B Y T

TRNG I HC DC H NI

BC CM MY

NGHIN CU C IM THC VT,

THNH PHN HA HC

V TC DNG KHNG KHUN

CA L CY HNG

KHA LUN TT NGHIP DC S

H NI 2014

B Y T

TRNG I HC DC H NI

BC CM MY

NGHIN CU C IM THC VT,

THNH PHN HA HC

V TC DNG KHNG KHUN

CA L CY HNG

KHA LUN TT NGHIP DC S

Ngi hng dn:

PGS.TS. V Vn in

Ni thc hin:

1. B mn Dc hc c truyn

2. B mn Vi sinh Sinh hc

H NI - 2014

LI CM N

Trong sut thi gian thc hin kha lun tt nghip, ti nhn c rt

nhiu s quan tm, gip nhit tnh t thy c, gia nh v bn b. Nhng s

quan tm gip qu bu gp phn gip ti hon thnh kha lun ny.

Trc ht, ti xin by t lng bit n su sc ti PGS.TS. V Vn in,

ngi thy trc tip hng dn, ch bo v cho ti nhiu li khuyn qu bu.

Thy dnh nhiu thi gian v cng sc tn tnh hng dn ti trong sut thi

gian hc tp v nghin cu.

Ti xin gi li cm n ti cc thy c, cc anh ch k thut vin B mn

Dc hc c truyn lun gip v to mi iu kin cho ti trong thi gian

nghin cu thc nghim ti B mn.

Ti cng xin gi li cm n chn thnh ti PGS.TS. Cao Vn Thu (B mn

Vi sinh Sinh hc) v TS. Nguyn Quc Huy (B mn Thc vt), nhng ngi

nhit tnh gip ti trong thi gian thc hin ti.

Cui cng, ti xin cm n gia nh v bn b, nhng ngi lun bn ti,

gip , h tr ti trong cng vic, ng thi cng l ngun ng vin khch l tinh

thn gip ti c gng hon thnh tt kha lun ca mnh.

Do hn ch v thi gian v phm vi ti, kt qu nghin cu ca ti mi

ch l bc u, ng thi trong qu trnh thc hin kh trnh khi thiu st, ti rt

mong nhn c nhng gp ca thy c v bn b. Ti xin chn thnh cm n.

H Ni, ngy 01 thng 05 nm 2014

Sinh vin

Bc Cm My

MC LC

T VN ........................................................................................................ 1

CHNG 1. TNG QUAN .................................................................................. 2

1.1. c im thc vt .......................................................................................... 2

1.2. Thnh phn ha hc ....................................................................................... 5

1.3. Tc dng sinh hc ca v thuc l cy Hng .................................................. 8

CHNG 2. I TNG V PHNG PHP NGHIN CU .................. 11

2.1. Nguyn vt liu v thit b nghin cu ......................................................... 11

2.2. Ni dung nghin cu ................................................................................... 13

2.3. Phng php nghin cu ............................................................................. 14

CHNG 3. THC NGHIM, KT QU V BN LUN ........................... 16

3.1. Nghin cu c im thc vt ca l cy Hng ............................................ 16

3.1.1. c im hnh thi ................................................................................ 16

3.1.2. c im vi phu .................................................................................. 17

3.1.3. c im bt l ..................................................................................... 19

3.2. Nghin cu thnh phn ha hc ca l cy Hng ......................................... 20

3.2.1. nh tnh cc nhm cht trong dc liu bng phn ng ha hc .......... 20

3.2.2. Chit xut, nh tnh v nh lng cc phn on ................................ 28

3.3. Th tc dng khng khun ca l cy Hng .................................................... 42

3.3.1. Th tc dng khng khun ca cao cn ton phn .................................... 42

3.3.2. Th tc dng khng khun ca phn on chloroform v ethyl acetat ....... 45

3.4. Bn lun ........................................................................................................ 47

KT LUN V KIN NGH ............................................................................. 49

TI LIU THAM KHO ................................................................................... 51

DANH MC CC K HIU, CC CH VIT TT

AST nh sng thng

D. Diospyros

EtOAc Ethyl acetat

EtOH Ethanol

MeOH Methanol

SKLM Sc k lp mng

TT Thuc th

VSV Vi sinh vt

DANH MC CC BNG

STT Tn bng Trang

1 Bng 1.1. Nng c ch ti thiu (MIC) ca kaempferol phn lp

t l Hng i vi mt s chng vi sinh vt 9

2 Bng 3.1. Kt qu nh tnh cc nhm cht trong l cy Hng 27

3 Bng 3.2. Hm lng cn cc phn on dch chit l Hng 31

4 Bng 3.3. Kt qu nh tnh cn cc phn on dch chit l Hng 32

5 Bng 3.4. Kt qu th hot tnh khng khun ca cao lng ton phn

cc nng 1:1, 1:2, 1:4 44

6 Bng 3.5. Kt qu th hot tnh khng khun ca 2 phn on

chloroform v ethyl acetat 45

DANH MC CC HNH V, TH

STT Tn hnh v, th Trang

1 Hnh 1.1. Hnh nh cy Hng v mt s b phn ca cy 4

2 Hnh 1.2. Cu trc ha hc ca 14 triterpenoid phn lp t l Hng 6

3 Hnh 1.3. Cu trc ha hc ca mt s flavonoid chnh trong l Hng 7

4 Hnh 3.1. Cnh mang l cy Hng 16

5 Hnh 3.2. Hnh thi l cy Hng 16

6 Hnh 3.3. nh chp vi phu gn l Hng 17

7 Hnh 3.4. nh chp vi phu phin l Hng 18

8 Hnh 3.5. c im bt dc liu l Hng 19

9 Hnh 3.6. S chit xut cc phn on t l cy Hng 30

10 Hnh 3.7. Sc k phn on n-hexan dch chit l Hng vi h

dung mi khai trin Toluen: EtOAc: Acid formic (8:3:1) 34

11 Hnh 3.8. Kt qu sc k phn on n-hexan dch chit l Hng

quan st di nh sng t ngoi bc sng 366 nm 35

12 Hnh 3.9 Sc k phn on chloroform dch chit l Hng vi h

dung mi khai trin Toluen: EtOAc: Acid formic (8:3:1) 37

13 Hnh 3.10. Kt qu sc k phn on chloroform dch chit l

Hng quan st di nh sng t ngoi bc sng 366 nm 38

14 Hnh 3.11. Sc k phn on ethyl acetat vi h dung mi khai

trin Toluen: EtOAc: Acid formic (9:6:1) 40

15 Hnh 3.12. Kt qu sc k phn on ethyl acetat quan st di

nh sng t ngoi bc sng 366 nm 41

16 Hnh 3.13. S sp xp khoanh giy lc cc mu th hot tnh

khng khun 43

17 Hnh 3.15. Vng v khun ca mu th phn on chloroform v

ethyl acetat vi vi khun Bacillus pumilus v Shigella flexneri 46

1

T VN

Cy Hng (Diospyros kaki L.f.) l mt loi cy thuc h Th (Ebenaceae), c

ngun gc t Trung Quc v Nht Bn, sau du nhp vo Vit Nam [10]. Hng l

mt ngun cung cp phong ph cc acid amin, carotenoid, flavonoid, protein,

vitamin[29] Trn th gii c nhiu nghin cu chng minh cc cht phn lp

t l Hng c nhiu tc dng sinh hc quan trng nh tc dng h huyt p [18],

khng khun, chng ung th [11], chng vim, h lipid mu [29], chng oxy ha

[23] iu ny cho thy tim nng pht trin ln ca dc liu ny.

Vit Nam, cy Hng c trng ch yu lm cy n qu. Mt s b

phn ca cy c dng lm thuc nh tai Hng (Th ), nc p t qu Hng

(Th tt), cht ng t qu Hng (Th sng) [6] L Hng cng c s dng

lm l tr cha bnh tng huyt p. Tuy nhin, vic s dng l Hng lm thuc

mi ch dng li kinh nghim dn gian, cha c c s khoa hc chng minh tc

dng v hng dn s dng loi dc liu ny. Cc ti liu ca Vit Nam v l

Hng cn rt hn ch, ch yu l thng tin v c im hnh thi thc vt. Vi mc

ch cung cp ci nhn tng qut v thc vt, ha hc v sinh hc cho dc liu l

Hng, t c s bc u cho nhng nghin cu chuyn su nhm pht huy ht

tim nng ca dc liu ny, chng ti tin hnh xy dng ti Nghin cu c

im thc vt, thnh phn ha hc v tc dng khng khun ca l cy Hng

vi cc mc tiu:

1. Nghin cu c im thc vt ca l cy Hng.

2. Nghin cu thnh phn ha hc ca l cy Hng.

3. Th hot tnh khng khun ca l cy Hng.

2

Chng 1. TNG QUAN

1.1. c im thc vt

1.1.1. V tr phn loi chi Diospyros

Cy Hng c tn khoa hc l Diospyros kaki L.f., tn ng ngha: Diospyros

chinensis Blume, l mt loi thuc chi Diospyros. Cy cn c cc tn gi khc nh

th inh, mc php (Ty) Tn nc ngoi: Kaki persimmon, Japanese

persimmon (Anh); kakier, plaqueminier kaki, plaqueminier du Japon, coing de

Chine (Php) [10], Shi Zi (Trung Quc) [19].

Theo h thng phn loi thc vt ca Takhtajan 1987 [1],[6], [24], v tr phn

loi ca chi Diospyros c tm tt nh sau:

Gii thc vt (Plantae)

Ngnh Ngc lan (Magnoliophyta)

Lp Ngc lan (Magnoliopsida)

Phn lp S (Dilleniidae)

Lin b quyn (Ericanae)

B Th (Ebenales)

H Th (Ebenaceae)

Chi Diospyros

1.1.2. c im thc vt v phn b chi Diospyros

1.1.2.1. c im thc vt

Cy g hoc cy bi, rng l hoc thng xanh. L mc so le, nguyn [7].

Thnh thong c cc vt m ri rc hoc cc vt r ln [22].

Hoa thng n tnh, khc gc hoc cng gc; him khi lng tnh. Hoa c

mc thnh xim nch l; nh t 4 n rt nhiu, thng kt hp thnh 2 vng xon.

Hoa ci thng n c, mc nch l; nh lp 1-16 hoc khng c; bu thng c

2 . i hoa thng c 3-7 thy. Trng hoa hnh chum, hnh chung, hoc hnh ng,

3-7 thy, rng sm. Qu tht, mang i to khng rng. Ht 1-10 (hoc hn), thng

hp theo b ngang [22].

3

1.1.2.2. Phn b

Chi Diospyros l chi ln nht trong h Ebenaceae, c khong 500 loi, phn

b ch yu khu vc nhit i ( chu c ti 200 loi) v mt s ni thuc khu

vc n i, trong a dng nht khu vc ng v ng Nam , ch c mt s

loi Ty , Nht Bn v ng Nam nc M [24]. Trung Quc c 60 loi, nhiu

nht ng Nam v Ty Nam Trung Quc [22]. nc ta, thng k c 70

loi [7], 9 loi cho g c gi tr s dng, 2 loi c qu n c l Hng (D.kaki) v

Cy (D.lotus) [10].

1.1.3. c im thc vt v phn b loi Diospyros kaki L.f.

1.1.3.1. c im thc vt

Cy g ln cao ti 15m. L mc so le; c cung ngn, di khng qu 1 cm.

Phin l thun hnh trng hoc tri xoan, di 6 18 cm, rng 3 9 cm, u c mi

li ngn, gc l nhn dn, mt trn mu lc sm, mt di nht c lng t, mp

nguyn hay hi ln sng.

Cm hoa mc k l, mu vng nht. Cy c, cy ci ring bit hoc c khi

hoa c, hoa ci c trn cng mt cy. Hoa c t hp thnh xim 3 hoa nch l,

c 2 l bc, 14-24 nh thng l 16. Hoa ci mc n c, i x 5 thy, bu c 4

vi nhy v 4 , thng c vch gi chia lm 8 ngn [8], [10], [22].

Qu mng, hnh cu hoc hnh trng, nhn, phng hoc c sng dc, mang

i tn ti cong ln, khi chn mu vng hoc . Ht dt, mu nu vng hoc nu

en. (Hnh 1.1)

Ma hoa: thng 3 7, ma qu: thng 8 10 [10].

1.1.3.2. Phn b

Diospyros kaki L.f. loi nguyn sn Nht Bn v Trung Quc, sau c du

nhp sang cc nc ng Nam khc nh vng Bc Thi Lan, o Java,

Sumatra (Indonesia); Malaysia. Italia, Israel, Brazil, M (California) cng c

trng loi hng ny.

Vit Nam, D. kaki l loi hng c trng ph bin. y l cy n qu lu

i cc tnh pha Bc t Thanh Ha tr ra. Cy thng trng vn nh, vn

4

trang tri t cao 500m tr xung. min Nam, hng mi c pht trin

Lt (Lm ng). Cc ging hng ang c trng hin nay v cng phong ph nh

hng Hc, hng Lng, hng vung, hng trng [10]

Hnh 1.1. Hnh nh cy Hng v mt s b phn ca cy

Ch thch:

1. Ton cy

2. Cy mang qu

3. Cnh mang l

4. Cnh mang hoa

5. Qu hng

6. Tai hng

5

1.2. Thnh phn ha hc

Trn th gii, nhiu hp cht c phn lp t cc b phn khc nhau ca

cy Hng, ch yu thuc cc nhm cht glycoside, flavonoid, tanin, saponin, sterol

v triterpenoid [20], [21].

Qu hng cha 79,6% nc, 0,8% protein, 0,2% cht bo, 0,4% cht

khong, 19% carbonhydrat, calci, phosphor, st, caroten, vitamin A, thiamin,

riboflavin, niacin v vitamin C. tm thy trong qu cc hp cht nh citrulin,

isodiospyrin, bisisodiospyrin, 2,3-butylen glycol, manitol, phytofluen, neodiospyrin,

zeaxanthin v lyeopin. Qu cn c tanin, pectin v polysaccharid. Tanin khi thy

phn vi kim thu c phloroglucinol v pyrocatechol [10]. Tanin c trong qu

hng xanh khin qu c v rt cht, khi chn v cht ny hu nh mt i, khi

lng ng khong 13 19% di dng glucose, saccharose v fructose [8].

Ht hng cha 0,8% acid citric, 4,76% ng kh v 5,9% ng t do

[10].

Tai hng cha acid oleanolic, acid ursolic, acid betulic, acid syringic, acid

vanillic, -sitosteryl- -glucoside, trifolin, hyperin v kaempferol. [6]

L Hng c cha nhiu hp cht hu c, ch yu thuc cc nhm cht:

pectic polysaccharid, flavonoid, triterpenoid, ng, protein, cc acid amin,

vitamin [12], [17], [29].

Triterpenoid

Nm 2008, cc nh khoa hc Vit Nam v Hn Quc (ti Hn Quc) phn

lp v xc nh cu trc ca 14 triterpenoid t l cy Hng (dch chit methanol,

phn on ethyl acetat). Trong c 2 triterpenoid mi v 12 triterpenoid bit.

Cu trc ca 2 triterpenoid mi c xc nh l 3R,19R-dihydroxyurs-

12,20(30)-dien-24,28-dioic acid (1) v 3R,19R-dihydroxyurs-12-en-24,28-dioic

acid (2).

12 triterpenoid bit c nhn din l: coussaric acid (3), barbinervic acid

(4), 24-hydroxy-3-epi-ursolic acid (5), rotungenic acid (6), pomolic acid (7), 24-

hydroxyursolic acid (8), ursolic acid (9), 24-dihydroxyurs-12-en-3-on-28-oic acid

6

(10),oleanolic acid (13), 24-hydroxy-3-epi-oleanolic acid (11), v spathodic acid

(14). [25]

Cng thc ha hc ca 14 triterpenoid c trnh by trong Hnh 1.2.

Hnh 1.2. Cu trc ha hc ca 14 triterpenoid phn lp t l Hng

Flavonoid

Nm 2010, mt nghin cu tng quan ca cc nh khoa hc Trung Quc cho

thy l ca loi Diospyros kaki L.f. c cc hp cht flavonoid chnh tng t nh l

ca loi Ginkgo biloba, gm cc cht sau: myricetin, quercetin, kaempferol,

astragalin, hyperin, isoquercitrin, rutin [29].

Cu trc ca cc flavonoid ny c trnh by trong Hnh 1.3.

7

Myricetin Quercetin Kaempferol

Astragalin Hyperin Isoquercitrin

Rutin

Hnh 1.3. Cu trc ha hc ca mt s flavonoid chnh trong l cy Hng

Ngoi ra, nhiu hp cht khc cng c phn lp t l Hng: gamnamoside

[4-(3-hydroxypropyl)-2-methoxyphenol -D-apiofuranosyl (16) -D-

glucopyranoside] (2009) [26]; 8-C-[-l-rhamnopyranosyl-(14)]--D-

glucopyranosylapigenin (2009) [13]; mt s pectic polysaccharid (2003, 2004,

2010) [15], [16], [14]; kaempferol 3-O-beta-D-galactopyranoside (TR),

8

isorhamnetin 3-O-beta-D-glucopyranoside (IS) (2011) [28]; hyperoside, trifolin,

chrysontemin, quercetin-3-O-(200-O--alloyl-D-glucopyranoside) (QOG),

kaempferol-3-O-(200-O--alloyl-D-glucopyranoside) (KOG) (2011); lupeol, -

sitosterol (2014) [11].

Ti Vit Nam, cha c nghin cu no c cng b v thnh phn ha hc ca

l cy Hng.

1.3. Tc dng sinh hc ca v thuc l cy Hng

1.3.1. Tc dng h huyt p

L Hng c s dng nh mt bi thuc dn gian iu tr bnh tng

huyt p ti nhiu nc chu nh Nht Bn, Trung Quc, Vit Nam[29].

Flavonoid t l Hng c tc dng c ch men chuyn angiotensin theo cch ph

thuc vo liu [10].

Ti Nht Bn, tc dng c ch men chuyn angiotensin c nghin cu

trn bn flavonoid phn lp t l Hng: astragalin (1), kaempferol -3 -O- (2" -O-

alloyl ) -glucoside (2), isoquercitrin (3) , v quercetin -3 -O- (2 " -O- alloyl )-

glucoside (4). Th nghim c tin hnh trn enzym chuyn angiotensin phn lp

t phi chut. Cc hp cht (1) (4) c kh nng c ch ln lt l 67%, 53%,

33%, v 48% nng 300 g/ml. Nng c ch 50% (IC50) ca (1) v (2) i

vi men chuyn tng ng l 180 g/ml v 280 g/ml. Mt khc, (2) v (4)

c chng minh l c hot tnh ca tanin, nhng (1) v (3) khng c hot tnh

tanin. Nhng kt qu ny cho thy khng c mi quan h gia s c ch men

chuyn vi hot tnh ca tanin trong bn hp cht [18].

1.3.2. Tc dng c ch vi sinh vt

Kh nng c ch VSV ca kaempferol phn lp t dch chit EtOAc ca l

Hng c kho st trn nhiu chng VSV. Cc th nghim c tin hnh theo

phng php khuch tn trn mi trng thch Saboroud, mu chun Amphotericin

B i vi nm; mi trng thch dinh dng, mu chun Penicillin G i vi vi

khun Gram (+) v Gentamycin i vi vi khun Gram (-). Kt qu c ghi trong

Bng 1.1.

9

Bng 1.1. Nng c ch ti thiu (MIC) ca kaempferol phn lp t l Hng

i vi mt s chng VSV [11]

Chng VSV th MIC (g/mL)

Kaempferol Mu chun

Nm:

Trichophyton mentagrophytes

Aspergillus fumigates

Candida albicans

Geotricum candidum

Penicillium marneffei

Syncephalastrum racemosum

3,9

15,3

31,25

0,98

3,9

31,25

Amphotericin B

3,9

0,98

0,06

0,12

0,98

0,007

Vi khun Gram (+):

Staphylococcus aureus

Staphylococcus epidermidis

Streptococcus pyogenes

Bacillus subtilis

Enterococcus faecalis

0,98

0,24

15,63

0,12

62,5

Penicillin G

0,015

0,12

0,06

0,007

0,98

Vi khun Gram (-):

Neisseria gonorrhoeae

Proteus vulgaris

Klebsiella pneumoniae

Shigella flexneri

Pseudomonas aeruginosa

125

0,98

0,12

1,95

62,5

Gentamycin

3,9

0,49

0,06

0,12

31,25

Nghin cu trn cho thy, kaempferol phn lp t l hng c hot tnh khng

VSV i vi c nm v vi khun, trong tc dng mnh trn nhiu vi khun Gram

(+) v Gram (-).

10

1.3.3. Tc dng chng ung th

Lupeol phn lp t l Hng cho kt qu r rng v kh nng chng ung th

th nghim trn t bo MCF-7 v t bo HeLa vi nng c ch 50% (IC50) ln

lt l 20,7 v 23,4 g/mL.

Uvaol, -amyrin v kampferol cng cho thy tc dng khng t bo MCF-7

vi IC50 tng ng l 46,2, 47,1 v 43,4 g/mL [11].

1.3.4. Tc dng chng oxy ha

Nghin cu flavonoid ton phn chit xut t l Hng trn t bo MC3T3-E1

vi cht chng oxy ha chun rutin cho thy dch chit flavonoid ton phn c tc

dng chng oxy ha mnh, c kh nng dn cc superoxide anion v cc gc

hydroxyl t do mc vt tri so vi rutin, kh nng dn cc gc 1,1-diphenyl-

2-picrylhydrazyl km hn so vi rutin [23].

11

Chng 2. I TNG V PHNG PHP NGHIN CU

2.1. Nguyn vt liu v thit b nghin cu

2.1.1. Nguyn liu nghin cu

Mu nghin cu l l cy Hng trn c thu hi ti x Yn M, huyn

Thanh Tr, H Ni vo thng 10 nm 2013 v thng 5 nm 2014.

Mu c thu hi v x l nh sau:

Mu cnh mang l v qu ti dng gim nh tn khoa hc.

Mu l ti c ra sch, dng nghin cu c im hnh thi v vi phu.

Mu l kh: L c ra sch, phi se, sy trong t sy nhit 60oC n kh.

- Mt phn mu c tn mn, dng nghin cu c im bt dc liu.

- Phn mu cn li c xay th, bo qun trong ti nilon kn, ni kh

ro, thong mt, dng chit xut nghin cu thnh phn ha hc v th

tc dng khng khun.

Mu nghin cu c TS. Nguyn Quc Huy, B mn Thc vt, trng i hc

Dc H Ni gim nh tn khoa hc l Diospyros kaki Thunb., h Th

(Ebenaceae). (Ph lc 1)

2.1.2. Phng tin nghin cu

2.1.2.1. Thuc th, dung mi, ha cht

Dung mi, ha cht s dng trong nghin cu c im thc vt

Nc ct

Nc ty Javen

Acid acetic 5%

Xanh methylen

carmin

Glycerin

Thuc th, dung mi, ha cht s dng trong nghin cu thnh phn ha

hc

- Dung mi: Ethanol, nc ct, n-hexan, chloroform, ethyl acetat, ether du ha,

toluen, aceton, acid formic, acid acetic

- Ha cht: NaOH, NH3 c, HCl, anhydrid acetic, H2SO4 c, FeCl3, ch acetat,

bt Magnesi, gelatin, KOH

12

- Thuc th: cc thuc th thng dng nh tnh cc nhm cht hu c trong

dc liu nh thuc th Mayer, Dragendorff, FeCl3 5%, Lugol

- Bn mng trng sn silicagel GF254 ca hng MERCK (c).

Cc thuc th, dung mi, ha cht t tiu chun ha cht kim nghim, c xut

x t Trung Quc.

Thuc th, ha cht s dng trong th tc dng khng khun

- Cc chng vi khun kim nh:

Vi khun Gram (-):

Escherichia coli ATCC 25922

Proteus mirabilis BV 108

Shigella flexneri DT 112

Salmonella typhi DT 220

Pseudomonas aeruginosa VM 201

Vi khun Gram (+):

Staphylococcus aureus ATCC 1128

Bacillus pumilus ATCC 10241

Bacillus subtilis ATCC 6633

Bacillus cereus ATCC 9946

Sarcina lutea ATCC 9341

- Cc mu chun :

Streptomycin sulfat: 20 IU/ml i vi vi khun Gram (-)

Benzathin penicillin: 20 IU/ml i vi vi khun Gram (+)

- Mi trng th nghim:

Mi trng canh thang nui cy vi khun kim nh

NaCl 0,5%

Pepton 0,5%

Cao tht 0,3%

Nc v 100 ml

Mi trng thch thng

NaCl 0,5%

Pepton 0,5%

Cao tht 0,3%

Thch 1,6%

Nc v 100 ml

13

Cc chng vi khun, mu chun v ha cht thuc b mn Vi sinh Sinh hc

trng i hc Dc H Ni.

2.1.2.2. Dng c v thit b

- Dng c:

Li dao lam, phin knh, l knh v cc dng c lm tiu bn vi phu khc.

Thc o ng knh vng v khun.

Thuyn tn, ci, ry, b dng c chit hi lu (bnh cu, ng sinh hn), bnh chit,

bnh nn, cc c m... v cc dng c th nghim khc.

- Thit b:

Knh hin vi LABOMED (M) Serial No. 070918442

My nh k thut s Sony Cybershot W370

T sy Memmert (c)

My soi n t ngoi VILBER LOURMAT CN-6.T (Php) Serial No.12 101537

My soi n t ngoi CAMAG (Thy S) Serial No.t 022.9120

T ht UNI-LAB

My ct quay BCHI Rotavapor R-200 (Thy S) Serial 803894010002

Cn phn tch Sartorius TE214S (c) serial 23808521

Cn k thut Precisa XB 320C (Thy S)

My ly tm K Universal Centrifuge PLC-012E (i Loan) serial 717902

B cch thy HHS4 (Trung Quc) Serial No. 211121618

Bp in Wisd Heating Mantle WHM12014 (Hn Quc) Serial No.0406452128

My o m Precisa XM120

H thng my chm sc k CAMAG LINOMAT 5, my chp sc k CAMAG TLC

VISUALIZE (Thy S) c kt ni vi my vi tnh c ci t phn mm

WinCATS, VideoScan.

Cc dng c, thit b thuc b mn Dc hc c truyn v mt s b mn khc,

trng i hc Dc H Ni.

2.2. Ni dung nghin cu

2.2.1. Nghin cu c im thc vt l cy Hng

14

M t c im hnh thi l nghin cu.

Nghin cu c im vi phu v c im bt l Hng.

2.2.2. Nghin cu thnh phn ha hc l cy Hng

nh tnh cc nhm cht trong dc liu bng phn ng ha hc.

Chit xut, nh tnh v nh lng mt s phn on.

2.2.3. Th tc dng khng khun ca l cy Hng

Th tc dng khng khun ca cao cn ton phn i vi 5 chng vi khun

Gram (-) v 5 chng vi khun Gram (+).

Th tc dng khng khun ca dch chit cc phn on chloroform v ethyl

acetat i vi cc chng vi khun trn.

2.3. Phng php nghin cu

2.3.1. Nghin cu v thc vt

M t hnh thi thc vt

Quan st trc tip mu nghin cu bng mt thng, m t cc c im hnh

thi ca l theo phng php ghi trong cc ti liu [1], [4].

Nghin cu c im vi phu

Ct tiu bn vi phu bng li dao lam, ty nhum bng phng php nhum

kp theo phng php vi phu thc vt thng quy [4]. Lm nhiu tiu bn, la

chn tiu bn mng, nhn r quan st v chp nh qua knh hin vi.

Nhn bit cc c im theo ti liu [4].

Nghin cu c im bt l

Soi bt l di knh hin vi, quan st cc c im. Lm nhiu tiu bn bt,

chp nh ghi li y cc c im ca bt l.

Nhn bit cc c im theo hng dn trong ti liu [2].

2.3.2. Nghin cu v ha hc

nh tnh cc nhm cht trong dc liu bng phn ng ha hc

Tin hnh theo cc phng php ha thc vt thng quy [2], [3], [9].

15

S dng dung mi thch hp chit dch chit ton phn ca mi nhm cht,

dng thuc th c hiu ca mi nhm cht nh tnh, c i chiu vi mu trng

nhn xt kt qu.

Chit xut, nh lng cc phn on dch chit t dc liu

S dng dung mi ethanol 70%, chit theo phng php ngm nhit phng

c 24h rt dch chit 1 ln, chit 3 ln, gp cc dch chit, thu hi dung mi thu

c cao lng nc. Loi tp. Dng cao lng ny chit ln lt vi cc dung mi c

phn cc tng dn: n-hexan, chloroform, ethyl acetat, sau bc hi dung mi

thu c cn cc phn on tng ng. Cn v tnh hm lng cc cn.

nh tnh cc phn on dch chit bng phn ng ha hc v sc k lp

mng

nh tnh cn cc phn on theo nh hng cc nhm cht chnh v phn tch

bng sc k lp mng theo cc phng php ha thc vt thng quy [2], [3], [9],

[27].

2.3.3. Th tc dng khng khun

Tin hnh th tc dng khng khun ca cao lng cn ton phn, 2 phn

on dch chit chloroform v ethyl acetat i vi cc chng vi khun Gram (+),

Gram (-) theo phng php khuch tn trn mi trng thch [5].

Nguyn tc: Mu th (c cha hot cht th) c t ln 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 vng v khun.

ng knh vng v khun c o bng thc Panmer c chnh xc

0,02 mm, so snh vi mu chun nh gi kt qu. Mi mu l 6 ln ly kt

qu trung bnh.

16

Chng 3. THC NGHIM, KT QU V BN LUN

3.1. Nghin cu c im thc vt ca l cy Hng

3.1.1. c im hnh thi

L Hng mc so le, n. Cung l di 0,5 1,5 cm. Phin l hnh bu dc

hoc hnh trng, di 6 20 cm, rng 3 9 cm; ngn l c mi li nhn, gc l

thun dn; mp l nguyn hoc hi ln sng; mt trn mu lc sm, mt di mu

nht, c lng. Gn l hnh lng chim.

Hnh 3.1. Cnh mang l cy Hng

Hnh 3.2. Hnh thi l cy Hng

17

3.1.2. c im vi phu

Phn gn l: Gn l pha trn li t, pha di li nhiu. Biu b trn (1) v

biu b di (7) l mt lp t bo hnh trn hay hnh bu dc, nh, xp u n,

mang lng che ch n bo (4), thnh thong c lng tit (8). St lp biu b l lp

m dy (5) cu to bi 3 4 lp t bo hnh trn hoc hnh trng, thnh dy. M

mm (6) gm cc t bo hnh trn hoc hnh trng, thnh mng, kch thc tng

i ln, sp xp ln xn. Mt b libe g xp thnh hnh cung; libe (3) di, bt

mu ; g (2) trn, gm nhiu mch g hnh trn, bt mu xanh. (Hnh 3.3)

Hnh 3.3. nh chp vi phu gn l Hng

Ch thch:

1. Biu b trn

2. G

3. Libe

4. Lng che ch n bo

5. M dy

6. M mm

7. Biu b di

8. Lng tit

18

Phn phin l: Phin l c cu to d th bt i xng. Biu b trn (1) v

biu b di (4) l mt hng t bo hnh ch nht, kch thc ln hn nhiu so vi

t bo biu b gn l, mang lng che ch n bo (5). Di lp biu b trn l m

giu (2) gm mt lp t bo di xp st nhau, thng ng vung gc vi b mt l.

M khuyt (3) c cu to bi cc t bo thnh mng, kch thc khng u, sp

xp ln xn to thnh cc khong trng. (Hnh 3.4)

Hnh 3.4. nh chp vi phu phin l Hng

Ch thch:

1. Biu b trn

2. M giu

3. M khuyt

4. Biu b di

5. Lng che ch n bo

19

3.1.3. c im bt l

Bt mu xanh nu, mi thm nh, khng v.

Quan st di knh hin vi thy cc c im: Mnh m mm phin l (1)

gm cc t bo hnh a gic. Mnh biu b mang nhiu l kh (2). Lng che ch n

bo thun di (3). Si thng tp trung thnh tng b si ln (4). Mnh mch gm

mch xon (5) v mch mng (6). Lng tit u a bo c mu , chn n bo.

Nhiu tinh th calci oxalat cc hnh dng khc nhau: hnh thoi, hnh khi (8)

nm c lp hoc nm trong cc m si (9). (Hnh 3.5)

Hnh 3.5. c im bt dc liu l Hng

Ch thch:

1. Mnh m mm

2. Mnh biu b mang l kh

3. Lng che ch n bo

4. B si

5. Mch xon

6. Mch mng

7. Lng tit

8. Tinh th calci oxalat

9. m si mang tinh th calci

oxalat

20

3.2. Nghin cu thnh phn ha hc ca l cy Hng

3.2.1. nh tnh cc nhm cht trong dc liu bng phn ng ha hc

3.2.1.1. nh tnh glycosid tim

Cho 10 g bt dc liu vo bnh nn, thm 100 ml ethanol 25%, ngm trong

24 gi. Gn ly dch chit, loi tp bng ch acetat d, lc qua giy lc gp np.

Chuyn dch chit vo bnh gn, lc vi hn hp chloroform - ethanol (4:1) 3 ln,

mi ln 10 ml. Gn ly lp chloroform, gp cc dch chit chloroform v loi nc

bng natri sulfat khan. Chia u dch chit vo 5 ng nghim nh c sy kh.

Bc hi dch chit trn bp cch thy n cn. Dng cn ny tin hnh cc phn

ng sau:

Phn ng xc nh khung steroid:

Phn ng Liebermann Burchardt

Ha tan cn vi 1 ml anhydrid acetic, 1 ml chloroform. Nghing ng 45o.

Cho t t theo thnh ng 1 ml acid sulfuric c, trnh xo trn cht lng trong ng.

Kt qu: mt tip xc gia hai lp cht lng xut hin mt vng mu

tm (phn ng dng tnh).

Phn ng xc nh vng lacton 5 cnh:

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.

Ha tan cn vi 1 ml ethanol 90%. Thm 1 ml thuc th Baljet mi pha.

Quan st mu ngay sau khi nh thuc th. So snh mu sc vi ng chng (ch cha

thuc th Baljet).

Kt qu: ng th c mu cam m hn ng chng (phn ng dng

tnh).

Phn ng Legal

Ha tan cn vi 1 ml ethanol 90%. Nh 1 git thuc th natri nitroprussiat

0,5% v 2 git dung dch NaOH 10%, lc u. Quan st mu ngay sau khi nh

thuc th. So snh mu sc vi ng chng (ng khng c cn).

21

Kt qu: ng th c mu cam m hn ng chng (phn ng dng

tnh).

Phn ng xc nh phn ng 2-desoxy:

Phn ng Keller - Kiliani

Ha tan cn vi 1 ml ethanol 90%. Thm vi git dung dch st (III) clorid

5% pha trong acid acetic. Lc u. Nghing ng 45o. Cho t t theo thnh ng 1 ml

acid sulfuric c, trnh xo trn cht lng trong ng.

Kt qu: mt tip xc gia 2 lp cht lng xut hin 1 vng mu cam, lp

cht lng pha trn c mu vng, khng c mu xanh l (phn ng m tnh).

Phn ng vi acid phosphoric m c

Ha tan cn vi 1 ml aceton. Thm 5 ml acid phosphoric m c, lc u,

t trn bp cch thy 15 pht, sau lm ngui.

Kt qu: Dung dch trong ng khng chuyn mu (phn ng m tnh).

Nhn xt: l Hng khng cha glycosid tim.

3.2.1.2. nh tnh flavonoid

Chit 10 g bt dc liu vi ethanol 70% bng cch un hi lu trong 1 gi.

Lc dch chit qua giy lc gp np, bc hi trn bp cch thy n kh. Loi tp

bng ether du ha. Ha tan li cn trong 10 ml ethanol 90%. Chia u dch chit

vo 5 ng nghim nh, em tin hnh cc phn ng sau:

Phn ng Cyanidin

Thm vo ng nghim cha dch chit mt t bt magnesi kim loi. Nh tng

git HCl m c (5 10 git). yn mt vi pht.

Kt qu: Dung dch mu vng chuyn sang mu hng (phn ng dng tnh).

Phn ng vi kim

Phn ng vi dung dch NaOH

Thm vo ng nghim cha dch chit vi git dung dch NaOH 10%.

Kt qu: Dung dch chuyn sang mu cam (phn ng dng tnh).

Phn ng vi hi amoniac

22

Nh mt git dch chit ln giy lc. Sy kh ri h trn ming l amoniac

c c m np. Quan st mu ca vt dch chit. So snh vi mu chng l

mt git dch chit khc khng c h trn l amoniac.

Kt qu: Mu vng ca vt dch chit th m hn so vi mu chng (phn

ng dng tnh).

Phn ng vi FeCl3

Thm vo ng nghim cha dch chit vi git FeCl3 5%.

Kt qu: Dung dch xut hin mu xanh en (phn ng dng tnh).

Phn ng diazo ha

Kim ha dch chit bng dung dch NaOH, thm vi git thuc th diazo

mi pha, lc u. un nng trn ni cch thy vi pht.

Kt qu: Dung dch chuyn thnh mu (phn ng dng tnh).

Nhn xt: L Hng c cha flavonoid.

3.2.1.3. nh tnh saponin

Quan st hin tng to bt

Cho vo ng nghim ln 0,1 g bt dc liu, thm 5 ml nc. Lc mnh theo

chiu dc trong 5 pht. yn v quan st hin tng to bt.

Kt qu: Bt cn bn sau 15 pht (phn ng dng tnh).

Cc phn ng mu

Chit 10 g bt dc liu vi ethanol 70% bng cch un hi lu trong 30

pht. Lc dch chit qua giy lc gp np, bc hi trn bp cch thy n kh. Loi

tp bng ether du ha. Dng cn lm cc phn ng sau:

Phn ng Salkowski:

Ha tan cn vi 1 ml ethanol 90%. Nghing ng nghim 45o. Cho t t theo

thnh ng 1 ml acid sulfuric c.

Kt qu: mt ngn cch gia 2 lp cht lng xut hin vng mu nu

(phn ng dng tnh).

23

Phn ng Liebermann Burchardt

Ha tan cn vi 1 ml anhydrid acetic, 1 ml chloroform. Nghing ng 45o.

Cho t t theo thnh ng 1 ml acid sulfuric c, trnh xo trn cht lng trong ng.

Kt qu: Vng ngn cch gia 2 lp cht lng c mu tm (phn ng

dng tnh).

Nhn xt: L Hng c cha saponin.

3.2.1.4. nh tnh coumarin

Chit 10 g bt dc liu bng ethanol 90% bng cch un hi lu trong 1

gi. Lc nng qua giy lc. Dch chit thu c em tin hnh cc phn ng sau:

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 c 2 ng nghim trn bp cch thy trong vi pht. ngui.

Kt qu: ng 1 c mu cam, ng 2 trong. Thm vo c 2 ng nghim mi

ng 2 ml nc ct. Khng c hin tng g xy ra (phn ng m tnh).

Phn ng diazo ha

Cho vo ng nghim 1 ml dch chit. Thm 1 ml dung dch NaOH 10%. un

cch thy trong 5 pht. Lm lnh di vi nc ri thm tng git thuc th diazo.

Kt qu: Dung dch xut hin mu (phn ng dng tnh).

Quan st hunh quang di nh sng t ngoi

Nh 1 git dch chit ln 1 t giy lc, ch kh dung mi v lp li nh trn

4 ln. Nh chng ln 1 git NaOH 5%, sy kh. Che vt bng 1 ming kim

loi ri em soi n UV trong vi pht. Ly ming kim loi ra v quan st vt dch

chit di nh sng t ngoi.

Kt qu: Vt dch chit sng ng u c na b che v khng b che (phn

ng m tnh).

Nhn xt: L Hng khng cha coumarin.

24

3.2.1.5. nh tnh tanin

Chit 10 g bt dc liu bng nc trn bp cch thy trong 15 pht.

ngui, lc qua giy lc. Dch lc em tin hnh cc phn ng sau:

Phn ng vi FeCl3

Ly 2 ml dch lc, thm 2 git dung dch FeCl3 5%.

Kt qu: Dung dch xut hin mu xanh en (phn ng dng tnh).

Phn ng vi ch acetat

Ly 2 ml dch lc, thm 2 git dung dch ch acetat 10%.

Kt qu: Trong ng nghim xut hin ta bng (phn ng dng tnh).

Phn ng vi dung dch gelatin

Ly 2 ml dch lc, thm 5 git dung dch gelatin 1%. So snh ng th vi

ng chng ch cha thuc th v nc ct.

Kt qu: Trong ng nghim xut hin ta bng trng (phn ng dng tnh).

Nhn xt: L Hng c cha tanin.

3.2.1.6. nh tnh anthranoid

Chit nng 10 g bt dcliu bng dung dch H2SO4 20% trong 1 gi. Lc

dch chit cn nng qua bng. Lm ngui dch lc. Lc dch lc vi ether (2 ln,

mi ln 8 ml). Lp ether thu c tin hnh cc phn ng nh sau:

Phn ng Borntraeger

Ly 1 ml dch chit, thm 1 ml dung dch NaOH 10%. Lc nh.

Kt qu: Lp nc khng c mu sim (phn ng m tnh).

Th nghim vi thng hoa

Tri bt dc liu thnh lp mng trong mt np nhm, t nh loi

nc. Sau y ln np nhm mt ming lam knh, bn trn c ming bng

thm nc, tip tc un nng trong khong 5 10 pht. Ly lam knh ra ngui,

soi knh hin vi.

Kt qu: Khng quan st thy cc tinh th hnh kim mu vng (phn ng m

tnh).

Nhn xt: L Hng khng cha anthranoid.

25

3.2.1.7. nh tnh alcaloid

Lm m 10 g bt dc liu bng NH4OH 25% trong 20 pht. Chit dc liu

vi chloroform bng Soxhlet trong 2 gi. Dch chit thu c lc vi dung dch acid

sulfuric 2%. Dich acid c dng nh tnh alcaloid bng cc phn ng sau:

Phn ng vi thuc th Mayer

Ly 2 ml dch chit, thm vi git thuc th Mayer.

Kt qu: Trong ng nghim khng xut hin ta (phn ng m tnh).

Phn ng vi thuc th Bouchardat

Ly 2 ml dch chit, thm vi git thuc th Bouchardat.

Kt qu: Trong ng nghim khng xut hin ta (phn ng m tnh).

Phn ng vi thuc th Dragendorff

Ly 2 ml dch chit, thm vi git thuc th Dragendorff.

Kt qu: Trong ng nghim khng xut hin ta (phn ng m tnh).

Nhn xt: L Hng khng cha alcaloid.

3.2.1.8. nh tnh polysaccharid, ng kh, acid hu c, acid amin

Chit 10 g bt dc liu vi nc trn bp cch thy trong 15 pht. Lc dch

chit qua giy lc gp np. Dng dch lc tin hnh cc phn ng sau:

nh tnh polysaccharid

Chun b 2 ng nghim:

ng 1: 4 ml dch chit + 5 git thuc th Lugol.

ng 2: 4 ml nc ct + 5 git thuc th Lugol.

Quan st mu dung dch trong 2 ng nghim.

Kt qu: Mu dch chit trong 2 ng nh nhau (phn ng m tnh).

Nhn xt: L Hng khng cha polysaccharid.

nh tnh ng kh

Ly 2 ml dch chit vo ng nghim, thm 1ml thuc th Fehling A v 1 ml

thuc th Fehling B. un cch thy trong vi pht.

Kt qu: Trong ng nghim xut hin ta gch (phn ng dng tnh).

Nhn xt: L Hng c cha ng kh.

26

nh tnh acid hu c

Ly 5 ml dch chit vo ng nghim. Thm vo dch chit mt t tinh th

Na2CO3.

Kt qu: Trong ng nghim xut hin bt kh bay ln (phn ng dng tnh).

Nhn xt: L Hng c cha acid hu c.

nh tnh acid amin

Ly 2 ml dch chit vo ng nghim. Thm vo dch chit vi git thuc th

Ninhydrin 3%, un cch thy vi pht.

Kt qu: Dung dch trong ng chuyn sang mu tm (phn ng dng tnh).

Nhn xt: L Hng c cha acid amin.

3.2.1.9. nh tnh cht bo, caroten, sterol

Chit 10 g bt dc liu vi ether du ha bng cch ngm qua m. Lc

dch chit qua bng. Dch lc em tin hnh cc phn ng sau:

nh tnh cht bo

Nh vi git dch chit ln 1 t giy lc. Sy nh cho bay hi ht dung mi.

Kt qu: Trn t giy lc khng c vt m (phn ng m tnh).

Nhn xt: L Hng khng cha cht bo.

nh tnh caroten

Ly 2 ml dch lc vo ng nghim, bc hi trn bp cch thy n kh.

Thm vo cn vi git H2SO4 c.

Kt qu: Trong ng nghim xut hin mu xanh l (phn ng dng tnh).

Nhn xt: L Hng c cha caroten.

nh tnh sterol

Ly 2 ml dch chit vo ng nghim, bc hi trn bp cch thy n kh.

Thm vo cn 1 ml anhydrid acetic, lc k cho tan ht cn. Nghing ng nghim

45o. Cho t t theo thnh ng 0,5 ml acid sulfuric c, trnh xo trn cht lng

trong ng.

Kt qu: Vng ngn cch gia 2 lp cht lng c mu tm (phn ng

dng tnh).

27

Nhn xt: L Hng c cha sterol.

Kt qu nh tnh cc nhm cht trong dc liu c tm tt Bng 3.1.

Bng 3.1. Kt qu nh tnh cc nhm cht trong l cy Hng

STT Nhm cht Phn ng nh tnh Kt qu Kt lun

1 Glycosid tim

Phn ng Liebermann Burchardt

Phn ng Baljet

Phn ng Legal

Phn ng Keller Killiani

Phn ng vi H3PO4 m c

+

+

+

-

-

Khng c

2 Flavonoid

Phn ng Cyanidin

Phn ng vi dung dch NaOH

Phn ng vi hi amoniac

Phn ng vi dung dch FeCl3 5%

Phn ng diazo ha

+

++

+

++

+

C

3 Saponin

Quan st hin tng to bt

Phn ng Salkowski

Phn ng Liebermann Burchardt

+

+

+

C

4 Coumarin

Phn ng m ng vng lacton

Phn ng diazo ha

Quan st hunh quang

-

+

-

Khng c

5 Tanin

Phn ng vi dung dch FeCl3 5%

Phn ng vi ch acetat

Phn ng vi dung dch gelatin 1%

++

++

+

C

6 Anthranoid Phn ng Borntraeger

Th nghim vi thng hoa

-

- Khng c

7 Alcaloid

Phn ng vi TT Mayer

Phn ng vi TT Bouchardat

Phn ng vi TT Dragendorff

-

-

-

Khng c

8 Polysaccharid Phn ng vi TT Lugol - Khng c

28

9 ng kh Phn ng vi TT Fehling + C

10 Acid hu c Phn ng vi tinh th Na2CO3 + C

11 Acid amin Phn ng vi TT Ninhydrin 3% + C

12 Cht bo Phn ng to vt m trn giy lc - Khng c

13 Caroten Phn ng vi H2SO4 c + C

14 Sterol Phn ng Liebermann Burchardt + C

Ch thch: (-) Phn ng m tnh (++) Phn ng dng tnh r

(+) Phn ng dng tnh

Nhn xt: Qua cc phn ng nh tnh trn, s b kt lun l cy Hng c

cha flavonoid, saponin, tanin, ng kh, acid hu c, acid amin, caroten v

sterol.

3.2.2. Chit xut, nh tnh v nh lng cc phn on

3.2.2.1. Chit xut phn on

Chun b bt dc liu:

L ti c ra sch, sy kh 60oC, xay thnh bt th.

Xc nh m ca bt dc liu bng my o m Precisa XM120 cho

kt qu: m dc liu: 10,15%.

Dung mi v phng php chit:

La chn dung mi chit l ethanol 70%. Chit theo phng php ngm

lnh, 24 gi rt dch chit 1 ln.

Chun b dng c chit:

Bnh gn 500 ml c nt mi kn, c ra sch, sy kh, lt bng y.

Chit xut cao lng ton phn:

Cn chnh xc 100,74 g bt dc liu, lm m bng dung mi chit 30

pht. Cho bt vo bnh gn, thm ethanol 70% ngp trn dc liu 2 3 cm, m

kha rt khong 10 ml dung mi, tr li, y kn bnh chit. Ngm nhit

phng, c 24 gi rt dch chit 1 ln, rt dch chit 3 ln. Gp cc dch chit (700

ml), ct thu hi dung mi thu c 200 ml dch chit. lng dch chit trong t

lnh 24 gi, lc b ta, ly tm thu c cao lng nc.

29

Chit xut phn on:

Chit ln lt vi cc dung mi theo th t tng dn phn cc: n-hexan,

chloroform, ethyl acetat.

- Chit phn on n-hexan: Cho dch chit ton phn vo bnh gn, thm n-

hexan (khong 100 ml), lc k, phn lp, gn ly lp n-hexan (lp trn). Thm n-

hexan vo dch chit ton phn chit tip. Chit nhiu ln n khi lp n-hexan

khng c mu (khong 500 ml), gp dch chit n-hexan, thu hi dung mi thu c

cn.

- Chit phn on chloroform v ethyl acetat: tin hnh tng t phn on n-

hexan, thu c 2 cn tng ng.

Cn cc phn on n-hexan, chloroform, ethyl acetat c k hiu ln lt l

H, C, E.

Quy trnh chit xut cc phn on c tm tt theo s Hnh 3.6.

30

Hnh 3.6. S chit xut cc phn on t l cy Hng

3.2.2.2. nh lng cc phn on

Cc cn H, C, E c em i cn xc nh khi lng (m).

Hm lng cn ca cc phn on (so vi dc liu kh tuyt i) c tnh

theo cng thc:

Bt th l Hng (100,74g)

EtOH 70%

Dch chit EtOH

Cao lng nc

Thu hi ethanol

EtOH

Cao nc ton phn

Ngm nhit phng,

24h/ln x 3 ln

lng, lc, ly tm

Lp nc

n-hexan

Lp n- hexan Cn H

chloroform

Cn C Lp nc Lp

Lp EtOAc Lp nc Cn E

EtOAc

31

H(%) = m

M. (1 h) .100

Trong :

H l hm lng cn (%)

m l khi lng cn (g)

M l khi lng bt dc liu (M = 100,74 g)

h l m ca dc liu (h = 10,15% = 0,1015)

Kt qu xc nh hm lng cn ca cc phn on c trnh by trong Bng 3.2.

Bng 3.2. Hm lng cn cc phn on dch chit l Hng

Khi lng

dc liu (g)

m

dc liu (%)

Phn on

dch chit

Khi lng cn

(g)

Hm lng cn

(%)

100,74 g 10,15%

n-hexan 0,25 g 0,28%

chloroform 0,76 g 0,84%

EtOAc 3,21 g 3,55%

Nhn xt: Phn on ethyl acetat c hm lng cn cao hn nhiu ln so vi

phn on n-hexan v chloroform.

3.2.2.3. nh tnh cn cc phn on bng phn ng ha hc

Tin hnh nh tnh mt s nhm cht chnh trong cn ca cc phn on

nh sau:

nh tnh flavonoid: Ha tan cn tng phn on trong ethanol 90% trn bp

cch thy, lc qua giy lc. Tin hnh cc phn ng ghi trong mc 3.2.1.2.

nh tnh saponin: S dng cn tng phn on tin hnh cc phn ng

ghi trong mc 3.2.1.3.

nh tnh tanin: Ha tan cn tng phn on trong nc ct trn bp cch thy,

lc qua giy lc. Tin hnh cc phn ng ghi trong mc 3.2.1.5.

Kt qu nh tnh cc phn on c trnh by trong Bng 3.3.

32

Bng 3.3. Kt qu nh tnh cn cc phn on dch chit l Hng

STT Phn ng nh tnh Kt qu

Cn H Cn C Cn E

1

Flavonoid:

Phn ng Cyanidin

Phn ng vi dung dch NaOH

Phn ng vi hi amoniac

Phn ng vi dung dch FeCl3 5%

Phn ng diazo ha

-

+

-

-

-

+

++

+

++

+

++

++

+

++

++

Kt lun Khng c C C

2

Saponin

Quan st hin tng to bt

Phn ng Salkowski

Phn ng Liebermann Burchardt

-

-

-

+

++

+

+

+

+

Kt lun Khng c C C

3

Tanin

Phn ng vi dung dch FeCl3 5%

Phn ng vi ch acetat

Phn ng vi dung dch gelatin 1%

-

-

-

++

+

+

++

+

+

Kt lun Khng c C C

Ch thch: (-) Phn ng m tnh (++) Phn ng dng tnh r

(+) Phn ng dng tnh

Nhn xt:

- Phn on n-hexan khng cha cc nhm cht chnh ca l Hng.

- Phn on chloroform v ethyl acetat cha c 3 nhm cht flavonoid, saponin,

tanin.

3.2.2.4. nh tnh cn cc phn on bng sc k lp mng

- Dch chm sc k: Cn tng phn on ha tan trong MeOH.

33

- Bn mng sc k: Bn mng trng sn silicagel GF254 (MERCK), hot ha

110oC trong 1 gi, bo qun trong bnh ht m.

- H dung mi khai trin: chun b v kho st mt s h dung mi i vi tng

cn la chn h dung mi tch tt nht.

- Tin hnh: chm dch chit methanol chun b trn ln bn mng. Sy nh

cho kh, t vo bnh sc k bo ha dung mi. Sau khi khai trin sc k, ly

bn mng ra khi bnh, sy nh 5 pht cho bay hi ht dung mi. Quan st sc

k thu c di nh sng thng, nh sng t ngoi v nhng thuc th

hin mu vanilin trong ethanol 96%.

Phn on n-hexan (cn H)

- H dung mi khai trin:

H I: Chloroform : MeOH (9:1)

H II: n-hexan : EtOAc : Acid formic (4:4:1)

H III: Toluen : EtOAc : Acid formic (8:3:1)

H IV: Chloroform : EtOAc : Acid formic (8:3:1)

H V: n-hexan : Aceton (9:2)

- Kt qu: Sau khi khai trin, quan st sc k di nh sng thng, nh sng

t ngoi bc sng 254nm, 366 nm, nhng thuc th hin mu vanilin trong

ethanol 96% thy h III cho kt qu tch tt nht, thu c sc k Hnh 3.7.

34

Hnh 3.7. Sc k phn on n-hexan dch chit l Hng

vi h dung mi khai trin Toluen: EtOAc: Acid formic (8:3:1).

Ch thch:

IIIa. Sc k quan st AST

IIIb. Sc k quan st bc sng 254nm

IIIc. Sc k quan st bc sng 366nm

IIId. Sc k sau khi nhng TT vanilin/ethanol 96%, quan st AST

Sc k c chp bi phn mm winCATS c nh dng .cna, sau

c chuyn thnh hnh nh c nh dng .cpf phn tch bng phn mm

VideoScan, cho th v bng biu din kt qu sc k tng ng. th v

bng biu din kt qu sc k phn on n-hexan bc sng 366 nm c

trnh by trong Hnh 3.8.

35

Hnh 3.8. Kt qu sc k phn on n-hexan dch chit l Hng

quan st di nh sng t ngoi bc sng 366 nm.

Nhn xt:

Sc k di nh sng thng khng quan st thy vt.

36

Sc k quan st bc sng 254 nm thy 6 vt, cc vt tch xa nhau, tuy

nhin cc vt mu nht, kh m.

Sc k quan st bc sng 366 nm thy 14 vt, cc vt tch u, nhiu mu

sc khc nhau, trong cc vt 7, 8, 13 m v sng nht.

Sc k sau khi nhng thuc th vanilin trong ethanol 96%, quan st di nh

sng thng thy 12 vt, cc vt u bt mu xanh en, m nht khc nhau.

Trong c 4 vt mu m, d quan st, cc vt cn li m hoc rt m.

nh tnh phn on chloroform (cn C):

- H dung mi khai trin:

H I: Chloroform : MeOH (19:1)

H II: n-hexan : EtOAc : Acid formic (4:4:1)

H III: Toluen : EtOAc : Acid formic (8:3:1)

H IV: Chloroform : EtOAc : Acid formic (7,5:3:1)

H V: Chloroform : Aceton : Acid formic (7:2,5:0,5)

H VI: EtOAc : MeOH : H2O (8:1:1)

- Kt qu: Sau khi khai trin, quan st sc k di nh sng thng, nh sng

t ngoi bc sng 254 nm v 366 nm, nhng thuc th vanilin trong ethanol

96% thy h III cho kt qu tch tt nht, thu c sc k trong Hnh 3.9.

37

Hnh 3.9 Sc k phn on chloroform dch chit l Hng

vi h dung mi khai trin Toluen: EtOAc: Acid formic (8:3:1)

Ch thch:

IIIa. Sc k quan st AST

IIIb. Sc k quan st bc sng 254nm

IIIc. Sc k quan st bc sng 366nm

IIId. Sc k sau khi nhng TT vanilin/ethanol 96%, quan st AST

Kt qu sc k sau khi phn tch bng phn mm VideoScan c trnh by

trong hnh 3.9.

38

Hnh 3.10. Kt qu sc k phn on chloroform dch chit l Hng

quan st di nh sng t ngoi bc sng 366 nm.

Sc k quan st di nh sng thng: thy 1 vt mu vng, r, tng ng

vi vt 11 trong sc k quan st bc sng 366 nm.

39

Sc k quan st bc sng 254 nm: thy 11 vt, cc vt tch u, m, r,

c 2 vt mu xanh lam.

Sc k quan st bc sng 366 nm: c 14 vt, trong cc vt 5, 6, 7 m

v sng nht.

Sc k sau khi nhng thuc th vanilin/ethanol 96%, quan st di nh sng

thng: thy 8 vt, tuy nhin cc vt tch nhau khng r. a s cc vt mu nu,

c 1 vt mu xanh m, r.

nh tnh phn on ethyl acetat (cn E):

- H dung mi khai trin:

H I: Chloroform: MeOH (9:1)

H II: Chloroform: EtOAc: Acid formic (8:3:1)

H III: Toluen: EtOAc: Acid formic (6:2:1)

H IV: Toluen: EtOAc: Acid formic (9:6:1)

H V: EtOAc: Acid acetic bng: Acid formic: H2O (100:11:11:26)

H VI: EtOAc: MeOH: H2O (8:1:1)

H VII: EtOAc: Butanon: Acid formic: H2O (5:3:1:1)

H VIII: EtOAc: Acid formic: H2O (8:1:1)

- Kt qu: Sau khi khai trin, quan st sc k di nh sng thng, nh sng

t ngoi bc sng 254 nm, 366 nm v nhng thuc th vanilin trong ethanol

96%, thy h IV cho kt qu tch tt nht, thu c sc k trong Hnh 3.11.

40

Hnh 3.11. Sc k phn on ethyl acetat vi h dung mi khai trin

Toluen : EtOAc : Acid formic (9:6:1)

Ch thch:

IVa. Sc k quan st di AST

IVb. Sc k quan st bc sng 254 nm

IVc. Sc k quan st bc sng 366 nm

IVd. Sc k sau khi nhng TT vanilin/ethanol 96%, quan st di AST.

Kt qu sc k sau khi phn tch bng phn mm VideoScan c trnh by

trong hnh 3.12.

41

Hnh 3.12. Kt qu sc k phn on ethyl acetat quan st di nh sng

t ngoi bc sng 366 nm.

42

Nhn xt:

Sc k quan st AST thy 6 vt, trong c 2 vt m, quan st r, tng

ng vi vt 7, 11 trn sc k quan st bc sng 366 nm.

Sc k quan st bc sng 254 nm thy 5 vt, cc vt u m, quan st r.

Sc k quan st bc sng 366 nm pht hin c 19 vt, m ca cc

vt tng i ng u, trong vt 11 sng nht.

Sc k sau khi nhng thuc th vanilin/ethanol 96%, quan st di AST thy

11 vt, trong c vt mu tm rt m.

th v bng biu din kt qu sc k cc phn on phn tch AST,

bc sng 254 nm v sau khi nhng TT hin mu c trnh by trong Ph lc 2.

3.3. Th tc dng khng khun ca l cy Hng

3.3.1. Th tc dng khng khun ca cao cn ton phn

- Chun b mu th: Cn chnh xc 10 g bt dc liu, chit vi ethanol 70% bng

phng php ngm lnh theo quy trnh phn 3.2.1.1, thu c cao lng ton phn.

Bc hi dung mi n cn. Khi lng cn cn c: 0,76 g. Ha tan cn trong 10

ml nc ct thu c cao lng 1:1 (tng ng vi hm lng cn 76mg/ml). T

cao lng 1:1, pha thnh cao lng cc nng 1:2, 1:4.

Mu th c nh s th t trnh nhm ln:

(1) Cao lng 1:1

(2) Cao lng 1:2

(3) Cao lng 1:4

Tm mu th ln cc khoanh giy lc sy kh. Mi khoanh giy tm 3 ln.

Sau mi ln tm mu th, cc khoanh giy u c sy cho bay ht dung mi.

- Chun b mi trng v cy VSV kim nh

Chun b mi trng canh thang, cy VSV kim nh v 37oC trong 18

24 gi.

Chun b mi trng thch thng, tit trng 118oC trong 30 pht, lm

ngui n 40 50oC , cy VSV kim nh vi t l 2,5 ml/100 ml, lc u VSV

43

phn tn trong mi trng thch thng, vo a petri c tit trng vi th

tch 20 ml/a. ngui cho thch ng li.

- Tin hnh kho st hot tnh khng khun: t cc khoanh giy lc ln a

thch chun b theo s Hnh 3.13, cc a petri trong t m nhit 37oC

trong 18 24 gi. Sau khi thi gian, ly cc a petri, o ng knh vng v

khun (nu c) bng thc kp Panmer nh gi mc khng khun.

i vi mi mu th, lm 6 th nghim song song.

Hnh 3.13. S sp xp khoanh giy lc cc mu th hot tnh khng khun

Ch thch:

- Cc s 1, 2, 3 tng ng vi mu (1), (2), (3).

- C: mu chun.

- nh gi kt qu: ng knh vng v khun c tnh theo cng thc:

=

=

()

Ch thch:

D: ng knh trung bnh vng v khun

Di: ng knh vng v khun

S: lch thc nghim chun c hiu chnh

n: S th nghim lm song song

Kt qu th hot tnh khong khun ca cao lng ton phn c trnh by trong

Bng 3.4.

44

Bng 3.4. Kt qu th hot tnh khng khun ca cao lng ton phn cc nng

1:1, 1:2, 1:4

VSV Mu D s

Escherichia

coli

1 - -

2 - -

3 - -

Chun 14,49 0,40

Salmonella

typhi

1 -

2 -

3 -

Chun 22,88 0,16

Proteus

mirabilis

1 - -

2 - -

3 - -

Chun 11,50 0,41

Shigella

flexneri

1 - -

2 - -

3 - -

Chun 18,78 0,57

Pseudomonas

aeruginosa

1 - -

2 - -

3 - -

Chun 11,50 0,50

VSV Mu D s

Bacillus

subtilis

1 - -

2 - -

3 - -

Chun 21,94 0,10

Bacillus

pumilus

1 8,02 0,16

2 7,82 0,48

3 - -

Chun 17,96 0,06

Sarcina lutea

1 - -

2 - -

3 - -

Chun 10,65 0,41

Bacillus cereus

1 - -

2 - -

3 - -

Chun 19,97 0,29

Staphylococcus

aureus

1 - -

2 - -

3 - -

Chun 25,13 0,15

Ch thch:

D: ng knh trung bnh ca vng v khun.

s: lch chun ( sai s thc nghim)

Nhn xt: Cao lng ton phn c 3 nng khng c tc dng i vi hu ht

cc chng vi khun kim nh. Cao lng 1:1 v 1:2 c tc dng trn vi khun

45

Bacillus pumilus, tuy nhin ng knh vng v khun rt nh. Trong , cao lng

1:1 cho ng knh vng v khun ln hn so vi cao lng 1:2.

3.3.2. Th tc dng khng khun ca phn on chloroform v ethyl acetat

- Chun b mu th: Cn cc phn on c ha tan vo nc ct vi nng 75

mg/ml (tng ng vi hm lng cn trong cao lng 1:1).

Mu th c nh s th t trnh nhm ln:

(1) Cao lng 1:1 phn on chloroform.

(2) Cao lng 1:1 phn on ethyl acetat.

Cc bc tin hnh v nh gi kt qu tng t nh i vi cao lng ton phn.

- Kt qu: Kt qu c trnh by trong Bng 3.5.

Bng 3.5. Kt qu th hot tnh khng khun ca 2 phn on

chloroform v ethyl acetat

Mu D s

Escherichia

coli

1 7,97 0,12

2 - -

chun 14,10 0,29

Salmonella

typhi

1 7,67 0,29

2 10,14 0,90

chun 22,60 0,40

roteus

mirabilis

1 - -

2 10,43 0,49

chun 17,35 0,05

Shigella

flexneri

1 9,57 0,27

2 11,46 0,18

chun 17,06 0,56

Pseudomonas

aeruginosa

1 - -

2 - -

chun 9,31 0,19

Mu D s

Bacillus

subtilis

1 7,42 0,27

2 9,94 0,32

chun 25,05 0,05

Bacillus

pumilus

1 8,08 0,30

2 12,13 0,80

chun 17,30 0,30

Sarcina lutea

1 - -

2 - -

chun 8,88 0,13

Bacillus cereus

1 7,57 0,09

2 - -

chun 23,61 0,51

Staphylococcus

aureus

1 7,54 0,04

2 - -

chun 25,13 0,13

46

Ch thch:

D: ng knh trung bnh ca vng v khun

s: lch chun ( sai s thc nghim)

Nhn xt: C 2 phn on u c hot tnh khng khun. Vng v khun ca cc

mu th u nh hn mu chun.

- Phn on chloroform c tc dng i vi 7 chng vi khun kim nh,

trong tc dng tt nht i vi cc chng: Shigella flexneri v Bacillus pumilus.

- Phn on ethyl acetat c tc dng i vi 5 chng vi khun kim nh, cc

vng v khun quan st r, kch thc kh ln, c bit i vi cc chng: Shigella

flexneri, Bacillus pumilus.

i vi nhng chng c 2 phn on u c tc dng, phn on ethyl acetat

cho thy tc dng mnh hn so vi phn on chloroform.

Hnh nh vng v khun ca mt s mu th trong hnh 3.15.

Bacillus pumilus(BP) Shigella flexneri (Shi)

Hnh 3.15. Vng v khun ca mu th phn on chloroform v ethyl acetat vi vi

khun Bacillus pumilus v Shigella flexneri.

Ch thch:

1. Mu th phn on chloroform

2. Mu th phn on ethyl acetat

47

3.4. Bn lun

Cy Hng l loi cy c tim nng ln khai thc lm dc liu. L Hng

l nguyn liu ph bin, sn c, d thu hi v bo qun.Ti nhiu nc trn th gii,

l Hng c nghin cu chng minh tc dng v s dng lm thuc. Tuy nhin,

Vit Nam, cy Hng ch c trng lm cy n qu, cha c nghin cu c

cng b v vic s dng l Hng lm thuc. Do , chng ti thc hin ti ny

nhm to c s bc u cho vic nghin cu v kh nng khai thc v s dng loi

dc liu ny. Sau thi gian nghin cu, chng ti nhn thy cc vn sau:

V thc vt

- V c im hnh thi: So snh cc c im quan st c ca mu nghin

cu vi cc c im ca l Hng c m t trong cc ti liu tham kho, thy hu

ht cc c im u trng khp. iu ny gp phn khng nh tn khoa hc ca

mu nghin cu. Tuy nhin, Hng l loi cy a dng v ging cng nh c im

thc vt, do , cn nghin cu k hn trnh nhm ln trong qu trnh khai thc

v s dng dc liu ny.

- V c im vi hc: Nghin cu c im vi hc cho thy hnh nh c

trng ca vi phu l Hng v hnh dng, cu to. c trng ca bt l: nhiu lng

che ch n bo thun di, lng tit c u a bo mu , cc tinh th calci oxalat

hnh khi a din. Nhng c im ny c th s dng kim nghim, phn bit

dc liu theo phng php vi hc.

V ha hc

- nh tnh s b cc nhm cht bng phn ng ha hc xc nh c l Hng

cha flavonoid, saponin, tanin, ng kh, acid hu c, acid amin, caroten, sterol.

Ring thnh phn polysaccharid, mt s nghin cu trc y trn th gii

phn lp v th tc dng ca cc polysaccharid t l Hng, tuy nhin kt qu nh

tnh s b cho thy mu nghin cu khng cha nhm cht ny. khng nh

chnh xc, cn tin hnh thm cc nghin cu su hn.

- Chit xut ngm lnh vi dung mi ethanol 70%, nhn thy dung mi ny chit

c phn ln cc nhm cht trong dc liu. Tuy nhin, s dng dung mi ny

48

khng loi c cc tp cht trong dc liu, gy nh hng ti qu trnh nh tnh.

Do , phi tin hnh loi b tp khi s dng quy trnh chit xut ny.

- Chit xut phn on v xc nh hm lng cn thu c so vi dc liu

kh tuyt i cho kt qu: hm lng cn n-hexan, chloroform, ethyl acetat ln lt

l 0,28%, 0,84% v 3,55%. Kt qu ny cho thy dung mi ethyl acetat cho hiu

sut chit cao nht. iu ny nh hng cho vic la chn dung mi chit xut

thch hp cho cc nghin cu ha hc chuyn su hn.

- nh tnh cc phn on bng phn ng ha hc cho thy cc nhm cht chnh

(flavonoid, saponin, tanin) ch yu tp trung trong phn on chloroform v ethyl

acetat. Phn on n-hexan khng thy s c mt ca cc nhm cht ny. Da vo

, c th la chn phn on cho qu trnh th tc dng sinh hc ca dc liu.

- nh tnh cc phn on bng sc k lp mng: C 3 phn on u s dng

h dung mi khai trin Toluen EtOAc Acid formic vi cc t l khc nhau. Cc

h dung mi ny u cho sc k c cc vt tch r, d quan st. X k bng

phn mm VideoScan thu c th c trng ca mi phn on. y l nghin

cu u tin v sc k lp mng ca dch chit l cy Hng. Cc kt qu ny c th

dng gp phn xy dng tiu chun kim nghim dc liu, ng thi to c s

la chn h dung mi trong qu trnh phn lp cht t cc phn on.

V tc dng khng khun

- Kho st hot tnh khng khun ca cao cn ton phn cc nng 1:1, 1:2,

1:4 cho thy cao ton phn hu nh khng c tc dng trn cc chng vi khun

kim nh. Trong , cao lng 1:1 c tc dng yu trn vi khun Bacillus pumilus.

D on nguyn nhn c th do cao long, khng th hin c tc dng, cn kho

st thm nhng nng m c hn nh cao lng 2:1, 3:1...

- Kt qu kho st hot tnh khng khun ca 2 phn on chloroform v ethyl

acetat cho thy c 2 phn on u c tc dng tt trn nhiu chng vi khun Gram

(+) v Gram (-). Kt qu ny chng minh l Hng c tc dng khng khun, t

c th nghin cu su hn tm ra cht c tc dng ny.

49

KT LUN V KIN NGH

Kt lun:

Trong khun kh kha lun tt nghip ny, chng ti thu c nhng kt

qu nh sau:

V nghin cu c im thc vt:

M t c chi tit v c im hnh thi l, c im vi phu v bt dc liu.

V nghin cu thnh phn ha hc:

- nh tnh s b bng phn ng ha hc cho thy l Hng c cha flavonoid,

saponin, tanin, ng kh, acid hu c, acid amin, caroten v sterol.

- s b xc nh c hm lng cn ca cc phn on so vi dc liu kh

tuyt i nh sau: Cn H (phn on n-hexan) 0,28%; cn C (phn on

chloroform) 0,51%; cn E (phn on ethyl acetat) 3,55%.

- nh tnh bng phn ng ha hc cho thy:

Phn on n-hexan khng cha cc nhm cht flavonoid, saponin, tanin.

Phn on chloroform v ethyl acetat c cha flavonoid, saponin, tanin.

- xc nh c cc h dung mi tch tt i vi cc phn on nh sau:

Phn on n-hexan: Toluen: EtOAc: Acid formic (8:3:1)

Phn on chloroform: Toluen: EtOAc: Acid formic (8:3:1)

Phn on ethyl acetat: Toluen: EtOAc: Acid formic (9:6:1).

V tc dng khng khun:

Cao lng chit cn 1:1 c tc dng khng khun yu.

Dch chit phn on chloroform v ethyl acetat u c tc dng khng

khun. Phn on chloroform c tc dng vi 7 chng vi khun kim nh; phn

on ethyl acetat c tc dng vi 5 chng vi khun kim nh. Trong , phn on

ethyl acetat c tc dng mnh hn phn on chloroform.

Kin ngh:

Do iu kin thi gian c hn, trong khun kh ca kha lun tt nghip ny,

cc nghin cu chng ti thc hin mi ch l bc u, mang li nhng nh gi

50

s b. c c s khoa hc nhm khai thc v s dng c hiu qu ngun dc

liu l cy Hng, chng ti a ra mt s xut sau y:

1. Tip tc nhng nghin cu su hn v thnh phn ha hc ca v thuc l

cy Hng nh phn lp cc cht t dch chit cc phn on nhm cung cp

c s tin hnh sc k vn tay cho kim nghim dc liu.

2. Tip tc nghin cu chit xut l Hng theo nh hng flavonoid v th cc

tc dng sinh hc c lin quan n thnh phn ny.

3. Trn c s cc cng dng ang c ng dng ca l cy Hng, tin hnh

nghin cu v mt s tc dng sinh hc nhm chng minh cng dng cha

bnh v to c s khoa hc cho vic s dng dc liu ny.

TI LIU THAM KHO

Ti liu ting Vit

1. L nh Bch, Trn Vn n (2007), Thc vt hc, NXB Y hc, H Ni, tr

390-394.

2. B mn Dc liu (2010), Thc tp dc liu, Trung tm Thng tin Th

vin i hc Dc H Ni, H Ni, tr. 61-79.

3. B mn Dc liu (2012), Phng php nghin cu dc liu, i hc Y

Dc TP. H Ch Minh, TP. H Ch Minh.

4. B mn Thc vt (2004), Thc tp Thc vt v nhn bit cy thuc, Trung

tm Thng tin Th vin i hc Dc H Ni, H Ni.

5. B mn Vi sinh Sinh hc (2005), Thc tp vi sinh k sinh, Trung tm

Thng tin Th vin i hc Dc H Ni, H Ni, tr. 48-55.

6. V Vn Chi (1997), T in cy thuc Vit Nam, NXB Y hc, TP. H Ch

Minh, tr. 579-580.

7. V Vn Chi (2003), T in thc vt thng dng tp I, NXB Khoa hc v k

thut, TP. H Ch Minh, tr. 974-980.

8. Tt Li (2004), Nhng cy thuc v v thuc Vit Nam, NXB Y hc, H

Ni, tr. 737-738.

9. Ng Vn Thu (1998) Bi ging dc liu tp I, Trung tm Thng tin Th

vin i hc Dc H Ni, H Ni.

10. Vin Dc liu (2006), Cy thuc v ng vt lm thuc Vit Nam tp I,

NXB Khoa hc v k thut, H Ni, tr 994-997.

Ti liu nc ngoi:

11. Hala Abozaid, Abeer Moawad, Elham Amin, Mona Hetta, Marawan

Shabana (2014), Phytochemical and biological study of Diospyros kaki L.

growing in Egypt, World Journal of Pharmaceutical Research, 3(2), 1786-

1795.

12. Chang Y.X. (2000), Taiwan Native Medicinal Plants, Committee on Chinese

medicine and pharmacy. Dept. Health, Executive Yuan, Taipei, Taiwan.

13. Chen G., Wei SH, Huang J, Sun J (2009), A novel C-glycosylflavone from

the leaves of Diospyros kaki, Journal of Asian Natural Products Research,

11(6),503-507.

14. Jinyou Duan, Vincent L. Chen, Qun Dong, Kan Ding, Jinian Fang (2010),

Chemical structure and immunoinhitory activity of a pectic polysaccharide

containing glucuronic acid from the leaves of Diospyros kaki, International

Journal of Biological Macromolecules, 46, 465 470.

15. Jinyou Duan, Xuesong Wang, Qun Dong, Ji-nian Fang, Xiaoyu Li (2003),

Structural features of a pectic arabinogalactan with immunological activity

from the leaves of Diospyros kaki, Carbonhydrate Research, 338(12),

1291-1297.

16. Jinyou Duan, Yun Zheng, Qun Dong, Jinian Fang (2004), Structural

analysis of a pectic polysaccharide from the leaves of Diospyros kaki,

Phytochemistry, 65(5), 609-615.

17. Duke J.A., Ayensu, E.S. (1985) Medicinal Plants of China. vol. 1 and 2,

Reference Publ. Inc. Algonac, Michigan.

18. Kenji Kameda, Takeshi Takaku, Hiromichi Okuda, Yoshiyuki Kimura

(1987), Inhibitory effects of various flavonoids isolated from leaves of

persimmon on angiotensin converting enzyme activity, Journal of Natural

Products, 50(4), 680-683.

19. Thomas S.C. Li (2002), Chinese and Related North American Herbs:

Phytopharmacology and Therapeutic Values, CRC Press, Florida USA.

20. Sunity Singh, Himanshu Joshi (2011), Diospyros kaki (Ebenaceae): A

Review, Asian Journal of Research in Pharmaceutical Sciences, 1(3), 55-

58.

21. B. N. Sinha, S. K. Bansal (2008), A review of phytochemical and

biological studies of Diospyros species used in folklore medicine of

Jharkhand, Journal of Natural Remedies, 8(1), 11-17.

22. Lee Shu-kang, Michael G. Gilbert, Frank White (1996), Ebenaceae, Flora

of China, 15, 215-234.

23. Lijun Sun, Jianbao Zhang, Xiaoyun Lu, Liyu Zhang, Yali Zhang (2011),

Evaluation to the antioxidant activity of total flavonoids extract from

persimmon (Diospyros kaki L.) leaves, Food and Chemical Toxicology, 49,

2689-2696.

24. Armen Takhtajan (2009), Flowering Plants, Sringer.

25. Phuong Thien Thuong, Chul Ho Lee, Trong Tuan Dao, Phi Hung Nguyen,

Wan Gi Kim, Sang Jun Lee, Won Keun Oh (2008), Triterpenoid from the

Leaves of Diospyros kaki (Persimmon) and Their Inhibitory Effects on

Protein Tyrosine Phosphatase 1B, Journal of Natural Products, 71, 1775-

1778.

26. Titto Varughese, Mozahidur Rahaman, No-Soo Kim, Soon-Chang Cho,

Surk-Sik Moon (2009), Gamnamoside, aPhenylpropanoid Glycoside from

Persimmon Leaves (Diospyros kaki) with an Inhibitory Effect against an

Alcohol Metabolizing Enzyme, Bulletin of the Korean Chemical Society,

3(5), 1035-1038.

27. H. Wagner, S. Bladt (1996), Plant Drug Analysis: A Thin Layer

Chromatography Atlas, Springer, New York.

28. Xue YL, Miyakawa T, Hayashi Y, Okamoto K, Hu F, Mitani N, Furihata K,

Sawano Y, Tanokura M (2011), Isolation and tyrosinase inhibitory effects

of polyphenols from the leaves of persimmon, Diospyros kaki, Journal of

Agricultural and Food Chemistry, 59(11), 6011-6017.

29. Ren-Jie Yin, Xue-Feng Xiao, Yuan-Yuan Xu, Xing-Hua Liu, De-Qin Wang,

Chu-Yuan Li, Chang-Xiao Liu (2010), Research information and review on

the leaves of Diospyros kaki L., Asian Journal of Pharmacodynamics and

Pharmacokinetics, 10(3), 181-207.

PH LC 1:

PHIU GIM NH TN KHOA HC

PH LC 2:

TH, BNG BIU DIN KT QU SC K CC PHN ON

DCH CHIT L HNG

2.1. th v kt qu sc k phn on n-hexan phn tch di nh sng t

ngoi bc sng 254 nm

2.2. th v kt qu sc k phn on n-hexan sau khi nhng thuc th

vanilin trong ethanol 96%, phn tch di nh sng thng

2.3. th v kt qu sc k phn on chloroform phn tch di nh sng

t ngoi bc sng 254 nm

2.4. th v kt qu sc k phn on chloroform sau khi nhng thuc

th vanilin trong ethanol 96%, phn tch di nh sng thng

2.5. th v kt qu sc k phn on ethyl acetat phn tch di nh sng

thng

2.6. th v kt qu sc k phn on ethyl acetat phn tch di nh sng

t ngoi bc sng 254 nm

2.7. th v kt qu sc k phn on ethyl acetat sau khi nhng thuc

th vanilin trong ethanol 96%, phn tch di nh sng thng

T VN Chng 1. TNG QUAN1.1. c im thc vt1.2. Thnh phn ha hc1.3. Tc dng sinh hc ca v thuc l cy Hng

Chng 2. I TNG V PHNG PHP NGHIN CU2.1. Nguyn vt liu v thit b nghin cu2.2. Ni dung nghin cu2.3. Phng php nghin cu

Chng 3. THC NGHIM, KT QU V BN LUN3.1. Nghin cu c im thc vt ca l cy Hng3.1.1. c im hnh thi3.1.2. c im vi phu3.1.3. c im bt l

3.2. Nghin cu thnh phn ha hc ca l cy Hng3.2.1. nh tnh cc nhm cht trong dc liu bng phn ng ha hc3.2.2. Chit xut, nh tnh v nh lng cc phn on

3.3. Th tc dng khng khun ca l cy Hng3.3.1. Th tc dng khng khun ca cao cn ton phn3.3.2. Th tc dng khng khun ca phn on chloroform v ethyl acetat

3.4. Bn lun

KT LUN V KIN NGH