Nghiên cứu thành phần hóa học và thử hoạt tính ức chế tế bào ung thư của lá cây mãng cầu xiêm ( Annona muricata L.) họ Annomaceae.pdf

B Y T TRNG I HC DC H NI

NGUYN THU TRANG

NGHIN CU THNH PHN HO HC V

TH HOT TNH C CH

T BO UNG TH CA L CY

MNG CU XIM (ANNONA MURICATA L.),

H ANNONACEAE

KHO LUN TT NGHIP DC S

H Ni, nm 2014

B Y T TRNG I HC DC H NI

NGUYN THU TRANG

NGHIN CU THNH PHN HO HC V

TH HOT TNH C CH

T BO UNG TH CA L CY

MNG CU XIM (ANNONA MURICATA L.),

H ANNONACEAE

KHO LUN TT NGHIP DC S

Ngi hng dn: 1. TS. Quyn 2. ThS, NCS H c Hng Ni thc hin: 1. B mn Dc liu, trng H Dc, H Ni 2. Vin Ha hc, Vin hn lm khoa hc v cng ngh Vit Nam

H Ni, nm 2014

LI CM N

u tin, ti xin by t lng bit n su sc n TS. Quyn, ngi

thy ng h, gip , tn tnh ch bo v to mi iu kin thun li cho

ti trong sut qu trnh lm kho lun.

Ti xin chn thnh cm n PGS., TS. Phm Gia in, TS. Bi Kim

Anh, Th.S, NCS. H c Hng gip , gp cho ti trong qu trnh

lm kha lun.

Ti xin chn thnh cm n cc cn b Phng Cng ngh cc hot

cht sinh hc, Vin Ho hc, Vin Hn lm Khoa hc v Cng ngh Vit

Nam to iu kin cho ti hon thnh kho lun ny.

Nhn dp ny, ti xin by t s knh trng v cm n ti Ban gim

hiu, Phng o to v cc thy c gio trng i hc Dc H Ni lun

ng hnh, to iu kin cho ti c hc tp, tip thu nhng kin thc v

kinh nghim qu bu trong sut 5 nm hc.

Xin cm n gia nh v bn b lun ng vin ti ti c thm ngh

lc v say m nghin cu khoa hc.

H ni, ngy 14 thng 4 nm 2014

Nguyn Thu Trang

MC LC

LI CM N i

NHNG CH VIT TT DNG TRONG LUN VN ii

DANH MC CC BNG V CC HNH iii

MC LC iv

T VN 1

CHNG I- TNG QUAN 2

1.1 V tr phn loi v c im thc vt 2

1.1.1. V tr phn loi ca loi Mng cu xim 2

1.1.2. c im thc vt v phn b ca chi Annona 2

1.1.3. c im thc vt v phn b ca loi Annona muricata L. 3

1.2. Thnh phn ha hc 3

1.2.1. Cc hp cht acetogenin 3

1.2.2. Cc hp cht alcaloit 6

1.2.3. Tinh du 7

1.2.4. Sterol 8

1.2.5. Carotenoit 8

1.2.6. Cc nhm cht khc 9

1.3. Tc dng sinh hc 9

1.4. Cng dng 12

CHNG 2- NGUYN LIU, THIT B, NI DUNG V

PHNG PHP NGHIN CU

14

2.1. Nguyn liu, ha cht v thit b 14

2.1.1. i tng nghin cu 14

2.1.2. Dung mi, ha cht 14

2.1.3. Thit b 14

2.1.3.1. Dng cho chit xut, nh tnh v phn lp 14

2.1.3.2. Dng cho xc nh thng s vt l v ha hc ca cc cht 15

2.1.3.3. Dng cho th hot tnh gy c t bo 15

2.2. Ni dung nghin cu 16

2.3. Phng php nghin cu 16

2.3.1. Nghin cu v ho hc 16

2.3.1.1. Chit xut 16

2.3.1.2. Phn lp 17

2.3.1.3. Nhn dng 17

2.3.2. Th tc dng gy c t bo 17

2.3.2.1. Cc dng t bo 17

2.3.2.2. Phng php th tc dng gy c t bo 17

CHNG 3- THC NGHIM, KT QU V BN LUN 19

3.1. Thc nghim v kt qu 19

3.1.1. Chit xut 19

3.1.2. Tc dng gy c t bo ung th 21

3.1.3. Phn lp cc cht t dch chit n-hexan 22

3.1.3.1. Kho st s b cn dch chit n-hexan bng SKLM 22

3.1.3.2. Phn lp cn dch chit n-hexan l Mng cu xim bng sc

k ct

24

3.1.3.3. Phn lp cht t phn on P27 27



3.1.4. Nhn dng cu trc 30

3.1.4.1. Nhn dng cu trc cht P27 30

3.1.4.2. Nhn dng cu trc Cht P71 31

3.1.4.3. Nhn dng cu trc Cht P55.7 33

3.2. Bn lun 36

3.2.1. Tc dng gy c t bo ca dch chit n-hexan t l cy

Mng cu xim

36

3.2.2. V thnh phn ho hc 37

KT LUN V XUT 39

TI LIU THAM KHO 40

PH LC 46

NHNG CH VIT TT DNG TRONG LUN VN

1H-NMR Proton Nuclear magnetic resonance

13C-NMR

Cacbon (13) Nuclear magnetic resonance

AST nh sng thng

ATCC American type culture collection

DEPT Distortionless Enhancement by Polarisation Transfer

DMEM Eagle's minimal essential medium

DMSO Dimethyl sulfoxide

EC50 The term half maximal effective concentration

EI-MS Electron ionization Mass sprectromery

IC50 The half maximal inhibitory concentration

J (Hz) Hng s tng tc tnh bng Hertz

KB Human epidermic carcinoma

LU Human lung carcinoma

MCF7 Human breast adenocarcinoma

RPMI Roswell Park Memorial Institute medium

SKLM Sc k lp mng

STT S th t

TMS Tetramethylsilane

UV Ultra violet

d Duplet

dd Duplet ca duplet

m Multiplet

t Triplet

(ppm) chuyn dch ha hc tnh bng ppm

ppm Phn triu

DANH MC CC BNG V CC HNH

Danh mc cc hnh

Hnh 1.1: Khung cu trc chung ca acetogenin 4

Hnh 1.2: Cu trc ho hc ca acetogenin mch thng 4

Hnh 1.3: Cu trc ho hc ca acetogenin c nhm epoxy 5

Hnh 1.4: Cu trc ho hc ca acetogenin c 1 nhm tetrahyrofuran 6

Hnh 1.5: Cu trc ho hc ca acetogenin c 2 nhm tetrahyrofuran 6

Hnh 1.6: Khung cu trc c bn ca 4 nhm alcaloit 7

Hnh 1.7: Khung cu trc c bn ca sterol 8

Hnh 3.1: S chit xut bt l cy Mng cu xim 20

Hnh 3.2: Sc k ca cn dch chit n-hexan ca l mng cu xim

trong cc h dung mi khc nhau quan st AST

22

Hnh 3.3: Sc k cn dch chit n-hexan trong h dung mi

Toluen:Etyl acetat:Acid acetic [40:10:1]

23

Hnh 3.4: Sc k ca cht P27 trong h dung mi CH2Cl2 : MeOH

[98:2], hin mu bng ceri sulfat

27



29

Hnh 3.6: Sc k ca cht P55.7 trong h dung mi CH2Cl2 : EtOAc

[8:2], quan st AST

30

Hnh 3.7: Cng thc cu to -sitosterol (P27) 31

Hnh 3.8: Cng thc cu to ca -sitosterol-3-O--D-glucopyranosit

(P71)

33

Hnh 3.9: Cng thc cu to ca lutein (P55.7) 35

Danh mc cc bng

Bng 3.1: Hm lng cc cn cc dch chit ca l cy Mng cu xim 19

Bng 3.2: Kt qu th hot tnh c t bo ca cn dch chit n-hexan t

l cy Mng cu xim

21

Bng 3.3: Kt qu SKLM ca cn dch chit n-hexan trong h dung mi

Toluen:Etyl acetat:Acid acetic [40:10:1]

24

Bng 3.4.1: S liu ph NMR ca hp cht P55.7 v lutein [10] 34

Bng 3.4.2: S liu ph NMR ca hp cht P55.7 v lutein [10] (tip) 35

1

T VN

Ngy nay, cc sn phm thin nhin c hot tnh sinh hc c ng dng

rt ln trong nhiu lnh vc khc nhau ca cuc sng. Nhiu hp cht thin

nhin hoc cc sn phm c tng hp, bn tng hp t cc hp cht thin

nhin c s dng mt cch hiu qu trong vic iu tr, phng nga mt

s bnh, c bit l cc bnh him ngho.

Theo thng k, mi nm ti Vit Nam, s bnh nhn ung th mc mi

l 150.000 ngi v c 75.000 ngi t vong [39]. Cc phng php iu tr

ung th thng thng nh ha tr v x tr hay li tc dng ph, nh hng

n cht lng cuc sng ca bnh nhn. V vy, tm ra cc liu php mi

trong iu tr ung th l mt nhu cu cp thit.

Hin nay, cy Mng cu xim (Annona muricata L.) ang thu ht s

quan tm ca nhiu nh khoa hc trn th gii. Vi kt qu thu c t cc

nghin cu tp trung theo hng nghin cu tc dng gy c t bo, Mng

cu xim c coi nh mt dc liu y tim nng trong iu tr ung th [7].

Vit Nam, cha c nghin cu no v Mng cu xim trong khi loi thc

vt ny c trng ph bin khai thc qu v em li hiu qu kinh t rt

ln. Cc b phn khc ca cy hu nh khng c tn dng. V vy, c

th ng gp thm nhng hiu bit gip lm tng gi tr ca ngun nguyn

liu ln, chng ti thc hin ti : Nghin cu thnh phn ho hc v th

hot tnh c ch t bo ung th ca l cy Mng cu xim (Annona muricata

L.), h Annonaceae vi hai mc tiu chnh:

- Nghin cu thnh phn ho hc ca l cy Mng cu xim.

- Th hot tnh c ch t bo ung th ca dch chit l cy Mng cu

xim.

2

CHNG I - TNG QUAN

1.1. V tr phn loi v c im thc vt

1.1.1. V tr phn loi ca loi Mng cu xim

Theo h thng phn loi ca Armen Takhtajan, loi Annona muricata

L. thuc chi Na (Annona), H Na (Annonaceae), Lin b Magnolianae, Phn

lp Ngc lan (Magnoliidae), Lp Ngc lan (Magnoliopsida), Ngnh Ngc lan

(Magnoliophyta) [32].

Ngnh Ngc lan (Magnoliophyta)

Lp Ngc lan (Magnoliopsida)

Phn lp Ngc lan (Magnoliidae)

Lin b Magnolianae

B Na (Annonales)

H Na (Annonaceae)

Chi Na (Annona)

1.1.2. c im thc vt v phn b ca chi Annona

M t: Cy g hay cy bi. V thn mng, c nhng vng lm rng v

nng, hoc nhng vt nt ni vi nhau, c vy, t nhng vt nt ny cc chi

cy mc ra. Phin l mng, mm do, c lng hoc khng. Cung hoa mc t

nch l, oi khi t nch chi, dng hoa n hay chm. Ba hay bn l i

rng sm thng nh hn cnh hoa pha ngoi v khng bao kn khi cn n.

Su hay tm cnh hoa nc mc thnh hai vng, cc cnh hoa mc vng

ngoi ln hn nhng khng bao kn; cc cnh hoa mc vng trong nh hn.

Nh nhiu, cun li, dng chu, hoc cong v c dng ti, hoc nhn u kia

bao phn. Nhu nhiu, gn trc tip phn y, ring r lin kt vi u nhu,

vi mt hoc hai non mi nhu; vi nhu v u nhu hnh chu hoc hnh

nn hp. Qu nc, hnh trng hoc hnh cu. Mi qu bao gm c nhiu qu

3

nh ring bit hoc qu t, vi mt qu t v ht mi nhu. Ht hnh ht u

vi lp o cng; nhn ht c [13].

Chi Na (Annona) c khong 125 loi phn b nhiu cc vng rng

ma nhit i thuc chu M, chu Phi Vit Nam c mt s loi thuc

chi Annona c pht hin l: Annona cherimolia, Annona glabra, Annona

muricata, Annona reticulata, Annona squamosa [1].

1.1.3. c im thc vt v phn b ca loi Annona muricata L.

M t: Cy cao 4-6m; nhnh cy c nhiu mu, nhn. L hnh mi mc-

trng ngc, hi nhn gc, nhn hay t ngn, 2 mt nhn, mt trn nht

mu v lng bng, di 12cm, rng 5 cm; gn l chia 8-10 cp, mng, c th

nhn thy c 2 mt l; gn con to thnh mng li; cung l chc, di

8mm. Cung hoa c 1 hoa n c, c lng t 2 mt. Cnh hoa ln, hnh

ovan, ngn hn bao hoa. L i hnh tam gic, ngn, dy, gn ging hnh tim

gc, c lng t 2 mt. Nhiu nh, ko di n trung i hnh vung, ct,

c gai tht; cc song song, lin k nhau; ch nh di gn bng cc . L non

c lng mu hung; vi nh di bng bu, c gai tht, hnh lng tr, ct. Qu

hnh cu hoc hnh tim, c rn gc, bao ph bi cc gai hnh nn t l non

hn li to thnh [38].

nc ta cy Mng cu xim thng c trng ly qu. Cy c

ngun gc vng nhit i chu M, c trng Min Nam Vit Nam v

cc nc nhit i chu , nc ta thng gp cc tnh Nam b v mt

phn Ty nguyn [2],[3].

1.2. Thnh phn ha hc

1.2.1. Cc hp cht acetogenin

Acetogenin l nhm cht ch c tm thy h Annonaceae, do vy

y l nhm cht c trng ca h thc vt ny. Cu trc thng gp ca

4

acetogenin l acid bo c cha 32 n 34 cacbon, mch thng khng phn

nhnh, kt thc bng mt -lactone, c th cha mt s nhm th c tnh oxy

ha nh hydroxyl, ketone, epoxide, tetrahydrofuran (THF), tetrahydropyran

(THP) cng cc lin kt i, lin kt ba [7],[12]. Hnh 1.1 trnh by khung cu

trc chung ca acetogenin.

Hnh 1.1: Khung cu trc chung ca acetogenin

Theo tng kt v acetogenin ca h Na (2005) [7], ti nay c 70

acetogenin c phn lp t cy Mng cu xim. Da trn cu trc ho hc

c th phn loi acetogenin c mt trong cy Mng cu xim lm 4 nhm:

acetogenin mch thng, acetogenin c nhm epoxy, acetogenin c 1 nhm

tetrahydrofuran, acetogenin c 2 nhm tetrahydrofuran.

- Acetogenin mch thng ( phn lp c 12 cht) bao gm cc loi:

giganin; muridienin-1 (hnh 1.2).

Acetogenin loi giganin

Acetogenin loi muridienin-1

Hnh 1.2: Cu trc ho hc ca acetogenin mch thng

O OOH

OH

O O

OH

n m

O ORR OH

OH

O O

L A

2/4

32/ 34

35/ 37

5

- Acetogenin c nhm epoxy ( phn lp c 8 cht) bao gm cc

loi: epoxymurin-A; diepomuricanin-A (hnh 1.3).

Acetogenin loi epoxymurin-A

Acetogenin loi diepomuricanin-A

Hnh 1.3: Cu trc ho hc ca acetogenin c nhm epoxy

- Acetogenin c 1 nhm tetrahydrofuran ( phn lp c 47 cht) bao

gm: mono-tetrahydrofuran , - dihydroxy acetogenin (gm cc loi:

uvariamicin-I, murisolin, annonacin, annomonicin); mono-tetrahydrofuran

/ monohydroxy acetogenin (gm cc loi: gigantetrocin-A, gigantriocin);

isoannonacin. (hnh 1.4)

- Acetogenin c 2 nhm tetrahydrofuran ( phn lp c 3 cht) bao

gm loi: goniodenin. (hnh 1.5)

O O

O E- A

cis

m

32 / 84

n

L -A

35 / 37

O O

R O O

n

L -A

35 / 37 m

E - B

p

R = H or = O

6

Acetogenin loi mono-tetrahydrofuran , - dihydroxy acetogenin

Acetogenin loi mono-tetrahydrofuran / monohydroxy acetogenin

Acetogenin loi isoannonacin

Hnh 1.4: Cu trc ho hc ca acetogenin c 1 nhm tetrahyrofuran

Acetogenin loi goniodenin

Hnh 1.5: Cu trc ho hc ca acetogenin c 2 nhm tetrahyrofuran

1.2.2. Cc hp cht alkaloid

n nay c 14 alcaloit c phn lp t cy Mng cu xim bao

gm: reticuline, coclaurine, coreximine, atherosperminine, anomurine,

anomuricine, stepharine [22]; annonaine, asimilobine, atherospermine,

muricine, muricinine [33]; annonamine [23]; nornuciferine [17]. Cc alcaloit

ny thuc 4 nhm: apomorphin, aporphin, benzyltetrahydroisoquinolin v

O O

O

R

OH

33 / 35 / 37

L - A

Rn

T - B

Rm

30 / 32 / 34

R = H or OH

O

O

OR

OH30 / 32 / 34

33 / 35 / 37

L - A

R = H or OH

R n

T - B

Rm

O

O

H

O

OOH OH

35 / 37

L - B1

Rn

T - A

m

32 / 34

O O

O O

R

OH

L -A

35 / 37 R n

T - E

R = H or = OH

Rm

32 / 34

7

tetrahydroprotoberberin. Hnh 1.6 trnh by khung cu trc c bn ca 4

nhm alcaloit.

1 2

3 4

Hnh 1.6: Khung cu trc c bn ca 4 nhm alcaloit

Trong : 1-apomorphin, 2-aporphin, 3-benzyltetrahydroisoquinolin,

4-tetrahydroprotoberberin

1.2.3. Tinh du

Tinh du lm l cy c mi thm du.

Trong mt nghin cu v thnh phn tinh du chit t l cy Mng cu

xim thu hi Benin, 82 hp cht c xc nh bng GC/MS, ch yu l cc

dn xut ca sesquiterpenes: -elemen, isocaryophyllene, -caryophyllene,

-humulene, -muurolene, -selinene, -muurolene, germacrene-A, -

cadiene; v alcol: elemol, (E)-nerolidol, spathulenol, globulol, epi-globulol, 1-

N

H

OH

OHN

CH3

NH

N

8

epi-cubenol, -epi-cadinol, -epi-muurolol, -cadinol. 4 hp cht c hm

lng cao nht l: -caryophyllene (13,6%), -cadiene (9,1%), -epi-cadinol

(8,4%) v -cadinol (8,3%) [8].

Nm 2012, Moses S. Owolabi v cng s nghin cu thnh phn

tinh du l cy Mng cu xim trng Nigeria v xc nh c 18 thnh

phn, cc thnh phn c hm lng ln nht l (E)-caryophyllene (38,9%),

eugenol (30,2%), -cadinene (6,0%), caryophyllene oxide (5,0%) v -

humulene (4,3%) [27].

1.2.4. Sterol

y l nhm cht khng c nghin cu nhiu trong cy Mng cu

xim. Theo ti liu tng quan v h Na [21], trong l cy mng cu xim c

cha sitosterol; trong ht c cha sitosterol, stigmasterol, campesterol,

cholesterol c xc nh hm lng. Khung cu trc ca sterol c trnh

by hnh 1.7.

Hnh 1.7: Khung cu trc c bn ca sterol

1.2.5. Carotenoit

L nhm cht ng vai tr nh mt cht dinh dng c trong qu Mng

cu xim, Berlowski A. v cng s xc nh hm lng carotenoit ton

phn trong cy mng cu xim trng Peru l 0.310 g/1 g [6]; ngoi ra

OH

9

xc nh c retinol (tin cht ca vitamin A) c trong v cng nh tht qu

Mng cu xim thu hi ng nam Nigeria [26].

1.2.6. Cc nhm cht khc

Mt s hp cht thuc cc nhm cht khc trong cy Mng cu xim l:

cacbohydrat (glucose trong l; sucrose, glucose, fructose trong qu;

galactomannan trong ht), lipid (du na kh vi cc acid bo trong ht v

l), acid amin (prolin, -amino-butyric acid trong qu), polyphenol (acid

caffeic, acid para-coumaric, procyanidin, tanins), vitamin (vitamin C trong

qu), glycosid cyano [21].

1.3. Tc dng sinh hc

- Tc dng gy c t bo l tc dng sinh hc ni bt ca cy Mng

cu xim, hin ang c tp trung nghin cu v khai thc. Cc th nghim

v hot tnh ca dch chit hay cc cht phn lp c t cy Mng cu xim

c tin hnh trn nhiu dng t bo ung th khc nhau v u cho hiu

qu r rt.

Tc dng ny ch yu do acetogenin gy ra. Mt s c ch tc dng

[33] l:

- c ch phc hp I ( NADH : ubiquinone oxidoreductase ) trong h

thng ti th vn chuyn in t, c ch phosphoryl ha oxy ha, kt qu lm

gim lng ATP, do c ch s pht trin ca t bo ung th.

- c ch NADH oxidase trong mng plasma ca t bo ung th.

Enzyme ny ch biu hin rt yu trong cc t bo bnh thng khe mnh.

Bng cch c ch enzyme ny, ATP ca t bo ung th s b cn kit.

- c ch t bo ung th a khng. Tng hot ng ca bm t bo, P-

glycoprotein, l yu t ng gp vo s a khng. Bm ny loi b cc hp

cht chng ung th trc khi n c hiu lc trn cc t bo ung th. Hai v tr

10

gn ATP ni bo c tm thy trn P-glycoprotein , v cc bm cn ATP

hot ng. Acetogenin , thng qua s suy gim ca ATP , c th lm gim

hoc dng hot ng ca cc bm ny.

- T bo ung th giai on S ca chu k t bo d b tn thng bi

annonacin acetogenin . Annonacin c th ngn chn chu k t bo trong giai

on G1 v c ch s pht trin n pha S. Ngoi ra annonacin cn tng

cng p53 v p21, cc protein kim sot chu k t bo.

- Cc annonacin acetogenin c th khin t bo cht do t hu. N tng

cng cc biu hin ca Bax v Bad , tr BCL- 2 hoc Bcl- xL.

Nghin cu ca trng i hc De Purdue a ra kt lun v mt s

acetogenin ca cy Mng cu xim c kh nng tiu dit c nhng t bo

ung th, ngay c nhng t bo ung th khng thuc iu tr ung th trc

[5]. Nghin cu ny ch ra rng trong 14 hp cht acetogenin ca Mng cu

xim c 13 cht c tc dng mnh i vi cc dng t bo ung th khng ba

loi thuc chng ung th l: adriamycine, vicristine v vinblastine [5],[12].

Cis-annonacin c IC50 trn dng t bo HT-29 l 1,0x10-8

g/ml, mnh gp

10000 ln so vi adriamycin [28]. Muricoreacin c tc dng trn dng t bo

PC-3 vi ED50= 0,025 g/ml trong khi adriamycin cho ED50= 0,11 g/ml

[18]. Dn xut acetonit ca annomuricin C v muricatocin C c ED50 trn

dng t bo MCF7 l 4,48x10-3 v 3,98x10-3 g/ml; mnh hn adriamycin vi

ED50 = 1,06x10-1

g/ml [36].

T qu kh, dch chit nc : aceton (1:1) c ch chn lc s pht trin

ca t bo ung th v MDA-MB-468 vi gi tr IC50= 4,8 g/ml [9].

T l, dch chit ethanol c kh nng gy c trn dng t bo bch cu

nguyn tu hng cu K562 vi nng gy c mnh nht l 2,5mg/ml [11];

dch chit DMSO khng t bo ung th ty vi gi tr IC50 trn t bo

FG/COLO357 v CD18/HPAF ln lt l 200 v 73 g/ml [34]; tinh du c

11

tc dng gy c t bo trn dng t bo MCF7 vi t l t bo cht nng

100 g/ml l 99,20.1% [27].

- Tc dng chng tng glucose huyt: Nghin cu ca nhm nh khoa

hc Nigeria chng minh tc dng h ng huyt ca chit xut

methanol t cy Mng cu xim trn chut mc tiu ng gy ra bi

streptozotocin [4].

- Tc dng ln tim v huyt p: Dch chit nc ca l tim tnh mch

gim huyt p trn chut. Dch chit nc ca v cy cho tc dng c ch tim

trn th [33].

- Tc dng chng lot: Dch chit ethanol t l c kh nng lm gim

vim lot trn chut b gy lot d dy bi 1ml ethanol 99,5% [15].

- Tc dng chng co tht v gin c: Dch chit ethanol v nc t l

v thn cy dng ng ung vi nng 0,033 ml/l trn ln guinea cho tc

dng chng co tht. th dch chit ethanol 3,3 ml/l lm gin c [33].

- Tc ng thn kinh

+ Dch chit ethanol ca cy Mng cu xim, vi liu dng 100 mg/kg

v 300 mg/kg, lm gim cn co git PZT-tonic v gim t l cht trn chut

c tim PZT (Lead zirconate titanate) [24].

+ Alcaloit t cy Mng cu xim gm asimilobine, nornuciferine,

annonaine c ch s lin kt ca cht ch vn tng phn [3H] rauwolscine vi

th th 5-HT1A, gi tr IC50 ( tng ng l 3, 9, 5 [17].

- Tc dng khng vi sinh vt v khng loi nguyn sinh:

+ Khng st rt: Cn dch chit tng ethanol v cc cn dch phn on

cloroform, methylene cloride, methanol ca cy Mng cu xim, vi hm

lng 10 g/ml, cho kt qu phn trm c ch s pht trin ca chng

Plasmodium falciparum FcB1 tng ng l 28%, 64%, 25%, 38 %. Cn dch

chit phn on cloroform cho hot tnh chng st rt mnh nht [25].

12

+ Khng khun: Chit xut t l, thn v v cy cn c tc dng khng

khun in vitro trn cc loi E. coli, P. aeruginosa, S. flexneri, S. spp, S.

marcescens, S. aureus, S. albus, S. Newport, B. Subtilis [33].

+ Khng virus: Mt phn on cht tan trong nc thu c t thn cy

c tc dng chng li s tng sinh ca t bo nhim HIV trong ng nghim ti

IC50 2 g/ml. Chit xut ethanol ca thn v v cy nng 1 mg/ml c

hot tnh khng vi rt Herpes simplex type 1 [33].

1.4. Cng dng

T lu Mng cu xim c s dng trong y hc truyn thng vi

nhiu trng hp bnh l khc nhau nh vim ph qun, ho, suyn, bnh gan,

bnh ti mt, kh tiu, l, co tht, tiu chy, i tho ng, cao huyt p,

nhim k sinh trng, cm, st cao, st rt Mng cu xim cn c s dng

nh thuc an thn, gim au. L cn c s dng vi mt s cng dng nh

lm thuc li tiu mnh khi b ph chn (Amazonia), tr bnh khp (thp

khp, vim khp), au dy thn kinh (Brazil), bnh da liu (ban da, nhim

trng da) (o Cook), nhp tim nhanh (Madagascar), lm thuc tr tim

(Guyana), gip se da v cm mu (Malaysia) [33].

Vit Nam, l cy Mng cu xim c dng cha bnh ngoi

da, giun, thp khp [1], st rt [2]. Tuy nhin cha c cng trnh no trong

nc nghin cu v thnh phn ha hc v hot tnh sinh hc ca cy Mng

cu xim. V th cn thit nghin cu su hn v thnh phn ha hc v tc

dng sinh hc ca Mng cu xim phc v vic khai thc, s dng hiu

qu loi cy ny Vit Nam.

T tng quan trnh by, nh hng nghin cu c a ra:

- V nghin cu ho hc, cc nhm cht c mt trong Mng cu xim

rt a dng, c cc lp cht t km phn cc n phn cc. V vy, la chn

13

dy dung mi c phn cc tng dn chit xut mu l, to thun li cho

vic phn lp v tinh ch sau ny.

- V nghin cu tc dng sinh hc, tc dng gy c t bo l tc dng

sinh hc c quan tm nhiu nht. V vy, la chn kho st tc dng c ch

t bo ung th ca dch chit l cy Mng cu xim s lm tng ngha ca

nghin cu.

14

CHNG 2 - NGUYN LIU, THIT B, NI DUNG V

PHNG PHP NGHIN CU

2.1. Nguyn liu, ha cht v thit b

2.1.1. i tng nghin cu

L cy Mng cu xim c chng ti thu hi vo thng 5/2013 ti x

Ho M, huyn Phng Hip, tnh Hu Giang, Vit nam. Tn cy c nh

thc vt hc Nguyn Kim o (Vin sinh thi v ti nguyn sinh vt) thm

nh c tn khoa hc l Annona muricata L., h Annonaceae. Tiu bn c

lu gi ti phng Cng ngh cc cht c hot tnh sinh hc, Vin Ha hc,

Vin Hn lm Khoa hc v Cng ngh Vit Nam.

Mu l cy Mng cu xim sau khi thu hi v c ra sch hong kh

ni thong mt, ri sy 45C n khi t hm m 10%, tip mu c

xay thnh bt mn ri chit xut nhit phng.

2.1.2. Dung mi, ha cht:

- Dng cho chit xut v phn lp: n-hexan, diclometan, etylaxetat,

methanol, toluen, acid acetic. Thuc hin dng trong sc k lp mng l dung

dch Ceri sulfat, dung dch Vanillin sulfuric/H2SO4.

- Dng cho th c tnh t bo ung th: huyt thanh phi b, mi

trng RPMI, mi trng DMEM, MTT, DMSO.

2.1.3. Thit b

2.1.3.1 Dng cho chit xut, nh tnh v phn lp

- Phu chit xut mu thc vt

- Phu chit phn lp dung tch 1 lt

- B my quay ct c chn khng thu hi dung mi (BUCHI waterbath

B480-c)

15

- B kho st sc k lp mng

- My sy

- Cn phn tch

- n t ngoi bc sng 254 v 360 nm

- Phu lc v cc bnh cha dung mi

- Ct sc k cao 80 cm ng knh 2,5 cm (Chy phn on)

- Ct sc k cao 50 cm ng knh 2,0 cm (Chy phn on)

- Ct sc k cao 30 cm ng knh 1cm (Tinh ch)

- Phu cha dung mi chy sc k ct

2.1.3.2. Dng cho xc nh thng s vt l v ha hc ca cc cht

- im nng chy c o trn my Buchi melting poit B-545

- Ph FT-IR c ghi trn my FT-IR Impact-410 ca hng Carl Zeiss

Jena ti Vin Ha Hc, Vin Hn lm KH & CN Vit Nam

- Ph EI-MS c o trn my HP 5989BMS

- Ph NMR c ghi trn my Bruker Avance 500MHz vi TMS l

cht ni chun

2.1.3.3. Dng cho th hot tnh gy c t bo

- Thit b nghin cu: t m CO2(INNOVA CO-170)

- T cy sinh hc an ton cp II

- My ly tm (Universal 320R)

- Knh hin vi ngc (Zeizz)

- T lnh su -250C, -80oC

- Bung m t bo (Fisher, Hoa K)

- My quang ph (Genios Tecan)

- Bnh nit lng bo qun t bo

- Cc dng c th nghim thng thng khc

16

2.2. Ni dung nghin cu

- Chit xut mu nghin cu vi cc dung mi c phn cc khc

nhau.

- Phn lp v nhn dng cht phn lp c t phn on dch chit n-

hexan.

- Th tc dng gy c t bo ca phn on dch chit n- hexan.

2.3. Phng php nghin cu

2.3.1. Nghin cu v ha hc

2.3.1.1. Chit xut

Bt l c chit xut vi cc dung mi c phn cc khc nhau theo

th t phn cc tng dn bng phng php ngm ngm kit ti nhit

phng vi thi gian mi ln l 24 gi, mi loi dung mi ngm chit lp li 3

ln. (Dung mi ngm chit phi ngp mu bt dc liu 3cm n 5cm). Dch

chit thu c em ct thu hi di p sut gim n cn. Bo qun cn trong

bnh ht m.

Hm lng cn cc dch chit (theo phng php cn) c tnh theo

cng thc sau:

a x 100%

m (%) = _________________

M x (1 h)

Trong : m (%) l hm lng cn thu c

a: khi lng cn (g)

M: khi lng dc liu (g)

h: m dc liu (%)

17

2.3.1.2. Phn lp

- phn lp cc cht, chng ti s dng cc phng php c bn nh:

+ Sc k ct m, cht hp ph l silica gel Merck c ht 40-63m

+ Sc k ct gel (sephadex LH-20)

+ Sc k lp mng kim tra cc cht c trong dch chit v cc phn

on dch chit. Sc k lp mng c thc hin trn bn mng trng sn

silica gel Merck 60 F254 c dy 0,2mm. Thuc hin l dung dch Ceri

sulfat, dung dch Vanillin sulfuric/H2SO4

- K thut tinh ch: kt tinh hay s dng sc k ct

2.3.1.3. Nhn dng

- Phn tch da trn d liu v tnh cht ho l, ph IR, ph ESI-MS,

ph 1H-NMR, ph 13C-NMR, ph DEPT.

2.3.2. Th tc dng gy c t bo

2.3.2.1. Cc dng t bo

Cc dng t bo cung cp bi ATCC gm: t bo ung th biu m KB, t

bo ung th phi LU, t bo ung th v MCF7.

2.3.2.2. Phng php th tc dng gy c t bo

Cc dng t bo ung th nghin cu c nui cy trong cc mi

trng nui cy ph hp c b sung thm huyt thanh phi b 10% (FBS) v

cc thnh phn cn thit khc iu kin tiu chun (5% CO2; 370C; m

98%; v trng tuyt i). Tu thuc vo c tnh ca tng dng t bo khc

nhau, thi gian nui cy chuyn cng khc nhau. T bo pht trin pha log

s c s dng th c tnh [14],[30].

Tin hnh: 200 dung dch t bo pha log nng 3x104 t bo/ml

vo mi ging (a 96 ging) trong mi trng RPMI 1640 cho cc dng t

18

bo MCF7, KB; mi trng DMEM cho LU-1, mu th c x l vi t bo

cc nng pha long khc nhau sao cho t n nng cui cng l 128

/ml, 32 /ml, 8 /ml, 2 /ml, 0,5 /ml, 0,125 /ml, 0,03125 /ml,

0,0078 /ml. 370C, 5% CO2 trong 3 ngy. Ging iu khin gm 200

dung dch t bo 3x104 t bo/ml; thm 50 MTT (1mg/ml pha trong mi

trng nui cy khng huyt thanh), 37oC trong 4h, loi b mi trng,

thm 100 DMSO lc u c kt qu bc sng 540 nm trn my

spectrophotometre Genios TECAN [14],[30].

Phn trm km hm s pht trin ca t bo c tnh ton da trn s

liu o mt quang hc OD trn my quang ph TECAN theo cng thc

sau:

ODmu th - ODcontrol(-)

IC= 100% ________________________

ODcontrol(+) - ODcontrol(-)

Trong

- ODcontrol(-) l gi tr thu c ging th ch c mi trng nui

cy m khng c t bo.

- ODcontrol(+) l gi tr thu c ging th c mi trng nui cy

cha t bo nhng khng x l vi mu cn dch chit.

Gi tr IC50 c tnh da trn kt qu s liu phn trm km hm s

pht trin ca t bo bng phn mm my tnh table curve.

19

CHNG 3 - THC NGHIM, KT QU V BN LUN

3.1. Thc nghim v kt qu

3.1.1. Chit xut

Np 1kg bt l Mng cu xim (hm m 10%) vo bnh ngm chit.

Bt l c ngm chit vi n-hexan 3 ln (3l/24h/ln). Dch chit c ct

loi dung mi di p sut thp thu c cn dch chit n-hexan. B dc liu

sau chit vi n-hexan c lm kh, tip tc ngm chit tng t nh trn vi

ln lt cc dung mi CH2Cl2, EtOAc v MeOH, t ln lt thu c cn

ca cc dch chit ny. S chit xut bt l Mng cu xim c trnh by

hnh 3.1.

Khi lng cn cc dch chit thu c trnh by trong bng 3.1.

Bng 3.1: Hm lng cc cn cc dch chit ca l cy Mng cu xim

Cn dch chit Khi lng thu c (g) Hm lng cn (%)

n-hexan 23,5 2,61

CH2Cl2 25,7 2,85

EtOAc 15,4 1,71

MeOH 65,8 7,31

Nhn xt:

Nhn thy cn dch chit EtOAc c hm lng nh nht, tng ng vi

1,71%. Cn dch chit n-hexan v CH2Cl2 c hm lng ln lt l 2,61% v

2,85%. Cn dch chit MeOH c hm lng ln nht (7,31%) so vi cc cn

dch chit cn li. nh gi s b trong l cy c nhiu cc hp cht phn

cc; cc cht km phn cc hay phn cc trung bnh chim t l thp hn.

20

Hnh 3.1: S chit xut bt l cy Mng cu xim

21

3.1.2. Tc dng gy c t bo ung th

Trong nghin cu ny, cn dch chit n-hexan ca l cy Mng cu

xim c s dng lm mu th kho st hot tnh c ch s pht trin t

bo ung th thng qua php th gy c t bo trn c 3 dng t bo ung th

KB, MCF7 v LU. Bng phng php th c trnh by phn 2.3.2

chng ti thu c kt qu trnh by bng 3.2.

Bng 3.2. Kt qu th hot tnh c t bo ca cn dch chit n-hexan t

l cy Mng cu xim

STT Tn mu Gi tr IC50 (g/ml)

KB LU MCF7

1. Cn dch n-

Hexan 0,08 0,7 6,9

2. Ellipticine 0,31 0,45 0,53

Nhn xt:

Cht i chng dng Ellipticin th hin hot tnh gy c i vi c 3

dng t bo ung th KB, LU, MCF7 vi gi tr IC50 ln lt l 0,31; 0,45 v

0,53 g/ml.

Cn dch n-hexan ca mng cu xim c tc dng gy c t bo trn

c 3 dng t bo th nghim KB, LU v MCF7 vi cc gi tr IC50 ln lt

tng ng l 0,08 g/ml, 0,7 g/ml v 6,9 g/ml. Tc dng ca cn dch chit

n-hexan trn dng t bo KB l mnh nht th hin gi tr IC50 nh nht l

0,08 g/ml, tt hn cht i chng Ellipticine vi gi tr IC50 ln hn; trong

khi cn chit ny c hiu lc trung bnh trn dng t bo MCF7.

T th nghim cho thy cn dch chit n-hexan c tc dng gy c t

bo ung th nn chng ti chn dch chit ny tin hnh phn lp cc hp

cht.

22

3.1.3. Phn lp cc cht t dch chit n-hexan

3.1.3.1. Kho st s b cn dch chit n-hexan bng SKLM

- Ly 0,5g cn dch chit n-hexan ho tan trong CH2Cl2, lc qua giy lc,

- Bn mng silicagel F254 c hot ho 1100C trong 1h

- Trin khai SKLM vi cc h dung mi sau :

H I: n-hexan:CH2Cl2 [7:3]

H II: CH2Cl2:EtOAc [6:4]

H III: EtOAc:MeOH [9:1]

Ta thu c hnh nh sc k nh hnh 3.2.

H (I) H (II) H (III)

Hnh 3.2: Sc k ca cn dch chit n-hexan ca l mng cu xim

trong cc h dung mi khc nhau quan st AST

Nhn xt:

Qua hnh nh bn mng nhn thy h I cc cht bt u tch vt.

h II, phn cc tng ln cc cht tip tc tch ra. H III l h phn cc cao,

23

cc vt tch ra v tch tt trn bn mng, y l c s la chn h dung

mi tch cc cht trong cn dch chit n-hexan trn SKLM v sc k ct.

- Trin khai SKLM vi h dung mi Toluen : Etyl acetate : Acid acetic

[40:10:1], quan st SKLM vi cc iu kin khc nhau (AST, UV254, hin

vt bng thuc th Vanillin sulfuric/H2SO4) thu c kt qu bng 3.3.

AST UV254 Hin mu

Hnh 3.3: Sc k cn dch chit n-hexan trong h dung mi

Toluen : Etyl acetat : Acid acetic [40:10:1]

24

Bng 3.3: Kt qu SKLM ca cn dch chit n-hexan trong h dung mi

Toluen : Etyl acetat : Acid acetic [40:10:1]

STT

Rf

iu kin quan st

AST UV245 Thuc th

Mu sc

m

Mu sc

m

Mu sc

m

1 0,06 - - en + - -

2 0.09 Xanh en + en + Xanh l +

3 0.18 Xanh en ++ en ++ Xanh l ++

4 0,20 Xanh en + en + Xanh l +

5 0,23 Xanh en + en + Nu +

6 0.26 Vng + - - Tm +

7 0,34 Vng +++ en ++ Tm +++

8 0.45 Vng xanh + en ++ Xanh l +

9 0.55 Xanh en + en ++ Xanh l +

10 0.66 Vng xanh + en + Xanh l +

11 0.70 Xanh tm + en ++ Xanh lam +

12 0.78 Vng xanh +++ en ++ Xanh l ++

13 0.84 Xanh tm +++ en +++ Xanh lam +++

Ghi ch: (+++) rt m, (++) m, (+) m, (-) khng hin vt

T hnh nh nhn thy c 8 vt cht chnh, mu m, cc vt 3,4,5,6 rt

gn nhau.

3.1.3.2. Phn lp cn dch chit n-hexan l Mng cu xim bng sc k

ct

Chun b ct: Cn 100g cht hp ph silica gel c ht 0,04-0,063 mm

c nhi vo ct sc k thu tinh (chiu cao = 80cm, ng knh = 2,5cm)

25

bng phng php nhi ct t. Di y ct nhi mt lp bng mng, lng

silicagel chun b c cho vo cc thy tinh 500 ml sau thm vo

dung mi n-hexan khuy u n khi to hn hp ng nht ri rt t t vo

ct n ht, n nh ct cho silica gel lng xung v m van dung mi

chy ra bnh hng n khi trn u ct vch dung mi cn khong 0,5 cm so

vi vch silicagel.

Chun b mu chy ct: Cn 10g cn dch chit n-hexan ha tan trong

dung mi n-hexan n khi ng nht, trn vi 1g silica gel khuy u sau

quay c chn khng loi dung mi n-hexan to dng bt, bt mu ny c

tri u ln b mt silicagel ca ct sc k.

Tin hnh chy sc k bng cc h dung mi nh sau:

- Chy sc k ct vi h dung mi n-hexan : CH2Cl2 vi t l nh sau:

n-hexan : CH2Cl2 100 : 0 500 ml

n-hexan : CH2Cl2 90 : 10 500 ml

n-hexan : CH2Cl2 80 : 20 500 ml

n-hexan : CH2Cl2 70 : 30 500 ml

n-hexan : CH2Cl2 60 : 40 500 ml

n-hexan : CH2Cl2 50 : 50 500 ml

n-hexan : CH2Cl2 40 : 60 500 ml

n-hexan : CH2Cl2 30 : 70 500 ml

n-hexan : CH2Cl2 20 : 80 500 ml

n-hexan : CH2Cl2 10 : 90 500 ml

Hng dch ra gii vo cc bnh hng (100ml/bnh hng). Kim tra

dch ra gii bng SKLM, gp cc dch c cc vt ging nhau trn sc k ,

ct thu hi dung mi, chuyn dch c vo cc ng nghim. Kt thc qu trnh

ra gii vi h dung mi n-hexan : CH2Cl2 thu c 30 phn on, k hiu l

P1 n P30.

26

- Chy sc k ct bng h dung mi CH2Cl2 : EtOAc vi t l nh sau:

CH2Cl2 : EtOAc 100 : 0 500 ml

CH2Cl2 : EtOAc 90 : 10 500 ml

CH2Cl2 : EtOAc 80 : 20 500 ml

CH2Cl2 : EtOAc 70 : 30 500 ml

CH2Cl2 : EtOAc 60 : 40 500 ml

CH2Cl2 : EtOAc 50 : 50 500 ml

CH2Cl2 : EtOAc 40 : 60 500 ml

CH2Cl2 : EtOAc 30 : 70 500 ml

CH2Cl2 : EtOAc 20 : 80 500 ml

CH2Cl2 : EtOAc 10 : 90 500 ml

Tin hnh tng t nh trn. Kt thc qu trnh ra gii vi h dung

mi CH2Cl2 : EtOAc thu c 30 phn on, k hiu l P31 n P60.

- Chy sc k ct bng h dung mi EtOAc : MeOH vi t l dung mi

nh sau:

EtOAc : MeOH 100 : 0 200 ml










27

Dch hng c chia u vo cc bnh hng (50ml/bnh hng). Tin

hnh tng t nh trn. Kt thc qu trnh ra gii vi h dung mi EtOAc :

MeOH thu c 18 phn on, k hiu l P61 n P78.

Tng cc phn on thu c l 78 (k hiu t P1 - P78).

3.1.3.3. Phn lp cht t phn on P27

T phn on P27 bay hi bt dung mi thy c cht kt ta mu

trng. Gn dch v ra ta nhanh bng n-hexan lnh thu c 13mg cht. Ho

tan ta trong lng n-hexan ti thiu c un nng trn bp cnh thy 60oC

n khi tan ht, sau ngui nhit phng n khi thy xut hin tinh

th, yn trong 24h. Sau gn b dch thu tinh th dng tinh th hnh kim,

ra tinh th bng n-hexan lnh v lm kh tinh th. nh lng thu c thu

c 9 mg cht sch (69% so vi ta thu c). K hiu l hp cht P27.

Sc k ca cht P27 trong h dung mi CH2Cl2 : MeOH [98:2], thy

vt cht P27 c Rf = 0,81; khng hp th UV; hin mu bng ceri sulfat cho

vt mu xanh tm (hnh 3.4).

Hnh 3.4: Sc k ca cht P27 trong h dung mi

CH2Cl2 : MeOH [98:2], hin mu bng ceri sulfat

28


T phn on P71 thy c xut hin cht rn mu trng. Kim tra

SKLM thy c mt vt chnh vi h dung mi CH2Cl2 : MeOH [9:1]. Quay

ct c chn khng loi dung mi thu c 20 mg cht rn. Cht rn ny c

tip tc tinh ch bng chy sc k ct nhanh.

Chun b ct: S dng ct sc k thu tinh (chiu cao = 30cm, ng

knh = 1cm) bng phng php nhi ct t. Di y ct nhi mt lp bng

mng, cn 15g silicagel cho vo cc thy tinh 200 ml sau thm vo dung

mi CH2Cl2 khuy u n khi to hn hp ng nht ri rt t t vo ct n

ht, n nh ct cho silicagel lng xung v m van dung mi chy ra

bnh hng n khi trn u ct vch dung mi cn khong 0,5 cm so vi vch

silica gel.

Chun b mu chy ct: Ly 20mg cht rn thu c t P71 c tri

u ln b mt silicagel ca ct sc k.

Tin hnh chy sc k: Ra gii vi h dung mi CH2Cl2 : MeOH

[9:1]. S dng 100ml h dung mi trn ra gii, dung mi c a ln

phu v rt t t vo ct sc k cho n khi gn y ct, lp h bm p va

phi y dung mi chy qua ct, ng thi hng dch ra gii. B 20ml

u v thu 50ml tip theo, phn ny c kim tra li bng SKLM vi h

dung mi CH2Cl2 : MeOH [9:1] thy mt vt cht sch. Quay ct c chn

khng loi dung mi thu c 14,8mg cht sch (74% so vi cht rn thu

c t phn on 71). Hp cht ny c k hiu l cht P71.

Sc k ca cht P71 trong h dung mi CH2Cl2 : MeOH [9:1], thy

vt cht P71 c Rf = 0,33; khng hp th UV; hin mu bng ceri sulfat cho

vt mu xanh tm (hnh 3.5).

29




T phn on P55, sau khi kim tra sc k lp mng thy c mt vt

cht chnh v c mu vng nht c ly tin hnh phn lp cht ny.

Phn on ny dc quay c chn khng loi dung mi thu c 30 mg cht

rn c s dng chy ct sc k phn lp cht.

Chun b ct: Cn 25g gel sephadex LH-20 cho vo ct sc k thy

tinh (chiu cao = 50cm, ng knh = 2cm) bng phng php nhi ct t.

Di y ct nhi mt lp bng mng, lng gel sephadex LH-20 chun

b c cho vo cc thy tinh 200 ml sau thm vo dung mi MeOH

khuy u n khi to hn hp ng nht ri rt t t vo ct n ht, n

nh ct cho gel sephadex LH-20 lng xung v m van dung mi chy ra

bnh hng n khi trn u ct vch dung mi cn khong 0,5 cm so vi vch

gel sephadex.

Chun b mu chy ct: 30mg cht rn thu t P55 c tri u ln b

mt gel sephadex ca ct sc k.

Tin hnh chy sc k: Ra gii vi dung mi MeOH. Lng dung mi

s dng l 200ml c a ln phu v cho chy t t qua ct sc k, phn

30

di ct c hng cc phn on (mi phn on 20 ml). Thu c 10 phn

on, k hiu t P55.1 - P55.10.

Kim tra SKLM cc phn on thu c vi h dung mi trin khai

CH2Cl2 : EtOAc [8:2]. Thy phn on P55.7 c mt vt cht sch, phn

on ny c quay ct c chn khng loi dung mi thu c 11mg cht rn

mu vng (36,6 % so vi cht rn thu c t P55). Hp cht ny c k

hiu l cht P55.7.

Sc k cht P55.7 trong h dung mi CH2Cl2 : EtOAc [8:2] cho Rf =

0,7 v vt cht cho mu vng nh sng thng (hnh 3.6).

Hnh 3.6: Sc k ca cht P55.7 trong h dung mi CH2Cl2 : EtOAc

[8:2], quan st AST

3.1.4. Nhn dng cu trc

3.1.4.1. Nhn dng cu trc cht P27

- Tnh cht: tinh th hnh kim, mu trng.

- Tnh tan: tan tt trong chloroform.

- Nhit nng chy: 140-142C.

- T ph 1H-NMR (CDCl3, 500MHz); thy cc tn hiu c trng ca

cu trc sterol:

+ Tn hiu proton ca 6 nhm methyl, (ppm): 1,01 (3H, s, CH3-19);

0,92 (3H, d, J = 6,4Hz, CH3-21); 0,85 (3H, t, J = 7,6Hz, CH3-29); 0,84 (3H, d,

31

J = 7,0Hz, CH3-26 or 27); 0,82 (3H, d, J = 7,0Hz, CH3-26 or 27); 0,68 (3H, s,

CH3-18).

+ Tn hiu ca proton olefin ti 5,35 ppm.

+ Tn hiu ca proton gn vi C3 (cacbon c nhm th -OH) ti

3,53ppm.

- T phn tch d liu ph 1H-NMR v c so snh vi ti liu [20], cho

php nhn dng cht P27 l - sitosterol.

OH

H

H

1

2

34

56

7

89

10

19 11

12

13

14 15

1617

18 20

21 22

2324

25

26

27

28

29

Hnh 3.7: Cng thc cu to -sitosterol (P27)

3.1.4.2. Nhn dng cu trc cht P71

- Tnh cht: cht rn mu trng.

- Tnh tan: tan tt trong chloroform, methanol.


- T ph 1H-NMR ca cht P71, ta thy c nhng im tng ng vi

ph ca cht P27:

+ Tn hiu ca 6 nhm metyl vi (ppm) l 0,77; 0,81; 0,82; 0,83; 0,90

v 0,96; trong c cp tn hiu dublet ti 0,82 ppm v 0,81 ppm cng c

32

hng s tng tc J =7,0 Hz l tng tc germinal ca 2 nhm metyl v tr C-

26 v C-27 gn vo nhm metin v tr 25.

+ Tn hiu dublet ti 5,33 ppm (J =5,0 Hz) l tn hiu ca proton olefin

v tr C6.

+ Tn hiu multiplet ti 3,46 ppm l tn hiu ca proton gn vi C3.

Nhng tn hiu trn cho thy P71 l dn xut ca sitosterol.

- Tn hiu ca proton anomer ti 4,22 ppm (J=7,0 Hz) cng vi vng

tn hiu proton 4,39-4,85 ppm cho thy s c mt ca 1 phn t ng

glucose trong phn t hp cht.

- Nhn nh trn cng c khng nh thng qua d liu trn ph

DEPT v v ph C13CPD ca P71 vi tn hiu ca 16 nhm CH, 6 nhm

CH3, 12 nhm CH2 v 3 C bc 4.

- Ph 13C-NMR cho thy tn hiu ca cacbon anomer ti 100,7 ppm v

5 tn hiu ca ng glucose ti C 73,3; 76,4; 70,1; 76,7 v 60,1 ppm.

- T cc phn tch trn cng vi s ph hp vi ti liu cng b [37]

cho php nhn dng cht P71 l -sitosterol-3-O--D-glucopyranosit hay

daucosterol.

* D liu ph 1H-NMR v ph 13C-NMR ca cht P71:

- Ph 1H-NMR (500MHz, DMSO-d6, TMS), (ppm): 0,77 (3H, s, CH3-

18), 0,81 (3H, d, J = 7,0 Hz, CH3-27); 0,82 (3H, d, J = 7,0 Hz, CH3-26); 0,83

(3H, t, J = 7,0 Hz, CH3-29); 0,90 (3H, d, J = 6,0 Hz, CH3-21); 0,96 (3H, s,

CH3-19); 3,46 (1H, m, H-3); 5,33 (1H, d, J = 5,0 Hz, H-6); 2,90 (1H, m, H-

2); 3,02 (1H, m, H-4); 3,07 (1H, m, H-5); 3,12 (1H, m, H-3); 3,41 (1H, m,

H-6a); 3,65 (1H, m, H-6b); 4,22 (1H, d, J = 7,0 Hz, H-1).

- Ph 13C-NMR (125MHz, DMSO-d6, TMS); , ppm: 11,7 (CH3-18),

19,0 (CH3-27); 19,7 (CH3-26), 11,73 (CH3-29); 18,6 (CH3-21); 18,90 (CH3-

19); 20,6 (C-11); 22,6 (C-28); 23,2 (C-15); 25,4 (C-23); 27,7 (C-16); 28,7 (C-

33

25); 29,2 (C-2); 31,3 (C-7); 31,4 (C-8); 33,3 (C-22); 35,4 (C-20); 36,2 (C-10);

36,8 (C-1); 38,3 (C-4); 39,2 (C-12); 41,8 (C-13); 45,1 (C-24); 49,6 (C-9);

55,4 (C-17); 56,1 (C-14); 76,9 (C-3); 121,1 (C-6); 140,4 (C-5); 60,1 (C-6);

70,1 (C-4); 73,3 (C-2); 76,4 (C-3); 76,7 (C-5); 100,7 (C-1).

H

OH

H

OH

H

H

O

1

2

34

56

7

89

10

19 11

12

13

14 15

1617

18 20

21 22

2324

25

26

27

28

29

O

H

H

H

OHOH

1'2'3

'

4'5'6

'

Hnh 3.8: Cng thc cu to ca -sitosterol-3-O--D-glucopyranosit

(P71)

3.1.4.3. Nhn dng cu trc cht P55.7

- Tnh cht: Cht rn mu vng m.

- Tnh tan: Tan tt trong chloroform, methanol.


- Ph ESI-MS: m/z 569,5 [M+H]-, s b nhn nh cng thc phn t

ca cht P55.7 l C40H56O2.

- Trn ph 13C-NMR v DEPT ca cht P55.7 cho thy tn hiu 40

cacbon bao gm 10 nhm metyl trong vng 12,7 - 29,5 ppm, hai nhm CH-

OH ti 65,1 ppm v 65,9 ppm, ngoi ra cn c tn hiu ca 22 cacbon lai ha

sp2 trong vng 124,02-138,50 ppm.

34

- Trn ph 1H-NMR ca hp cht P55.7 cho thy tn hiu proton c

trng ca su nhm metyl trong vng 0,84 - 1,73 ppm v bn nhm metyl

trong vng 1,90 - 1.96 ppm. Ngoi ra cn c cc tnh hiu proton ca nhm

CH-OH ti 4,00 ppm v 4,25 ppm; tn hiu ca proton olefin ti vng 5,43

6,62 ppm.

Bng 3.4.1: S liu ph NMR ca hp cht P55.7 v lutein [10]

V tr C

Lutein [10] Cht P55.7

Ca,b

Ha,c

(J, Hz) Ca, d

DEPT Ha, e

1 37,1 - 37,1 C -

2 48,4 2,48; m 48,5 CH2 2,38; m

3 65,0 4,06; m 65,1 CH 4,00; m

4 42,5 2,48; m 42,6 CH2 2,38; m

5 126,2 - 126,2 C -

6 137,5 - 137,6 C -

7 125,6 6,20; d (16,0) 125,6 CH 6,16; s

8 138,5 6,13; m 138,5 CH 6,13; m

9 135,7 - 135,7 C -

10 131,3 6,13; m 131,3 CH 6,13; m

11 124,9 6,72; d (16,0) 124,9 CH 6,62; m

12 137,6 6,41; d (16,0) 137,7 CH 6,35; d (12,5)

13 136,5 - 136,5 C -

14 132,6 6,32; d (10,0) 132,6 CH 6,25; d (10)

15 130,1 6,69; d (10,0) 130,1 CH 6,62; m

16 28,7 1,15; s 28,7 CH3 1,07; s

17 30,2 1,15; s 30,3 CH3 1,07; s

18 21,6 1,80; s 21,6 CH3 1,73; s

19 12,8 2,02; s 12,8 CH3 1,96; s

20 12,8 2,04; s 12,8 CH3 1,96; s a o trong CDCl3,

b 100 MHz,

c 400 MHz,

d500 MHz,

e125 MHz

35

Bng 3.4.2: S liu ph NMR ca hp cht P55.7 v lutein [10] (tip)

V tr C

Lutein [10] Cht P55.7

Ca,b

Ha,c

(J, Hz) Ca, d

DEPT Ha, e

1 34,0 - 34,0 C -

2 44,6 2,48; m 44,6 CH2 2,38; m

3 65,9 4,30; m 65,9 CH 4,25; s

4 124,8 5,60; br.s 124,8 CH 5,54; br.s

5 137,7 - 137,7 C -

6 54,9 2,48; m 55,0 CH 2,38; m

7 128,7 5,51; dd (5,1; 15,0) 128,7 CH 5,43; dd (5,5; 15,5)

8 137,8 6,13; m 137,8 CH 6,13; m

9 135,0 - 135,0 C -

10 130,8 6,13; m 130,8 CH 6,13; m

11 124,5 6,72; d (16,0) 124,5 CH 6,62; m

12 137,7 6,41; d (16,0) 137,7 CH 6,35; d (12,5)

13 136,4 - 136,4 C -

14 132,6 6,32; d (10,0) 132,6 CH 6,25; d (10)

15 130,0 6,69; d (10,0) 130,0 CH 6,62; m

16 24,2 0,92; s 24,3 CH3 0,85; s

17 29,5 1,07; s 29,5 CH3 0,99; s

18 22,9 1,72; s 22,9 CH3 1,67; s

19 13,1 1,94; s 13,1 CH3 1,90; s

20 12,7 2,04; s 12,7 CH3 1,96; s a o trong CDCl3,

b 100 MHz,

c 400 MHz,

d500 MHz,

e125 MHz

T d liu ph thu c so snh vi ti liu tham kho [9] cht P55.7

c xc nh l lutein.

Hnh 3.8: Cng thc cu to ca lutein (P55.7)

36

3.2. Bn lun

3.2.1. Tc dng gy c t bo ca dch chit n-hexan t l cy Mng cu

xim

Th hot tnh sinh hc c tin ti phng Ha sinh ng dng -Vin

Ha hc - Vin Hn lm khoa hc v Cng ngh Vit Nam.

Cc t bo ung th l nhng t bo km bit ha do s phn chia qu

nhanh chng v din ra lin tc. Chng c kh nng vt qua s kim sot

ca h min dch ca c th v tng cng xm ln vo cc t chc sng ln

cn. Nhng cht c kh nng phng chng v iu tr ung th l nhng cht

c kh nng ngn nga s hot ng v bt gi cc tc nhn gy ung th, c

ch qu trnh tin trin bnh thng qua s c ch hot ng mt s enzyme

hay tc ng gy bit ha t bo. Php th sinh hc sng lc v tm kim

cc hot cht cha tr bnh ung th s dng chnh t bo ung th lm i

tng nghin cu.

Trong nghin cu ny, dch chit n-hexan ca l cy Mng cu xim

c kho st hot tnh c ch s pht trin t bo ung th thng qua php

th gy c t bo trn c 3 dng t bo ung th KB, MCF7 v LU. Kt qu

cho thy cn dch n-hexan c kh nng gy c t bo KB vi gi tr IC50 l

0,08 g/ml; kh nng gy c t bo MCF7 vi gi tr IC50 l 6,9 g/ml v LU

l 0,7 g/ml. Tc dng ca cn dch chit n-hexan rt mnh trn dng t bo

KB, mnh hncht i chng dng ellipticine; tc dng tt trn dng t bo

LU; tc dng trn dng t bo MCF7 yu nht trong s 3 dng t bo ung th

th nghim v mc trung bnh.

T cc gi tr ny c th s b kt lun trong dch chit n-hexan c

cha cc hp cht c tc dng n l hoc cc hp cht c hip ng tc

dng gy c t bo. Trong thc t ngi ta thng b qua dch chit ny bi

lp cht trong dch chit n-hexan c tnh phn cc thp, ng dng ca chng

37

c phn hn ch hn so vi lp cht phn cc mnh hn do vn v sinh

kh dng. Mc d vy, vic sng lc v tm kim cc hot cht trong dch

chit ny l cn thit c c hiu bit ton din, y nht v cy Mng

cu xim, ng thi c th c nhng pht hin mi, nhng cht mi tim nng

trong iu tr ung th.

3.2.2. V thnh phn ho hc

V chit xut

Trong nghin cu ny chng ti tin hnh chit xut l cy Mng cu

xim s dng cc dung mi c phn cc tng dn. Vic s dng phng

php chit ny tuy tn thi gian v ho cht song gip phn chia cc lp cht

theo phn cc tng dn hiu qu v d x l hn.

V phn lp

Chng ti tin hnh phn lp cc hp cht t cn dch chit l Mng

cu xim bng sc k ct v s dng h dung mi gradient nng bt u t

dung mi khng phn cc l n-hexan n dung mi phn cc mnh l

methanol. Vi h dung mi ra gii tng phn cc dn, cc lp cht phn b

vo cc phn on tng phn cc dn theo h dung mi ra gii, s phn tch

hiu qu hn.

V xc nh cu trc

Cht P27, cht P71 v cht P55.7 c xc nh l -sitosterol, -

sitosterol-3-O--D-glucopyranosit v lutein da vo cc tnh cht vt l, ph

hng ngoi, ph khi lng v ph cng hng t ht nhn cng vi so snh

ti liu tham kho khng nh.

c nghin cu ch ra tc dng c ch t bo ung th ca -sitosterol

[19],[29], ca -sitosterol-3-O--D-glucopyranosit [19] v lutein

[10],[16],[31]. V vy s b kt lun 3 cht c phn lp c th c vai tr

38

trong tc dng c ch t bo ung th ca cn dch chit n-hexan ca l Mng

cu xim

39

KT LUN V XUT

KT LUN

Nghin cu ny thu c nhng kt qu sau:

- Bng phng php ngm kit, s dng ln lt cc dung mi n-hexan,

CH2Cl2, EtOAc, MeOH thu c cn cc dch chit vi hm lng tng ng

ln lt l: 2,61%, 2,85%, 1,71%, v 7,31% t 1kg bt l Mng cu xim.

- th hot tnh c ch t bo ung th ca cn dch chit n-hexan ca

l Mng cu xim, kt qu cho thy cn dch chit n-hexan c tc dng c ch

t bo ung th v gi tr IC50 trn dng t bo ung th biu m KB, dng t

bo ung th v MCF7 v dng t bo ung th phi LU tng ng l: 0,08

g/ml, 6,9 g/ml v 0,7 g/ml.

- phn lp c 3 cht P27, P71, P55.7 bng phng php sc k

ct m, sc k ct nhanh, sc k lc gel t cn dch chit n-hexan ca l

Mng cu xim.

- Bng sc k lp mng, s b kt lun 3 cht phn lp c tinh khit.

- nhn dng 3 cht phn lp c ln lt l - sitosterol,

daucosterol v lutein da trn phn tch tnh cht vt l, ph IR, ph ESI-MS,

ph 1H-NMR, ph 13C-NMR v so snh ti liu tham kho.

XUT

- Tip tc phn lp v xc nh cu trc cc hp cht c trong cn dch

chit n-hexan ca l cy Mng cu xim.

- Th hot tnh c ch t bo ung th ca cc cht phn lp c t cn

dch chit n-hexan.

40

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Guillaumin, J.-D. Hooker. H. Lecomte, F. Pellegrin, G.J. Pitard.,( 1907-1912)

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boulevard saint-germain (VIe)

45

Ti liu internet :

[39]http://www.nihbt.org.vn/tin-trong-nuoc/sung-sot-vi-benh-nhan-ung-thu-tang-nhanh/p124i6718.html

46

PH LC

Ph lc 1a: Ph 1H-NMR ca cht P27 (-sitosterol)

47

Ph lc 1b: Ph 1H-NMR ca cht P27 (-sitosterol)

48

Ph lc 2a: Ph 1H-NMR ca cht P71 (-sitosterol-3-O--D-glucopyranosit)

49

Ph lc 2b: Ph 1H-NMR ca cht P71 (-sitosterol-3-O--D-glucopyranosit)

50

Ph lc 2c: Ph 1H-NMR ca cht P71 (-sitosterol-3-O--D-glucopyranosit)

51

Ph lc 3a: Ph 13C-NMR ca cht P71 (-sitosterol-3-O--D-glucopyranosit)

52

Ph lc 3b: Ph 13C-NMR ca cht P71 (-sitosterol-3-O--D-glucopyranosit)

53

Ph lc 4a: Ph DEPT ca cht P71 (-sitosterol-3-O--D-glucopyranosit)

54

Ph lc 4b: Ph 13C-DEPT ca cht P71 (-sitosterol-3-O--D-glucopyranosit)

55

Ph lc 5a: Ph 1H-NMR ca cht P55.7 (lutein)

56

Ph lc 5b: Ph 1H-NMR ca cht P55.7 (lutein)

57

Ph lc 5c: Ph 1H-NMR ca cht P55.7 (lutein)

58

Ph lc 6a: Ph 13C-NMR ca cht P55.7 (lutein)

59

Ph lc 6b: Ph 13C-NMR ca lutein

60

Ph lc 6c: Ph 13C-NMR ca cht P55.7 (lutein)

61

Ph lc 7: Ph DEPT ca cht P55.7 (lutein)

62

Ph lc 8: Ph ESI-MS ca cht P55.7 (lutein)

9:5

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569.5

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0.6

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1.0

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450

500

550

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650

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Documents

Nghiên cứu thành phần hóa học và thử hoạt tính ức chế tế bào ung thư của lá cây mãng cầu xiêm ( Annona muricata L.) họ Annomaceae.pdf