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.