BỘ Y TẾ TRƯỜNG ĐẠI HỌC DƯỢC HÀ NỘI ĐINH THỊ XUÂN NGHIÊN CỨU ĐẶC ĐIỂM THỰC VẬT, THÀNH PHẦN HÓA HỌC VÀ TÁC DỤNG KHÁNG KHUẨN CỦA VỊ THUỐC LÁ CÂY THỊ KHÓA LUẬN TỐT NGHIỆP DƯỢC SĨ HÀ NỘI - 2014
Sep 04, 2015
B Y T
TRNG I HC DC H NI
INH TH XUN
NGHIN CU C IM THC VT, THNH PHN HA HC V TC DNG
KHNG KHUN CA V THUC
L CY TH
KHA LUN TT NGHIP DC S
H NI - 2014
B Y T
TRNG I HC DC H NI
INH TH XUN
NGHIN CU C IM THC VT, THNH PHN HA HC V TC DNG
KHNG KHUN CA V THUC L CY TH
KHA LUN TT NGHIP DC S
NGI HNG DN:
PGS.TS. V VN IN
NI THC HIN:
B MN DC HC C TRUYN
H NI - 2014
MC LC
T VN ................................................................................................................................... 1
CHNG I: TNG QUAN ............................................................................................................ 2
1.1. c im chung ca h Th (Ebenaceae) ............................................................................ 2
1.2. Tng quan v chi Diospyros ................................................................................................. 2
1.2.1. V tr phn loi ca chi Diospyros .................................................................................. 2
1.2.2. c im chung ca chi Diospyros ................................................................................ 2
1.2.3. Mt s loi thuc chi Diospyros...................................................................................... 3
1.3. c im ca loi Diospyros decadra Lour. ........................................................................ 6
1.3.1. c im thc vt ............................................................................................................ 6
1.3.2. Phn b, sinh thi ........................................................................................................... 6
1.3.3. B phn dng .................................................................................................................. 6
1.3.4. Thnh phn ha hc ....................................................................................................... 6
1.3.5. Tc dng dc l............................................................................................................ 10
1.3.6. Tnh v, cng nng ........................................................................................................ 11
1.3.7. Cng dng v liu dng ................................................................................................ 11
1.3.8. Cc bi thuc c th ....................................................................................................... 11
CHNG II: I TNG V PHNG PHP NGHIN CU ......................................... 13
2.1. Nguyn vt liu, thit b ...................................................................................................... 13
2.1.1. Nguyn liu .................................................................................................................... 13
2.1.2. Ha cht v dng c ..................................................................................................... 13
2.1.3. Thit b, my mc .......................................................................................................... 14
2.2. Ni dung nghin cu ........................................................................................................... 14
2.2.1 Nghin cu v thc vt .................................................................................................. 14
2.2.2. Nghin cu v ha hc ................................................................................................. 15
2.2.3. Nghin cu v tc dng khng khun ......................................................................... 15
2.3. Phng php nghin cu .................................................................................................... 15
2.3.1. Nghin cu v thc vt ................................................................................................. 15
2.3.2. Nghin cu v ha hc ................................................................................................. 15
2.3.3. Nghin cu v tc dng khng khun ......................................................................... 16
CHNG III. THC NGHIM V KT QU ........................................................................ 17
3.1. Nghin cu v c im thc vt ....................................................................................... 17
3.1.1. c im l th............................................................................................................... 17
3.1.2. Xc inh tn khoa hc ca mu nghin cu ............................................................... 18
3.1.3. c im vi phu l cy th ........................................................................................... 18
3.1.2. c im bt l .............................................................................................................. 19
3.2. Nghin cu thnh phn ha hc l cy Th ...................................................................... 21
3.2.1. nh tnh cc nhm cht .............................................................................................. 21
3.2.2. Chit xut, nh lng, nh tnh cn mt s phn on ............................................ 28
3.3. Nghin cu tc dng khng khun ca dch chit l cy Th.......................................... 36
3.3.1. Chun b ........................................................................................................................ 36
3.3.2. Kt qu ........................................................................................................................... 38
3.4. Bn lun ............................................................................................................................... 39
3.4.1. V thc vt ..................................................................................................................... 39
3.4.2. V ha hc ..................................................................................................................... 39
3.4.3. V tc dng khng khun ............................................................................................. 40
CHNG 4: KT LUN V KIN NGH ................................................................................ 41
4.1. Kt lun ................................................................................................................................ 41
4.1.1. V thc vt .................................................................................................................... 41
4.1.2. V ha hc ..................................................................................................................... 41
4.1.3. V tc dng khng khun ............................................................................................ 41
4.2. Kin ngh. ............................................................................................................................. 41
TI LIU THAM KHO
3
DANH MC CC K HIU, CC CH VIT TT
EtOH: ethanol
EtOAC: ethylacetat
TT: Thuc th
SKLM: Sc k lp mng
D.: Diospyros
VSV: Vi sinh vt
A. formic: acid formic
NC: Nghin cu
DANH MC CC BNG
STT Tn bng Trang
1 Mt s loi thuc chi Diospyros 3
2 Kt qu nh tnh cc nhm cht hu c trong l Th 27
3 Hm lng cn cc phn on theo dc liu kh tuyt i 31
4 Kt qu nh tnh mt s nhm cht trong 3 phn on n-hexan,
chloroform, ethylacetat 31
5 Kt qu th tc dng khng khun ca dch chit cao lng l Th 38
DANH MC CC HNH V, TH
STT Tn hnh nh Trang
1 Cy Th X Tam Cng 17
2 L v cnh mang l Th 17
3 Ht th 17
4 Qu th 17
5 Dc liu kh 17
6 Vi phu gn l Th 19
7 Vi phu phin l 19
8 c im bt l Th 20
9 S chit xut 30
10 Sc k cn dch chit n-hexan di AST, UV254, UV366 32
11 Sc k cn dch chit Chloroform di AST, UV254, UV366 33
12 Sc k cn dch chit Chloroform di AST, UV366, UV366 33
13 Sc k cn dch chit Ethylacetat UV254 vi h dung mi
Toluen - Ethyl acetat - A.formic 34
14 Sc k cn dch chit Ethylacetat UV254 vi h dung mi
Toluen - Ethyl acetat - A.formic 34
15
H sc k ca 3 cn n-hexan, ethylaceta, chloroform trong cng
mt iu kin vi cng mt h dung mi Toluen - Ethyl acetat -
A.formic
35
16 S t mu nghin cu th tc dng khng khun 37
LI CM N
Trong sut qu trnh lm kha lun tt nghip, ti nhn c rt
nhiu s quan tm gip t cc thy, c gio, gia nh v bn b ng
nghip.
Vi lng knh trng v bit n su sc, ti xin chn thnh cm n:
PGS.TS.V Vn in, ngi tn tnh ch bo, to mi iu kin, trc tip
hng dn ti trong sut thi gian thc hin v hon thnh kha lun.
Ti xin chn thnh cm n PGS.TS. Cao Vn Thu v cc anh ch k
thut vin b mn Vi sinh - Sinh hc gip ti trong nghin cu tc dng
khng khun ca l th.
Ti xin chn thnh cm n TS. Nguyn Quc Huy gip ti gim
nh tn khoa hc ca mu nghin cu.
Ti cng xin gi ti li cm n ti ton th cc thy c, cc k thut
vin B mn Dc Hc C Truyn gip v to iu kin cho ti trong
sut qung thi gian ti thc hin nghin cu ti b mn.
Trong phm vi hn ch ca kha lun, nhng kt qu thu c mi ch
l bc u v trong qu trnh lm vic kh trnh khi nhng thiu xt, ti rt
mong nhn c s gp ca cc thy c gio v bn b ng nghip.
H Ni, ngy 14 thng 5 nm 2014
Sinh vin
inh Th Xun
1
T VN
Vit Nam l nc c kh hu nhit i gi ma, vi a hnh a dng, ko di
trn nhiu v . V vy Vit Nam l mt trong 16 quc gia c tnh a dng sinh hc
cao nht th gii vi khong 12.000 loi thc vt bc cao gm 485 loi cy c tinh
du, 473 loi c du bo, 800 loi c tanin, 113 loi c nha thm. Trong s hn
4000 loi thc vt c xc nh c hot tnh sinh hc v c s dng cha
bnh . Mt khc, nhn dn ta c truyn thng lu i s dng cy c lm thuc cha
bnh v bo v sc khe, do tch ly c kho tng tri thc qu gi v nhng
dc liu c ngun gc t nhin. Nhng iu kin trn a Vit Nam tr thnh
mt quc gia c ngun ti nguyn dc liu v cng phong ph [2].
Trong s , nhiu cy thuc c nghin cu v xy dng tiu chun
a vo Dc in Vit Nam. Tuy nhin vn cn c nhng cy thuc hin nay
ang c nhn dn s dng theo kinh nghim dn gian m cha c nghin cu
hoc nghin cu cha y . V vy vic nghin cu cy thuc nhm lm sng t
kinh nghim dn gian v nng cao gi tr s dng ca dc liu l rt cn thit.
Cy th c tn khoa hc l Diospyros decandra Lour, thuc h th
Ebenaceae rt gn gi vi chng ta c trng ri rc trong cc vn gia nh,
nh, cha, miu mo ly qu. Theo kinh nghim dn gian, hu ht cc b phn
ca cy th u c dng lm thuc cha bnh nh cha giun kim tr nh, cha
y bng, to bn, vim tinh hon, mn nhn... Cy th c nhiu b phn dng lm
thuc nh qu, ht, v thn, v r v l, trong mi c cng trnh nc ngoi
nghin cu thnh phn ha hc ca v cy. Tuy nhin, hin nay Vit Nam c t
cng trnh nghin cu v cy th. Vi mc tiu tm hiu kh nng s dng dc liu
ny, chng ti tin hnh thc hin ti:" Nghin cu c im thc vt, thnh
phn ha hc v tc dng khng khun ca v thuc l cy th" vi mt s ni
dung sau:
1. Nghin cu v c im thc vt l th.
2. Nghin cu v thnh phn ha hc ca l th.
3. Th tc dng khng khun ca dch chit l th.
2
CHNG I: TNG QUAN
1.1. c im chung ca h Th (Ebenaceae)
L n thng mc cch, khng c l km. Hoa thng n tnh khc gc,
mu 3 hoc 4 - 5, nh ri nhau, ng s v xen k vi thy trng hoc gp i s
cnh nhng xp thnh hai vng, bao phn i khi m bng l nh, bu thng, 2
- 16 y . H th c trng bi i bn, ng trng theo qu: cnh hoa xp
vn, xp lp hay xp van; qu mng; ht c ni nh sng v thng nhn nheo [1].
Phn b rng nhit i v cn nhit i chu , chu Phi v chu M [1].
1.2. Tng quan v chi Diospyros
1.2.1. V tr phn loi ca chi Diospyros
Chi Diospyros c v tr phn loi nh sau: [7]
Gii thc vt: Plantae
Ngnh Ngc lan: Magnoliophyta
Lp Ngc lan: Magnoliopsida
Phn lp S: Dilleniida.
Lin b quyn: Ericanae
B Th: Ebenale
H Th: Ebenaceae
Chi: Diospyros
1.2.2. c im chung ca chi Diospyros
Cy g hay cy bi. L sm rng, mc so le, l nguyn.
Hoa a tnh hay khc gc, 4 l i tn ti, 4 cnh hoa hp gc, nh 10 - 15
i, bu 4 - 5 . Qu mng c khi n c [6].
Gm khong 475 loi phn b cc vng nhit i, chu c ti 2000
loi. nc ta thng k c 70 loi [6]. Trong ng ch c nhng loi
cho g qu nh Mun (Diospyros mun A.Chev.), qu n c nh hng (Diospyros
kaki L.f) v cy th (Diospyros decandra Lour.) [12].
3
Cc thnh phn chnh c phn lp t cc loi thuc chi Diospyros gm
naphthaquinones, triterpenoids v steroid c tc dng khng khun, khng nm,
antiprotozoal, antimolluscocidal, chng vim v c t bo [13].
1.2.3. Mt s loi thuc chi Diospyros
Bng 1: Mt s loi thuc chi Diospyros [13]
STT Tn loi
Tn thng gi
B
phn
NC
Thnh phn ha hc Tc dng sinh
hc
1 - D.tricolor
Isodiospyrin
Diosquinone Khng khun
2
- D.morrisiana
Hance.
- Th Morris, La
ph th, Th ni
[7].
R
Thn
Isodiospyrin
b-amyrin
Olean-12-en-3-on
Bi-naphthoquinone
c t bo
3 D.leucomelas L
Betuline
Acid Betuline
Acid ursolic
Chng vim
4 D.usambarensis V r
7-methyljuglone
Mamegakinone
Isodiospyrin
Diosindigo A
7-methyluglone
Diosindigo B
Molluscocidal
Khng nm
5 - D.Mollis Griff
- Mcna [8]. Qu
Lupeol
a-amyrine
b-sitosterol
Diospyrol
1,8-dihydroxynaphthalene
4
6
- D.peregrina
Gurcke,
- Th u heo,
Cm th [7].
L
Qu
Triterpenes
Anthrocyanin
Taraxerone
Sitosterol
Acid galic
Peregrinol
H ng
huyt
Khng khun
R
Dihydroflavonol glycoside
5,7,3,5'- Tetra hydroxyl-3'-
methoxy flavone
Antiprotozoal
Khng vi rt
7 D. melanoxlon
Roxb G
-sitosterol terpenoid
Lupeol
Betulin
acid Betulinic
2-methyl-5-methoxy-1,4
naphthaquinone
3-methyl-8-methoxy-
1,9,naphthaquinone
2-methyl-3-hydroxy-5-
methoxy-1,4-
naphthaquinone
2-methyl-5,6-di methoxy-
1,4-
naphthaquinone
L
-sitosterol
monohydroxy
monocarboxylic acid
monohydroxy triterpene
Bauererys acetat
ursolic
5
Betulinic acid
Baurenol
Diospyric acid
obanerenol
Methyl betulinate
8 D. ismailli Ng G ti
coumarin
Ismallin
4-hydroxy-5-methyl
coumarins
9
- D.lotus L
- Th b, Th sen,
cy [7].
Taraxerol,
Isodiospyrin,
7-methyljugulone
acid betulinic
Xallobetulin
10 D. canaculata De
Wild
Coumarin
Ismailin
Canaculation
11 D.chloroxlon
7-methyljuglone
Diospyrin
Isodiospyrin
Xylospyrin
2-methyl-3,6-dihydroxy-
4,5-dimethoxy
naphthalenes
2-methyl-3,4,5,6-tetra
methoxy-naphthalene
6
1.3. c im ca loi Diospyros decadra Lour.
1.3.1. c im thc vt
Tn khoa hc: Diospyros decandra Lour.
Tn Vit Nam: Th, Th mun, Th sp [12]. Th rng, Th mi nh [9].
Cy to, cao 7-10 m, phn cnh nhiu. Cnh non c lng t mm mu hung.
L mc so le, cung ngn, hnh tri xoan - thun, gc hnh nm, u t hi nhn,
hai mt gn nh cng mu [12], gn ph 6 cp, mnh [8].
Cm hoa mc thnh xim k l, ngn hn l nhiu; hoa mu trng; hoa c
c ng di ngn, c lng; hoa tp tnh gm nhng hoa sinh sn gia, hoa khng
sinh sn mt ngoi, tt c u ph nhiu lng mm, bu nhiu non [12].
Qa mng, hnh cu, t trn, to bng qu cam, khi chn m vng, rt thm,
i tn ti nh gm 4 thy cong ln v c lng; ht cng, dt [12].
Ma ra qu thng 6 9 [12].
1.3.2. Phn b, sinh thi
Th c ngun gc vng ng (L Trn Chn et al,1999); c ti liu cho
rng th l c hu ca Vit Nam, Lo, Campuchia (V Vn Chi, 1997), hoc va
thy mc hoang di va c trng nhng nc ny v Thi Lan (Vidal,J.E.,
Martel, G.& Lewitz, S.,1969). Vit Nam, th l cy trng quen thuc, i vo
cc cu chuyn c tch t xa xa. Cy c nhiu vn gia nh, nh, cha cc tnh
ng bng, trung du v vng ni thp min Bc [12].
Th lc nh chu bng, sau tr ln a sng; ra chi v l non trong ma xun
- h; qu chn gia ma thu, ti sinh t nhin ch yu t ht [12].
1.3.3. B phn dng
V r, qu v l th. [14]
V r thu hi quanh nm, phi kh. Qu thu hi thng 8-9 [12].
1.3.4. Thnh phn ha hc
V qu cha mt t tinh du mi gn ging mi ete amyl valerianic.
7
Tht qu th: Theo kt qu phn tch ca Peirier (1932) c 86,20% nc,
0,16% cht bo, 0,67% cht protit, 12% gluxit, 0,33% tanin, 0,47% xenluloza,
0,50% tro. [9]
Tanin trong th thuc loi tanin pyrocatechic, khi b oxy ha th cho cht mu
nu. Peirier cho l hot cht chnh [9].
Cc thnh phn t v cy
Tin hnh sc k ct dch chit chloroform v cy D.decandra thu c 5
hp cht mi, cng vi hp cht bit trc l acid betulinic [14].
- Hp cht 1: acid diospyric A, c cng thc ha hc l 2-oxo-3, 19-
dihydroxy-24-nor-urs-12-en-28-oic acid [14].
(1a) R=R"=H, R'=COCH3 (2-oxo-3-O-acetyl-19-hydroxy-24-nor-urs-12-en-28-
oic acid)
(1b) R=H, R'=COCH3, R"=CH3 (2-oxo-3-O-acetyl-19-hydroxy-24-nor-urs-12-
en-28-oic acid methyl ester)
8
- Hp cht 2: acid diospyric B, c cng thc cu to l 2-oxo-3, 19, 22-
trihydroxy-24-nor-urs-12-en-28-oic acid [14].
2 R=OH, R'=R"=H
(2a) R=OCOCH3, R'=COCH3, R"=H (2-oxo-3, 22-di-O-acetyl-19-hydroxy-
24-nor-urs-12-en-28-oic acid
(2b) R=OCOCH3, R'=COCH3, R"=CH3 (2-oxo-3, 22-di-O-acetyl-19-hydroxy-
24-nor-urs-12-en-28-oic acid methyl ester).
- Hp cht 3: acid diospyric C, c cng thc cu to l 3-oxo- 2, 19,22-
trihydroxy-24-nor-urs-1,4,12-trien-28-oic acid [14].
- Hp cht 4: acid diospyric D, c cng thc cu to l 4-oxo-19, 22-
dihydroxy-3,24-dinor-2,4-seco-urs-12-en-2,28-dioic acid [14].
9
(4) R=H
(4a) R=CH3 (4-oxo-19, 22-dihydroxy-3, 24-dinor-2,4-seco-urs-12-en-2,28-dioic
acid dimethyl ester)
- Hp cht 5: acid diospyric E, cng thc cu to l 19, 22-dihydroxy-24-
nor-2,3-seco-urs-12-en-2,3,28-trioic acid trimethyl ester [14].
Cc hp cht ca acid betulinic 1-4 c th nghim cho thy tc dng
khng vi khun lao, khng nm. Acid betulinic v hp cht 1 cho thy kh nng
khng vi khun lao trung bnh v yu vi MIC25, tng ng 200mg/ml. Acid
betulinic v hp cht 2 cho thy kh nng khng nm Candida albicans vi gi tr
IC50 l 27,2 tng ng vi 42,6mg/ml. Trong c hp cht 1,3 c kh nng gy
c t bo chng li bnh ung th v (BC), ung th mi hng (KB) v dng t bo
phi ngi NCI-H187 mc yu, kh nng chng li dng t bo NCI-H187
vi IC50 l 12,6mg/ml, trong khi cht 1 v 2 khng c tc dng trn dng t bo ny
20mg/ml [14].
10
1.3.5. Tc dng dc l
Tc dng trn giun t invitro: dng 20g bt tht qu th phi kh, tn nh,
chit bng 200ml nc. Kt qu cho thy liu va phi lm giun b t lit,
liu cao lm cht giun v liu cng cao, giun cht cng nhanh, c tc gi cho
rng tc dng ny do tanin glucosidic, dn cht pyrocatechic phlobaphen
[12].
Tc dng trn rut th ti ch: m bng th, bc l rut quan st s co
bp rut bng mt thng. Nc sc l th 1:1 (c 1g l th kh sc ri c
cn 1ml) tim vo tnh mch vnh tai liu 2, 3 v 5 ml/kg thy rut tng co
bp, nht l t trng, trc trng, c bit trc trng, c hin tng cng
phng ln, nhu ng u v tng phn ra ngoi [12].
Tc dng trn rut th c lp: mt on hi trng th c lp theo phng
php Magnus, c nui bng dung dch Tyrode. Nh 5, 8, 10 git nc sc
l th 1:1 vo dch nui s lm tng co bp c v tn s ln bin [12].
Tc dng trn tim mch, h hp: liu nh dch chit l th s lm tng bin
tim, liu cao lm yu tim, lon nhp tim v ngng tim, liu nh t nh
hng ti huyt p, nhng liu cao gy gin mch, h huyt p, h hp hi
tng c v tn s ln bin [12].
Tc dng trn c vn ca b xng: dng c nng chn ch c lp v
phng php Claude bernard trn c th ton vn th u thy l th lm
cho c phn ng nhy hn i vi cc kch thch in trc tip trn c v dy
thn kinh hng ch. Tc dng ny cng ging tc dng ca prostigmin [12].
c tnh: dng nc sc l th kh cho kh, th, chut lang, chut nht trng,
ch ung, tim di da, tim tnh mch hoc tim vo ti bch huyt cho ch
u thy t c, tr khi dng liu cao. V d th, liu nc sc tnh theo
dc liu kh l 16 g/kg tim tnh mch mi thy biu hin c, trong khi
liu c tc dng kch thch lm tng bin tim l 0,4 g/kg [12].
Th lm sng bnh vin Ph Th: nc sc l th 1:1 mi ngy cho ung
10, 20, 30 ml, ng thi ly bng tm nc sc ny p vo rn gy trung
11
tin sau khi m, kt qu rt tt, thi gian trung tin nhanh hn so vi l
chng [12].
1.3.6. Tnh v, cng nng
V r cy th c v ng, tnh hi lnh, c tc dng gii nhit c, tr giun.
Tht qu th c tc dng tr giun, an thn. V qu tiu c, tiu vim, l h
kh, gy trung tin, tiu vim, gim au [12].
1.3.7. Cng dng v liu dng
Tht qu thi c dng an thn v ty giun (nht l giun kim) tr em, hng
ngy n 2-3 qu. V qu phi kh, t thnh than tn bt mn, ha vi du
vng hoc m ln, bi cha rp da cho con gii leo, rn cn, hoc trn vi
than chiu ci v inh hng cha l r hu mn [12].
V r th, ch ly lp mng trng trong (40g), sc ung cha tr nn i, u
mnh nng [12].
V thn cy th, co ly lp t trng trong, gi vi mui, p vo vt
thng lm gim au v rt gai, dm [12].
L th thng hi, gy trung tin, cha ph thng, ngy 30 - 50 g. Dng
ngoi, ly l ti gi p, cha mn nht, vt thng, vt bng la [12].
Campuchia, ngi ta dng qu Th tr bnh mt ng v dng ch thuc
iu kinh [7].
1.3.8. Cc bi thuc c th
Cha l lot, su qung: V r th sc ly nc hoc l th sc c ri ra.
Kt hp ly v thn t thnh than tn mn, rc ln vt lot [12].
Thuc gy trung tin cha trng bng y hi [12].
- L th, thi nh, phi kh, cun vo giy nh iu thuc l, ngy ht 3 ln.
- L th ti khong 100g, gi nt, p mt na vo rn v mt na vo hu
mn.
- L th kh 100g, sc cn li 100ml, cho bnh nhn sau khi m ung mi ln
20 - 30 ml, ngy 2 ln. Kt hp ly bng thm nc sc ny p vo rn.
12
Cha vim tinh hon (Thin try), mn nht mi pht:
L th ti, gi nt, thm t ru, cht ung v b p [12].
Cha d ng: L th 100 g, thn r cy ry 50 g, thi nh, phi kh, nu vi
nc, ri xng [12].
Cha ph thng: L th, l u d, l tru khng v l lc mi mi v 50g,
phi kh, sc ung. Kt hp ly l ti ca 4 th vi lng nh trn, gi nt,
gi bng l chui, nng chn ri rt vo rn, bng li [12].
13
CHNG II: I TNG V PHNG PHP NGHIN CU
2.1. Nguyn vt liu, thit b
2.1.1. Nguyn liu
- L cy th c thu hi ti x Tam Cng, huyn Vnh Bo, t.p Hi Phng.
- Thi gian ly mu: Thng 12 nm 2013.
- X l mu: L th ra sch, loi b cnh, sy kh 60oC. Nghin thnh bt
th v c bo qun trong ti ni lng kn, ni kh ro nghin cu.
2.1.2. Ha cht v dng c
Thuc th, ha cht, dung mi dng trong nghin cu thc vt:
Javen, cloralhydrat, acid acetic 5%, xanh methylen, son phn.
Thuc th, ha cht, dung mi dng trong nghin cu thnh phn ha hc:
- Mg, HCl, TT Mayer, TT Dragendorff, TT Bouchardat, TT diazo mi pha,
FeCl3 5%, genlatin 1%, TT Lugol, TT ninhydrin 3%...
- Ethylacetat, n-hexan, cloroform, ethanol, methanol, toluen, acid formic.
- Thuc th vanilin 1%/H2SO4 5%.
- Bn mng Silicagel F254 trng sn (Merck).
Tt c c mua ti H Ni, tiu chun lm thc nghim theo DVN
IV.
Vi sinh vt kim nh do b mn Vi sinh - Sinh hc trng i hc Dc H
Ni cung cp:
- Vi khun Gram m:
Pseudomonas aeruginosa VM201 (Pseu)
Escherichia coli ATCC25922 (EC)
Proteus mirabilis BV108 (Pro)
Shigella flexneri DT112 (Shi)
Salmonella typhi DT220 (Sal)
- Vi khun Gram dng:
Staphylococcus aureus ATCC 1128 (Sta)
Bacillus pumilus ATCC 10241 (Bp)
14
Bacillus subtilis ATCC 6633 (Bs)
Bacillus cereus ATCC 9946 (Bc)
Sarcina lutea ATCC 1128 (Sar)
Mi trng th nghim
- Mi trng canh thang:
NaCl 0,5%
Pepton 0,5%
Cao tht 0,3%
Nc va 100ml
- Mi trng thch thng:
NaCl 0,5%
Pepton 0,5%
Cao tht 0,3%
Thch 1,6%
Nc va 100ml
Dng c th nghim:
- Bnh gn, bnh cu, cc c m, ng nghin, pipet, ng ong, phin knh,
lam knh,... ca b mn Dc Hc C Truyn.
2.1.3. Thit b, my mc
T sy
Knh hin vi quang hc
Ni cch thy, bp in
My ct quay thu hi dung mi
n t ngoi
Cn k thut, cn phn tch
2.2. Ni dung nghin cu
2.2.1 Nghin cu v thc vt
M t c im hnh thi l v xc nh tn khoa hc ca mu nghin cu.
M t c im bt v vi phu l ca cy th.
15
2.2.2. Nghin cu v ha hc
nh tnh s b cc nhm cht chnh trong l cy th bng cc phn ng ha
hc.
Chit xut, nh lng, nh tnh mt s phn on chnh ca l cy th.
2.2.3. Nghin cu v tc dng khng khun
Th tnh khng khun ca cao lng l Th chit bng Ethanol 70% 3 nng
khc nhau: 1:1, 1:2, 1:4.
2.3. Phng php nghin cu
2.3.1. Nghin cu v thc vt
Quan st trc tip v m t hnh thi l th theo phng php ghi trong ti
liu [2], [5].
Nghin cu c im vi hc theo phng php c ghi trong cc ti liu
[3], [10].
2.3.2. Nghin cu v ha hc
2.3.2.1. nh tnh cc nhm cht hu c trong l cy Th bng cc phn ng
ha hc
nh tnh s b cc nhm cht bng cc thuc th v phn ng c hiu ca
tng nhm cht c ghi trong cc ti liu [3], [4].
2.3.2.2. Chit xut, nh tnh v nh lng cn cc phn on
Xc nh hm m bt dc liu.
Chit xut dc liu bng dung mi EtOH, thu hi EtOH, dung mi cn li
c lc vi cc dung mi c phn cc tng dn n-Hexan, Chloroform,
Ethylacetat. Thu hi dung mi, sy cn n khi lng khng i, cn cn v
tnh hm lng theo dc liu kh tuyt i.
nh tnh cn phn on bng cc phn ng c hiu ca cc nhm cht
chnh c trong dc liu.
Phn tch cn bng SKLM: Kho st trn mt s h dung mi, chn h dung
mi cho kt qu tch tt nht. Dng phn mm videoscan v wincats phn
tch pic v tnh Rf.
16
2.3.3. Nghin cu v tc dng khng khun
Nguyn tc: Th tc dng khng khun tin hnh theo phng php khuch
tn trn thch. Mu th (c cha hot cht th) c t trn lp thch dinh
dng cy VSV kim nh, hot cht t mu th khuch tn vo mi
trng thch s c ch s pht trin ca VSV kim nh to thnh cc vng
v khun. Hot lc ca cht th c so snh vi cht chun theo phng
php thng k.
Thuc th gm cao lng chit bng dung mi ethanol 70% vi 3 nng
1:1, 1:2, 1:4.
17
CHNG III. THC NGHIM V KT QU
3.1. Nghin cu v c im thc vt
Hnh1: Cy Th X Tam Cng Hnh2: L v cnh mang l Th
Hnh 3: Ht Th Hnh 4: Qu Th Hnh 5: Dc liu kh
3.1.1. c im l th
Dc liu ti: L mc so le, cung ngn c lng. Phin l hnh bu dc, gc
t, u hi nhn, mp l nguyn. Gn ph c 6 cp, mnh. Cnh v l non c lng
mu hung c 2 mt, lng mnh v rng khi l gi.
Dc liu kh: mu nu m, nhn nheo, phin l hnh bu dc, mp
nguyn, di 7 - 9 cm, cung ngn. Dc liu dai v nhiu x.
18
3.1.2. Xc inh tn khoa hc ca mu nghin cu
Qua cc c im hnh thi trn, kt hp so snh i chiu vi kha phn
loi, ti liu chuyn su v thc vt, cng vi s gip ca TS. Nguyn Quc
Huy kt lun mu nghin c thu hi x Tam Cng, huyn Vnh Bo, thnh
ph Hi Phng c tn khoa hc l Diospyros decandra Lour, thuc h th
Ebenaceae.
3.1.3. c im vi phu l cy th
Tin hnh:
Ct vi phu l Th bng my ct cm tay, ty v nhum theo phng php
nhm kp vi cc thuc nhum xanh methylen, son phn. Chn mt ct
ngang ca l mang y c im dc liu, v tr ct cch cung l 1/3
chiu di ca l. Quan st di knh hin vi m t c im vi phu v
chp li bng my nh k thut s.
Phn gn l
Gn l li c 2 mt trn v di, gn di li nhiu hn. Biu b trn (8) v
biu b di (1) cu to l mt lp t bo trn, tng i nh, xp u n.
St lp biu b trn v di l lp m dy (2) gm 4-5 hng t bo hnh trn,
thnh dy. M mm (3) gm nhng t bo hnh trng, thnh mng, kch
thc ln, khng u nhau, xp ln xn. Nm ri rc trong m mm c cc
tinh th calci oxalat hnh a gic (7). gia gn l l mt b libe-g to hnh
cung bao bc bi vng si (4). Libe (5) xp thnh vng bao quanh g, g (6)
cu to bi nhng mch g ln xp tp trung thnh b.
Phn phin l
Biu b trn (8') v biu b di (1') l mt hng t bo nh hnh ch nht xp
u n. Di lp biu b l hng m giu (9) cu to bi mt lp t bo
hnh ch nht xp thng ng vung gc vi b mt l, m mm (3') l cc t
bo thnh mng, kch thc ln.
19
Hnh 6: Vi phu l Th
1, 1': Biu b di 6: G
2: M dy 7: tinh th calci oxalat
3, 3': M mm 8, 8': Biu b trn
4: Si 9: M giu
5: Libe
3.1.2. c im bt l
Tin hnh:
Nghin cu c im bt: L cy th c sy kh, em tn thnh bt mn
bng my xay c lm sch v sy kh, ry qua ry to bt mn, ln tiu
bn. Soi tm c im ca bt qua knh hin vi v chp li bng my nh k
thut s.
Kt qu
20
Bt mu xanh nht. Soi di knh hin vi thy cc c im: mnh m mm
(1), lng che ch n bo (8), mnh biu b mang l kh v cc mnh mang
mu (2), mnh mch xon (7), mng mch cha mch dn (5), tinh th calci
oxalat hnh a gic (4), mnh mch (1), mnh mang mu (6), si (3), mnh
m mm cha tinh th calcioxalat (9).
Hnh 8: c im bt l Th
1: M mm 6: Mnh mang mu
2 Mnh biu b mang l kh 7: Mnh mch xon
3: si 8: Lng che ch n bo
4: Tinh th calci oxalat 9: M mm
5: Mng mch cha mch dn
21
3.2. Nghin cu thnh phn ha hc l cy Th
3.2.1. nh tnh cc nhm cht
nh tnh Glycosid tim
Cn 10g dc liu tn nh cho vo mt bnh nn dung tch 250ml. Thm
100ml EtOH 25% ri ngm trong 24 gi. Gn dch chit vo cc c m dung tch
100ml. Thm vo dch chit 3ml ch acetat 30%, khuy u. Lc qua giy lc gp
np vo 1 cc c m dung tch 100ml. Nh vi git dch lc u tin vo mt ng
nghim, thm mt git ch acetat. Nu xut hin ta th ngng lc, thm khong
1ml ch acetat 30% vo dch chit, khuy u, lc li v tip tc th n khi dch
lc khng cn ta vi ch acetat.
Chuyn ton b dch lc vo bnh gn dung tch 100ml. Chit glycosid tim
bng cch lc vi chloroform - EtOH (4:1) 2 ln, mi ln 8ml. Gn lp chloroform
vo mt cc c m c sy kh. Gp cc dch chit chloroform v loi nc
bng natri sulfat khan.
Chia u dch chit vo 4 ng nghim nh c sy kh. t cc ng
nghim ln gi v bc hi trn ni cch thy n kh. Cn thu c em tin hnh
lm cc phn ng nh tnh.
Phn ng Liebermann - Burdchardat
Cho vo ng nghim c cha cn glycosid tim 1ml anhydrid acetic, lc u
cho tan ht cn. Nghing ng 45o. Cho t t thnh ng 0,5ml acid sulfuric
c, trnh xo trn cht lng trong ng.
Kt qu: mt tip xc gia 2 lp cht lng khng xut hin vng mu tm
(Phn ng m tnh)
Phn ng Baljet
Pha thuc th Baljet: cho vo ng nghim to 1 phn dung dch acid picric
1% v 9 phn dung dch NaOH 10%, lc u.
22
Cho vo ng nghim c cha cn glycosid tim 0,5ml EtOH 90%. Lc u
cho tan ht cn. Nh tng git thuc th Baljet cho n khi xut hin mu
da cam. So snh mu vi ng chng l ng khng c cn glycosid tim.
Kt qu: ng th c mu cam khng m hn ng chng (phn ng m
tnh).
Phn ng Legal
Cho vo ng nghim c cha cn glycosid tim 0,5ml EtOH 90%. Lc u
cho tan ht cn. Nh tng 1 git thuc th Natri nitroprussiat 0,5% v 2 git
dung dch NaOH 10%. Lc u s xut hin mu cam. So snh mu vi
ng chng l ng khng c cn glycosid tim.
Kt qu: ng th c mu cam khng m hn ng chng (Phn ng m
tnh).
Nhn xt: L cy th khng cha Glycosid tim.
nh tnh Flavonoid
Cho 2g bt dc liu vo bnh nn c dung tch 50ml, thm 20ml EtOH
90%. un cch thy si 5 pht. Lc nng, dch lc thu c dng lm cc
phn ng.
Phn ng Cyanidin (Phn ng Shinoda)
Cho vo ng nghim nh 1ml dch chit. Thm mt t bt Magnesi kim loi
(khong 10mg). Nh tng git HCl m c (3-5 git). yn mt vi pht.
Kt qu: Dung dch chuyn t mu vng sang mu (Phn ng dng
tnh).
Phn ng vi kim
Cho vo ng nghim 1 ml dch chit. Thm vi git dung dch NaOH 10%.
Kt qu: xut hin kt ta vng.
Thm 1ml nc ct
Kt qu: Ta tan v mu vng ca dung dch tng ln (Phn ng dng tnh).
Phn ng vi FeCl3
23
Cho vo ng nghim 1ml dch chit EtOH 90%. Thm 2-3 git dung dch
FeCl3 5%
Kt qu: Xut hin kt ta xanh en (Phn ng dng tnh).
Nhn xt: L cy Th c cha Flavonoid.
nh tnh Coumarin
Cho 2g bt dc liu vo bnh nn c dung tch 50ml, thm 20ml EtOH
90%. un cch thy si 5 pht. Lc nng, dch lc thu c dng lm cc
phn ng.
Phn ng m ng vng lacton
Cho vo 2 ng nghim mi ng 1ml dch chit.
ng 1: thm 0,5 ml dung dch NaOH 10%,
ng 2: nguyn.
un 2 ng nghim n si. ngui ri quan st.
ng 1: c mu vng hoc ta c mu vng
ng 2: trong
Thm vo 2 ng nghim mi ng 2ml nc ct. Lc u ri quan st.
ng 1: trong sut
ng 2: c ta c
Acid ha ng 1 bng vi git HCl c, ng 1 s tr li c nh ng 2
Kt qu: phn ng m tnh.
Phn ng diazo ha
Cho vo ng nghim nh 1ml dch chit. Thm vo 2ml dung dch NaOH
10%. un cch thy n si ri ngui. Nh vi git thuc th Diazo
Kt qu: Khng c ta mu gch (phn ng m tnh)
Quan st hunh quang ca cc vt coumarin di nh sng t ngoi khi
tc dng vi dung dch kim (phn ng chuyn t ng phn dng cis sang
ng phn dng trans di tc dng ca tia t ngoi).
Nh vi git dch chit ln giy thm. Nh tip vi git dung dch NaOH 5%.
Sy nh, che mt phn din tch dch chit ln giy lc bng mt ng xu
24
kim loi ri chiu tia t ngoi trong mt vi pht. B ming kim loi ra, quan
st hunh quang di n t ngoi trong mt vi pht.
Kt qu: phn khng b che khng sng hn phn b che (phn ng m tnh).
Nhn xt: L cy Th khng cha Coumarin.
nh tnh tannin
Ly khong 1g bt dc liu cho vo bnh nn dung tch 50ml, thm 20ml
nc ct, un si trong 2 pht. ngui, lc, dch lc dng nh tnh.
Phn ng vi gelatin 1%
Ly 2ml dch lc, thm 5 git dung dch gelatin 1%
Kt qu: thy xut hin kt ta bng trng (phn ng dng tnh).
Phn ng vi ch acetat 10%
Ly 2ml dch lc, thm 2 git ch acetat 10% (TT).
Kt qu: thy xut hin kt ta bng (phn ng dng tnh)
Phn ng vi FeCl3 5%
Ly 2ml dch lc, thm 2 git dung dch FeCl3 5% (TT)
Kt qu: Thy xut hin ta xanh en (phn ng dng tnh)
Nhn xt: L cy Th cha tannin.
nh tnh Saponin
Quan st hin tng to bt
Cho 0,5g bt dc liu vo ng nghim ln, thm 10ml nc ct. Lc mnh
trong 5 pht theo chiu dc ng nghim, yn.
Kt qu: Ct bt khng bn sau khi ngng lc trong 15 pht (phn ng m
tnh).
Phn ng Salkowski:
Cho vo ng nghim to 2g dc liu, thm vo 20ml EtOH 90%, un si
cch thy. Lc ly dch lc cho vo ng nghim khc, nghing ng
nghim 450, cho t t theo thnh ng nghim 1 - 2 git H2SO4 c.
Kt qu: mt phn cch gia hai lp cht lng khng xut hin vng
nu (phn ng m tnh).
25
Nhn xt: L cy th khng cha Saponin.
nh tnh Alcaloid
Cn 0,5g bt dc liu, cho vo bnh nn dung tch 50ml. Thm 15ml dung
dch acid sulfuric 1N. un n si, ngui, lc dch lc vo bnh gn dung
tch 125ml. Kim ha dch lc bng dung dch amoniac 6N m pH=9 - 10.
Chit alcaloid base bng cloroform (chit 3 ln, mi ln 5ml). Gp cc dch
chit chloroform, loi nc bng Natrisulfat khan, dch chit chloroform em
lm phn ng nh tnh. Ly mt phn dch chit chloroform c chun
b trn em lc vi acid sulfuric 1N hai ln, mi ln 5ml. Gp cc dch
chit nc. Chia u vo 4 ng nghim nh, mi ng 1ml. Nh vo tng ng
nghim 2 - 3 git ln lt cc thuc th sau:
ng 1: thuc th Mayer
Kt qu: Xut hin kt ta trng (phn ng dng tnh)
ng 2: thuc th Bouchardat
Kt qu: Xut hin ta nu (phn ng dng tnh).
ng 3: thuc th Dragendorff
Kt qu: Xut hin ta vng (phn ng dng tnh).
Nhn xt: L cy Th c cha Alcaloid
nh tnh Anthranoid
Phn ng Borntrager: nh tnh anthranoid ton phn
Ly 3 g bt dc liu cho vo bnh nn 100ml. Thm 40ml dung dch
H2SO4 1N. un trc tip trn ngun nhit n soi khong 15 pht. Lc dch
chit cn nng qua giy lc vo bnh gn dung tch 125ml. Lm ngui dch
lc, thm 5ml chloroform, lc nh. Gn b lp nc, gi lp chloroform
lm phn ng.
Ly 1ml dch chit chloroform, cho vo ng nghim nh. Thm 1 ml dung
dch NaOH 10%, lc nh.
Kt qu: Lp nc khng xut hin mu . (Phn ng m tnh).
Vi thng hoa:
26
Rc mt t bt dc liu thnh lp mng trong mt np chia bng nhm,
t nh trn n cn loi nc. Sau y np nhm bng mt lam
knh, bn trn c ming bng thm nc, tip tc un nng trong khong
5 - 10 pht. Ly lam knh ra ngui, soi di knh hin vi.
Kt qu: khng thy tinh th hnh kim (Phn ng m tnh).
Nhn xt: L cy Th khng cha Anthranoid.
nh tnh acid hu c, acid amin, ng kh, polysaccarid
Cho 10g bt dc liu vo bnh nn, thm 50ml nc ct. un si 10 pht
trn ngn la n cn, lc bng giy lc gp np, dch lc lm cc phn
ng:
nh tnh acid hu c
Ly khong 2ml dch chit, thm vo dch lc mt t tinh th Na2CO3
Kt qu: Khng thy c bt kh bay ln (phn ng m tnh)
Nhn xt: L cy Th khng cha acid hu c
nh tnh acid amin
Ly 2ml dch chit cho vo ng nghim khc, thm vo 3 git thuc th
Ninhydrin 3%, un si cch thy 10 pht.
Kt qu: khng thy dung dch chuyn sang mu tm. (phn ng m tnh)
Nhn xt: L cy Thi khng cha acid amin
nh tnh ng kh
Cho 2ml dch lc vo ng nghim, thm 3 git thuc th Fehling A v 3
git thuc th Fehling B. un si cch thy 10 pht.
Kt qu: C ta gch di y ng nghim.(phn ng dng tnh)
Nhn xt: L cy Th c cha ng kh.
nh tnh Polysaccharid
Ly 10 ml dch chit cho vo 2 ng nghim:
ng 1: 2ml dch chit + 5 git dung dch Lugol
ng 2: 2m nc ct + 5 git dung dch Lugol
Kt qu: mu ng 1 khng m hn mu ng 2 (phn ng m tnh)
27
Nhn xt: L cy Th khng cha Polysaccharid
nh tnh sterol, cht bo, caroten
Ngm 10g bt dc liu trong bnh nn bng ether du ha, qua m.
Lc thu c dch lc lm cc phn ng.
nh tnh cht bo
Nh 2 git dch chit ether du ha ln giy lc, h nng cho bay ht dung
mi.
Kt qu: Khng c vt c m trn giy lc (phn ng m tnh)
Nhn xt: L cy Th khng cha cht bo
nh tnh sterol (Phn ng Liberman)
Ly 1ml dch chit ether du ha, cho vo ng nghim, bc hi dung mi
n kh. Thm vo ng nghim 1ml anhydridacetic, lc k. nghing 45o,
Nh t t acid sulfuric c theo thnh ng nghim.
Kt qu: khng thy xut hin vng mu tm mt phn cch
Nhn xt: L cy th khng cha Sterol.
nh tnh caroten
Cho vo ng nghim 2ml dch chit ether du ha, bc hi cch thy n
cn. Thm 2 git H2SO4 c vo cn.
Kt qu: khng thy xut hin mu xanh.
Nhn xt: L cy th khng cha caroten.
Kt qu nh tnh cc nhm cht trong l cy Th c trnh by bng:
Bng 2: Kt qu nh tnh cc nhm cht trong l Th
STT Nhm cht Tn phn ng Kt qu Kt lun
1
Alcaloid
Phn ng vi TT Mayer (+)
Khng c
Phn ng vi TT Dragendorff (+)
Phn ng vi TT Bouchadart (+)
2
Glycosid tim
Phn ng Liebermann- Budchardt (-) Khng c
Phn ng Legal (-)
28
Phn ng Baljet (-)
3
Flavonoid
Phn ng vi dung dch kim (+)
C Phn ng vi dd FeCl3 5% (+)
Phn ng Cyanidin (+)
4 Anthranoid Phn ng Borntraeger (-) Khng c
5 Courmarin Phn ng m, ng vng Lacton (-) Khng
C Quan st hunh quang (-)
6 Saponin Quan st hin tng to bt (-) Khng c
7
Tanin
Phn ng vi dd FeCl3 5% (+ )
C Phn ng vi dd Gelatin 1% (+)
Phn ng vi dd ch acetat 10% (+)
8 Acid hu c Phn ng vi Na2CO3 (-) Khng c
9 Acid amin Phn ng vi TT Ninhydrin (-) Khng c
10 ng kh Phn ng vi TT Fehling (+) C
11 Polysaccharid Phn ng vi TT Lugol (-) Khng c
12 Cht bo To vt m trn giy (-) Khng c
13 Sterol Phn ng Liebermann (-) Khng c
14 Caroten Phn ng vi H2SO4 c (-) Khng c
T kt qu nh tnh s b bng phn ng ha hc trnh by bng cho thy
l cy Thi cha cc nhm cht: Flavonoid, alcaloid, tanin, ng kh.
3.2.2. Chit xut, nh lng, nh tnh cn mt s phn on
3.2.2.1. Xc nh m dc liu
Ly khong 1 g bt dc liu xc nh m. Bt my o m, iu
chnh nhit 1100C. dc liu ln a cn v tri ln mt a, y a cn v
i my t ng hin kt qu ln mn hnh, sau 10 pht c kt qu. m ca
dc liu l 8,72%.
29
3.2.2.2 Chit xut
Chun b bt dc liu: l c sy kh 600C, tn thnh bt th ng thi
xc nh hm m. Cn 100,0g bt dc liu.
Dng c chit: Dng bnh gn 500ml c kha kn, c ra sch, sy kh.
Dung mi chit: ethanol 70%, n-hexan, chloroform, ethylacetat.
Chit bng ethanol 70% theo phng php ngm lnh. Lm m dc liu,
dc liu trng n trong 2 - 3 gi, thm dung mi cho ngp dc liu,
ngm dc liu trong 24 gi. Rt dch chit v b sung dch mi. Chit cho
n khi dch khng cn mu xanh. Gp cc dch chit li, ct thu hi dung
mi, dch chit nc cn li lnh 12 gi, em li tm loi b ta. Dch sau
li tm c chit vi cc dung mi c phn cc tng dn.
Chit phn on n-hexan: Cho dch chit vo bnh gn, thm n-hexan vi
lng bng 2/3 dch chit, lc k, phn lp, gn ly lp n-hexan, thm
dung mi vo chit tip, chit nhiu ln cho n khi lp n-hexan khng c
mu. Gp dch chit n-hexan, thu hi dung mi c cn. em cn i sy
n khi khi lng khng i. Cn cn thu c.
Chit cc phn on chloroform v ethylacetat tng t nh phn on n-
hexan, thu c cn chlorform v ethylacetat.
Cn thu c tnh hm lng cc cht trong phn on v nh tnh bng
sc k lp mng. Cng thc tnh hm lng cc cht trong phn on:
=
(100 ) 10
Trong : X: l hm lng cht (%)
a: khi lng cn (g)
M: khi lng dc liu em cn (g)
x: m ca dc liu (%)
Quy trnh chit xut cc phn on c tm tt s hnh 9.
Hm lng cn cc phn on c ghi bng 3.
30
Hnh9: S chit xut cc phn on
Chloroform
Dch chit chloroform Dch chit nc
Chloroform thu hi
Cn CHCl3
Ethyl acetat
Dch chit Ethyl acetat Dch chit nc
Erthyl acetat thu hi
Cn EtOAc
Dch chit ethanol
Dch chit nc
Dch chit N-hexan
Ethanol 70%
Ethanol thu hi
n-hexan
Dch chit nc
n-hexan thu hi
Bt dc liu
Cn n-hexan
- t lnh qua m
- Li tm loi b cn
31
Bng3: Hm lng cn cc phn on theo dc liu kh tuyt i
STT Phn on Khi lng cn (g) % so vi nguyn liu kh
1 n-hexan 0,1676 0,18
2 Chloroform 1.1652 1,28
3 Ethyl acetat 2.0688 2,27
Nhn xt: Trong cc phn on dch chit l cy Th, khi lng kt ta
Ethyl acetat cao nht (2,27%). Khi lng phn on n-hexan l t nht (0,18%).
3.2.2.3. nh tnh cn cc phn on bng phn ng ha hc.
Xc nh s c mt ca mt s nhm cht trong phn on n-hexan,
chloroform, ethyl acetat bng phn ng ha hc. Kt qu th hin bng sau:
Bng 4: Kt qu nh tnh mt s nhm cht trong 3 phn on n-hexan,
chloroform, ethylacetat
STT
Nhm
cht nh
tnh
Phn ng nh tnh Cn n-
hexan
Cn
chloroform Cn
ethylacetat
1 Flavonoid
-P. Cyanidin
-P. vi NaOH
-P. vi dd FeCl35%
-
-
-
+
+
+
+
+
+
2 Tanin
-P. vi dd FeCl3 5%
-P. vi dd ch acetat
10%
-P. vi dd gelatin 1%
-
-
-
+
+
+
+
+
+
3 ng
kh -P. vi TT Fehling -
+
+
4 Alcaloid
-P. vi TT Mayer
-P. vi TT
Bouchardat
-P. vi Dragendroff
-
-
-
+
+
+
+
+
+
32
3.2.2.4. nh tnh cn cc phn on bng SKLM
Chun b dch chit chm sc k: Cn thu c mi phn on em ha tan
vi methanol chm sc k.
Chm sc k trn bn mng Silicagel F254 (Merck), hot ha 110oC trong
1 gi.
Tin hnh SKLM trn cc h dung mi thm d:
H I: Chloroform - Methanol (9:1)
H II: Chloroform - Methanol (4:1)
H III: Chloroform - Ether (3:7)
H IV: Ether - Ethyl acetat (7:3)
H V: Toluen - Ethyl acetat- Acid formic (6:2:1)
H VI: Toluen - Ethyl acetat- Acid formic (5:4:1)
Quan st vt nh sng t ngoi (UV254, UV366) v phun thuc th hin
mu. Kt qu cho thy h V tch tt nht vi c dch chit 3 phn on.
Cn phn on n - hexan
A B
Hnh 10: Sc k cn dch chit n - hexan di UV254, UV366 vi h dung mi
Toluen - Ethyl acetat - A.formic (6:2:1)
A: Sc k di UV254 B: Sc k di UV366
33
Nhn xt:
Ti UV254 quan st c 7 vt, trong vt th 7 (Rf = 0,88) c mu r nht
Ti UV366 quan st c 13 vt, trong cc vt th 3, 5 c Rf ln lt l
0,14; 0,26 cho hunh quang sng r nht.
Cn phn on Chloroform
Hnh 11: Sc k cn dch chit Chloroform di UV254 vi h dung mi
Toluen - Ethyl acetat - A.formic (6:2:1)
Nhn xt:
Ti UV254 cc vt tch r, ta quan st c 12 vt. Trong vt th 3, 6, 8,
12 c gi tr Rf ln lt l 0,16; 0,42; 0,59; 0,88 c mu r nht.
Hnh 12: Sc k cn dch chit Chloroform di UV366 vi h dung mi
Toluen - Ethyl acetat - A.formic (6:2:1)
34
Nhn xt:
Ti UV366: Quan st c 13 vt, trong vt th 4, 6, 7, 8, 11 c gi tr Rf
ln lt l 0,20; 0,31; 0,35; 0,39; 0,68 cho hunh quang sng r nht.
Cn phn on Ethylacetat
Hnh 13: Sc k cn dch chit Ethylacetat UV254 vi h dung mi Toluen -
Ethyl acetat - A.formic (6:2:1)
Nhn xt: Ti UV254 quan st c 9 vt, trong vt th 1, 4, 6, 9 c gi tr Rf ln
lt l 0,05; 0,29; 0,42; 0,88 c mu r nht.
Hnh 14: Sc k cn dch chit Ethylacetat UV254 vi h dung mi Toluen -
Ethyl acetat - A.formic (6:2:1)
35
Nhn xt: Ti UV366 quan st c 11 vt, trong vt th 3, 5, 9 c gi tr Rf ln
lt l 0,18; 0,32; 0,67 cho hunh quang sng r nht.
Khai trin h sc k ca 3 cn trong cng mt iu kin vi cng mt h
dung mi Toluen - Ethyl acetat - A.formic (6:2:1)
Hnh 15: H sc k ca 3 cn n-hexan, ethylaceta, chloroform trong cng mt
iu kin vi cng mt h dung mi Toluen - Ethyl acetat - A.formic (6:2:1)
A: Sau khi nhng thuc th vanilin/H2SO4 H: Cn N-hexan
B: Sc k UV254 C: Cn chloroform
C: Sc k UV366 E: Cn ethylacetat
Nhn xt:
UV254 sc k ca c 3 cn c 3 vt tng ng vi m nht khc
nhau.
UV366 sc k ca c 3 cn c 6 vt tng ng nhau. Sc k cn C
v E c 10 vt tng ng.
Sau khi phun thuc th hin mu sc k ca c 3 cn c 6 vt tng
ng.
36
3.3. Nghin cu tc dng khng khun ca dch chit l cy Th
Tc dng khng vi sinh vt c th bng phng php khuch tn, o vng
v khun bng thc panmer c chnh xc 0,02 mm trn a thch, c thc
hin ti b mn Vi sinh - Sinh hc Trng i hc Dc H Ni.
3.3.1. Chun b
a. Chng vi sinh vt kim nh do b mn Vi sinh - Sinh hc Trng i hc Dc
H Ni cung cp:
b. Mi trng th nghim
- Mi trng canh thang nui cy vi khun kim nh.
- Mi trng thch thng.
c. Tin hnh
Chun b mu th:
Mu th dng cao lng 1:1 c nh s 1
Mu th dng cao lng 1:2 c nh s 2
Mu th dng cao lng 1:3 c nh s 3
Khng sinh chun i vi vi khun Gr(+) th s dng dung dch
Benzathin Penicillin G: 28,8 IU/ml, vi cc vi khun Gr(-) th s dng
ding dch Gentamicin 20 g Gentamicin base/ml.
Cc khoanh giy v trng v c sy kh c tm 3 ln vi dung
dch mu th, sau mi ln tm cc khoanh giy lc c cha mu th
u c sy nhit 500C n kh ht dung mi.
Chun b mi trng v cy vi sinh vt kim nh.
Vi sinh vt kim nh c cy vo mi trng canh thang, ri c
nui cy v cho pht trin trong t m 370C trong thi gian t 18-24 gi
n nng 107 t bo/ml (kim tra bng pha long v dy dch chun).
Mi trng thch thng v trng (tit trng 1180C/30 pht c lm
ngui 40-500C v c cy ging vi sinh vt kim nh vi t l 2,5
ml/100ml lc trn vi sinh vt kim nh phn tn u trong mi
37
trng thch thng, ri vo a petri v trng vi th tch 20ml/a
v cho thch ng li.
Th tnh khng khun
Mi vi khun lm trn 5 a petri
t khoanh giy lc: Khoanh giy lc c tm cht th v x l
nh trn, c t trn b mt mi trng thch thng, cha vi sinh
vt kim nh theo s sau:
Hnh 16: S t mu nghin cu th tc dng khng khun
Trong :
K: Khoanh giy tm khng sinh
1: Khoanh giy tm dch chit cao lng 1:1 l Th
2: Khoanh giy tm dch chit cao lng 1:2 l Th
3: Khoanh giy tm dch chit cao lng 1:3 l Th
Sau khi cy, cc a petri c mu th c t nh trn trong t m c
nhit 370C trong 18-24 gi, khi thi gian ly ra c kt qu, o ng knh
vng v khun nu c ( thc kp panmer o chnh xc 0,02mm).
nh gi kt qu:
o ng knh vng v khun bng thc kp Panmer c chnh xc n
0,02 mm. nh gi ng knh vng v khun c x l theo cng thc:
1
1
3 2
2 3
K
38
=
s=
()
D: ng knh trung bnh vng v khun
Di: ng knh vng v khun th i
s: lch thc nghim chun c hiu chnh
n: S th nghim lm song song
3.3.2. Kt qu
Bng 5: Kt qu th tc dng khng khun ca dch chit cao lng l Th
STT
Mu
kim
nh
Vi sinh vt kim nh- ng knh vng v khun [mm] (D,s)
EC Pro Shi Sal Pseu
SA Bp Bs Bc Sar
1 Pen - - - - - 11.90
0.10
11.55
0.55
12.05
0.05
11.49
0.09
23.05
0.05
2 Gen 15.90
0.10
14.80
0.30
14.9
0.10
11.30
0.10
10.75
0.25 - - - - -
3
Cao
lng
1:1
- 6.49
0.07
7.12
0.11
10.94
1.05
9.75
0.31 - - - - -
4
Cao
lng
1:2
- - - - - - - - - 8.53
0.24
5
Cao
lng
1:3
- - - - - - - - - -
Ch thch
Gr (-) Gr (+)
EC: Escherichia coli ATCC25922 Sta: Staphylococcus aureus ATCC 1128
Pro: Proteus mirabilis BV108 Bp: Bacillus pumilus ATCC 10241
39
Shi: Shigella flexneri DT 112 Bs: Bacillus subtilis ATCC 6633
Sal: Salmonella typhi DT 220 Bc: Bacillus cereus ATCC 9946
Pseu: Pseudomonas aeruginosa VM201 Sar: Sarcina lutea ATCC 1128
Nhn xt:
Trong 3 mu th, ch c mu 1 c kh nng khng khun v ch tc dng trn vi
khun Gr(-), trong c Pseudomonas aeruginosa.
3.4. Bn lun
3.4.1. V thc vt
Mu nghin cu l l cy Th c thu hi ti huyn Vnh Bo- Hi Phng,
c TS. Nguyn Quc Huy xc nhn tn khoa hc l Diospyros decandra Lour,
h th Ebenaceae. ti tin hnh nghin cu c im bt v vi phu l cy
Th. L th khi cn non c lng mu hung c 2 mt l v gn chnh, nhng lng
mnh v rt d rng khi vut nh, khi l trng thnh th lng khng cn na,
chng ti tin hnh ct vi phu trn l cy trng thnh nn khng thy lng vi
phu. Tuy nhin cung l th trng thnh c lng nn trong bt l th vn cha
lng che ch n bo.
3.4.2. V ha hc
Cn phn on n-hexan c mu xanh nht, khi lng rt nh. Kt qu nh
tnh bng phn ng ha hc u cho kt qu m tnh vi cc nhm cht. Kt qu sc
k lp mng cho thy phn on ny c nhiu chlorophyll (c hunh quang
bc sng t ngoi =366 nm), cc vt cn li rt m. C th hm lng cc thnh
phn khc trong dch chit n-hexan rt thp nn c kt qu m tnh vi cc phn
ng ha hc hoc do chlorophyll lm che m cc phn ng ha hc. Hn na,
flavonoid trong dc liu tn ti c hai dng t do v kt hp. Dng t do tan
trong dung mi km phn cc, dng kt hp tan trong dung mi phn cc. Kt qu
nh tnh cn n-hexan c th gii thch l dng t do trong dc liu thp nn lng
flavonoid trong cn n-hexan rt thp nn cho kt qu m tnh.
Cn phn on chloroform c mu hi vng. Kt qu nh tnh bng phn
ng ha hc u dng tnh vi cc nhm cht c kho st trong dch chit
ton phn. Sc k lp mng cho kt qu tch tt vi h dng mi Toluen-
40
Ethylacetat-Acid formic (6:2:1). Cc vt tch nhau v cho mu r v m. Hin
mu bng thuc th valinin/H2SO4 cho di nhiu mu.
Cn phn on ethylacetat c mu vng m, khi lng cn ln nht. Kt
qu nh tnh bng phn ng ha hc u dng tnh vi cc nhm cht c
kho st trong dch chit ton phn. c bit cho hin tng rt r vi phn ng
Cyanidin (phn ng c trng ca nhm cht Flavonoid). Sc k lp mng cho kt
qu tch tt, cc vt tch nhau v cho mu kh r. Hin mu bng thuc th dd
KOH cho cc vt mu vng m (c th l ca flavonoid). C th trong phn on
ny cha nhiu flavonoid.
3.4.3. V tc dng khng khun
Kt qu th tc dng khng khun cho thy dch chit cao lng 1:1 ca l
Th c kh nng khng khun i vi mt s vi khun, tuy nhin tc dng khng
khun cn yu. Dch chit cao lng 1:2 v 1:4 th khng cho kt qu khng khun
vi bt k chng vi khun th nghim no. iu ny c th l do hm lng cc
cht c tc dng khng khun trong dch chit cao lng qu t nn cha to ra cc
vng khng khun. Kin ngh nn th tc dng khng khun vi dch chit tng
phn on chloroform v ethylacetat.
Cc kt qu ca ti ch l kt qu bc u, to c s cho nhiu nghin
cu su hn v l cy Th, phc v cho lnh vc kim nghim sau ny.
41
CHNG 4: KT LUN V KIN NGH
4.1. Kt lun
Sau khi thc hin ti "Nghin cu c im thc vt, thnh phn ha
hc v tc dng khng khun ca v thuc l cy th" chng ti c cc kt lun
sau:
4.1.1. V thc vt
m t hnh thi ca mu nghin cu v xc nh tn khoa hc ca cy l
Diospyros decandra Lour, h Th (Ebenaceae).
m t c c im vi phu v bt l cy Th.
4.1.2. V ha hc
nh lng cn cc phn on theo dc liu kh tuyt i.
Bng cc phn ng ha hc xc nh c l cy Th c cha cc thnh
phn l Flavonoid, Tanin, alcaloid, ng kh. Trong phn on Chloroform v
ethylacetat ca l cy Th c cha Flavonoid, tanin, alcaloid, ng kh.
Bng phng php sc k lp mng xc nh c c 3 phn on dch
chit n-hexan, chloroform, ethylacetat u cho kt qu tch tt vi h dung mi
Toluen-ethylacetat-acid formic (6:2:1).
Phn on n-hexan cho 7 vt UV254, 13 vt UV366.
Phn on chloroform cho 12 vt UV254, 13 vt UV366.
Phn on ethylacetat cho 9 vt UV254, 11 vt UV366.
4.1.3. V tc dng khng khun
Dch chit cao 1:1 c tc dng khng khun trn mt s chng vi khun Gr-.
4.2. Kin ngh.
Tip tc nghin cu su hn khng nh kh nng s dng ca l th nh:
Th mt s tc dng sinh hc lin quan n cng dng dng trong dn gian
ca l th.
Xc nh thnh phn, hm lng v phn lp cc flavonoid trong l th c
hng khai thc.
TI LIU THAM KHO
Ting vit
1. Nguyn Tin Bn (1997), Cm nang tra cu v nhn bit cc h thc vt ht
kn Vit Nam. NXB Nng Nghip, trang 28.
2. L nh Bch, Trn Vn n (2007), Thc vt hc, NXB Y hc.
3. B mn dc liu (2005), Thc tp dc liu, Trung tm thng tin th vin
trng i hc Dc H Ni.
4. B mn Dc liu - Trng i hc Dc H Ni (2006), Bi ging dc
liu tp I, II, NXB Y hc.
5. B mn Thc vt - Trng i hc Dc H Ni (2012). Thc tp thc vt
v nhn bit cy thuc, Trung tm thng tin - Th vin trng i hc Dc
H Ni.
6. V Vn Chi , T in thc vt thng dng, NXB Khoa hc v k thut, tp I,
trang 974.
7. V Vn Chi (1997), T in cy thuc vit nam, NXBY hc.
8. Phm Hong H (1999), Cy c Vit Nam, NXB tr, Thnh ph H Ch
Minh, quyn I, trang 641.
9. Tt Li (1999), Nhng cy thuc v v thuc Vit Nam, NXB Y hc,
trang 424.
10. Nguyn Vit Thn (2003), Kim nghim dc liu bng phng php hin
vi, NXB Khoa hc & K thut H Ni.
11. Hp Trn, Bi Qunh Nguyn (1993), Cy g kinh t Vit Nam, NXB
Nng Nghip, trang 168.
12. Vin dc liu (2006), Cy thuc v ng vt lm thuc Vit Nam, NXB
Khoa hc v k thut, tp II. trang 8852.
Ting anh
13. M.Maridass (2008), "Phytochemicals From Genus Diospyros (L.) and their
Biological Activities", Ethnobotanical Leaflets,(12: 231-244), 1-9.
14. Parichat et al (2006), " Novel 24-nor-, 24-nor-2,3-seco-, and 3,24-dinor-2,4-
seco-ursane triterpenes from Diospyros decandra: evidences for ring A
biosynthetic transformations",Tetrahedron 62 (2006), 5519-5526.
Trang Bia.pdfKhoa Luan Tot Nghiep_Xuan_full.pdfT VN CHNG I: TNG QUAN 1.1. c im chung ca h Th (Ebenaceae) 1.2. Tng quan v chi Diospyros 1.2.1. V tr phn loi ca chi Diospyros 1.2.2. c im chung ca chi Diospyros 1.2.3. Mt s loi thuc chi Diospyros
1.3. c im ca loi Diospyros decadra Lour. 1.3.1. c im thc vt 1.3.2. Phn b, sinh thi 1.3.3. B phn dng 1.3.4. Thnh phn ha hc 1.3.5. Tc dng dc l 1.3.6. Tnh v, cng nng 1.3.7. Cng dng v liu dng 1.3.8. Cc bi thuc c th
CHNG II: I TNG V PHNG PHP NGHIN CU 2.1. Nguyn vt liu, thit b 2.1.1. Nguyn liu 2.1.2. Ha cht v dng c 2.1.3. Thit b, my mc
2.2. Ni dung nghin cu 2.2.1 Nghin cu v thc vt 2.2.2. Nghin cu v ha hc 2.2.3. Nghin cu v tc dng khng khun
2.3. Phng php nghin cu 2.3.1. Nghin cu v thc vt 2.3.2. Nghin cu v ha hc 2.3.3. Nghin cu v tc dng khng khun
CHNG III. THC NGHIM V KT QU 3.1. Nghin cu v c im thc vt 3.1.1. c im l th 3.1.2. Xc inh tn khoa hc ca mu nghin cu 3.1.3. c im vi phu l cy th 3.1.2. c im bt l
3.2. Nghin cu thnh phn ha hc l cy Th 3.2.1. nh tnh cc nhm cht 3.2.2. Chit xut, nh lng, nh tnh cn mt s phn on
3.3. Nghin cu tc dng khng khun ca dch chit l cy Th 3.3.1. Chun b 3.3.2. Kt qu
3.4. Bn lun 3.4.1. V thc vt 3.4.2. V ha hc 3.4.3. V tc dng khng khun
CHNG 4: KT LUN V KIN NGH 4.1. Kt lun 4.1.1. V thc vt 4.1.2. V ha hc 4.1.3. V tc dng khng khun
4.2. Kin ngh.