B GIO DC V O TO

TRNG I HC BCH KHOA H NI

H Th H

NGHIN CU HOT TNH SINH HC CA MT S HP

CHT CHIT TCH T L U (Carica papaya Linn)

Chuyn ngnh: Cng ngh sinh hc

M s: 62420201

TM TT LUN N TIN S CNG NGH SINH HC

H Ni - 2014
 Cng trnh c hon thnh ti:
Trng i hc Bch khoa H Ni

Ngi hng dn khoa hc:

PGS.TS. Th Hoa Vin
TS. L Quang Ha

Phn bin 1: PGS.TS. Nguyn Th Hong Anh

Phn bin 2: PGS.TS. V nh Hong

Phn bin 3: PGS.TS. H Huy K

Lun n s c bo v trc Hi ng chm lun n tin s cp Trng

hp ti Trng i hc Bch khoa H Ni

Vo hi .. gi, ngy .. thng .. nm

C th tm hiu lun n ti th vin:

1. Th vin T Quang Bu - Trng HBK H Ni
2. Th vin Quc gia
 DANH MC CC CNG TRNH CNG B

1. Do Thi Hoa Vien, Ho Thi Ha (2013). Research on anti-cancer

activity of some extracts from Vietnamese carica papaya leaves.
Proceeding of the 7th SEATUC Symposium, 4-6 March 2013,
Bangdung, Indonesia, ISSN 2186 7631, OS4-4.

2. H Th H, L Quang Ha, Phm Vn Cng, on Th Mai

Hng, Nguyn Thy Linh, Chu Vn Minh, Th Hoa Vin
(2013) Bc u nghin cu thnh phn ha hc l cy u
carica papaya h u (caricaceae). Tp ch ha hc, T. 51
(6ABC), ISSN 0866 - 7144, pp 59 63.

3. H Th H, Th Hoa Vin, L Quang Ha (2014) Nghin cu

hot tnh sinh hc ca mt s hp cht chit tch t l u
(Carica papaya L.). Tp ch khoa hc cng ngh Vit Nam, b
Khoa hc v Cng ngh, s 2+3, ISSN 1859 4794, pp 119-122.

4. H Th H, Th Hoa Vin, L Quang Ha, Phm Vn Cng,

on Th Mai Hng, Nguyn Thy Linh, Chu Vn Minh (2014)
Carpainone: alkaloid mi t l cy u . Tp ch khoa hc v
cng ngh, vin Hn lm Khoa hc v Cng ngh Vit Nam, tp
52, s 5, ISSN 0866708X, pp593 - 598.
 T VN
1. Tnh cp thit ca ti
Cy u (Carica papaya Linn) l mt loi cy n qu c trng
cc nc vng nhit i. Vit Nam, cy u c trng hu ht cc tnh
min Bc v min Nam. L u c chng minh l c kh nng chng oxy
ha rt mnh, c hot tnh khng khun tt. Ngoi ra, l u cn c kh
nng khng vim, gim au. c thng bo dch chit nc l cy u c
tc dng c ch mt s dng t bo ung th ngi nh ung th d dy, ung
th phi, ung th mu. Ngoi ra, nc l cy u cn c tc dng iu ha
min dch. Tuy nhin, nhng cng b u ch s lc dng dch chit v
cha xc nh c thnh phn no l hot cht. V vy, vic chng minh
c thnh phn hot cht c th ca l u l mt vic lm ht sc cn
thit, to c s khoa hc cho vic ng dng ngun nguyn liu sn c Vit
Nam lm thuc iu tr bnh ung th. Do , ti la chn ti: Nghin
cu hot tnh sinh hc ca mt s hp cht chit tch t l u (Carica
papaya Linn
2. Mc tiu ca ti
- Phn lp v xc nh cu trc mt s hp cht c hot tnh sinh hc v hp
cht mi c trong l u .
- nh gi hot tnh sinh hc ca cc hp cht phn lp lm c s khoa hc
chng minh cho vic ngi dn mt s nc trn th gii s dng l u
cha tr mt s loi bnh trong c bnh ung th.
3. Ni dung nghin cu
- Nghin cu phn lp mt s hp cht c trong l u .
- Xc nh cu trc ca mt s hp cht phn lp c.
- nh gi hot tnh gy c t bo ung th ca cc phn on cn chit t l
u .
- nh gi kh nng gy c t bo ca cc hp cht phn lp c.
1
- nh gi kh nng kch hot enzyme caspase 3/7 ca hp cht carpaine v
pseudocarpaine trn dng t bo ung th phi LU-1.
- nh gi kh nng chng oxy ha ca cc hp cht phn lp t l u
bng cc phng php: loi b gc t do 1,1-diphenyl-2-picrylhydrazyl
(DPPH), th nghim malonyl dialdehyd (MDA), chng oxy ha in vitro trn
t bo gan chut phn lp.
- nh gi kh nng khng vi khun v nm ca cc hp cht phn lp t l
u .
- nh gi kh nng kch thch min dch ca cc hp cht phn lp t l u
.
4. Tnh mi ca ti
Phn lp v xc nh cu trc ca mt hp cht mi t l u (Carica
papaya Linn) c t tn l carpainone.
xc nh hot tnh gy c ln mt s dng t bo ung th ca cc
hp cht phn lp t l u , trong hp cht carpaine v pseudocarpaine
th hin hot tnh gy c t bo ung th r rt.
Ln u tin xc nh c kh nng kch hot enzyme caspase 3/7 ca
hp cht carpaine v pseudocarpaine trn t bo ung th phi LU-1.
nh gi c kh nng chng oxy ha, khng vi khun, khng nm
v kh nng kch thch min dch ca mt s hp cht phn lp t l u .
5. B cc ca lun n
Lun n gm 113 trang: t vn (3 trang), chng 1: tng quan (29
trang). Chng 2: vt liu v phng php nghin cu (12 trang). Chng 3:
kt qu v tho lun (56 trang vi 6 bng v 54 hnh). Kt lun v kin ngh
(2 trang). Danh mc cc cng trnh cng b (1 trang). Ti liu tham kho (10
trang gm 20 ti liu ting Vit, 86 ti liu ting Anh).

2
 CHNG 1: TNG QUAN
1.1. Gii thiu v cy u
H u (Caricaceae) trn th gii gm c 4 chi v 45 loi, phn b
cc vng nhit i v cn nhit i. nc ta c 1 chi v mt loi. Mt s
ging u hin nay ang c trng Vit Nam bao gm: ging u ta,
u Mhico, u So Lo, u Trung Quc, u Thi Lan, u i
Loan. Ti H Ni, ch c duy nht ging u i Loan c trng cc
huyn: Gia Lm, ng Anh, Sc Sn, Thch Tht,
1.2. Nghin cu v thnh phn ha hc ca l u
Trong l u c cc cht: tannin, alkaloid, saponin, glycosis,
phytosterol, phenol, flavonoid, steroid, terpenoid,. Alkaloid c khung
piperidin, ch yu l carpaine, pseudocarpaine, dehydrocarpaine I,
dehydrocarpaine II, choline, . Ngoi ra cn c vitamin C, E, nguyn t
khong Ca, K, Mg, Zn, Mn, Fe,...
H H
H H H CH3
N N
(C H 2)7 C C CH3 (C H 2)7 C C H

H 2C C H 2C C
CH2 CH2 O
O
C=O C=O
C=O C=O

O O H
H
CH2 CH2
C CH2 C CH2
CH3 (C H 2)7 CH3 C C (C H 2)7
C C N
N H H
H H
H H

Carpaine Pseudocarpaine
1.3. Nghin cu v hot tnh sinh hc ca l u
Mt s nhm cht trong l u c hot tnh chng ung th. V d:
nhm glucosise bao gm cc cht benzyl glucosinolate, benzyl isothiocyanate
c hot tnh chng nhiu dng t bo ung th khc nhau. Nhm cc cht
phenol bao gm cc cht: 5,7-dimethoxy coumarin, axi protocatechui, axit -
coumaric, axit caffeic, kaempferol, quercetin. Nhm cc cht ny c
chng minh l c hot tnh: c ch s tng sinh t bo, tng cng chc nng
min dch, c ch enzyme pha I v II trong chu k phn bo, c ch s kt
dnh t bo v xm ln, cm ng qu trnh t cht. Nhm cc cht carotenoid
cc cht: -carotene, lycopene, -cryptoxanthin. Nhm cc cht ny c tc
3
 dng phng ung th phi, ung th tin lit tuyn, ung th v, ung th d
dy, ung th gan,
Trong l u c cha protein bt hot ribosome (RIPs). RIPs c kh
nng gy c t bo ung th v T47D vi IC50 = 2,8 g/ml. Cht chit bng
nc t cc phn khc nhau ca u c tc dng ngn nga, tiu dit hoc
c ch nhiu loi t bo ung th nh: d dy, phi, tuyn ty, gan, mu,
lymphoma, bnh bch cu,..Dch chit l u bng nc (nng 0,625-20
g/ml) c tc dng c ch nhiu dng t bo ung th khc nhau. Tc gi
chng minh dch chit t l u cn c tc dng h tr h min dch tn
cng vo cc t bo ung th. Bng cch thc y s gia tng cc sn phm
cytokine dng Th1 nh l IL-12p40, IL-12p70, INF- v TNF-, cc cytokine
ny c kh nng chng li khi u. Sau tc gi s dng mng lc tch
thnh 2 phn c trng lng phn t khc nhau. Cc cht c hot tnh c ch
t bo ung th v iu ha min dch c xc nh l nm phn c trng
lng phn t nh hn 1000.
Cht chit l u c hot tnh chng oxy ha. Cc ging u khc
nhau c tng hm lng phenol khc nhau v hot tnh chng oxy ha cng
khc nhau. iu chng t rng cc cht phenol gy ra hot tnh chng oxy
ho. Cc b phn khc nhau ca cy u c hot tnh chng oxy ha gim
dn theo th t: l non qu xanh qu chn ht. Tuy nhin, cc hot
cht c tc dng chng oxy ha cn cha c phn lp.
Dch chit l u c kh nng c ch nhiu loi vi khun v nm. Cao
l u c tc dng khng khun i vi Typhimurium mentagrophytes, T.
rubrum v Staphylococcus aureus. Ngoi ra, dch chit l u cn c kh
nng khng vim, khng virut st xut huyt,.

4
 CHNG 2: VT LIU V PHNG PHP NGHIN CU
2.1. Mu thc vt
Ging u i Loan (Carica papaya L.) c trng ti Nam Hng
ng Anh H Ni. Thu hi l trn cy u ci, dng l bnh t, khng b
su bnh, khng b dp nt. L thu hi vo thng 10 nm 2012. L cy u
thu hi v c sy nhit 40 - 500C cho n kh ri nghin thnh bt.
2.2. Phng php nghin cu
2.2.1. Cc phng php phn lp v xc nh cu trc hp cht:
dng phng php chit ngm, chit bng sng siu m thu cn chit t l
u . S dng sc k ct, sc k lp mng phn lp cc cht tinh khit.
S dng ph hng ngoi, ph khi lng, ph cng hng t ht nhn xc
nh cu trc ca cc hp cht phn lp c.
2.2.2. Cc phng php th hot tnh sinh hc
2.2.2.1. Phng php th c t bo (cytotoxic assay): Php th tin hnh
xc nh hm lng protein t bo tng s da vo mt quang hc (OD
Optical Density) o c khi thnh phn protein ca t bo c nhum bng
Sulforhodamine B (SRB). Gi tr OD my o c t l thun vi lng SRB
gn vi phn t protein, do lng t bo cng nhiu (lng protein cng
nhiu) th gi tr OD cng ln. Php th c thc hin trong iu kin c th
nh sau:
- Cht th (10 l) pha trong DMSO 10% c a vo cc ging ca khay
96 ging c nng 100 g/ml; 20g/ml; 4 g/ml; 0,8 g/ml.
- Trypsin ha t bo th nghim lm ri t bo, thm lng t bo ph
hp v chng pht trin trong vng t 3-5 ngy.
- Mt khay 96 ging khc khng c cht th nhng c t bo ung th s
c s dng lm i chng ngy 0.
- Sau giai on pht trin trong t m CO2, t bo c c nh vo y
ging bng axit trichloracetic trong 30 pht, c nhum bng SRB trong
1 gi 37 0C.
5
 - Cui cng, s dng 10 mM unbuffered Tris base ha tan lng SRB
bm v nhum cc phn t protein, a ln my lc a lc nh trong
10 pht v s dng my ELISA Plate Reader c kt qu v hm lng
mu ca cht nhum SRB qua ph hp ph bc sng 515 nm.
- Ellipticine lun c s dng nh l cht i chng dng.
- DMSO10% lun c s dng nh i chng m. Cht th no c IC50 <
20 g/ml (vi cht chit th, hoc vi phn on ha hc) hoc IC50 4
g/ml (vi hot cht tinh khit) s c xem l c hot tnh gy c t bo
v c kh nng c ch s pht trin hoc dit t bo ung th.
2.2.2.2. Phng php xc nh hot tnh enzyme caspase 3 / 7: c thc
hin da trn b kit Apo-ONE Homogeneous Caspase-3/7 Assay (Promega)
theo ng hng dn ca nh sn xut.
2.2.2.3. Phng php xc nh hot tnh chng oxy ha bng loi b gc t
do DPPH
Nguyn tc: 1,1-diphenyl-2-picrylhydrazyl (DPPH) l gc t do c dng
sng lc tc dng chng oxy ha ca cc cht nghin cu. Hot tnh chng
oxy ha th hin qua vic lm gim mu DPPH ca cht th, c xc nh
bng php o hp th bc sng 517 nm trn my quang ph.
2.2.2.4. Phng php xc nh hot tnh chng oxy ho in vitro bng th
nghim malonyl dialdehyd (MDA test)
Xc nh kh nng c ch peroxy ho lipid thng qua vic xc nh
hm lng MDA theo phng php ca Stroev E A, Makarova V G (1989),
Vin Dc liu - B Y T (2006). Khi cho phn ng vi axit thiobarbituric,
mt phn t MDA phn ng vi hai phn t axit thiobarbituric to phc mu
hng hp thu cc i bc sng 532 nm. Phn ng thc hin mi trng
pH 2 - 3, nhit 90 - 100 0C trong vng 10 - 15 pht. o cng mu
ca phc suy ra lng MDA c trong mu.
2.2.2.5. Phng php xc nh hot tnh chng oxy ho in vitro trn t bo
gan chut
6
 Chut BALB/c kho mnh c s dng tch t bo t bo gan. T
bo gan sau phn lp c nui n nh 1-2 ngy. Thm hot cht nghin cu
hoc cucurmin (i chng dng). Thm 100 M H2O2 vo mi ging,
trong 2 gi. Cho 50 l/ging MTT nng 1mg/ml, trong 4 gi. Loi b
dch ni, thm vo mi ging 100 l DMSO 100%. o mt quang hc
(OD) bc sng 492 nm.
2.2.2.6. Phng php th hot tnh khng vi khun v nm
Hot tnh khng vi khun v nm c thc hin da trn phng php
pha long a nng (Multimicrodilution assay).
2.2.2.7. Phng php th hot tnh kch thch min dch
ng vt th nghim: Th 3 thng tui khe mnh, c nui ti
chung nui ca vin Cng ngh sinh hc.
Hot tnh kch thch min dch ca cc mu c xc nh theo phng
php MTT (Mosmann (1983)).
T bo lympho c a vo cc ging ca a 96 ging vi nng
2x106 t bo/ml. B sung cht th pha trong DMSO 10%. Concavalin A c
s dng lm i chng. Mu c trong thi gian 48h 37 oC, 5% CO2.
Sau giai on pht trin trong t m CO2, thm vo mi ging 50l MTT
1mg/ml. Sau khi a t bo 37o C trong 4h, loi b mi trng v thm
vo mi ging 100 l DMSO. a t bo c a ln my lc a lc nh
trong 10 pht v s dng my ELISA Plate Reader (Bio-Rad) c kt qu
v hm lng mu qua ph hp ph bc sng 490nm.

7
 CHNG 3: KT QU V THO LUN
3.1. Phn lp cn chit phn on CH2Cl2
T 101,90 gam cn chit CH2Cl2, tin hnh sc k ct silica gel vi h
dung mi CH2Cl2/MeOH gradient (bt u t 0% MeOH n 50% MeOH).
Cui cng dng dung mi MeOH 100% di ct nhm ra ton b cc cht
cn b gi li trn ct sc k.
S dng sc k lp mng kim tra cc phn on, soi UV bc
sng 254 nm v 365 nm, hin mu bn mng bng thuc th Ce(SO4)2 v
vanilin. Kt qu, thu c 13 phn on chnh nh sau:
Bng 3.1: Khi lng cc phn on t cn chit CH2Cl2 ca l u (bng
3.2 ca lun vn)
TT K hiu phn on Khi lng (g)
1 F1 1,0530
2 F2 2,4254
3 F3 2,3174
4 F4 3,6284
5 F5 2,2090
6 F6 3,0837
7 F7 1,2896
8 F8 5,2600
9 F9 13,8602
10 F10 10,0700
11 F11 20,5137
12 F12 4,2901
13 F13 27,6800
3.2. Phn lp cc hp cht t mt s phn on
T phn on F5, sau khi tinh ch bng ct silica gel vi h dung mi
CH2Cl2/MeOH gradient, thu c 6 phn on nh k hiu F5.1-F5.6 Phn
on F5.2 c tinh ch bng ct sephadex vi dung mi MeOH thu c
cht sch CP1 (139,6 mg).
8
 T phn on F8, sau khi tinh ch bng ct silica gel vi h dung mi
Hx/EtOAc gradient thu c 5 phn on nh k hiu F8.1-F8.5 Phn on
F8.3 c tinh ch bng ct sephadex vi dung mi MeOH v ct silica gel
h dung mi Hx/EtOAc xut hin tinh th. Lc ra bng Hx/CH2Cl2 thu c
cht sch CP2 (6,7 mg).
T phn on F9.I, sau khi chy ct silica gel vi h dung mi
CH2Cl2/EtOAc gradient v HCOOH (1%) thu c 11 phn on nh k hiu
F9.I.1-F9.I.11 Phn on F9.I.3 c tinh ch bng ct sephadex vi dung
mi EtOH v ct silica gel vi h dung mi CH2Cl2/EtOAc gradient thu c
cht sch CP3 (6 mg). Phn on F9.I.11 c tinh ch bng ct sephadex
vi dung mi MeOH v ct silica gel vi h dung mi CH2Cl2/axeton
geadient thu c cht sch CP4 (16,4 mg).
T phn on F11.II, sau khi chy ct silica gel vi h dung mi
CH2Cl2/EtOAc/NH4OH v CH2Cl2/MeOH/NH4OH thu c 7 phn on nh
k hiu F11.II.1- F11.II.7. Kt tinh phn on F11.II.4 bng h dung mi
Hx/CH2Cl2 thu c cht sch CP5 (210,1 mg). Dch ci tip tc c tinh
ch bng sc k ct silica gel vi h dung mi CH2Cl2/EtOAc/NH4OH v sc
k ct sephadex vi dung mi MeOH thu c cht sch CP6 (18,7 mg).
3.3. Xc nh cu trc ca cc hp cht c phn lp
3.3.1. Hp cht CP1
Cht rn mu trng, nhit nng chy 145 1460C
1
H-NMR (500 MHz, CDCl3): (ppm) 3,58 (1H, t, J = 4,5 Hz, OH), 3,94 (6H,
s, 2 x OCH3), 4,83 (2H, d, J = 4,5 Hz, CH2-2), 6,21 (1H, s, OH), 7,18 (1H, s,
H-2 + H-6).
13
C-NMR (125 MHz, CDCl3): (ppm) 56,5 (2 x OCH3), 64,9 (C-2), 105,0 (C-
2 + C-6), 124,8 (C-1), 140,8 (C-4), 147,1 (C3 + C5), 196,6 (C=O).
Phn tch chi tit cc ph 1H-NMR, 13C-NMR v DEPT v so snh vi
ti liu tham kho cho php xc nh cht CP1 l danielone. Hp cht ny
ln u tin c tch ra t l u .

9
 O
1 2'
HO 1' OCH3
2 3'

6' 4'
5'
OH
OCH3

Hnh 3.1: Cu trc ha hc ca hp cht danielone (hnh 3.3 ca lun vn)

3.3.2. Hp cht CP2
Cht rn mu trng, nhit nng chy 135 - 1370C. ESI-MS: m/z 274
[M+Na]+. HR-ESI-MS (+) m/z: 274,1411.
1
H-NMR (500 MHz, CD3OD): (ppm): 1,37 (6H, m, CH2-4+ CH2-5+ CH2-
6); 1,06 (2H, quint, J= 7,5 Hz, CH2-7); 1,69 (2H, quint, J= 7,5 Hz, CH2-3);
2,28 (2H, t, J= 7,5 Hz, CH2-8); 2,30 (3H, s, CH3); 2,71 (2H, t, J= 7,5 Hz,
CH2-2); 5,97 (1H, d, J= 3,5 Hz, H-4); 6,92 (1H, d, J= 3,5 Hz, H-5)
13
C-NMR (125 MHz, CDCl3): (ppm) 12,8 (CH3); 26,1 (C-7); 27,1 (C-3);
30,1 (C-6); 30,2 (C-5); 30,3 (C-4); 35,1 (C-8); 38,4 (C-2); 110,3 (C-4);
119,7 (C-5); 132,2 (C-2); 138,6 (C-3); 177,0 (C-9); 192,1 (C-1)..

Hnh 3.2: Ph HR-ESI-MS (+) ca hp cht CP2 (hnh 3.4 ca lun vn)

Hnh 3.3: Ph 13C-NMR ca hp cht CP2 (hnh 3.6 ca lun vn)

10
Hnh 3.4: Ph 1H-NMR ca hp cht CP2 (hnh 3.7 ca lun vn)

Hnh 3.5: Ph COSY ca hp cht CP2 (hnh 3.9 ca lun vn)

Hnh 3.6: Ph HMBC ca hp cht CP2 (hnh 3.10 ca lun vn)

11
 Hnh 3.7: Ph HSQC ca hp cht CP2 (hnh 3.11 ca lun vn)
Phn tch d liu ph 1H-NMR, 13
C-NMR, COSY, HSQC, HMBC
chng ti xc nh c cht CP2 l axit 9-(3-methyl-pyrrol-2-yl)-9-
oxononanoic. y l mt hp cht mi c t tn l carpainone. Trn
th gii v Vit Nam cha c cng b v hp cht ny trong l u cng
nh cc loi thc vt khc.

4
3
2' 4' 6' 8'
5 2 1'
N 3' 5' 7' COOH
9'
H O

Hnh 3.8: Cu trc ha hc ca hp cht carpainone (hnh 3.12 ca lun

vn)
3.3.3. Hp cht CP3
Cht rn mu trng, nhit nng chy 192 1930C. ESI-MS: m/z 167 [M-
H]-.
1
H-NMR (500 MHz, CDCl3): (ppm) 3,91 (3H, s, OCH3), 6,83 (1H, d, J= 8,0
Hz, H-6), 7,55 (1H, dd, J = 1,5; 8,0 Hz, H-5), 7,58 (1H, dd, J = 1,5 Hz, H-3).
Sau khi so snh cc s liu ph ca cht CP3 vi ti liu tham kho
chng ti i n kt lun cht CP3 l axit pluchoic.
C OOH
1
OCH 3
6
2

5 3
4

OH

12
 Hnh 3.9: Cu trc ha hc ca hp cht axit pluchoic (hnh 3.15 ca lun
vn)
3.3.4. Hp cht CP4

Cht rn mu trng, nhit nng chy 100 - 1040C

[]25D +288 (c=0,25 g/100ml trong MeOH)
1
H-NMR (500 MHz, CDCl3): (ppm) 0,97 (3H, s, CH3-12), 1,01 (3H, s, CH3-
13), 1,26 (3H, d, J = 6,5 Hz, CH3-10), 2,10 (1H, d, J = 17,0 Hz, H-2a), 2,38
(1H, d, J = 17,0 Hz, H-2b), 2,56 (1H, d, J = 8,5 Hz, H-6), 4,11 (1H, dd, J =
1,5; 17,0 Hz, H-11a), 4,20 (1H, dd, J = 1,5; 17,0 Hz, H-11b), 4,20 (1H, quint,
J = 6,0 Hz, H-9), 5,57 (dd, J = 8,5; 15,5 Hz, H-7), 5,66 (1H, dd, J = 5,5; 15,5
Hz, H-8), 6,17 (1H, s, H-4).
13
C-NMR (125 MHz, CDCl3): (ppm) 23,5 (CH3-10), 27,0 (CH3-12), 27,7
(CH3-13), 36,2 (C-1), 48,1 (C-2), 50,8 (C-6), 63,8 (C-11), 68,0 (C-9), 122,3
(C-4), 126,3 (C-8), 138,6 (C-7), 165,9 (C-5), 199,6 (C=O).
Phn tch chi tit cc ph 1H-NMR, 13C-NMR v DEPT COSY, HSQC,
HMBC v so snh vi ti liu tham kho, chng ti xc nh c cht CP4
l Apocynol A. Hp cht ny ln u tin c tch ra t l u .
OH
7 9
2 1 6
8 10
3 5
O 4

OH

Hnh 3.10: Cu trc ha hc ca hp cht Apocynol A (hnh 3.19 ca lun

vn)
3.3.5. Hp cht CP5
Cht rn mu trng, nhit nng chy 118 1200C.
ESI-MS: m/z 479 [M+H]+, 240 [M/2+H]+
1
H-NMR (500 MHz, CDCl3): (ppm) 1,05 (3H, d, J = 7,0 Hz, CH3-2), 1,19
(1H, m, H-5a), 1,32 (9H, m, CH2-8+ CH2-9 + CH2-10 + CH2-11 + H-7a), 1,47
(2H, m, H-7b + H-5b), 1,65 (3H, m, CH2-12 + H-4a), 2,01 (1H, m, H-4b),
2,32 (1H, m, H-13a), 2,42 (1H, m, H-13b), 2,59 (1H, m, H-6), 2,87 (1H, dq,
J=1,0, 7,0 Hz, H-2), 4,77 (1H, br, s, H-3).
13
 13
C-NMR (125 MHz, CDCl3): (ppm) 18,6 (CH3-2), 25,4 (C-12), 25,4 (C-
8), 26,2 (C-5), 28,7 (C-11), 29,1 (C-4), 29,7 (C-9), 29,7 (C-10), 34,6 (C-13),
37,2 (C-7), 53,6 (C-2), 50,6 (C-6), 70,3 (C-3), 173,5 (C=O).
T cc d liu ph 1D, 2D-NMR, MS v so snh vi ti liu tham kho
cho php xc nh y l mt alkaloid c cu trc i xng hai bn c tn
carpaine.
H
H
6' N 2'
(C H 2)7 CH 3
3'
5'
4'
O
C=O

C O
O
4
5
3
H 3C (C H 2)7
2 N 6
H
H

Hnh 3.11: Cu trc ha hc ca hp cht carpaine (hnh 3.24 ca lun vn)

3.3.6. Hp cht CP6
Cht rn mu trng, nhit nng chy 72 730C
ESI-MS: m/z 479 [M+H]+, 240 [M/2+H]+
1
H-NMR (500 MHz, CDCl3): (ppm) 1,02 (3H, d, J = 6,5 Hz, CH3), 1,06
(3H, d, J = 6,5 Hz, CH3), 2,50 (2H, m, H-6 + H-6), 2,69 (1H, m, H-2a), 2,86
(1H, dq, J=1,5, 6,5 Hz, H-2), 4,33 (1H, m, H-3), 4,83 (1H, br s, H-3),
13
C-NMR (125 MHz, CDCl3): (ppm) 18,5 (CH3), 19,1 (CH3), 24,7 (CH2),
24,9 (CH2), 25,1 (CH2), 25,4 (CH2), 26,7 (CH2), 27,8 (CH2), 28,0 (CH2), 28,1
(CH2), 28,4 (CH2), 28,5 (CH2), 28,8 (CH2), 29,1 (CH2), 29,5 (CH2), 30,6
(CH2), 30,9 (CH2), 34,5 (CH2), 34,8 (CH2), 35,4 (CH2), 37,3 (CH2), 53,8
(CH), 54,9 (CH), 55,7 (CH), 56,6 (CH), 70,1 (CH), 75,8 (CH), 173,3 (C=O),
173,7 (C=O).
Kt hp cc d liu ph v so snh vi ti liu tham kho cho php xc
nh cht CP6 l ng phn ca cht CP5, ch khc nhm CH3 C-2 di
dng axial. Hp cht ny c tn pseudocarpaine v c phn lp t l cy
u .

14
 H
CH 3
6' N 2'
(C H 2)7 H
3'
5'
4'
O
C=O

C O
O
4
5
3
H 3C (C H 2)7
2 N 6
H
H

Hnh 3.12: Cu trc ha hc ca hp cht pseudocarpaine (hnh 3.28 ca

lun vn)
Kt lun: T cn chit CH2Cl2 ca l u , chng ti tch chit c 6
hp cht. Trong , hp cht danielone (CP1) c tch ra t phn on F5
ca cn chit. Hp cht carpainone (CP2) c tch ra t phn on F8 ca
cn chit, Hp cht axit pluchoic (CP3), apocynol A (CP4) c tch ra t
phn on F9 ca cn chit. Hp cht carpaine (CP5), pseudocarpaine (CP6)
c tch ra t cc phn on F11 ca cn chit.
3.4. nh gi tc dng gy c t bo ung th ca cc cn chit t l u

Cn chit cc phn on em th hot tnh gy c t bo ung th: ung

th biu m ngi KB, ung th phi ngi LU-1, ung th v ngi MCF-7.
Kt qu thu c hnh 3.13:
Trong 5 phn on cn chit th 4 loi cn chit (Hx, EtOAc, BuOH v
cn nc cn li) khng c hot tnh gy c t bo ung th KB, LU-1, MCF-
7 (vi IC50>100 g/ml). Ch c duy nht cn chit CH2Cl2 l c hot tnh gy
c t bo trn c 3 dng t bo ung th th nghim: KB, LU-1, MCF-7 vi
gi tr IC50 (g/ml) ln lt l: 18,44; 18,21; 19,16.

Hnh 3.13: Tng hp kt qu th hot tnh gy c t bo ung th ca cc

cn chit t l u (Hnh 3.32 ca lun vn)

15
 3.5. nh gi tc dng gy c t bo ung th biu m KB ca cc phn
on phn lp t cn chit CH2Cl2 t l u
Sau khi phn lp cn CH2Cl2 bng sc k ct silica gel thu c 13
phn on F1- F13. Cc phn on F1- F13 c em th hot tnh gy c
t bo ung th biu m KB, kt qu nh sau:

Hnh 3.14: Kt qu gy c t bo trn dng t bo ung th biu m KB ca

cc phn on cn chit CH2Cl2 t l u (Hnh 3.33 ca lun vn)
Kt qu trn cho thy: Cc phn on F8, F9, F10, F11 c th hin hot
tnh gy c t bo ung th biu m KB vi IC50 t 15,41 n 6,86 g/ml.
c bit 2 phn on F10, F11 th hin hot tnh gy c t bo ung th biu
m KB rt mnh (IC50 tng ng l 7,17 v 6,86 g/ml).
Cc phn on cn li khng c hoc c hot tnh c ch t bo ung th
biu m KB yu (IC50 t 23,93 n >100 g/ml). i chng dng l
ellipticine hot ng n nh.
3.6. nh gi tc dng gy c t bo ung th ca cc hp cht phn lp
t l u
Chng ti tin hnh xc nh kh nng dit hoc km hm s pht trin
t bo ung th ca cc hp cht phn lp c t l u trn bn dng t
bo ung th khc nhau. Ngoi ra, cc mu nghin cu cn c th nghim
trn t bo thng NH3T3 ca ngi v xem nh l dng t bo i chng
kim tra kh nng gy c t bo vi t bo bnh thng. Kt qu c th nh
sau:

16
 Hnh 3.15: Tng hp kt qu th hot tnh gy c t bo ca cc hp cht
phn lp t l u (hnh 3.49 ca lun vn)
Bn hp cht (danielone, carpainone, axit pluchoic, apocynol A) khng
c hot tnh gy c t bo i vi 04 dng t bo ung th v 1 dng t bo
thng th nghim.
Hai hp cht carpaine v pseudocarpaine th hin hot tnh rt tt trn
c 4 dng t bo ung th nghin cu (ung th biu m KB, ung th mu LH-
60, ung th phi LU-1, ung th v MCF-7) vi cc gi tr IC50 t 1,13 n
3,49 g/ml.
3.7. nh gi kh nng kch hot enzyme caspase 3/7 ca mt s hp cht
ln t bo ung th phi LU-1
Xc nh kh nng kch hot enzyme caspase 3/7 ca 2 hp cht
(carpaine v pseudocarpaine) tch chit c t l u trn dng t bo ung
th phi LU-1. Kt qu c th nh sau:
3.7.1. nh gi kh nng kch hot enzyme caspase 3/7 ca hp cht
carpaine ln t bo ung th phi LU-1
Hp cht carpaine tch chit c t l u c nh gi kh nng
kch hot enzyme caspase 3/7 trn dng t bo ung th phi LU-1.

Hnh 3.16: Kt qu kch hot enzyme caspase 3/7 ca hp cht carpaine ln

t bo ung th phi LU-1(hnh 3.50 ca lun vn)

17
 Hp cht carpaine th hin kh nng kch hot enzyme caspase 3/7
nng th cao nht (20 g/ml) l 386,5 RFU. Cht i chng dng
tamoxifen hot ng n nh trong th nghim vi hot tnh caspase nng
20 g/ml l 3100 RFU.
Thng qua th nghim xc nh c ca hot cht ln dng t bo
nui cy bng phng php nhum SRB, kt qu t l t bo sng st so vi
i chng ca hot cht nng th nghim nm trong khong 84,67 n
90,44% cho thy hp cht carpaine khng gy cht t bo trn din rng
nhng cng c tc ng nht nh ln t bo.
3.7.2. nh gi kh nng kch hot enzyme caspase 3/7 ca hp cht
psuedocarpaine ln t bo ung th phi LU-1
Hp cht pseudocarpaine tch chit t l u c nh gi kh nng kch
hot enzyme caspase 3/7 trn dng t bo ung th phi LU-1.

Hnh 3.17: Kt qu kch hot enzyme caspase 3/7 ca hp cht

pseudocarpaine ln t bo ung th phi LU-1(hnh 3.51 ca lun vn)
Kt qu trn cho thy hp cht pseudocarpaine th hin kh nng kch
hot enzyme caspase 3/7 nng th cao nht (30g/ml) l 778 RFU. Cht
i chng dng tamoxifen hot ng n nh trong th nghim vi hot tnh
caspase nng 20 g/ml l 3100 RFU.
Thng qua th nghim xc nh c ca hot cht ln dng t bo
nui cy bng phng php nhum SRB, kt qu t l t bo sng st so vi
i chng ca hot cht nng th nghim nm trong khong 89 n 96
18
% cho thy hp cht pseudocarpaine khng gy cht t bo trn din rng
nhng cng c tc ng nht nh ln t bo.
3.8. nh gi kh nng chng oxy ha ca cc hp cht phn lp t l u

3.8.1. Kt qu chng oxy ha in vitro bng phng php loi b gc

t do DPPH ca cc hp cht phn lp t l u
Xc nh kh nng chng oxy ha ca 6 hp cht tch chit c t l
u bng phng php th DPPH.

Hnh 3.18: Kt qu th hot tnh chng oxy ha bng DPPH ca cc hp

cht chit tch t l u (hnh 3.52 ca lun vn)
Kt qu cho thy: 6 hp cht th nghim u c EC50 > 128 g/ml, ln
hn nng cao nht ca php th. Cht tham kho l resveratrol c EC50 l
8,3 g/ml. Kt qu ny chng t, c 6 hp cht tch chit c t l u
cha th hin hot tnh chng oxy ha nng th nghim.
3.8.2. Kt qu chng oxy ha in vitro bng th nghim MDA ca cc hp
cht phn lp t l u
Xc nh kh nng chng oxy ha ca 6 hp cht tch chit c t l
u bng phng php th nghim MDA.

19
Hnh 3.19: Kt qu th hot tnh chng oxy ha bng php th MDA ca cc
hp cht chit tch t l u (hnh 3.53 ca lun vn)

Bng th nghim MDA kim tra kh nng chng oxy ha in vitro

ca cc hp cht: danilone, carpainone, axit pluchoic, apocynol A, carpaine,
pseudocarpaine chng ti nhn thy ch c duy nht hp cht pseudocarpaine
nng th nghim cao nht 100 g/ml cng ch c hot tnh chng oxi
ho khong 33,59%, cho thy c hot tnh yu. Cc hp cht cn li bao gm:
danilone, carpainone, axit pluchoic, apocynol A v carpaine u khng th
hin hot tnh chng oxi ha trn th nghim MDA cc nng nghin
cu.
3.8.3. Kt qu chng oxy ha in vitro trn t bo gan chut ca cc hp
cht phn lp t l u
Xc nh kh nng chng oxy ha ca 6 hp cht tch chit c t l
u trn t bo gan chut.

Hnh 3.20: Kt qu xc nh hot tnh chng oxi ha trn t bo gan chut

ca cc hp cht chit tch t l u (hnh 3.54 ca lun vn)

20
 Qua qu trnh nghin cu kh nng chng oxy ha in vitro trn t bo
gan phn lp ca 6 hot cht cho thy cc cht u khng th hin hot tnh
nng nghin cu. Cucurmin hot ng n nh trong th nghim.
3.9. nh gi kh nng khng vi khun v nm ca cc hp cht phn
lp c
Bng 3.2: Kt qu th hot tnh khng vi khun v nm ca cc hp cht
phn t l u (bng 3.5 ca lun vn)
Nng c ch 50% s pht trin ca vi khun v
Cht th nm - IC50 (g/ml)
Vi khun gram dng Vi khun gram m Nm
1 2 3 4 5 6 7
Danielone >128 >128 >128 >128 >128 >128 >128
Carpainone >128 >128 >128 >128 >128 >128 >128
Axit pluchoic >128 >128 >128 >128 >128 >128 >128
Apocynol A >128 >128 >128 >128 >128 >128 >128
Carpaine >128 >128 >128 >128 >128 >128 >128
Pseudocarpaine 80 >128 >128 >128 >128 >128 >128
Ampicilin 1,0 1,05 1,02 - 1,2 - -
Stretomycin - - - 1,25 - 12 -
Amphotericin B - - - - - - 0,8
K hiu:
1: Staphylococcus aureus, 2: Bacillus subtilis, 3: Lactobacillus
fermentum, 4: Salmonella enterica, 5: Escherichia coli, 6: Pseudomonas
aeruginosa, 7: Candida albican,(-): khng th
Qua s liu bng 3.2 thy rng, c 5 trong tng s 6 hp cht th
nghim u khng c kh nng khng cc chng vi khun gram dng, gram
m v nm nng cht th cao nht l 128 g/ml.

21
 Ring hp cht pseudocarpaine ch th hin hot tnh khng vi khun
gram dng Staphylococcus aureus rt yu vi IC50 l 80 g/ml. Cn i vi
cc chng vi khun v nm cn li u c IC50 > 128 g/ml.

3.10. nh gi kh nng kch thch t bo lympho ca cc hp cht phn

lp t l u
Tin hnh nh gi kh nng kch thch t bo lympho ca su hp cht
phn lp t l u . Kt qu thu c nh sau:
Bng 3.3: Kt qu kch thch t bo lympho ca cc hp cht phn lp t l
u (bng 3.6 ca lun vn)
Nng SI (ch s kch thch t bo lympho
(g/ml) pht trin so vi i chng m)
Cht th 100 20 4 0,8
Danielone 1,15 1,04 1,05 1,01
Carpainone 0,95 0,93 0,93 0,92
Axit
pluchoic 0,98 1,00 1,03 1,00
Apocynol A 1,11 1,05 1,03 0,99
Carpaine 0,95 1,08 1,11 1,05
Pseudocarpa 1,04
ine 0,99 0,98 1,00
Concavalin 1,44 1,15 1,04 1,01
A (1 (0,5 (0,25 (0,125
g/ml) g/ml) g/ml) g/ml)

Kt qu trn cho thy cc hot cht nghin cu khng th hin hot tnh
kch thch t bo lympho. Concavalin A c cho thy kh nng kch thch t
bo lympho tng sinh. Do vy, chng ti khng xc nh c gi tr SD50
ca tt c cc mu nghin cu.
22
 KT LUN V KIN NGH
Kt lun
1. Chit phn on dch chit MeOH t l u bng cc dung mi c
phn cc tng dn (n-hexan, CH2Cl2, EtOAc, buthanol). T cn chit CH2Cl2
phn lp c 6 hp cht k hiu t CP1 n CP6 v xc nh c cu trc
nh sau: danielone (CP1), carpainone (CP2), axit pluchoic (CP3), apocynol
A (CP4), carpaine (CP5), pseudocarpaine (CP6). Trong carpainone l
mt hp cht mi. Hai hp cht danielone v apocynol A ln u tin
c tch ra t l u .
2. xc nh c phn on cn chit CH2Cl2 ca l u c kh nng
gy c t bo ung th biu m KB (IC50 = 18,44 g/ml), ung th phi LU-1
(IC50 = 18,21 g/ml) v ung th v MCF-7 (IC50 = 19,16 g/ml).
3. Hai hp cht carpaine v pseudocarpaine phn lp t cn CH2Cl2
ca l u ln u tin c chng minh c hot tnh gy c mnh
trn c bn dng t bo ung th ngi: ung th biu m KB, ung th mu
HL-60, ung th phi LU-1, ung th v MCF-7 (IC50 t 1,13 n 3,49 g/ml).
ng thi hai hp cht ny cng gy c vi t bo thng ca ngi (t bo
NIH 3T3) (IC50 t 1,40 n 1,88 g/ml). Cc hp cht cn li (danielone,
carpainone, axit pluchoic v apocynol A) u khng th hin kh nng gy
c t bo i vi 4 dng t bo ung th v 1 dng t bo thng (IC50 >100
g/ml).
4. Hai hp cht carpaine v pseudocarpaine ln u tin c chng
minh kh nng kch hot enzyme caspase 3/7 (tng ng l 386,5 v 778
RFU) nng th nghim cao nht (tng ng 20 v 30 g/ml) nhng
khng mnh khi so vi cht i chng l tamoxifen (l 3100 RFU nng
th 20 g/ml).
5. Cc hp cht phn lp c t l u khng th hin kh nng chng
oxy ha cc nng nghin cu trong cc th nghim DPPH (EC50 > 128

23
 g/ml), MDA (ED50 > 100 g/ml) v trn t bo gan chut phn lp (ED50 >
100 g/ml).
6. Hp cht pseudocarpaine cho thy kh nng khng vi khun gram
dng Staphylococcus aureus vi IC50 = 80 g/ml, khng th hin hot tnh
khng cc chng vi khun gram dng, gram m v nm khc nng cht
th cao nht l 128 g/ml (vi IC50 > 128 g/ml). Cc hp cht cn li
(danielone, carpainone, axit pluchoic, apocynol A v carpaine) u khng th
hin kh nng khng vi khun v nm ti nng th nghim (vi IC50 > 128
g/ml).
7. Cc hp cht phn lp c t l u cha cho thy kh nng kch
thch t bo lympho ti cc nng th nghim. Khng xc nh c gi tr
SD50 ca cc hp cht ti cc nng th nghim t 0,8 n 100 g/ml.
Kin ngh
1. Tip tc nghin cu mt s hot tnh sinh hc ca hp cht carpaine v
pseudocarpaine nh: kh nng hn ch s di cn ca khi u, kh nng hn ch
s hnh thnh mch mu trong khi u,..
2. Nghin cu ti u ha quy trnh chit tch carpaine v
pseudocarpaine t l u Carica papaya Linn.

24