Khe, s 101; tr.10-11.

61

7. Nguyn Kim Phi Phng, 2000-2001. Cc phng php nhn danh; trch ly c
lp cc hp cht hu c. Trng i Hc Khoa Hc T Nhin.
8. Nguyn Vn Thanh. Vi sinh hc. Nh Xut Bn Y hc.
9. Nguyn Th Minh Thu, Trn Cng Khnh, Trn Vn Hin, T Th Phng, Trn
L Du, 1999. Th c tnh cp din v tc dng bo v t bo gan ca cy Xun
Hoa. Tp Ch Dc Hc, s 9; tr.15-17.
10. Bi ging chit xut dc liu. Trng i Hc Y Dc TpHCM.
11. Bi ging dc liu, 2002. Trng i Hc Dc H Ni, B Mn Dc Liu.
Nh Xut Bn Y Hc.
12. Vi sinh vt y hc. B Y T, V Khoa Hc- o To.
TING NC NGOI
13. Dejian Huang, Boxin Ou and Ronald L. Prior, 2005. The chemistry behind
antioxidant capacity assays. Journal of Agricultural and Food Chemistry, 53;
p.41-56.
14. Hunh Kim Diu, Chu B Lc, Seishi Yamasaki and Yutaka Hirata, 2006. The
effects of Pseuderanthemum palatiferum a new medicinal, on growth
performances and diarrhea of piglets.JARQ, No.40; P.85-91.
15. Irina I. Koleva, Teris A. van Beek, jozef P. H. Linssen, Aede de Grt and Lyuba
N. Evstatieva, 2002. Screening of plant extract for antioxidant activity: a

62

comparative study on three testing methods. Phytichemical analysid, No.13;

p.8-17.

16. Phan Minh Giang, Ha Viet Bao, Phan Tong Son. 2000. Phytochemical study on
Pseuderanthemum palatiferum (Nees) Raldk, Acanthaceae. Joural of chemistry,
Vol.41, No.2, trang 115-118.
TI LIU TRN MNG
17. http:// www.jireas. Affre.go.jp.

18. http:// www.cancerweb.ncl.ac.uk/cgi-bin/omd=antioxidants.

19. http:// www.unabridged.merriam-webster.com.

Ph lc 1 : Ph IR ca cht XH-K13.

Ph lc 2 : Ph 1H- NMR ca cht XH-K13

Ph lc 2a: Ph 1H- NMR ca cht XH-K13.

Ph lc 2b : Ph 1H- NMR ca cht XH-K13.

Ph lc 3: Ph 13C- NMR ca cht XH-K13.

Ph lc 3a: Ph 13C- NMR ca cht XH-K13.

Ph lc 3b: Ph 13C- NMR ca cht XH-K13.

Ph lc4: Ph Dept ca cht XH-K13

Ph lc 5: Ph HSQC ca cht XH-K13

Ph lc 5a: Ph HSQC ca cht XH-K13.

Ph lc 5b: Ph HSQC ca cht XH-K13.

Ph lc 6: Ph HMBC ca cht XH-K13.

Ph lc 6a: Ph HMBC ca cht XH-K13.

Ph lc 6b: Ph HMBC ca cht XH-K13.

Ph lc 6c: Ph HMBC ca cht XH-K13.

Ph lc 7: Ph COSY ca cht XH-K13.

Ph lc 7a: Ph COSY ca cht XH-K13.

Ph lc 7b: Ph COSY ca cht XH-K13.

Ph lc 8: Ph MS ca cht XH-K13.

Ph lc 9: Ph 1H ca cht XH-K11.

Ph lc 10: Ph 13CNRM v DEPT ca cht XH-K11.

Ph lc11: Ph

13

CNMR ca cht XH-K11

Ph lc 12: Ph HSQC ca cht XH-K11

Ph lc 12a: Ph HSQC cht XH-K11.

Ph lc 12b: Ph HSQC ca cht XH-K11

Ph lc 13: Ph HMBC ca cht XH-K11

Ph lc 13a: Ph HMBC ca cht XH-K11

Ph lc 13b: Ph HMBC ca cht XH-K11

Ph lc 14: Ph COSY ca cht XH-K11

Ph lc 14a: Ph COSY ca cht XH-K11

Ph lc 14b: Ph COSY ca cht XH-K11.

Ph Lc14c: Ph COSY ca cht XH-K11

Ph lc 15: D liu ph 13C NMR v DEPT ca cht XH-K13.

V tr C
(ppm)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
1'
2'
3'
4'
5'
6'

13

C NMR

36,80
29,24
76,94
38,29
140,43
121,29
31,34
31,39
49,59
36,18
20,56
39,30
41,82
56,15
23,82
27,74
55,42
11,62
18,91
35,44
18,58
33,34
25,47
45,14
28,71
19,65
19,05
22,59
11,74
100,79
73,44
76,69
70,09
76,75
61,08

Dept 135
CH2
CH2
CH=O
CH2
C=
CH=
CH2
CH
CH
C
CH2
CH2
C
CH
CH2
CH2
CH
CH3
CH3
CH
CH3
CH2
CH2
CH
CH
CH3
CH3
CH2
CH3
CHOH
CHOH
CHOH
CHOH
CHOH
CH2OH

Tn hiu mi
Mi m, mt trong Dept 90
Mi m, mt trong Dept 90
Mi dng
Mi m, mt trong Dept 90
Bin mt
Mi dng
Mi m, mt trong Dept 90
Mi dng
Mi dng
Bin mt
Mi m, mt trong Dept 90
Mi m, mt trong Dept 90
Bin mt
Mi dng
Mi m, mt trong Dept 90
Mi m, mt trong Dept 90
Mi dng
Mi dng, mt trong Dept 90
Mi dng, mt trong Dept 90
Mi dng
Mi dng, mt trong Dept 90
Mi m, mt trong Dept 90
Mi m, mt trong Dept 90
Mi dng
Mi dng
Mi dng, mt trong Dept 90
Mi dng, mt trong Dept 90
Mi m, mt trong Dept 90
Mi dng, mt trong Dept 90
Mi dng
Mi dng
Mi dng
Mi dng
Mi dng
Mi m, mt trong Dept 90

Ph lc 16: D liu cc ph ca cht XH-K13

V tr 1H NMR
C
( ppm, J = Hz)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
1'
2'
3'

13

C NMR
COSY
HMBC
1
( ppm )
H - 1H
HC

1,77; 0,987; m
1,804; 1,508; m
3,43, m
4a:2,12 / 4b:2,37
5,32; s; 1H
1,946; 1,97; t
1,512; m; 1H
0,9075; d; 1H
11a:1,4; 11b:1,5; m
12a:1,17; 12b:1,97; m
0,86; m; 1H
15a:1,13; 15b:1,52; m
16a:1,23; 16b:1,78; m
1,157; m; 1H
0,67; s; 3H
0,96, s; 3H
1,3; m; 1H
0,91, d; 3H
22a:1,4; 22b:1,24
1,18; m; 2H
0,93; m; 2H
1,609; m; 1H
0,78; s; 3H
0,80; s; 3H
28a:1,20; 28b:1,22
0,82; t; 3H
4,22; d, J=8
2,90; m; 1H
OH-2'=4,84
3,12; m; 1H
OH-3' = 4,85
3,02; m; 1H

36,80
29,24
76,94
38,29
140,43
121,29
31,34
31,39
49,59
36,18
20,56
39,30
41,82
56,15
23,82
27,74
55,42
11,62
18,91
35,44
18,58
33,34
25,47
45,41
28,71
19,65
19,05
22,59
11,74
100,79

C3, C5, C10

H3/H4a;H3/H4b
H4a/H4b
C2,C10,C3 /C6,C5
H6/H7
H7/H8

C7,C8,C10, C4

C13, C17,C14
C9, C10, C5
H20/H21
H21/H20

C17,C20,C22

H1'/ H2'

C3'

73,44

OH-2'/ H2'

76,69

OH-3'/ H3'

4'
5'

OH-4'= 4,82
3,07; m; 1H

70,09
76,75

OH-4'/ H4'

Ph lc 17: D liu ph 13C-NMR v DEPT ca XH-K11

V tr C

2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
1
2
3
4
5
6

13

C-NMR
( ppm )

164,30
102,98
181.96
161,09
99,50
162,93
94,83
156,92
105,31
120,77
128,60
116,06
162,31
116,06
127,60
Glucose
99,90
73,09
77,16
69,56
76,42
60,60

DEPT 90

DEPT 135

Kt lun

Bin mt
Mi dng
Bin mt
Bin mt
Mi dng
Bin mt
Mi dng
Bin mt
Bin mt
Bin mt
Mi dng
Mi dng
Bin mt
Mi dng
Mi dng

Bin mt
Mi dng
Bin mt
Bin mt
Mi dng
Bin mt
Mi dng
Bin mt
Bin mt
Bin mt
Mi dng
Mi dng
Bin mt
Mi dng
Mi dng

>C=O
CH=
>C=O
=C-OH
CH=
=C-O
CH=
=CO
=C<
>C=
CH=
CH=
=COH
CH=
CH=

Mi dng
Mi dng
Mi dng
Mi dng
Mi dng
Bin mt

Mi dng
Mi dng
Mi dng
Mi dng
Mi dng
Mi m

>CH k O
CH OH
CH OH
CH OH
CH OH
CH2OH

Ph lc 18: D liu ph 13C-NMR, 1H-NMR, COSY, HMBC ca cht XHK11.

V trC
( ppm )

13

164,30
3
102,98
4
181.96
5
161,09
6
99,5
7
162,93
8
94,83
9
156,92
10
105,31
1
120,77
2
128,60
3
116,06
4
162,31
5
116,06
6
127,60
Glucose
1
99,9
2
73,09
3
77,16
4
69,56
5
76,42
6
60,60

C-NMR

H-NMR
1
H

COSY
1

HMBC
1

13

6,86 (1H,s)

6,45 (1H, d, J = 2,0 Hz)

H6

H8

6,83 (1H, d, J = 2,0 Hz)

H8

H6

7,95 (1H, d, J = 9,0 Hz)

6,94 (1H, d, J = 8,5 Hz)

H2
H3

H3
H2

6,94 (1H, d, J = 8,5 Hz)

7,95 (1H, d, J = 9,0 Hz)
5,06 (1H,d, J=7,5Hz)

H1

C7

H8

H3

C4, C2

H6

C8, C10

C6, C7, C9, C10