PHNG PHP TCH CHIT, NH TNH, NH LNG FLAVONOID TRONG DC

LIU CAO KIM TIN THO

A. TNG QUAN V FLAVONOID
1. nh ngha, cu trc
Flavonoid l nhm hp cht phenol c cu to khung theo kiu C6-C3-C6 hay ni cch khc l khung c bn
gm 2 vng benzen A v B ni vi nhau qua mt mch 3 carbon. l nhm hp cht t nhin thng gp trong
dc liu ngun gc thc vt.
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2. Phn loi flavonoid
C nhiu cch phn loi khc nhau, cch phn loi thng dng l da vo v tr ca gc aryl (v vng B) v cc
mc oxy ho ca mch 3C.
Euflavonoid
isoflavonoid
neoflavonoid

cc flavonoid c gc aryl v tr C-2

cc flavonoid c gc aryl v tr C-3

cc flavonoid c gc aryl
v tr C-4

anthocyanidin, flavan*, flavan 3Isoflavan*, Isoflav-3-ene, Isoflavan-4ol*, flavan 4-ol*, flavan 3,4ol*, Isoflavon*, rotenoid, pterocarpan,
diol*, flavanon*, 3-hydroxy
Coumestan, 3-phenylcoumarin,
flavanon*, flavon*, flavonol*,dihydr coumaronochromen,
ochalcon*, chalcon*, auron.
coumaronochromon
Ngi ta cn phn bit biflavonoid l nhng flavonoid dimer, triflavonoid cu to bi 3 monomer flavonoid,
flavolignan l nhng flavonoid m phn t c mt phn cu trc lignan.
Phn ln cc flavonoid c th xem l cc dn cht c gc phenyl ca cc nhn trn. nh s th t bt u t
d vng, s 1 t d t oxy ri tip n vng A, vng B nh s ph. Trng hp khng c vng C (ngha l
mch 3C h) v d trng hp chalcon th nh s bt u t vng B, vng A nh s ph.
3. Phn b flavonoid trong thc vt
- Flavonoid tp trung ch yu vo ngnh ht kn lp 2 l mm. C rt nhiu h cha flavonoid v cc
loi flavonoid. Tuy nhin cng c mt vi nt c trng cho mt s h v d h Asteraceae l mt h ln vi
15.000 loi, 1000 chi, c rt nhiu dn cht thuc cc nhm khc nhau. Tuy nhin, mt s chi c nt c
trng ring ca n, v d trong cc chi Carthamus, Coreopsis, Cosmos, Dahlia th hay gp cc dn cht
chalcon v auron. Chi Gymnosperma, Ageratum th gp cc dn cht flavon v flavonol c nhiu nhm th
c oxy (c th n 8 nhm). H Fabaceae th hay gp cc cht thuc nhm isoflavonoid. H Rutaceae thng
gp cc flavon v flavonol c nhiu nhm methoxy. H Theaceae hay gp cc flavan-3-ol. H Ranunculaceae
v Paeoniaceae hay gp cc dn cht flavonol 3,7 diglycosid. H Rosaceae chi Rubrus v Prunus trong qu
hay gp cc anthocyanin c mch ng phn nhnh. H Polygonaceae chi Hydropiper hay gp cc flavon
v flavonol sulfat.
- ng vt khng tng hp c flavonoid nhng trong mt vi loi bm khi phn tch thy c flavonoid l
do chng ly t thc n thc vt.

- Hm lng v c thnh phn flavonoid trong cy ph thuc vo ni mc. Cy mc vng nhit i v ni

cao th hm lng cao hn ni cy thiu nh sng.
4. Phng php trch flavonoid ra khi cy
Cc flavonoid c ha tan khc nhau ty theo s lng nhm hydroxyl v cc nhm th khc c trong cu
trc ha hc nn rt kh c mt phng php chung trch ly flavonoid ra khi cy.
- Flavonoid no mang nhiu nhm metoxi - OCH3 v t nhm hidroxy - OH, c tnh phn cc yu, tan
tt trong dung mi t phn cc nh: benzen, clorofom, etyl acetate, i khi tan mt t trong hexan.
- Flavonoid mang nhiu nhm hidroxi s c tnh phn cc mnh s ha tan trong cc dung mi c tnh
phn cc nh: aceton, etanol, butanol, nc...
- Cc flavonoid glycosid, antocyanidin khng tan trong dietyl ete, nhng tan trong nc nng, etanol
nng. Ring antocyanidin kh bn vi nhit nn c th c chit tch di dng clorua bng
dung dch 0,01N HCl/metanol.
Cc phng php sau c dng tch chit flavonoid:
Phng php trng lng:
Nguyn tc: ch acetat kim c kt ta mu vi hu ht cc flavonoid phenol cn ch acetat trung tnh
to kt ta vi cc dn cht c nhm o-ihidroxyphenol. Da vo nguyn tc ny, ta s dng ch
acetat ta flavonoid sau dng Na2SO4 loi ch khi flavonoid.
Phng php hp ph:
Nguyn tc: hp ph flavonoid bng than hot tnh v dng dung mi hu c thch hp chit cc
flavonoid khc nhau.
Cho than hot tnh vo dung dch cao chit nhm hp th flavonoid. Lc ly than, s dng cc dung
mi hu c t phn cc yu n phn cc mnh nh etyl ete, etyl acetat, metanol, nc nng, phenol
7% ... chit cc flavonoid c tnh phn cc khc nhau.
Phng php tch phn on:
Nguyn tc: cc flavonoid c c nhiu nhm -OH phenol nn c th tan trong dung dch kim. da
vo nguyn tc ny, c th s dng dung dch kim long hoc nc vi chit flavonoid khi cy
c. sau dung dch c axit ha tr v dng -OH t do.
B. S LC V KIM TIN THO
Tn khoa hc: Herba Jinqiancao, Desmodium styracifolium (Osbeck) Merr. H Cnh Bm (Fabaceae)
M t: cy tho, sng lu nm, b st t, di khong 1m. L mc so le, gm 3 l cht hnh trn. Hoa t hnh
chm. Trng hoa hnh bm, mu ta. Qu loi u, di 14-16 mm, cha 4- 5 ht. mc hoang trn vng i
trung du, vng ni.
B phn dng: ton cy.
Thnh phn ha hc: Kim tin tho c cha polysaccharid, saponin triterpenic, cc flavonoid nh
isovitexin, vicenin glycosid, isoorientin v cc cht khc nh desmodimin, desmodilacton, lupenon, lupcol,
tritriacontan, acid stearic,..
Tc dng dc l: Theo dc hc c truyn, kim tin tho v ngt, tnh bnh, c cng dng thanh nhit, li
thp, li niu thng lm, tiu thng bi thch, thng c dng cha cc chng bnh nh vim ng
tit niu, si tit niu, si mt, ph thng do vim thn, hong n (vng da), tch t, ung thng,...
C. QUY TRNH NH LNG TNG FLAVONOID BNG PHNG PHP UV-VIS
1. X l mu:
- Chun b mu: cn 0,5g cao kim tin tho cho vo cc, ha tan bng 10ml etanol 80% trong b iu
nhit ( khong 500C), lc ly dung dch, nh mc n 10ml etanol 80%. Thu c dung dch mu
nng Cm.

Tin hnh to phc: chun b trong bnh nh mc 10ml, cho vo bnh nh mc 10ml: 0,1 ml mu
nng C0, 0,1ml cht chun nng C1, C2, C3, C4, C5 (g/ml); 0,3ml NaNO2 5%; 0,3 ml AlCl3; 2
ml NaOH 10%. nh mc n vch, 5 pht.
2. Kho st cc iu kin ti u:
- Xc nh bc sng ti u:
+ Cn chnh xc mt lng mu cao ( khong 30g) cho vo bese 100 ml, thm tip 20ml etanol 80%,
lc u trong b iu nhit 500C khong 5 pht, ngui, lc ly dung dch, nh mc n 25ml bng
etanol 80%. Thu c mu dung dch nng C0.
+ Tin hnh:
Mu cao: cho vo bnh nh mc 50 ml: 0,1 ml mu; 0,1 ml chun (100g/ml), 10ml nc ct, 3ml
NaNO2, 3ml AlCl3, 2ml NaOH 10%. nh mc n 50ml bng nc ct.
Cht chun: tin hnh phn ng tng t vi 0,2ml dung dch chun.
Kho st hp th trong khong 200 - 500 nm. Thu c kt qu max cao kim tin tho l 382 nm.
- Kho st nng dung mi nh hng n hp th:
Cn 0,5g mu cao cho vo 5 cc bese 100ml, ln lt thm vo 10ml etanol 40%, 60%, 70%, 80%,
90% trong b iu nhit 500C khong 5 pht. ngui, lc ly dung dch, nh mc n 10 ml
etanol nng tng ng. thc hin phn ng nh trn, kho st ti max 382 nm nh kho st
trn.
40
60
70
80
90
A (Kim tin
0,1625
0,1687
0,1712
0,1784
0,1325
tho)
Ta thy, ti nng etanol 80%, phc c hp th ln nht. iu ny cho thy, flavonoid chit tt
trong etanol 80%.
- Kho st nng AlCl3 nh hng n hp th:
+ Chun b dung dch mu: Cn 15g mu kim tin tho cho vo bese 100 ml, thm 20ml etanol 80%,
lc trong b iu nhit 5 pht 500C. ngui, lc, ly dung dch nh mc n 25ml bng etanol
80%. Thu c dung dch mu nng Cm.
+ Tin hnh: cho vo bnh nh mc 50ml: o,1 ml mu nng Cm, 10ml nc ct, 3ml AlCl3 nng
t 4 - 10%, 2ml NaOH 10%, nh mc n vch bng nc ct. o, ghi nhn hp th ti bc
sng 382 nm (nh kho st) trong 5 pht.
CAlCl3 (%)
5
6
7
8
9
10
11

hp th A
0,4103
0,4165
0,4216
0,4284
0,4325
0,4518
0,4389

T kt qu trn, ta thy ti nng AlCl3 l 10%, phc t hp th cao nht. Nng AlCl3 10%
l ti u.
Kho st thi gian phn ng nh hng n hp th
Cho vo bnh nh mc 50 ml: 0,1 ml mu nng Cm, 10ml nc ct, 3ml AlCl3 nng t 10%,
2ml NaOH 10%, nh mc n vch bng nc ct. o hp th ngay sau khi thc hin phn ng.
Ghi nhn hp th cc im thi gian.

Nhn thy sau 15 pht, hp th bt u gim dn. Vy thi gian phn ng l 15 pht l ti u.
3. Xy dng quy trnh nh lng tng flavonoid bng phng php thm chun
Cao dc liu
Etanol 80%

Ta

Dung dch
V (ml) chun AlCl3, NaNO2, NaOH

Dung dch mu
o hp th max

y=ax+ b

Dng pt

Ghi nhn A

Sau khi chun b dung dch phn tch, tin hnh o hp th cng vi mu trng bc sng 382 nm. Ghi
nhn hp th, v phng trnh ng thng y = ax+b (y: hp th, x l nng ). Kt qu c tnh theo
cng thc:
F = (Cm.V.a)/M
Trong :
F l hm lng tng flavonoid (mg/g)
Cm l nng flavonoid suy ra t phng trnh
V l th tch mu
a l h s pha long
M l khi lng mu
Phng php thm chun s dng cht chun Quercetin
Sau khi chun b mu tin hnh cho vo bnh nh mc 10 ml: 0,1 ml mu nng C0, 1ml quercetin nng
ln lt 10, 20, 30, 40, 50 g/ml; 0,3 ml NaNO2 5%; 0,3 ml AlCl3, 2ml NaOH 10%. o hp th ti
bc sng 382 nm trong 5 pht.
Nng
(g/ml)

C0+10

C0+20

C0+30

C0+40

C0+50

0,591

0,6903

0,7944

0,9004

1,0092

Hnh. th biu din hp th ca cao Kim tin tho - Quercetin

th biu
din trn
2
th hin tuyn tnh cao gia nng v hp th (R =0,997)
Phng php thm chun s dng cht chun Rutin
Sau khi chun b mu tin hnh cho vo bnh nh mc 10 ml: 0,1 ml mu nng C0, 1ml Rutin nng ln
lt 10, 20, 30, 40, 50 g/ml; 0,3 ml NaNO2 5%; 0,3 ml AlCl3, 2ml NaOH 10%. o hp th ti bc sng
382 nm trong 5 pht.
Nng
(g/ml)

C0+10

C0+20

C0+30

C0+40

C0+50

0,379

0,4527

0,5196

0,6016

0,6692

Hnh. th biu din hp th ca cao Kim tin tho - Rutin

th biu din trn th hin tuyn tnh cao gia nng v hp th (R2=0,9992)
T hai phng trnh ng chun trn, ta suy ra c gi tr Cm = b/a, t suy ra tng flavonoid (mg/g)
- Tnh theo quercetin: 0,9228
- Tnh theo Rutin: 0,8384
D. KT LUN
T hai kt qu trn nhn thy hm lng tng flavonoid tnh theo quercetin cao hn rutin do cu trc
rutin c cu trc ng glycoside, chnh phn ng ny lm nh hng n i lc ca phn
aglycone vi mi trng xung quanh nn lm cho hunh quang ca Rutin gim i rt nhiu so vi
Quercetin.