Phân lập một số hợp chất từ phân đoạn Chloroform chiết xuất từ Cỏ seo gà.pdf

B Y T

TRNG I HC DC H NI

NGUYN TH TUYT NHUNG

PHN LP MT S HP CHT

T PHN ON CHLOROFORM

CHIT XUT T C SEO G

KHA LUN TT NGHIP DC S

H NI 2014

B Y T

TRNG I HC DC H NI

NGUYN TH TUYT NHUNG

PHN LP MT S HP CHT

T PHN ON CHLOROFORM

CHIT XUT T C SEO G

KHA LUN TT NGHIP DC S

Ngi hng dn:

1. NCS. Nguyn Duy Ch

2. Triu Th Nhung

Ni thc hin:

1. B mn dc liu - i hc Dc H Ni

2. Trung tm Kim nghim Dc Qun i

H NI 2014

LI CM N

Trong sut qu trnh lm kha lun tt nghip, ti nhn c rt

nhiu s gip t thy c, gia nh v bn b. Nhng s gip qu bu y

gip ti hon thnh kha lun ny, ng thi cng cho ti hiu bit thm

nhiu iu v cch t duy trong nghin cu khoa hc. Nhn dp ny ti xin

by t li cm n su sc ti:

PGS.TS. Nguyn Thi An, c tn tnh ch bo, to mi iu kin gip

ti trong qu trnh thc hin kha lun.

Ths.Nguyn Duy Ch, DS. Triu Th Nhung tn tnh ch bo, to

mi iu kin v ha cht, trang thit b trong qu trnh thc hin v trc

tip hng dn ti hon thnh kha lun.

Ti cng xin chn thnh cm n cc thy c, cc k thut vin B mn

Dc liu, cc anh, ch trong Trung tm Kim nghim Dc Qun i, cc

phng ban trong Trung tm Kim nghim Dc Qun i gip to iu

kin cho ti thc hin kha lun ny.

Cui cng, ti xin cm n gia nh, bn b ng vin, khch l ti rt

nhiu ti c thm s nhit tnh v say m trong nghin cu khoa hc.

H ni, ngy 14 thng 5 nm 2014.

Sinh vin

Nguyn Th Tuyt Nhung

MC LC

Danh mc cc k hiu, ch vit tt

Danh mc bng

Danh mc hnh

T VN ..1

CHNG 1: TNG QUAN3

1.1. V TR PHN LOI, C IM THC VT V PHN B.3

1.1.1. V tr phn loi ca chi Pteris L...3

1.1.2. c im chung ca h C lung (Pteridaceae)......3

1.1.3. c im thc vt chi Pteris L3

1.1.4. c im thc vt v phn b ca c Seo g (Pteris multifida

Poir.)...4

1.2. THNH PHN HA HC..5

1.2.1. Thnh phn ha hc ca chi Pteris L...5

1.2.2. Thnh phn ha hc ca c Seo g (Pteris multifida Poir.).6

1.3. TC DNG DC L..11

1.4. TNH V - CNG DNG...14

1.5. MT S N THUC..15

CHNG 2: I TNG V PHNG PHP NGHIN CU..16

2.1. NGUYN LIU NGHIN CU.....16

2.2. PHNG TIN NGHIN CU ............ 16

2.2.1. Cc trang thit b dng trong nghin cu ... 16

2.2.2. Ha cht ..... 17

2.3. PHNG PHP NGHIN CU17

2.3.1. nh tnh......17

2.3.2. Chit xut v phn lp .................................................................. 17

2.3.3. Nhn dng cht tinh khit ............................................................. 18

CHNG 3: THC NGHIM, KT QU V BN LUN ..................... 19

3.1. CHIT XUT ...................................................................................... 19

3.1.1. Xc nh m dc liu ............................................................. 19

3.1.2. Chit xut ..................................................................................... 19

3.1.3. nh tnh cn phn on chloroform bng SKLM ........................ 22

3.2. PHN LP ........................................................................................ 25

3.2.1. Chun b ct ................................................................................. 25

3.2.2. Tin hnh ..................................................................................... 26

3.2.3. Kim tra tinh khit cht phn lp ............................................. 28

3.3. NHN DNG CC CHT PHN LP .............................................. 30

3.4. BN LUN .......................................................................................... 32

KT LUN V XUT ......................................................................... 35

KT LUN ........................................................................................... 35

XUT .............................................................................................. 35

TI LIU THAM KHO

PH LC

DANH MC CC K HIU, CH VIT TT

Ch vit tt Ch vit y

AST nh sng thng

BHA Butylated hydroxyanisol

Dd Dung dch

DEPT Detortionless Enhancement by Polarization

Transfer

DPPH 2,2-diphenyl picrylhydrazyl

C Chloroform

CC Column chromatography

CK Creatinin kinase

H n-hexan

IC50 Half maximal inhibitory concentration

KHV Knh hin vi

LPS Lipopolisaccharid

MeOH Methanol

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

tetrazolium bromid

NMR Nuclear Magnetic Resonance

13C-NMR Carbon (13) Nuclear Magnetic Resonance

1H-NMR Proton (1) Nuclear Magnetic Resonance

PCA Protocatechu aldehyd

Rf H s di chuyn

ROS Reactive Oxygen Spcecies

SKLM Sc k lp mng

TT Thuc th

UV254 nh sng t ngoi bc sng 254nm

UV365 nh sng t ngoi bc sng 365nm

XDT Xanh da tri

DANH MC BNG

STT K hiu Tn bng Trang

1 Bng 1.1 Mt s hp cht phn lp t c Seo g (Pteris

multifida Poir.) 8

2 Bng 1.2 Nng c ch ti thiu ca mt s hp cht

phn lp t c Seo g 13

3 Bng 3.1

Hm lng % ca cc cn so vi khi lng

bt dc liu 20

4 Bng 3.2 Kt qu nh tnh mt s nhm cht trong cn

chloroform 22

5 Bng 3.3 Kt qu nh tnh cn phn on chloroform

bng SKLM 24

6 Bng 3.4 Kt qu SKLM ca PM12 vi 3 h dung mi

AST v UV254 29

7 Bng 3.5 D liu ph NMR ca PM12 32

DANH MC HNH

STT K hiu Tn hnh Trang

1 Hnh 3.1 S chit xut cn cc phn on t c Seo g 21

2 Hnh 3.2 Sc k ca cn C vi h dung mi IV cc

iu kin quan st 23

3 Hnh 3.3 S phn lp cc cht t phn on

chloroform chit xut t c Seo g 27

4 Hnh 3.4 Hnh nh SKLM ca PM12 vi 3 h dung mi

UV254 28

5 Hnh 3.5 Hnh nh SKLM ca PM12 vi 3 h dung mi

nh sng thng 29

6 Hnh 3.6 Sc k so snh PM12 vi cn chloroform 30

7 Hnh 3.7 nh chp tinh th PM12 di KHV vt knh 20 31

8 Hnh 3.8 Cu trc ha hc ca hp cht PM12 32

1

T VN

Vi v tr a l v thin nhin u i, Vit Nam nm trong vng kh hu

nhit i gi ma, nng v m, thch hp cho vic pht trin h thc vt phong

ph v a dng, trong c bit k n nhm ti nguyn cy thuc. Theo kt

qu iu tra ca Vin Dc liu nm 2006, Vit Nam c 3948 loi thc vt bc

cao, bc thp v nm ln c dng lm thuc [1]. Tuy nhin phn ln cc cy

cn c s dng theo kinh nghim dn gian hoc theo Y hc c truyn m cha

c nghin cu k v y .

C Seo g l mt trong 280 loi thuc chi Pteris L. [39], [41], c phn

b rng ri cc vng nhit i v cn nhit i, c ngun gc t Trung Quc

v Nht Bn [15]. Trong Y hc c truyn Trung Quc, c Seo g c s dng

iu tr cc bnh: eczema, hong n, th huyt, vim rut, l trc khun

[24], bn cnh c Seo g cn c bit n vi tc dng chng ung th v

khng khun [25]. Vit Nam, c Seo g v ng nht, tnh mt, c tc dng

thanh nhit, gii c, gim au c dng cha kit l mn tnh, l trc

khun, vim gan, vim ng tit niu, vim hng, vim tuyn nc bt, sng

v, mn nht, l nga, bnh ngoi da [15].

Nhn thy, c Seo g l mt cy thuc dn gian cn c quan tm, tuy

nhin nhng nghin cu tng th v c Seo g cn t v cha thc s y . T

nm 2012, Nguyn Duy Ch v cng s tin hnh kho st, nghin cu thnh

phn ha hc ca c Seo g v thu c cc kt qu bc u. Nhm khai

thc ngun nguyn liu sn c ng dng trong phng v tr bnh song song vi

vic chng minh kinh nghim s dng c Seo g trong dn gian gp phn b

sung vo kho tng cy thuc Vit Nam mt dc liu mi, chng ti tin

2

hnh thc hin ti Phn lp mt s hp cht t phn on chloroform

chit xut t c Seo g. vi nhng mc tiu sau:

1. Chit xut, phn lp mt s hp cht t phn on chloroform chit xut

t c Seo g.

2. Nhn dng cht phn lp da trn cc d liu ph.

thc hin c mc tiu ra, ti c tin hnh vi cc ni

dung sau:

1. Gim nh tn khoa hc mu nghin cu.

2. Chit xut phn on chloroform.

3. Phn lp cht tinh khit t phn on chloroform.

4. Nhn dng cht phn lp c da trn cc d liu ph.

3

CHNG I

TNG QUAN

1.1. V TR PHN LOI, C IM THC VT V PHN B.

1.1.1. V tr phn loi ca chi Pteris L.

Theo [1], [8], [9] c Seo g thuc chi Pteris L., h C lung (Pteridaceae),

b C lung (Pteridales), phn lp Dng x (Polypodiidae), lp Dng x

(Polypodiopsida), ngnh Dng x (Polypodiophyta).

Ngnh Dng x (Polypodiophyta)

Lp Dng x (Polypodiopsida)

Phn lp Dng x (Polypodiidae)

B C lung (Pteridales)

H C lung (Pteridaceae)

Chi Pteris L.

1.1.2. c im chung ca h C lung (Pteridaceae).

H c Lung cn gi l h c Seo g [1]. H c Lung gm 23 chi, phn b

rng ri trn th gii. Vit Nam c 12 chi vi khong 50 loi, mc hoang. C

mt loi thng lm thuc l c Seo g (Pteris multifida Poir.) [1].

Cy mc rt gn t, c nhiu lng vy [1]. L c hai loi: bt th v hu

th. L kp lng chim, chia thy u n, t khi x ngn. Gn ni vi nhau thnh

hnh vnh khuyn [1].

ti bo t mp l hay gia vnh khuyn, c o do mp l gp li, lin nhau

thnh mt ng lin tc. Vng c gii khng y , i qua chn [1].

1.1.3. c im thc vt chi Pteris L.

4

Thn r mc b, c vy mu en [1]. L ging nhau hoc c 2 loi. L kp

lng chim, gn l ni vi nhau to hnh vnh khuyn [41], cung mang 3 l ph

to hoc l ph vi thy di, t hoc thy u, nhiu [13].

ti bo t mp l hoc gia vnh khuyn, c o do mp l gp li,

lin nhau thnh di lin tc [41]. Bo t hnh t din hoc hnh cu, mu nu

nht hoc en, c u sn [41].

Chi Pteris L. c khong 280 loi [39], [41], l mt chi ln, phn b khp

cc nc thuc vng nhit i v cn nhit i nh Trung Quc, Nht Bn, i

loan, hoc mt s nc chu Phi, chu c, chu M, tr khu vc a Trung

Hi [15], [7].

1.1.4. c im thc vt v phn b ca c Seo g (Pteris multifida Poir.)

Tn ng ngha: Pteris serrulata L. f. [12], [15].

Tn khc: Phng v tho, c Lung [11], [15], [12]; Rng seo g s nhiu

[12].

Tn nc ngoi: Spider brake (Anh) [15].

Cy nh, thn c, cao trung bnh 20-25cm, c cy cao hn [12]. Thn r

nh, ngn chng 3-4cm [12], [15], hnh cong queo, sn si, nhiu mu, hi cng,

v hi ngt, ng v t, mi thm hc [12].

L mc thng t thn r, x su hnh lng chim hai ln, gn l r, c 2 loi

l: l khng sinh sn ngn, mu lc nht hi vng, cc thy to nh khng u

mc i nhau, mp hi kha rng, c u trn, ring thy tn cng thun di

thnh mi nhn; l sinh sn di gm cc thy hnh di thun un o, mc i,

u nhn hot, mp l gp li mang ti bo t dy c pha trong. Cung l rt

di, mu nu nht gc, hi vng pha trn [15]. Ma sinh sn: thng 5-10

[15].

5

Bo t hnh bn cnh, hi trn, mu vng nht, c nhiu u sn nh [15].

C Seo g c ngun gc t Trung Quc, Nht Bn [15]. C Seo g c vng

phn b t nhin khp khu vc ng v ng Bc . nc ta, c Seo g mc

ph bin cc tnh min ni, trung du v i khi c vng ng bng. C Seo

g l cy sng dai, c th gp trn nhng vch vi, vng t cn ci, xung

quanh b ging, ven ng i, nhng ni thong m v mt [12], [15].

1.2. THNH PHN HA HC

1.2.1. Thnh phn ha hc ca chi Pteris L.

Theo mt s ti liu cho thy thnh phn ha hc ca chi Pteris L. bao gm:

flavonoid, diterpenoid, diterpenoid glycosid, sesquiterpenoid [21]; flavonoid

glycosid [21], [33], [36]; cc hp cht phenolic [9]; kauran [46]; pterosin-

sesquiterpen [26], [31].

T. Hakamatsuka v cng s phn lp t l v thn r ca Pteris cretica Linni.,

c cc hp cht ent-kauran glycosid l: 2, 6, 15-trihydroxy-ent-kaur-16-ene-2-O-

-D-allopyranosid; 2,6,15-trihydroxy-ent-kaur-16-ene; 2,6,15-trihydroxy-ent-

kaur-16-ene-2-O--D-glucopyranosid; 2,16-dihydroxy-ent-kaurane [39]. Nm 1994,

t Pteris cretica Linnin., F. Imperato chit tch v phn lp mt s flavonoid

glycoside l: luteolin 8-C-rhamnosid-7-O-rhamnosid; luteolin-7-O-glucopyranosid v

luteolin-7-O-gentiobiosid [20].

T dch chit ethylacetat ca Pteris ensiformis Burm., Chen YH v cng s

phn lp c: 2R,3R-pterosin L-3-O--D-glucopyranosid, -D-xylopyranosyl(12)-7-

O-benzoyl--D-glucopyranosid, 4-O-benzoyl--D-xylopyranosyl(12)-7-O-benzoyl--

D-glucopyranosid, 5-[2-hydroxyethylidene]-2(5H)-furanone, -D-glucopyranosyl

benzoic acid ester, pterosin B, benzoic acid, 5-O-coumaroylquinic acid, coumaric acid,

cyclolaudenol, -sitosterol-3-O--D-glucosid v -D-sitosterol [47].

6

Nm 2010, t Pteris semipinnata L., Shi v cng s phn lp c mt ent-

kauran diterpenoid glucosid l pterisid v 3 sesquiterpenoid l (2R)-pterosin B; (2S, 3S)-

pterosin C; pterosin C-3-O--D-glucosid [34]. Bn cnh , Zha-Jun Zhan v cng s

phn lp c 3 sesquiterpenoid l (2R)-norpterosin B, (2R)-12-O--D-

glucopyranosyl- norpterosin B v semipterosin A [53].

1.2.2. Thnh phn ha hc ca c Seo g (Pteris multifida Poir.)

Theo [14] trong c Seo g c cha: flavonoid, coumarin, acid hu c,

ng kh, sterol; khng cha glycosid tim, saponin, tanin, anthranoid, alcaloid,

acid amin, cht bo, caroten, polysaccharid.

Theo [15], t c Seo g, Okuno Masaaki v cng s phn lp c -

sitosterol v -sitosterol--glucosid; Woerdenbag Herman J. v cng s phn

lp c 2 cht diterpen l ent-kauran-2, 16-diol v ent-kaur-16-en-2,15-

diol.

Theo B Trung dc T hi, c Seo g cha cc pterosin B, F, O, S, C;

pterosin C-3-O--D-glucosid; 2,15-dihydroxy-ent-kaur-16-en v dn cht 2-

O--D-glucosid; 2-16-dihydroxy-ent-kauran; apigenin-7-O--D-glucosid;

luteolin-7-O--D-glucosid [15].

Mt s nh khoa hc phn lp v xc nh cu trc ca cc pterosin

sesquiterpen v ent-kauran diterpenoid t dch chit ethanol ca Pteris multifida

Poir. l: multifidosid A, multifidosid B, multifidosid C v pterokauran M1,

pterokauran M2, pterokauran M3 [47].

T r c Seo g, chit xut hai hp cht xanthon O-glycosid: 1-hydroxy-

4,7-dimethoxy-8-(3-methyl-2-butenyl)-6-O--L-rhamnopyranosyl-(12)-[-D-

glucopyranosyl-(13)]--D-glucopyranosylxanthon; 1,3-dihydroxy-7-methoxy-

7

8-(3-methyl-2-butenyl)-6-O--L-rhamnopyranosyl-(12)-[-D-glucopyranosyl-

(13)]--D-glucopyranosylxanthon [50].

Theo Wang WS v cng s, t dch chit methanol ca Pteris multifida

Poir., phn lp c mt sesquiterpen glycosid l pterosin C-3-O--D-

glucosid v cc flavonoid l: apigenin, luteolin, apigenin-7-O--D-glucosid,

apigenin-7-O--D-glucosyl-4-O--L-rhamnosid, apigenin-4-O--L-rhamnosid,

luteolin-7-O--D-glucosid [43].

Nm 2008, Harinantenaiana L v cng s phn lp c 4-caffeoyl

quinic acid 5-O-methyl ether, (2R,3R)-pterosin L-3-O--D-glucopyrannosid, -

sitosterol--D-glucopyrannosid, apigenin-7-O--D-glucopyrannosid, luteolin-7-

O--D-glucopyrannosid, sucrose, caffeic acid, pterosid A, pterosin C-3-O--D-

glucopyranosid, 4,5-dicaffeoyl quinic acid, (2S)-5,7,3,5-tetrahydroxyflavanon,

pterosid C, wallichosid [31].

T dch chit n-BuOH, Xu-dong Zheng, Hao-bin Hu xc nh c

scaphopetalon, 6,7-dihydroxy-3'-methoxy-4',5'-methylenedioxyisoflavon 6-O--

D-xylopyranosyl-(16)--D-glucopyranosid, (-)-isolariciresinol-3--O--

apiofuranosyl-(12)-O--glucopyranosid, polyporusteron [48] v hai glycosid

neolignan mi: (7S,8S)-2,9-dihydroxy-5-methoxy-7,3-dioxy-8,4-neolignan-

4-O--D-apiofuranosyl-(16)--D-glucopyranosid (multifidosid A) v (7S,8S)-

2,9,9-trihydroxy-7,3-dioxy-7,3-dioxy-8,4-neolignan-4-O--D-apiofuranosyl -

(16)--D-glucopyranosid [49].

Nm 2011, Jianqun Liu v cng s phn lp c hai Pterosin dimers l

ng phn ca nhau: bimutipterosin A v bimutipterosin B [25].

8

Vit Nam, nm 2014 Hong L Sn v Trn Hunh Phc Tho cng b

2 hot cht chit xut t dch chit ethanol 96% ca c Seo g l : rutin v

apigenin-7-O--D-glucopyranosid [22].

Bng 1.1. Mt s hp cht phn lp t c Seo g (Pteris multifida Poir.)

STT Tn hp cht Cng thc TLTK

1 2R,3R-pterosin L-3-O--D-

glucopyranosid

OHO

O

OH

O

OH

HOHO OH

[31]

2

(2R,3S)-pterosin C (1);

(2S,3S)-pterosin C (2)

1: R1= -CH3; R2= H

2: R1= -CH3; R2= H

O

HO

R1

OR2

[52]

3 Pterosin C-3-O--D-

glucopyranosid

HOO

OO

OH

HO

HO OH

[31]

4 Apigenin O

O

HO

OH

OH

[43]

5 Apigenin-7-O--D-

glucopyranosid

O

O

O

OH

OH

O

OH

HOHO OH

[31]

9

6 Luteolin-7-O--D-

glucopyranosid O

O

O

OH

OH

O

OH

HOHO OH

OH

[31]

7 Acid caffeic

OH

HO CH CH COOH

[31]

8 -sitosterol

HO

H

H

HH

[15]

9

Pterokauran M1, M2

M1: R = H

M2: R = OH

RH

H

HO

OH

HO

[47]

10 Pterokauran M3

H

H

HO OH

OH

OH

[47]

11

Multifidosid A: R1 = M, R2 =

-CH3, R3 = OH

Multifidosid B: R1 = M, R2 =

-CH3, R3 = OH

Multifidosid C: R1 = H, R2 =

-CH3, R3= M

R

O

O

OH

OH

OHO

HO

O

M

R3

2

R1O

[25]

[49]

10

12 4-caffeoyl quinic acid 3-O-

methyl ether HHO

H

HOOC

OH

OCH3

OHOH

O

O

[31]

13 Rutin

O OH

OHO

O

O

O

OH

HO

OH

HO

HO

OHOH

OH

HO

[22]

14

Apigenin 7-O--D-

glucopyranosyl-4-O--L-

rhamnopyranosid

R1= -D-glucopyranosyl

R2= -L-rhamnopyranosid

OR1O 7 92 1'

4'

45

OH O

OR2

[22]

15

Scaphopetalon (1),

(-)-isolariciresinol 3--O--

apiofuranosyl-(12)-O--

glucopyranosid (2)

1: R1=H; R2 = Me

2: R1= Glc-Api; R2= H

1

MeO

OH

OMe

OH

R2OOR

[48]

11

16

6,7-dihydroxy-3'-methoxy-4',5'-

methylenedioxyisoflavon 6-O--

D-xylopyranosyl-(16)--D-

glucopyranosid

O

OO

HO

OMe

O

GlcXyl O

[48]

1.3. TC DNG DC L.

Cho n nay c nhiu nghin cu ghi nhn nhng hot tnh sinh hc ca

c Seo g:

Tc dng chng oxy ha:

Dch chit Pteris multifida Poir. c tc dng thu dn gc t do DPPH,

hydroxyl v c kh nng kh [28], [36], [37]. Theo Wang TC v cng s (2007),

mc d dch chit Pteris multifida Poir. c tc dng chng oxy ha yu hn so

vi BHA (butylated hydroxyanisol) nhng tc dng thu dn gc t do mnh hn

v kh nng kh ln hn mt s dch chit t tho dc khc i Loan [36].

Theo Tzu-Ching Wang v cng s, dch chit c seo g c tc dng chng oxy

ha th hin kh nng thu dn cc gc anion superoxid, 2,2'-azinobis (3-

ethylbenzothiazoline-6-sulfonic acid); to phc chelat vi cc ion kim loi

chuyn tip (Fe2+, Cu2+...); chng li s kch thch ca FeCl2/H2O2 ln qu trnh

peroxyd ha acid linoleic, ph v phn ng dy chuyn ca s peroxyd ha lipid

nn c tc dng chng peroxyd ha lipid [40]. Vit Nam, nh gi hot ng

chng oxy ha bng cch s dng cc gc t do n nh 2,2-diphenyl

picrylhydrazyl (DPPH), Hong L Sn v Trn Hunh Phc Tho chit c

rutin t dch chit c seo g c tc dng chng oxy ha mnh [22].

12

Tc dng chng gy t bin:

Bng phng php nghin cu s dng h Salmonella v microsom, v

cht gy t bin l acid picrolonic v benzopyren, cao c Seo g chit bng

cch sc vi nc c kt qu c ch mnh s gy t bin do acid picrolonic v

c tc dng yu hn i vi benzopyren [15].

Tc dng chng li ph i tuyn tin lit lnh tnh:

T dch chit ethanol 20% ca c Seo g, B.Qin v cng s phn lp c

pterosides P, luteolin, acid palmitic v apigenin-4-O--L-rhamnopyranosid.

Th tc dng trn in vitro cho thy cc cht ny c tc dng c ch s tng

trng ca t bo biu m tuyn tin lit chut trong ng nghim tng t vi

epristerid [16].

Tc dng khng khun:

Khi th tc dng khng khun ca 12 loi trong chi Pteris L. cho thy cc

chng vi khun chu tc dng bao gm: Agrobacterium tumefaciens, Escherichia

coli, Salmonella arizonae, Salmonella typhi v Staphylococcus aureus [32]. Kt

qu tng t cng tm thy trong ti liu v 3 loi trong chi Pteris L. [30].

Tc dng lm h lipid mu:

Bt ng kh ca Pteris multifida Poir. c kh nng lm h lipid mu

trn chut n ch giu cholesterol (so snh vi nhm dng -sitosterol). Kt

qu cng cho thy mu nghin cu khng nhng c kh nng lm gim

cholesterol v triglicerid mu m cn tng o thi v chuyn ha lipid qua

ng tiu ha [42].

Tc dng h ng huyt:

13

Pterosin A (hot cht c trong Pteris multifida Poir.), dng ng ung

trong 4 tun, c hiu qu gim bt s tng ng huyt, dung np glucose trong

ch n ung giu cht bo v trn chut b tiu ng. Khng thy c tc

dng ph trn chut bnh thng hay chut b bnh tiu ng ang c iu

tr bng pterosin A trong 4 tun. Trong nghin cu ny, ln u tin chng minh

pterosin A c kh nng lm h ng huyt thng qua c ch c ch

gluconeogenesis gan [19].

Tc dng chng ung th:

C nhiu nghin cu c tin hnh th tc dng ca c Seo g nh:

+ Kh nng gy c i vi dng t bo HL60 th trn in vitro (dng

t bo ung th bch cu ngi): bng phng php MTT, mt s hp cht

pterosin secquiterpenes chit t Pteris multifida Poir. c chng minh c tc

dng gy c i vi dng t bo HL60 th hin trong bng 1.2.

Bng 1.2. Nng c ch ti thiu ca mt s hp cht phn lp t c Seo g

STT Tn cht IC50 (M)

TLTK

1 2R,3R-13-hydroxy-pterosin L-3-O--d-glucopyranosid 14,6

[26] 2 2R,3S-acetyl pterosin C 48,3

3 2S,3S-acetyl pterosin C 35,7

4 Bimutipterosin A 12,8 [25]

5 Bimutipterosin B 26,6

+ Kh nng gy c i vi dng t bo HepG2 (t bo ung th biu

m gan): Multifidosid A v multifidosid B c phn lp t dch chit ethanol

95% c hot tnh gy c i vi dng t bo HepG2. Nng gy c cho 50%

t bo ln lt l 8,69M v 9,26M [47].

14

+ Kh nng chng li dng t bo MCF-7 (t bo ung th v): Theo

[22], t dch chit c Seo g, nghin cu chit xut ra apigenin-7-O--D-

glucopyranosid ch ra rng c Seo g c hot ng chng ung th mnh m

chng li dng t bo MCF-7 vi IC50 = 22,62 0,59 mg/ml.

+ Kh nng c ch s di cn ca t bo MG-63 (t bo to xng):

Cc flavonoid trong Pteris multifida Poir. c kh nng c ch s di cn ca t

bo MG-63. C ch tc ng c lin quan n s c ch mc protein vim

interleukin-6 (IL-6) trong t bo MG-63 [27].

+ c tnh c Seo g: Nm 2007, nghin cu c tnh ca dch chit

nc ca c Seo g trn chut, T.C.Wang v cng s cho thy khi s dng dch

chit nc theo ng ung khng th hin c tnh ti liu th. Th nghim

c tin hnh bng cch a dch chit theo ng ung vi liu 100, 500 v

1000 mg/kg trng lng trong vng 28 ngy [38].

1.4. TNH V - CNG DNG

Ton cy c Seo g c v ngt nht, hi ng, tnh lnh, c tc dng thanh

nhit, lng huyt, li thp, ch l [15]. R c v ngt, ng, hi t, mi thm

hc [15].

Mt s loi trong chi Pteris L. c s dng lm thuc cha bnh nh:

Pteris cretica L. dng iu tr nht, bnh ngoi da, tr, tiu chy; Pteris

semipinnata L. dng iu tr nhng vim sng c m; Pteris vittata L. c

dng iu tr thp khp [12], [13].

Ring c Seo g, ton cy c dng cha l mn tnh, l trc khun, vim

rut, vim ng tit niu, vim hng, vim tuyn nc bt, l amip, l trc

khun, vim gan, tr, chy mu, sng v, mn nht, l nga, bnh ngoi da [15].

15

Y hc c truyn Trung Hoa c nhiu nghin cu cng b chng minh c Seo

g lin quan n tc dng iu tr ung th [46], [47].

R v l c Seo g c dng lm thuc cha tiu chy, i ngoi ra mu, l

di dng thuc sc hay thuc ngm ru; nc sc i khi cn thy ra giun

[12].

R, l c Seo g sao vng, tn nh un vi du vng thnh thuc du bi

cha mt s bnh ngoi da cho tr em [12]. Nc sc l c Seo g c s dng

cha bng [15].

1.5. MT S N THUC

Cha kit l: c Seo g (ton thn), dy m lng, r c tranh, r phn en,

mi v 20g; gng sng 3 lt, sc ung lm 2-3 ln trong ngy, vo lc i (Nam

dc thn hiu). C th dng c Seo g 30g, v sn thuyn 12g, cm go rang

vng 6g hoc u en rang chy 20g, sc ung, chia lm 3 ln trong ngy [15].

Cha kit l (trc trng): r v l c Seo g sao cho thm 24g, nc

100ml, un si, gi si trong vng 30 pht. Chia 2 hay 3 ln ung trong ngy

cha l trc trng [12].

Ch ti 100g, c Seo g kh 24g, nc 150ml, un si gi si trong 30

pht. Chia hai hay ba ln ung trong ngy cha l trc trng [12].

Cha l lot, bnh ngoi da: ton thn c Seo g t thnh than, tn bt,

trn vi du vng ri bi. C th dng c Seo g ti gi p [15].

Tr xut huyt: c Seo g 60g, r cy mui 60g, sc ung trong ngy [7].

16

CHNG 2

I TNG V PHNG PHP NGHIN CU

2.1. NGUYN LIU NGHIN CU

Mu nghin cu c Seo g bao gm:

Mu cy ti mang b phn sinh sn, gim nh tn khoa hc.

Ly cy ti, phi se, sy trong t sy 50C n kh, lm nh, bo

qun trong ti nilon kn, ch mt lm nguyn liu nghin cu v

mt ha hc.

Ni thu hi: Ba V - H ni.

Thi im thu hi mu: 10/2012 - 1/2013.

Mu nghin cu c TS Th Xuyn - Vin Sinh thi v Ti nguyn

Sinh vt gim nh tn khoa hc l Pteris multifida Poir., h c Lung

(Pteridaceae).

2.2. PHNG TIN NGHIN CU

2.2.1. Cc trang thit b dng trong nghin cu

Bn mng Silicagel GF254 (Merck).

B cch thy n nhit WNB14 - MEMMERT.

Cn phn tch Metler Toledo.

Ct sc k.

n t ngoi.

Knh hin vi 2 mt YS100.

Knh hin vi quang hc Mocros MCX 100.

My nh Canon IXY 430F.

My ct quay chn khng Buchi Rotavapor R-200.

17

My xc nh hm m Sartorius.

T sy chn khng Shellab 1430D-2F.

T sy UNB 400 Memmert.

T ht c.

My o ph cng hng t ht nhn (NMR): Bruker AM500 FT-NMR

Spectrometer, Vin Ha hc cc hp cht thin nhin - Vin Hn lm

Khoa hc v Cng ngh Vit Nam.

My o im nng chy: Kofler micro-hotstage, Vin Ha hc cc hp

cht thin nhin - Vin Hn lm Khoa hc v Cng ngh Vit Nam.

2.2.2. Ha cht

Cc thuc th, dung mi, ha cht s dng trong nghin cu t tiu chun

phn tch theo tiu chun ca Dc in Vit Nam IV.

2.3. PHNG PHP NGHIN CU

2.3.1. nh tnh

nh tnh mu nghin cu bng sc k lp mng tham kho cc ti liu

sau:

- Bi ging Dc liu I [5].

- Bi ging Dc liu II [6].

- Thc tp Dc liu - Phn ha hc [3].

- Phng php nghin cu ha hc cy thuc [10].

2.3.2. Chit xut v phn lp

- Chit xut bng phng php ngm nhit phng vi dung mi

methanol. Tip tc chit p dng phng php chit lng - lng ln lt vi cc

dung mi c phn cc tng dn gm n-hexan, chloroform. Thu c 2 phn

18

on dch chit. Cc dch chit c ct thu hi dung mi n cn, tng ng l

cn H, cn C.

- Cn C c dng lm mu nghin cu phn lp cht tinh khit bng

phng php sc k ct (CC) vi cht hp ph l silicagel.

- Kim tra tinh khit cht phn lp bng SKLM vi mt s h dung mi

khc nhau.

2.3.3. Nhn dng cht tinh khit

Cc cht phn lp c dng tinh khit c kho st cc c trng vt

l: mu sc, dng th hnh, tan, nhit nng chy. Nhn dng cht phn lp

da trn cc d liu ph.

19

Chng 3

THC NGHIM, KT QU V BN LUN

3.1. CHIT XUT

3.1.1. Xc nh m dc liu

Ly khong 2g bt dc liu xc nh m. Bt my o m, iu

chnh nhit 1100C. Tri u dc liu ln a cn, y a cn v i my t

ng hin kt qu.

m ca dc liu l 15,93 %.

3.1.2. Chit xut

Cn chnh xc khong 950,00 gam bt dc liu kh, chit bng phng

php ngm nhit phng vi methanol 80% (ngm 3 ln, mi ln 2 ngy).

Lc, dch lc c gp chung, em ct thu hi dung mi v c thu c 58,39

gam cn. Cn c ha tan vo nc ct nng 60C. Tip tc chit bng phng

php chit lng - lng ln lt vi cc dung mi c phn cc tng dn n-

hexan, chloroform. Thu c 2 phn on dch chit. Cc dch chit c ct thu

hi dung mi n cn, k hiu tng ng l cn H, cn C. S chit xut c

m t nh hnh 3.1.

Tnh hm lng % ca cc cn so vi khi lng bt dc liu, kt qu

c tng hp trong bng 3.1.

- Tnh hm lng % ca cn so vi khi lng bt dc liu:

Hm lng % ca cn so vi khi lng bt dc liu c tnh theo cng

thc sau:

X% =)1(

%100.

xM

a

20

trong :

X: Hm lng (%)

M: khi lng dc liu em chit (g)

x: m dc liu (%)

a: khi lng cn phn on (g)

Bng 3.1. Hm lng % ca cc cn so vi khi lng bt dc liu

STT Phn on Khi lng

cn a(g)

% so vi cn ton

phn

% so vi nguyn

liu kh X(kl/kl)

1 n- hexan 16,67 28,55 2,09

2 Chloroform 10,00 17,13 1,25

Phn on chloroform c la chn tin hnh nh tnh bng sc k

lp mng v phn lp cht tinh khit.

21

Hnh 3.1. S chit xut cn cc phn on t c Seo g

Dc liu

Dch chit methanol

Methanol. Lc

Cn ton phn

Methanol thu hi

Dch chit nc

Nc ct 600C

n-hexan

Dch chit n-hexan

n-hexan thu hi

Cn H

Dch chit nc

Dch chit chloroform

Chloroform

Cn C

Chloroform thu hi

Dch chit nc

22

3.1.3. nh tnh cn phn on chloroform bng SKLM

- Dch chm sc k: Cn mu nghin cu ha tan hon ton trong

methanol.

- Bn mng silicagel GF254 (Merck) trng sn c hot ha 1h 1100C.

ngui.

- Tin hnh thm d trn cc h dung mi khai trin sau:

H I: Chloroform - Methanol (9:1)

H II: Ethylacetat - Methanol - H2O (10:1,35:1)

H III: Toluen - Ethylacetat - Acid formic (5:4:1)

H IV: Toluen - Ethylacetat - Acid formic (6:2:1)

H V: Ethylacetat - Chloroform - Acid formic (3:3:1)

- Hin mu vi thuc th vanillin trong H2SO4 10%, quan st di nh

sng thng.

- Tin hnh: Chm dch chit methanol ln trn bn mng, sy nh cho

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

mng ra khi bnh, sy nh cho bay ht dung mi. Pht hin vt bng n t

ngoi hai bc sng 254nm v 365nm. Sau phun thuc th l dung dch

vanillin trong H2SO4 10% ln bn mng, sy kh nhit 1100C ri quan st

di nh sng thng.

Sau nhiu ln trin khai sc k thy h IV cho kt qu tch tt nht. H IV

tip tc c trin khai SKLM cho cn chloroform. Kt qu th hin hnh 3.2

v bng 3.3.

Nhn xt:

Cn chloroform khai trin vi h dung mi IV cho kt qu nh sau:

23

Khi cha phun TT:

nh sng thng: 9 vt

nh sng t ngoi bc sng 254nm: 11 vt

nh sng t ngoi bc sng 365nm: 15 vt

Khi phun TT:

nh sng thng: 12 vt

a) b) c) d)

Hnh 3.2. Sc k ca cn C vi h dung mi IV cc iu kin quan st.

a) Khng phun TT, AST b) Khng phun TT, UV254nm

c) Khng phun TT, UV365nm d) Phun TT, AST

24

Bng 3.3. Kt qu nh tnh cn phn on chloroform bng SKLM.

STT Khng c thuc th C thuc th

Rf x 100 AST UV254 UV365 AST

1 en

+

hng

++ 3,81

2 Vng

+

hng

++

Tm

+ 9,52

3 en

+++

Hng

+

Tm

+++ 13,33

4 Vng

+ 16,19

5 Xanh l

+++

Tm

++ 20,95

6 Vng xanh

++

en

++

Hng

++ 22,85

7 en

++ 26,67

8 Hng sng

+++

Tm

+++ 29,52

9 Vng

++

en

++

Hng

++ 32,38

10 Nu

++ 34,29

11 Vng

+

en

+

Hng

++ 37,14

25

12 Nu

++ 40,00

13 hng

+++

Nu

++ 48,57

14 en

+++

50,00

15 Vng xanh

+++

en

++

Hng

++

Nu

++ 61,43

16 Vng xanh

++

en

++

Xanh sng

++++

Tm

++++ 63,33

17 Vng

+++

en

+++

hng

++++

Nu

+ 69,05

18 Vng

+++

en

++

Tm hng

+++ 72,86

19 Hng

++

Tm

+ 79,25

20 XDT

++++ 84,91

21 Xanh tm

+++ 91,51

3.2. PHN LP

3.2.1. Chun b ct

Ct thy tinh c nt mi v kha, chiu di ct 40cm, ng knh 30mm.

Ra sch, lm kh, lp c nh ct vo gi theo chiu thng ng.

Ghi ch: +: rt m ++: m +++: r ++++: rt r

26

Dng a thy tinh di lt mt lp bng (loi bng thm nc) ln trn

ng thot dch ca ct.

Cn mt lng silicagel cn dng vo cc c m. Thm dung mi ra gii

vo, dng a thy tinh khuy u cho ti khi ht bt kh.

M vi, rt hn dch trn vo ct, cho dung mi chy v silicagel lng

t nhin xung y ct. Khi dung mi chy gn ht trong ct, tip tc rt hn

dch trn vo ct. Ch khng kh dung mi ct. Tip tc dng dung mi

hng c rt ln ct v cho chy lin tc 1 thi gian. n nh ct trong 12 gi.

3.2.2. Tin hnh

Cn chloroform c ha tan bng lng dung mi chloroform/ methanol

ti thiu. Cn chloroform c tin hnh phn tch bng sc k ct nhi silicagel

pha thng (c ht 240 - 430 mesh, 0,040 - 0,063 mm), ra gii bng h dung

mi gradient n-hexan/ aceton vi phn cc tng dn (t 10/1 - 0/1, v/v), cui

cng l methanol. Chm kim tra, gp cc phn ging nhau thu c 5 phn

on chnh l F4 (1g), F5 (0,34g), F6 (0,67g), F7 (1,34g), F8 (2,34g).

Phn on F8 (2,34 g) tip tc c tin hnh phn tch trn ct silicagel

pha thng vi h dung mi ra gii l n-hexan/aceton: 2,5/1 thu c 4 phn l:

F8A D.

Phn F8B tin hnh chy trn ct silicagel pha thng, h dung mi

dicloromethan/aceton: 2/1, thu c cht 3 (10 mg).

27

Hnh 3.3. S phn lp cc cht t phn on chloroform chit xut

t c Seo g

Cn chloroform

30g

F8C F8D

Cht 3 (PM12)

CC, Silicagel,

gradient n-hexan - aceton

Methanol

CC, Silicagel,

n-hexan/aceton: 2,5/1

CC, Silicagel,

dicloromethan/aceton:

2/1

F4 F5 F6 F7 F8

10/1 5/1 Aceton 2/1

F8B F8A

28

3.2.3. Kim tra tinh khit cht phn lp

Hp cht PM12

PM12 c kim tra tinh khit bng SKLM trin khai vi 3 h dung mi:

H I: Toluen: Ethylacetat: Acid formic (5:4:1)

H II: Toluen: Ethylacetat: Acid formic (6:2:1)

H III: Ethylacetat: Methanol (6:1)

Kt qu : Quan st nh sng thng PM12 ch xut hin mt vt mu

vng nu nht, nh sng UV254 PM12 ch xut hin mt vt mu en. Sau khi

phun TT H2SO4 10% v quan st nh sng thng PM12 ch xut hin mt vt

mu vng nu. V vy, s b kt lun PM12 l mt cht tinh khit (hnh 3.4, 3.5

v bng 3.4).

H I H II H III

Hnh 3.4. Hnh nh SKLM ca PM12 vi 3 h dung mi UV254

29

H I H II H III

Hnh 3.5. Hnh nh SKLM ca PM12 vi 3 h dung mi nh sng thng.

Bng 3.4. Kt qu SKLM ca PM12 vi 3 h dung mi AST v UV254.

H dung mi I II III

AST

Rf x 100 45,59 26,47 50,00

Mu sc Vng nu Vng nu Vng nu

UV254

Rf x 100 45,59 26,47 50,00

Mu sc en en en

30

Ngoi ra cn tin hnh sc k so snh PM12 vi cn chloroform trn cng

mt bn mng, h dung mi khai trin l chloroform: ethylacetat: acid formic

(5:4:1). Kt qu th hin hnh 3.6.

1 2

Hnh 3.6. Sc k so snh PM12 vi cn chloroform

1. Cn phn on chloroform 2. PM12

3.3. NHN DNG CC CHT PHN LP

- Tnh cht: PM12 dng tinh th hnh khi. Nhit nng chy l: 150 - 1570C.

Tan trong chloroform v methanol.

- PM12 c cng thc phn t C7H6O3, M = 138.

31

Hnh 3.7. nh chp tinh th PM12 di KHV vt knh 20

Ph cng hng t ht nhn: Trn ph 1H-NMR xut hin tn hiu ca 3

proton thm H 7,34 (H-2, d, J = 1,5 Hz), 6,94 (H-5, d, J = 8,0 Hz) v 7,32 (brs)

gi mt vng benzen th kiu 1, 3, 4. Tn hiu ca c trng ca nhm aldehyd

(CHO) quan st thy ti v tr cng hng H 9,71 (s).

Ph 13C-NMR v DEPT cho tn hiu ca 7 carbon, trong c 6 tn hiu

carbon vng thm ti cc v tr cng hng C 130,84 (C), 119,46 (CH), 153,74

(C), 147,19 (C), 116,26 (CH) v 126,40 (C); v 1 nhm carbonyl ca nhm

aldehyd ti C 193,11. Cc d kin ph trn cho php ta ngh n cu trc mt

vng benzen th 1, 3, 4; trong c 2 nhm hydroxy v mt nhm aldehyd. So

snh cu trc d on vi cht 3 trong ti liu [45] thy hon ton ph hp ti

cc v tr tng ng, cho php khng nh PM12 chnh l 3,4-dihydroxy

benzaldehyd.

32

Bng 3.5. D liu ph NMR ca PM12

No.

*C [45]

(H, mult., J in Hz) Ca Hb (mult., J in Hz)

1 130,6 130,84

2 118,4 119,46 7,34 (d, 2,0)

3 152,0 153,74

4 146,4 147,19

5 111,3 116,26 6,94 (d, 8,0)

6 124,3 126,40 7,32 (brs)

CHO 190,9 193,11 9,71 (s)

o trong MEOD: a) 125 MHz, b) 500 MHz

5

OH

OH

OH

12

3

4

6

Hnh 3.8. Cu trc ha hc ca hp cht PM12

3.4. BN LUN

C Seo g l mt dc liu c s dng kh ph bin trn th gii. Ti

Trung Quc, c Seo g c dng iu tr vim ng tit niu, tr tr em

33

kinh phong, a chy, l amip, l trc khun, vim gan, tr, hong n cp tnh,

phong thp but au, st rt, nht, bnh ngoi da [8], [24]. Ring Vit Nam,

c Seo g c bit n vi cc bi thuc tr xut huyt, l trc trng, bnh

ngoi da [7], [12], [15]. V vy, ti nghin cu c tin hnh l cn thit

gp phn nng cao gi tr s dng ca dc liu ny.

Hin nay, c nhiu phng php chit xut c p dng trong nghin cu

quy m phng th nghim. Phng php ngm nhit phng l mt trong

nhng phng php ti u c la chn trong phn lp cc cht c trong dc

liu vi nhng u im: l phng php n gin nht, d thc hin, thit b n

gin, r tin. Mu nghin cu c chia nh lm tng din tch tip xc gia

dc liu v dung mi, lm tng h s khuch tn t lm tng hiu sut chit.

Cc nh khoa hc trn th gii phn lp c mt s hp cht khc

nhau: -sitosterol--glucosid [15], multifidosid A, B, C v pterokauran M1, M2,

M3 [47]. T phn on chloroform ca c Seo g, ti phn lp c hp

cht PM12 c xc nh cu trc l: 3,4-dihydroxy benzaldehyd da vo d

liu ph NMR. Kt qu ny cng ph hp vi cc nghin cu trn th gii.

3,4-dihydroxy benzaldehyd hay protocatechu aldehyd c cc nhm nh

khoa hc khc nhau trn th gii phn lp v chng minh tc dng dc l. Nm

2007, mt s nh khoa hc phn lp v tinh ch c 3,4-dihydroxy

benzaldehyd t qu Xanthium strumarium bng phng php sc k ct vi cht

nhi ct l silicagel v chng minh c n c tc dng c ch hot ng CKII

vi IC50 khong 783 M. nng 300 M, 3,4-dihydroxy benzaldehyd c kh

nng c ch 50% s tng trng ca t bo ung th U937 ngi [29]. Tng

t, bng phng php sc k ct silicagel kt hp sc k lng hiu nng cao, Jin

Boo Jeong v cng s phn lp c cht 3,4-dihydroxy benzaldehyd t ht

34

la mch Hordeum vulgare v chng minh n c tc dng thu dn gc DPPH,

gc hydroxyl, ROS ni bo, to phc chelat vi Fe2+, c ch s oxy ha ph hy

DNA v c ch gy cht t bo gy ra bi H2O2 [23]. Theo [18], protocatechu

aldehyd (PCA) l mt hp cht polyphenol t nhin c phn lp t gc r ca

cc loi tho dc S. miltiorrhiza v cy tr la mch. PCA lm gim tc dng

ca -catenin v cyclin D1 (tin gen gy bnh ung th v) nn c tc dng ngn

chn s tng sinh ca t bo ung th v ngi. Thm vo , protocatechu

aldehyd c cho l hp cht phenolic chnh ca Phellinus gilvus c tc dng

thu dn gc t do DPPH v c ch s sn xut NO c hot ho bng LPS

trong i thc bo RAW264 [17]. Ngoi ra, 3,4-dihydroxy benzaldehyd cn c

tc dng chng vim, bo v v phc hi t bo c tim sau chn thng do thiu

mu cc b [44], chng nhim trng huyt [51], h tr tc dng ca aspirin trn

tiu cu [35]. Nhn thy vai tr ngy cng quan trng ca 3,4-dihydroxy

benzaldehyd, con ngi ang tch cc u t cho nghin cu phn lp hp cht

ny t cc ngun dc liu khc nhau. V vy, vic phn lp ra 3,4-dihydroxy

benzaldehyd t c Seo g l nh hng cho vic nghin cu cc hp cht c kh

nng iu tr cc bnh ung th, bnh lin quan n gc t do Tm li, y l

mt ng gp mi ca kha lun, gp phn nghin cu su hn v thnh phn

ho hc ca c Seo g, nghin cu c phn no mi quan h gia cc thnh

phn ho hc vi cc tc dng sinh hc, m ra hng mi nghin cu su hn

v tc dng sinh hc c Seo g.

35

KT LUN V XUT

KT LUN

Sau thi gian tin hnh lm thc nghim, kha lun thu c mt s

kt qu sau y:

1. Chit xut dc liu bng phng php ngm nhit phng vi

methanol, sau chit lng - lng vi ln lt vi cc dung mi c phn cc

tng dn n-hexan, chloroform thu c 10,00g cn phn on chloroform. Hm

lng % cn phn on so vi khi lng bt dc liu l 1,25%. nh tnh cn

chloroform bng SKLM cho thy: cn chloroform cho 15 vt c mu sc,

m nht, Rf khc nhau, khi quan st cc iu kin AST, UV365nm, UV254nm,

trc v sau khi phun thuc th.

2. T cn chloroform phn lp c PM12. Sau khi kim tra bng

SKLM cho thy cht phn lp c l tinh khit. Cu trc c xc nh da

trn cc d liu ph NMR. Cu trc ha hc c nhn dng l: 3,4-dihydroxy

benzaldehyd.

XUT

Do thi gian thc hin ti c hn nn nhng kt qu nghin cu trn

ca chng ti mi ch ng gp phn no trong cng trnh nghin cu v c Seo

g. C Seo g l mt dc liu c s dng ph bin trn th gii v c

nghin cu chng minh c nhiu tc dng dc l ng ch . V vy chng ti

xin a ra mt s xut:

- Tip tc nghin cu thnh phn ha hc ton cy c Seo g.

36

- Nghin cu tc dng sinh hc ca c Seo g (tc dng chng ung th,

chng oxy ha, tc dng chng vim )

- Nghin cu dng bo ch ph hp, tin s dng, hiu qu trong iu tr

t c Seo g.

TI LIU THAM KHO

Ti liu ting Vit

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207-213.

2. B mn Dc liu - Trng i hc Dc H Ni (2006), Thc tp dc

liu phn hin vi.

3. B mn Dc liu - Trng i hc Dc H Ni (2006), Thc tp dc

liu phn ha hc.

4. B mn Thc vt - Trng i hc Dc H Ni (2004), Thc tp thc

vt v nhn bit cy thuc.

5. B mn Dc liu - Trng i hc Dc H Ni (2011), Bi ging dc

liu, tp 1.

6. B mn Dc liu - Trng i hc Dc H Ni (2011), Bi ging dc

liu, tp 2.

7. V Vn Chi (1997), T in cy thuc Vit Nam, Nh xut bn Y hc, tr

275.

8. V Vn Chi (1999), Cy c c ch Vit Nam, tp 1, Nh xut bn Gio

dc, tr 150-156.

9. V Vn Chi (2004), T in thc vt thng dng, Nh xut bn Khoa hc

v k thut, tr 138, tr 2063-2067.

10. Nguyn Vn n - Nguyn Vit Tu (1985), Phng php nghin cu

ha hc cy thuc, Nh xut bn Y hc.

11. L Trn c (1997), Cy thuc Vit nam: trng hi, ch bin, tr bnh

ban u, Nh xut bn Nng nghip, tr 748-749.

12. Tt Li (1999), Nhng cy thuc v v thuc Vit Nam, Nh xut bn

Y hc, tr 207-208.

13. Phm Hong H (1999), Cy c Vit Nam, Quyn 1, Nh xut bn tr, tr

61-68.

14. Nguyn Th Thanh Huyn (2013), Nghin cu c im thc vt v thnh

phn ha hc ca cy c Seo g, Kha lun tt nghip Dc s i hc.

15. Vin Dc liu (2004), Cy thuc v ng vt lm thuc Vit nam, tp

2, Nh xut bn khoa hc v k thut, tr 729-730.

Ti liu ting Anh

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X.F.Shao (2006), Chemical constituents of Pteris multifida and their

inhibitory effects on growth of rat prostatic epithelial cells in vitro,

Chinese Journall of Natural Medicines, 5(6), 431-438.

17. Chang ZQ1, Gebru E, Lee SP, Rhee MH, Kim JC, Cheng H, Park SC

(2011), In vitro antioxidant and anti-inflammatory activities of

protocatechualdehyde isolated from Phellinus gilvus, J Nutr Sci

Vitaminol (Tokyo), 57 (1), 118-122.

18. Choi J1, Jiang X, Jeong JB, Lee SH. (2014), Anticancer Activity of

Protocatechualdehyde in Human Breast Cancer Cells, Journal of

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19. Feng-Lin Hsu, Chun-Fa Huang, Ya-Wen Chen, Yuan-Peng Yen, Cheng-

Tien Wu, Biing-Jiun Uang, Rong-Sen Yang and Shing-Hwa Liu (2012),

Antidiabetic Effects of Pterosin A, a Small Molecular Weight Natural

Product, on Diabetic Mouse Models, Published online October 15, 2012,

628- 638.

20. F.Imperato (1994), Luteolin 8-C-rhamnoside-7-O-rhamnoside from

Pteris cretica, Phytochemistry, 37(2), 589-590.

21. Gong XL, Chen ZH, Liang NC (2007), Advances in study on chemical

constituents and pharmacological activities of plants of genus Pteris,

Zhongguo Zhong Yao Za Zhi, 32(14), 1382-1387.

22. Hoang Le Son and Tran Huynh Phuoc Thao (2014), In vitro Antioxidant

and Anticancer Properties of Active Compounds from Methanolic Extract

of Pteris multifida Poir. Leaves, European Journal of Medicinal Plants,

292-302.

23. Jeong JB, Hong SC, Jeong HJ (2009), 3,4-dihydroxy benzaldehyde

purified from the barley seeds (Hordeum vulgare) inhibits oxidative DNA

damage and apoptosis via its antioxidant activity, Phytomedicine, 16 (1),

85-94.

24. Jiang Su new college of Medicine (1985), Dictionary of chinese

traditional medicine, Shang Hai Technology Press, 487-488.

25. Jianqun Liu, Jicheng Shu, Rui Zhang, Wei Zhang (2011), Two new pterosin

dimers from Pteris multifida Poir, Fitoterapia, 82(8), 1181-1184.

26. Jicheng Shu, Jianqun Liu, Youquan Zhong, Jinghang Pan, Lifang Liu,

Rui Zhang (2012), Two new pterosin secquiterpens from Pteris multifida

Poir., Phytochemistry, 5(2), 276-279.

27. Kong Weixin, Yang Yongming, Zou Jun, Qu Jie, Tian Yue (2013),

Effect of the Pteris Multifida Flavones on the Metastasis Ability of

Human Osteosarcoma Cell Line MG-63 Cells, Journal of Liaoning

University of TCM, Vol.15, No.1, 42-45.

28. Kuang-Ping Lan, Ying-Pei Shen, Shui-Huei Lee, Tzu-Ching Wang, Tzu-

Linh, Wang-Mccall, Chung-Ching Lin and Chi-Ching Yang, Antioxidant

and Free Radical - Scavenging Activities of Pteris Multifida Poiret

Aqueous Extract , Journal of Food Quality 34, 252-258.

29. Lee BH, Yoon SH, Kim YS, Kim SK, Moon BJ, Bae YS (2008),

Apoptotic cell death through inhibition of protein kinase CKII activity by

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Prod Res., 22(16), 1441-1450.

30. Liliana Cristina Soare, Mariana Ferdes, Ionica Deliu, Alexandru Gibea

(2012), Studies Regarding The Antibacterial Activity of some Extracts of

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31. Liva Harinantenaina, Katsuyoshi Matsunami, Otsuka H. (2008),

Chemical and biologically activie constituents of Pteris multifida,

Joural of Natural medicines, 62(4), 452-455.

32. Pradeep Parihar, Leena Parihar1 and Achaleshwar Bohra (2010) In vitro

antibacterial activity of fronds (leaves) of some important pteridophytes,

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33. Sahatino M.L., Prado F. (1998), Flavonoid glycosides of Pteridaceae

from Brazil, J.Bio.System.Eco, 26, 761-769.

34. Shi, Lin-Mei, Bai, Hai-Bo (2010), A new diterpenoid glucoside from

Pteris semipinnata, Journal of chemical research, 34(4), 206-207.

35. Sun S, Hao H, Gong P, Tang Z, Li F, Chen X, Shi H, Wang G. (2011),

Protocatechualdehyde synergizes with aspirin at the platelet

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36. T.C.Wang, M.C.Ti, S.C.Lo, C.C.Yang (2007), Free radical - scavenging

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78(3), 248-249.

37. T.C.Wang, Y.P.Su, T.Y.Hsu, C.C.Yang, C.C.Lin. (2007), 28-Day oral

toxicity study of the aqueous extract from spider brake (Pteris multifida

Poiret.) in rats, Food and Chemical toxicology, 45(9), 1757-1763.

38. T.Hakamatsuka, D.Tamaka, Y.Namatame, H.Wada, N.Tamaka (1997),

Four new ent-kaurane glucosides from Pteris cretica, Natural

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Chemical and chemotaxonomical studies of the Pteris family and related

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Chem. Pharm. Bull., 26, 3580-3582.

40. Tzu-Ching Wang, Hou-I Lee and Chi-Yang (2009), Evaluation of In

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diet, Pharm Biol, 48(2), 221-226.

43. Wang WS, Wang ZQ, Zhou YW (2008), Studies on chemical

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31(8), 1165-7.

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Jing-Hua Yang (2013), Chemical Constituents of Pteris multifida,

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Shan (2010), Three New Illudalane Sequiterpenoids from Pteris

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PH LC

PH LC 1. KT QU GIM NH TN KHOA HC V BIN SON

T LIU LOI THC VT

PH LC 2. KT QU PH CA CHT PM12 (1H-NMR, 13C-NMR,

DEPT)

PH LC I

KT QU GIM NH TN KHOA HC V BIN SON T

LIU LOI THC VT

PH LC II

KT QU PH CA CHT PM12:

- 1H-NMR

- 13C-NMR

- DEPT

PM12MeOD1H

13 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm

2

.

1

7

4

3

.

3

2

7

3

.

3

3

0

3

.

3

3

3

3

.

3

6

9

4

.

5

8

0

4

.

8

5

8

5

.

2

3

2

6

.

7

6

0

6

.

8

7

3

6

.

9

2

4

6

.

9

4

0

7

.

3

2

0

7

.

3

3

6

7

.

3

3

9

9

.

7

0

8

0

.

3

0

6

7

.

2

6

3

0

.

1

7

9

0

.

3

6

2

0

.

1

7

8

1

.

0

1

5

2

.

0

0

3

1

.

0

0

0

PC 1.00GB 0LB 0.30 HzSSB 0WDW EMSF 500.1300014 MHzSI 32768F2 Processing parameters

SFO1 500.1335009 MHzPL1 1.00 dBP1 10.50 usecNUC1 1H======== CHANNEL f1 ========

MCWRK 0.01500000 secMCREST 0.00000000 secD1 1.00000000 secTE 300.0 KDE 6.00 usecDW 50.000 usecRG 128AQ 3.2769001 secFIDRES 0.152588 HzSWH 10000.000 HzDS 0NS 16SOLVENT MeODTD 65536PULPROG zg30PROBHD 5 mm MultinuclINSTRUM spectTime 15.19Date_ 20130911F2 Acquisition Parameters

PROCNO 1EXPNO 1NAME 11K_PM12Current Data Parameters

PM12MeOD1H

6.76.86.97.07.17.27.37.4 ppm

6

.

7

6

0

6

.

8

7

3

6

.

9

2

4

6

.

9

4

0

7

.

3

2

0

7

.

3

3

6

7

.

3

3

9

0

.

3

6

2

0

.

1

7

8

1

.

0

1

5

2

.

0

0

3


SFO1 500.1335009 MHzPL1 1.00 dBP1 10.50 usecNUC1 1H======== CHANNEL f1 ========

MCWRK 0.01500000 secMCREST 0.00000000 secD1 1.00000000 secTE 300.0 KDE 6.00 usecDW 50.000 usecRG 128AQ 3.2769001 secFIDRES 0.152588 HzSWH 10000.000 HzDS 0NS 16SOLVENT MeODTD 65536PULPROG zg30PROBHD 5 mm MultinuclINSTRUM spectTime 15.19Date_ 20130911F2 Acquisition Parameters


PM12MeODC13CPD

200 180 160 140 120 100 80 60 40 20 ppm4

8

.

4

9

2

4

8

.

6

6

3

4

8

.

8

3

3

4

9

.

0

0

3

4

9

.

1

7

4

4

9

.

3

4

4

4

9

.

5

1

4

4

9

.

8

5

3

1

0

4

.

8

7

3

1

1

4

.

8

2

8

1

1

5

.

4

0

7

1

1

5

.

8

3

6

1

1

6

.

2

5

7

1

1

9

.

4

5

8

1

2

6

.

4

0

0

1

3

0

.

8

3

7

1

3

1

.

1

0

9

1

4

6

.

1

2

6

1

4

6

.

6

7

1

1

4

7

.

1

8

9

1

5

3

.

7

3

8

1

9

3

.

1

1

3


SFO2 500.1320005 MHzPL13 22.00 dBPL12 16.64 dBPL2 1.00 dBPCPD2 80.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========

SFO1 125.7716224 MHzPL1 1.00 dBP1 5.00 usecNUC1 13C======== CHANNEL f1 ========

MCWRK 0.01500000 secMCREST 0.00000000 secDELTA 1.89999998 secd11 0.03000000 secD1 2.00000000 secTE 300.0 KDE 6.00 usecDW 15.900 usecRG 32768AQ 1.0420883 secFIDRES 0.479836 HzSWH 31446.541 HzDS 2NS 256SOLVENT MeODTD 65536PULPROG zgpg30PROBHD 5 mm MultinuclINSTRUM spectTime 15.37Date_ 20130915F2 Acquisition Parameters


PM12MeODC13CPD

110115120125130135140145150155 ppm

1

0

4

.

8

7

3

1

1

4

.

8

2

8

1

1

5

.

4

0

7

1

1

5

.

8

3

6

1

1

6

.

2

5

7

1

1

9

.

4

5

8

1

2

6

.

4

0

0

1

3

0

.

8

3

7

1

3

1

.

1

0

9

1

4

6

.

1

2

6

1

4

6

.

6

7

1

1

4

7

.

1

8

9

1

5

3

.

7

3

8


SFO2 500.1320005 MHzPL13 22.00 dBPL12 16.64 dBPL2 1.00 dBPCPD2 80.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========

SFO1 125.7716224 MHzPL1 1.00 dBP1 5.00 usecNUC1 13C======== CHANNEL f1 ========

MCWRK 0.01500000 secMCREST 0.00000000 secDELTA 1.89999998 secd11 0.03000000 secD1 2.00000000 secTE 300.0 KDE 6.00 usecDW 15.900 usecRG 32768AQ 1.0420883 secFIDRES 0.479836 HzSWH 31446.541 HzDS 2NS 256SOLVENT MeODTD 65536PULPROG zgpg30PROBHD 5 mm MultinuclINSTRUM spectTime 15.37Date_ 20130915F2 Acquisition Parameters


PM12MeODC13CPD

2030405060708090100110120130140150160170180190200210 ppm

2030405060708090100110120130140150160170180190200210 ppm

2030405060708090100110120130140150160170180190200210 ppm

DEPT90

DEPT135

C13CPD

CH&CH3

CH2

&DEPT

PM12MeODC13CPD

105110115120125130 ppm

105110115120125130 ppm

105110115120125130 ppm

DEPT90

DEPT135

C13CPD

CH&CH3

CH2

&DEPT

ni dung.pdf1.1. V TR PHN LOI, C IM THC VT V PHN B. 1.1.1. V tr phn loi ca chi Pteris L. 1.1.2. c im chung ca h C lung (Pteridaceae). 1.1.4. c im thc vt v phn b ca c Seo g (Pteris multifida Poir.)

1.2. THNH PHN HA HC 1.2.1. Thnh phn ha hc ca chi Pteris L. 1.2.2. Thnh phn ha hc ca c Seo g (Pteris multifida Poir.)

1.5. MT S N THUC Chng 3 THC NGHIM, KT QU V BN LUN 3.1. CHIT XUT 3.1.1. Xc nh m dc liu Ly khong 2g bt dc liu xc nh m. Bt my o m, iu chnh nhit 1100C. Tri u dc liu ln a cn, y a cn v i my t ng hin kt qu. 3.1.2. Chit xut

3.2. PHN LP 3.2.1. Chun b ct 3.2.2. Tin hnh Hp cht PM12

ti liu tham kho.pdf11K_PM12_1H.pdf11K_PM12_1Hex.pdf11K_PM12_13C.pdf11K_PM12_13Cex.pdf11K_PM12_DEPT.pdf11K_PM12_DEPTex.pdf

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