CHIẾT XUẤT VÀ PHÂN LẬP FLAVONOID TỪ CÂY DIẾP CÁ

L/O/G/O
CHIT XUT V PHN LP

FLAVONOID T CY DIP C
Houttuynia cordata Thunb. Saururaceae.
Nhm 3
Tiu nhm 4
Ging vin hng dn:
PGS.TS: V TH BCH HU
TS: NGUYN VIT KNH
Th.S : VN MI
Th.S : NGUYN TH NH NGUYT
www.trungtamtinhoc.edu.vn
Tng quan v thc v t hoc
I. TNG QUAN
Tng quan v hoa hoc

Tng quan v tac dung cng dung
II.TNG QUAN FLAVONOID
2
3
III.THC NGHIM
4
IV. KT LUN V NHN NH
Khai nim
Cu trc v phn loi
Tac dung
Chit xut nhom flavonoid
Phn lp nhom flavonoid
2
I. Tng quan
1.1. Tng quan v thc vt hoc.
Chi Dip C
(Houttuynia
Thunb)
H L Gip
(Saururace
ae)
Gii thc
vt
(Plantae)
Ngnh Ngc
Lan
(Magnoliophy
ta)
DIP C
Houttuynia cordata
Thunb.Saururaceae
B H Tiu
(Piperales)
Lp Ngc Lan
(Magnoliopsid
a)
Phn lp Ngc
Lan
(Magnoliidae)
3
Phn b, sinh thi v thu hi.

Loi ca luc a chu , phn b t n qua Trung Quc, Nht
Bn, Thai Lan, cac nc ng Nam .
Vit Nam, Dip Ca moc hoang ch m t, thung lng, ven
sui, b mng, khp cac tnh min ni. Thng c trng lm
rau v lm thuc.
4
1.2. Tng quan v thnh phn ha hc
Text in here
Flavonoid
Text in here
Tinh du
Text in here
Acid amin
Alkaloid
Thnh phn
khac
5
1.3. Tng quan v tc dng, cng dng

1.3.1. Cng dng
Rau Dip Ca l mt loi rau rt quen thuc trong ba n hng ngy
ca ngi Vit Nam. Ngoi cng dung c xem nh mt loi rau
ngon, dip ca cn co rt nhiu cng dung khac.
Quc gia
B phn dng
Tc dng
Vit Nam, Trung

Quc
R, la
Rau
Nht Bn
Ton cy
Nc ung, Kh mi
Hn Quc
Ton cy
Kim chi, Nc tng, Siro,

Nc ung co gas
Thi Lan
La non
Rau
6
Tac dung:
Theo ng y, Dip ca co v ng, tnh n, tac dung vo cac kinh mch
i trng v Ph, v vy co tac dung:
7
1.3.2. Tc dng dc l v cc
nghin cu lm sng
Khang khun
Khang vim
Tc dng dc l
Trn h min dch

Li tiu
Chng Oxy hoa
Khang t bo ung th
8
1.4. Cc ch phm c cha Dip c trn

th trng.
9
II. TNG QUAN V FLAVONOID
2.1. Khi nim chung v Flavonoid.

Flavonoid l mt trong nhng nhom hp cht
thng gp nht trong dc liu thc vt nht l
cac loi rau qu.
Cho n nay co hn 4000 cht xac nh
cu trc, ch ring hai nhom flavon v flavonoid
vi nhom th l OH hoc OCH3 .
Phn ln cac cht cha flavonoid co mu vng,
cng co mt s cht mu xanh, tm hoc
khng co mu.
10
2.2. Cu trc ha hc ca flavonoid.
2.2.1. Khung flavonoid.

Flavonoid l mt chui polyphenolic gm 15 nguyn t cacbon v
hai vng benzen lin kt bi mt ng thng co 3 cacbon. Khung
co h thng C6 C3 C6
C
C
11
2.2.2. Phn loi Flavonoid

Euflavonoid
Isoflavonoid
Neoflavonoid
Bi-flavonoid
Anthocyanidin
Flavon
Flavan 3-ol
Flavan 3,4-diol
Flavanon
Chalcon, dihydrochalcon
Auron
Isoflavon
Pterocarpan
Rotenoid
Isoflavanon
3-arylcoumarin
4-arylchroman.
Dalbergion.
t quan trng, ch yu gp ngnh ht

trn.
12
2.3. Tnh cht:

Cac cht dn flavon co mu vng rt nht co khi khng mu flavon
vng nht n vng
Chalcon v auron vng m n cam.
Cac cht dn anthocyanidin th mu thay i ty theo pH ca mi
trng.
tan khng ging nhau, thng flavonoid glycosid v flavonoid
sulfat l nhng hp cht phn cc nn khng tan hoc t tan trong
dung mi hu c, tan c trong nc tt nht l cn nc.
13
Tc dng
Tc dng enzyme
Tc dng khng sinh, vim nhim.
Tc dng i vi ung th.
Tc dng i vi cc bnh tim mch.
Tc dng chuyn ha v trn lm sng.
Tc dng chng oxy ha.
14
III. PHN THC NGHIM

3.1.Vi phu.
Cc bc tin hnh nhum vi phu

B1: Dng dao lam ct vi phu thn, cung, la dip ca sao cho dy
tng lat ct khong <1mm. Dao lam khi ct t thng goc vi mu vt
B2: Ngm vi phu trong nc Javel cho n khi vi phu trng
B3: Loi ht Javel ra ra bng nc ct ( khong 3-4 ln )
B4: Ngm vi phu ra trong dung dch acid acetic 10% trong 10
pht
B5: Loi b ht acid acetic
B6: Ngm vi phu trong thuc nhum 3 pht
B7: Ra thuc nhum bng nc ct
B8: Bo qun trong nc ct ri xem
15
3.1.1.Vi phu thn
Vi phu thn
T bo biu b Tit Thn
Vi phu hnh a giac.

Biu b t bo hnh ch
nht, cutin rng ca, ri
rac co l kh
T bo biu b mu vng
sm, l phin nm ngang
vi t bo biu b.
Lng che ch - Thn

Trn t bo biu b co t
lng che ch a bo 1
dy (2 4 t bo)
16
3.1.2.Vi phu l
Vi phu l: Gn gia:
mt di li, mt trn
hi lm hay phng.
Vi phu phin l: ri rac

biu b trn v biu b
di co t bo biu b tit
mu vng, l kh ch co
biu b di.
Vi phu cung l: vi
phu lm mt trn,
li mt di, mt
di co 4 cnh.
17
3.1.3. c im bt dc liu
Mnh m mm Bt
dc liu.
Ht tinh bt Bt dc
liu.
Lng che ch - Bt dc
liu
Mnh m mm, t
bo hnh a giac vach
mng.
Ht tinh bt hnh trn

hay a giac, t r hay
khng.
Lng che ch a bo co
nhng vn doc.
18
Mnh mch mng
Mnh mch xon
Mnh biu b ca la bc, t

bo vach hi un ln
19
20
3.2. Quy trnh chit xut:
Bt Dip ca (6 kg) c lm m vi cn 96% trong 2 gi, np

vo bnh ngm kit, thm cn vo bnh chit, ngm trong 12 gi.
Rt dch chit tc 10 12 giot/pht cho n khi dch chit
nht mu, g p ton b dch chit cn, c thu hi dung mi di
ap sut gim c dch chit m c (k hiu A).
21
3.3. Qui trnh phn lp

3.3.1. Phng php sc k ct
3.3.1.1. Phn tch cc phn
on bng phn b lng-lng
22
S : chit xut v phn tch cc phn
on
nh tnh flavonoid trong cac cao A1, A2, A3 bng phn ng

cyanidin v sc k lp mng.
Kt qu: phn on 2 - cao A2 giu flavonoid nht so vi phn
on 1 v phn on 3, do o A2 c dng phn lp cac
flavonoid bng sc k ct.
3.3.1.2.Phn lp flavonoid t cao A2 bng SKC chn khng.
a) Thm d h dung mi trn sc k lp mng
b) Phn lp flavonoid bng sc k ct chn khng (Ct 1)
c) Phn lp flavonoid bng sc k ct chn khng (Ct 2)
23
a) Thm d h dung mi trn sc k lp mng

STT
H dung mi
Kh nng tch
Butyl acetat-acid formic (15:1)
++
Ethyl acetat
Ethyl acetat-MeOH-nc (10:0,5:1)
++
n-BuOH-ethyl acetat-nc (6:2:0,5)
Ethyl acetat-aceton-nc (8:1,8:0,2)
+++
Cloroform-ethyl acetat (2:8)
Butyl acetat-aceton (30:1)
Cloroform-aceton (8:2)
Butyl acetat-nc-acid formic (15:5:5)
10
Butyl acetat-nc (15:5)
++
++++
+++
24
Kt qua: H butyl acetat-nc (15:5) co kh nng

tach tt, Rf thch hp nn c dng tin hnh
trin khai trn sc k ct chn khng. Tuy nhin do
khng co mt ca acid formic nn cac vt tach ko
ui, do o silica gel s c tm 5% acid formic
cac vt tach gon hn.
25
b) Phn lp flavonoid bng sc k ct chn khng

(Ct 1)
iu kin sc k :Ct thy tinh xp 5 x 30 cm, x l bng
sulfocromic, ra sch, sy kh.
Pha tinh: Silica gel 60, c ht 0,015-0,04 mm, khi lng
150 g, c tm vi 7 ml acid formic, lc u, sy 120 oC/2
gi trong hp kn.
Pha ng: Butyl acetat nc (15:5)
Nhi ct: Nhi c t kh, sau o cho 150 ml pha ng chy
qua ct, ht chn khng n khi khng cn dung mi.
Khi lng mu: 25g cao A2.
26
Np mu dng dung dch. Mu c np u ln b mt silica gel, ht chn

khng n khi mu thm ht xung silica gel, sau o tin hnh hng phn on.
Th tch hng mi phn on l: 150 ml.
Kim tra cac phn on trn bng sc k lp mng trang sn vi h dung mi
butyl acetat nc acid formic (15:5:5), phat hi n di anh sang thng, UV
254 nm, UV 365 nm, thuc th FeCl3 5%.
Kt qua 5 phn on c ghi nhn:
Phn on
V (ml)
P (g)
Thnh phn
450
1,1
B C D
1200
3,7
E F
3300
4,2
750
2400
H
H
I J
2,3
G H
I J
11,6
I J
27
Kt qu phn lp flavonoid bng dung mi butyl

acetat-nc (15:5)
A
B
C
D
E
F
G
H
I
J
TP
5
28
Kt lun:
Phn on 1 (1,1 g) c kt tinh trong methanol thu c
tinh th tinh khit mu vng chanh, t tn l DC1 (34 mg).
Tinh th c kim tra trn sc k lp mng qua 4 h dung
mi khac nhau ethyl acetat-nc-acid formic-acid acetic
(100:10:5:5), EtOAc-aceton-H2O (4:5,5:0,5), CHCl3-HCOOHCH3COOH (6:2:0,5), butyl acetat-nc-acid formic (15:5:5),
phat hin bng TT. vanilin-sulfuric, TT. FeCl3, soi UV 365 nm,
UV 254 nm
29
Phn on 2 (3,7 g) co t nht 3 vt phat quang mu tm di n UV 365 nm

v cho mu xanh en vi TT. FeCl3, trong o co 1 vt chim lng ln, tin
hnh kt tinh phn on ny nhiu ln trong methanol bng cach ha tan hon
ton phn on ny trong lng va methanol, loc qua phu thy tinh xp.
Dch loc c vo t lnh, cho bay hi dung mi t t, loc v ra tinh th
bng methanol lnh. Tinh th c kim tra trn sc k lp mng qua 4 h
dung mi khac nhau EtOAc-H2O-HCOOH-CH3COOH (100:10:5:5),
EtOAc-aceton-H2O (4:5,5:0,5), CHCl3-HCOOH-CH3COOH (6:2:0,5),
BuOAc-H2O-HCOOH (15:5:5), phat hin bng TT. vanilin-sulfuric, TT.
FeCl3, soi UV 365 nm, UV 254 nm, tinh th thu c mu vng ti t tn l
DC2(450mg).
Phn on 4 (2,3 g) v phn on 5 (11,6 g) kha ging nhau nn gp chung,
tin hnh kt tinh 2 phn on ny trong methanol nhng khng thu c tinh
th, c thu hi dung mi 2 phn on, kim tra trn sc k lp mng nhn thy
vt G b phn hu thnh 2 vt l G v G (co th l do s co mt ca acid
formic). Do o phn lp c cht G th cn phi kho sat li h dung mi
30
khai trin m khng co mt ca acid.
Phn on 4 (2,3 g) v phn on 5 (11,6 g) kha ging nhau nn

gp chung, tin hnh kt tinh 2 phn on ny trong methanol
nhng khng thu c tinh th, c thu hi dung mi 2 phn on,
kim tra trn sc k lp mng nhn thy vt G b phn hu thnh
2 vt l G v G (co th l do s co mt ca acid formic). Do o
phn lp c cht G th cn phi kho sat li h dung mi
khai trin m khng co mt ca acid.
Cac phn on cn li do khi lng t v co nhiu vt nn khng
tip tuc nghin cu.
Cht chit xut thu c t cac phn on ct 1
Phn on
Cht tinh khit
c im
Khi lng (mg)
DC1
Vng chanh
34
DC2
Vng ti
450
31
c) Phn lp flavonoid bng sc k ct chn

khng (Ct 2)
Mc ch: tip tuc phn lp cht G hay cac flavonoid

cn li trong cao A2, tin hnh kho sat li mt s h dung
mi khai trin. Kt qu h ethyl acetat-aceton-nc (8:1,9:0,1)
c chon lm dung mi khai trin v co kh nng tach tt
nht v Rf thch hp.
32
iu kin sc k:
Ct thy tinh xp 5 x 30 cm, x l bng sulfocromic, ra
sch, sy kh.
Pha tinh: Silica gel 60, c ht 0,015-0,04 mm, khi lng 150 g
Pha ng: Ethyl acetat aceton H2O (8:1,9:0,1)
Khi lng mu: 25 g cao A2.
Np mu: Np mu kh
Th tch hng mi phn on l: 150 ml.
Kim tra cac phn on trn bng sc k lp mng trang sn vi
h dung mi butyl acetat nc acid formic (15:5:5), phat
hi n di anh sang thng, UV 254 nm,
UV 365 nm, thuc th FeCl3 5%.
33
Kt qu: thu c cc cng on c ghi nhn

Phn on
Dung mi
V (ml)
P (g)
Thnh phn
1 (1-5)
Ethyl acetat 100%
750
1,3
Tp km phn cc
300
2,2
600
4,7
1200
5,2
3600
4,3
500
6,2
2 (6-7)
3 (8-11)
4 (12-19)
Ethyl acetat-aceton-nc
(8:1,9:0,1)
5 (20-43)
6
MeOH 100%
G
G
Tp phn cc
34
Kt qu phn lp:
A
B
C
D
E
F
G
H
I
J
TP
6
35
Nhn xt:
Phn on 2 (2,2 g) c kt tinh nhiu ln trong methanol thu c tinh th
tinh khit mu vng ti l DC2 (220 mg). Tinh th c kim tra trn sc k
lp mng qua 4 h dung mi khac nhau EtOAc-H2O-HCOOH-CH3COOH
(100:10:5:5), EtOAc-aceton-H2O (4:5,5:0,5), CHCl3-HCOOH-CH3COOH
(6:2:0,5), butyl acetat-H2O-acid formic (15:5:5), phat hin bng TT. vanilinsulfuric, TT FeCl3, soi UV 365 nm, UV 254 nm.
Phn on 4 (5,2 g) co t nht 1 vt phat quang mu tm di n UV 365 nm
v cho mu xanh vi TT FeCl3, tin hnh kt tinh phn on ny nhiu ln
trong methanol bng cach ha tan hon ton phn on ny trong lng va
methanol, loc qua phu thy tinh xp. Dch loc vo t lnh, cho bay hi
dung mi t t, loc v ra tinh th bng methanol lnh. Tinh th c kim tra
trn sc k lp mng qua 4 h dung mi khac nhau EtOAc-H2O-HCOOHCH3COOH (100:10:5:5), EtOAc-aceton-H2O (4:5,5:0,5), CHCl3-HCOOHCH3COOH (6:2:0,5), butyl acetat-H2O-acid formic (15:5:5), phat hin bng TT.
vanilin-sulfuric, TT FeCl3, soi UV 365 nm, UV 254 nm, tinh th thu c v
nh hnh mu vng ti t tn l DC3 (800 mg).
36
Cac phn on cn li co nhiu vt nn khng tip tuc nghin cu.
Cht chit xut thu c t cac phn on ct 2

Phn on
Cht tinh khit
c im
Khi lng (mg)
DC2
Vng ti
220
DC3
Vng ti
800
d) Tm tt qu trnh phn lp cht

T 50 g cao A2 tin hnh phn lp cac flavonoid bng sc k ct chn khng (ct 1
v ct 2) thu c 3 hp cht tinh khit l DC1, DC2, DC3 vi khi lng tng
ng 34 mg, 670 mg, 800 mg. Tin hnh xac nh cu trc cho kt qu DC1 l
quercetin, DC2 l quercetin-3-O--L-rhamnopyranosid vi tn thng goi l
quercitrin, DC3 l quercetin-3-O--D-galactopyranosid.
37
3.3. Phng php HPLC iu ch

a) La chn pha tnh
K thut sc k pha o vi ct C8 hay C18 l s la chon u tin.
Vic la chon loi ct ph hp vi hp cht quan tm phu thuc nhiu
vo iu kin th nghim.
Do o, s tht kho khn nu thiu s hiu bit v nhom hp cht cn
quan tm.
Vic chy th vi 2 hay 3 iu kin ct pha o khac nhau s mang li
nhiu li ch cho ta.
b) S la chn dung mi
i vi sc k pha o, nc c s dung nh mt dung mi yu
trong khi cac dung mi hu c nh MeOH, MeCN hay THF l cac
dung mi mnh.
38
c) iu kin HPLC iu ch quercetin
C t sc k: Chromegabond C8.
Pha n g: CH3CN-H2O (30:70)
Thm 3 giot H3PO4 1% trong 250 ml dung mi
pha n g.
Bc song phat hi n: 370 nm.
Tc dng: 2ml/pht.
Th tch bm: 20ml.
Nhi t c t : nhit phng.
Thu gom cac phn on theo hnh dng peak.
39
IV. Phn kt lun v nhn nh

4.1. Kt lun
Chit xut: t 6 kg dc liu chit ngm kit vi cn 96% thu
c 54 g cao A2, cao A2 cha hm lng ln cac flavonoid.
Phn lp: T 50 g cao A2 tin hnh phn lp cac flavonoid bng sc
k ct chn khng (Ct 1 v ct 2) thu c 3 hp cht tinh khit
l DC1, DC2, DC3 vi khi lng tng ng 34 mg, 670 mg, 800
mg.
Cu trc ca cac cht c xac nh ln lt l: quercetin (DC1),
quercitrin (DC2), quercetin-3-O--D-galactopyranosid (DC3)
4.2. Nhn nh
Co th chit xut nhom Flavonoid ton phn t Dip ca bng
phng phap ngm kit vi cn 96% v phng phap siu m.
Co th phn lp quercetin, quercitrin, quercetin-3-O--Dgalactopyranosid bng sc k ct chn khng hoc HPLC
iu ch.
40
Cac cht phn lp c co th c xem nh

cht i chiu cho cac muc ch nghin cu v
Dip ca.
Phn lp bng HPLC co nhiu u im hn so
vi phng phap sc k ct chn khng, tuy
nhin cn phi co may moc hin i, iu ny
khng phi c s sn xut Dc phm no
cng ap ng c, nn kho ap dung rng ri
hn so vi phng phap sc k ct chn
khng.
41
Ti liu tham kho
B mn Dc liu i hoc Y Dc TPHCM- B mn Dc liu i hoc Dc H Ni

(1998), Bi ging Dc liu-tp 1, B Y t v B Giao duc o to, tr.259-289.
B mn Thc vt i hoc Y Dc TPHCM (2005), Gio trnh Phn loi thc vt, tr.51.
B Y t (2002), Dc in Vit Nam III, Nh xut bn Y Hoc, tr.350.
Tt Li (2000), Nhng cy thuc v v thuc Vit Nam, Nh xut bn Y Hoc, tr.40-41.
Hong Thanh Hng, Trn Qunh Hoa, H Vit Bo, Nguyn Danh Thuc (2002), Gop
phn nghin cu thnh phn flavonoid chit xut t la cy Dip ca Houttuynia cordata
Thunb. ca Vit Nam, Tp ch Dc hc, Nh xut bn B y t, H Ni, 9, tr.13-15.
Phm Hong H (1999), Cy c Vit Nam-quyn 1, Nh xut bn Tr, tr.288.
Phm Xun Sinh, Cao Vn Thu, inh Th Thanh Thy (1997), Nghin cu thnh phn
hoa hoc cy Dip ca, Tp ch Dc hc, Nh xut bn B y t, H Ni, 7, tr.7-9.
Trn Vit Hng (1998), Thuc nam trn t M-tp 1, Nh xut bn HK publishing Co.
V Vn Chi (2004), T in thc vt thng dng-tp 2, Nh xut bn Khoa hoc v k
thut, tr. 1386-1387.
42
CM N THY C V CC
BN LNG NGHE !
43
DANH SCH THNH VIN
V HUNH NH
MSSV :12D720401050
CHU HNG M
MSSV :12D720401040
TRNG KHNH LINH MSSV :12D720401033
NGUYN TH NGC M
MSSV :12D720401044
NGUYN THO TRANG MSSV :12D720401079
PHM VN MINH TNG
MSSV :12D720401074
V TH N
MSSV :12D720401002
44

CHIẾT XUẤT VÀ PHÂN LẬP FLAVONOID TỪ CÂY DIẾP CÁ

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CHIẾT XUẤT VÀ PHÂN LẬP FLAVONOID TỪ CÂY DIẾP CÁ

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L/O/G/O

CHIT XUT V PHN LP

Tng quan v thc v t hoc

Tng quan v hoa hoc

Phn b, sinh thi v thu hi.

1.2. Tng quan v thnh phn ha hc

1.3. Tng quan v tc dng, cng dng

Vit Nam, Trung

Kim chi, Nc tng, Siro,

Trn h min dch

1.4. Cc ch phm c cha Dip c trn

II. TNG QUAN V FLAVONOID

2.1. Khi nim chung v Flavonoid.

2.2. Cu trc ha hc ca flavonoid.

2.2.1. Khung flavonoid.

2.2.2. Phn loi Flavonoid

t quan trng, ch yu gp ngnh ht

2.3. Tnh cht:

III. PHN THC NGHIM

Cc bc tin hnh nhum vi phu

3.1.1.Vi phu thn

T bo biu b Tit Thn

Vi phu hnh a giac.

Lng che ch - Thn

Vi phu phin l: ri rac

Ht tinh bt hnh trn

Mnh mch mng

Mnh mch xon

Mnh biu b ca la bc, t

3.2. Quy trnh chit xut:

Bt Dip ca (6 kg) c lm m vi cn 96% trong 2 gi, np

3.3. Qui trnh phn lp

nh tnh flavonoid trong cac cao A1, A2, A3 bng phn ng

a) Thm d h dung mi trn sc k lp mng

Butyl acetat-acid formic (15:1)

Ethyl acetat-MeOH-nc (10:0,5:1)

n-BuOH-ethyl acetat-nc (6:2:0,5)

Ethyl acetat-aceton-nc (8:1,8:0,2)

Cloroform-ethyl acetat (2:8)

Butyl acetat-aceton (30:1)

Butyl acetat-nc-acid formic (15:5:5)

Butyl acetat-nc (15:5)

Kt qua: H butyl acetat-nc (15:5) co kh nng

b) Phn lp flavonoid bng sc k ct chn khng

Np mu dng dung dch. Mu c np u ln b mt silica gel, ht chn

Kt qu phn lp flavonoid bng dung mi butyl

Phn on 2 (3,7 g) co t nht 3 vt phat quang mu tm di n UV 365 nm

Phn on 4 (2,3 g) v phn on 5 (11,6 g) kha ging nhau nn

Cht tinh khit

Khi lng (mg)

c) Phn lp flavonoid bng sc k ct chn

Mc ch: tip tuc phn lp cht G hay cac flavonoid

Kt qu: thu c cc cng on c ghi nhn

Ethyl acetat 100%

Cht chit xut thu c t cac phn on ct 2

Cht tinh khit

Khi lng (mg)

d) Tm tt qu trnh phn lp cht

3.3. Phng php HPLC iu ch

c) iu kin HPLC iu ch quercetin

IV. Phn kt lun v nhn nh

Cac cht phn lp c co th c xem nh

Ti liu tham kho