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B GIO DC V O TO

B Y T

TRNG I HC DC H NI

PHM TH PHNG DUNG

XY DNG PHNG PHP IU CH


CAO KH V PHN LP PUERARIN
T SN DY
LUN VN THC S DC HC

H NI - 2014

B GIO DC V O TO

B Y T

TRNG I HC DC H NI

PHM TH PHNG DUNG

XY DNG PHNG PHP IU CH


CAO KH V PHN LP PUERARIN
T SN DY
LUN VN THC S DC HC
C

G G

BO C THUC

: 60720402

gi hng dn khoa hc: TS. Nguyn Vn Hn

H NI 2014

LI CM N

Vi lng knh trng v bit n su sc, ti xin gi li cm n chn


thnh ti thy gio TS. Nguyn Vn Hn, l ngi tn tnh hng dn v
gip ti nghin cu v hon thnh lun vn ny.
Ti xin cm n cc thy c v anh ch k thut vin ca b mn Cng
nghip Dc to iu kin v gip ti v mi mt trong qu trnh
nghin cu hon thnh lun vn.
Ti cng xin gi ti Ban gim hiu, phng o to sau i hc, cc thy
c gio trng i hc Dc

i lng bit n v s quan tm, gip

trong thi gian hc tp v nghin cu ti trng.


Cui cng, ti xin gi li cm n ti gia nh, ngi thn v bn b
ng vin, h tr ti trong sut thi gian qua.
i, thng 07 nm 2014
c vin

Phm Th Phng Dung

MC LC
DANH MC CC K HI U, CH VIT TT
DANH MC CC BNG, BIU
DANH MC CC HNH V, TH
T V .............................................................................................................1
Chng 1. TNG QUAN .........................................................................................2
1.1.

Vi nt v cy sn dy .................................................................................2

1.1.1.

c im thc vt v phn b ca cy sn dy .....................................2

1.1.2.

Thnh phn ha hc ca r c sn dy ..................................................2

1.1.3.

Tc dng dc l ....................................................................................4

1.1.4.

Cng dng ...............................................................................................6

1.1.5.

Cc nghin cu chit xut isoflavonoid t sn dy ................................6

1.2.

Puerarin ......................................................................................................12

1.2.1.

Cng thc ha hc ................................................................................12

1.2.2.

Tnh cht l ha ....................................................................................12

1.2.3.

K thut phn tch .................................................................................13

1.2.4.

Tc dng sinh hc ca puerarin ...........................................................15

1.2.5.

Cc nghin cu tinh ch puerarin hin nay..........................................19

1.3.

Cao thuc ....................................................................................................20

1.3.1.

Khi nim ..............................................................................................20

1.3.2.

Phn loi ...............................................................................................20

1.3.3.

K thut iu ch ..................................................................................21

1.3.4.

Cc ch tiu cht lng ca cao thuc .................................................22

Chng 2. NGUYN VT LI U, TRANG THIT B V

NGHIN CU ..........................................................................................................23
2.1.

Nguyn vt liu, thit b ............................................................................23

2.1.1.

Nguyn liu ...........................................................................................23

2.1.2.

Dng c .................................................................................................23

2.1.3.

Phng tin v my mc.......................................................................23

2.2.

Ni dung nghin cu..................................................................................24

2.2.1.

Chit xut isoflavonoid t nguyn liu kh ..........................................24

2.2.2.

Chit xut isoflavonoid t nguyn liu ti..........................................24

2.2.3.

iu ch cao kh...................................................................................25

2.2.4.

Phn lp puerarin v daidzin ...............................................................25

2.3.

Phng php nghin cu ..........................................................................25

2.3.1.

Phng php nh lng isoflavonoid ton phn v puerarin ............25

2.3.2.

Phng php nh tnh cc isoflavonoid bng sc k lp mng ..........29

2.3.3.

Phng php chit xut isoflavonoid t sn dy kh ...........................30

2.3.4.

Phng php chit isoflavonoid t sn dy ti ..................................30

2.3.5.

Phng php tinh ch dch chit sn dy ti .....................................31

2.3.6.

Phng php iu ch cao kh.............................................................32

2.3.7.

Phng php phn lp puerarin v daidzin .........................................35

2.3.8.

Phng php xc nh cu trc puerarin v daidzin ...........................35

2.3.9.

Phng php xc nh khi lng cn trong dch chit ......................35

2.3.10. Phng php xc nh mt khi lng do lm kh ..............................36


Chng 3. KT QU NGHIN CU....................................................................37
3.1.

nh lng isoflavonoid ton phn trong nguyn liu ...........................37

3.1.1.

ng chun isoflavonoid ..................................................................37

3.1.2.

nh lng isoflavonoid trong nguyn liu .......................................38

3.2.

nh lng puerarin trong nguyn liu...................................................39

3.3.

Chit xut isoflavonoid t sn dy kh ...................................................40

3.3.1.

Kho st cc dung mi chit .................................................................40

3.3.2.

Tinh ch dch chit sn dy kh ............................................................44

3.4.

Chit xut isoflavonoid t sn dy ti ...................................................45

3.4.1.

Chit isoflavonoid t sn dy ti ........................................................45

3.4.2.

Tinh ch dch chit sn dy ti ...........................................................46

3.5.

iu ch cao kh t dch chit sn dy ti ............................................47

3.6.

Phn lp puerarin v daidzin ...................................................................49

3.6.1.

Phn lp daidzin ...................................................................................49

3.6.2.

Phn lp puerarin .................................................................................53

3.6.2.1. Tin hnh ..............................................................................................53


Chng 4. B L N ............................................................................................57
4.1.

V nguyn liu chit xut isoflavonoid ....................................................57

4.2.

V phng php bo ch cao kh ............................................................57

4.3.

V phng php phn lp puerarin v daidzin ......................................58

KT LU V XUT ......................................................................................60
KT LUN ...........................................................................................................60
XUT..............................................................................................................61
TI LI
LC

T A

K O

DANH MC CC K HIU, CH VIT TT


AMPK

5'-adenosine monophosphat-activated protein kinase

ATP

Adenosine triphosphat

CV

Coefficient of variation
( s phn tn)

DEPT

Distortionless Enhancement by Polarization Transfer

DV

Dc in Vit am

ESI MS

ElectroSpray Ionization - Mass spectrometry


(Khi ph - Ion ha bng phng php phun m in t)

GOT

Glutamic oxaloacetic transaminase

HDL

High-density lipoprotein

HPLC

High Performance Liquid Chromatography


( c k lng hiu nng cao)

HSCCC

High Speed Counter Current Chromatography


( c k ngc dng tc cao)

IL

Interleukin

IR

Infrared
( ng ngoi)

LDL

Low-density lipoprotein

LPS

Lipopolysaccharid

mTOR

mammalian Target of Rapamycin


(ch tc dng trn ng vt c v ca Rapamycin)

MAPK

Mitogen-activated-protein-kinase

NMR

Nuclear magnetic resonanc


(Cng hng t ht nhn)

H-NMR

13

C-NMR

h cng hng t ht nhn proton

h cng hng t ht nhn carbon 13

PG

Pueraria glycosid

RSD

Relative standard deviation

( lch chun)
TLC

Thin Layer Chromatography


( c k bn mng)

TNF

Tumor necrosis factors


(Yu t hoi t khi u)

UV

Ultraviolet
(T ngoi)

DANH MC CC BNG, BIU


Bng 1.1. Cng thc cu to ca mt s isoflavonoid thng gp trong sn dy ......3
Bng 1.2. o snh ba phng php chit xut sn dy thng dng..........................9
Bng 2.1. Ha cht s dng ......................................................................................23
Bng 3.1. Mt quang ca dy dung dch cht i chiu ti bc sng 250 nm. .37
Bng 3.2. Kt qu nh lng isoflavonoid trong nguyn liu .................................39
Bng 3.3. Din tch pic sc k ca dy dung dch chun ..........................................39
Bng 3.4. m lng puerarin trong nguyn liu thu mua Ph Th.....................40
Bng 3.5. Kt qu kho st cc dung mi ethanol c nng khc nhau ................41
Bng 3.6. Kt qu kho st chit isoflavonoid t sn dy kh bng dung mi nc
...................................................................................................................................43
Bng 3.7. Kt qu tinh ch dch chit sn dy kh ...................................................44
Bng 3.8. Kt qu chit isoflavonoid t sn dy ti ...............................................45
Bng 3.9. Hiu sut v hm lng cao c sau tinh ch (thu c t 500 ml dch
chit, tng ng khong 150 g dc liu) ................................................................46
Bng 3.10. Dch tinh ch em vo phun sy .............................................................47
Bng 3.11. Mt s ch tiu cht lng ca cao kh phun sy...................................48
Bng 3.12. Kt qu o ph hng ngoi ca daidzin .................................................51
Bng 3.13. Kt qu o ph khi lng (ESI-MS) ca daidzin .................................52
Bng 3.14. Kt qu o ph NMR ca daidzin ..........................................................52
Bng 3.15. Kt qu o ph hng ngoi ca puerarin ................................................54
Bng 3.16. Kt qu o ph khi lng (ESI-MS) ca puerarin ...............................54
Bng 3.17. Kt qu o ph NMR ca puerarin .........................................................55

DANH MC CC HNH V, TH
nh 1.1. Sn dy ........................................................................................................2
nh 2.1. chit isoflavonoid t sn dy ti ...................................................31
nh 2.2. Quy trnh iu ch cao kh .......................................................................34
Hnh 3.1. Ph UV-VIS ca dung dch cht i chiu ...............................................37
nh 3.2. ng chun biu th mi tng quan gia nng puerarin v mt
quang .........................................................................................................................38
nh 3.3. th biu din mi tng quan gia nng puerarin v din tch pic
sc k .........................................................................................................................40
nh 3.4. Biu so snh lng isoflavonoid chit c bi cc dung mi ethanol
c nng khc nhau. ...............................................................................................42
nh 3.5. Bt cao kh phun sy ................................................................................48
nh 3.6. nh nh sc k lp mng di n t ngoi 254 nm ca dch chit .......49
nh 3.7. Quy trnh phn lp daidzin v puerarin .....................................................50
Hnh 3.8. Hnh nh sc k lp mng di n t ngoi 254nm ca daidzin ...........51
nh 3.9. Cng thc cu to ca daidzin ..................................................................52
Hnh 3.10. Hnh nh sc k lp mng di n t ngoi 254nm ca puerarin ........53
nh 3.11. Cng thc cu to ca puerarin ...............................................................55

T VN
Sn dy t lu c s dng trong dn gian. Theo y hc c truyn, r sn
dy l mt v thuc mt, c tc dng gii nhit, sinh tn, chng kht, lm ra m hi,
d tiu, ch t, cha cc chng st nng, nhc u, mn nga, rm sy, vim rut,
au d dy... Gn y trn th gii c kh nhiu cng trnh khoa hc chng minh
tc dng ca dch chit r sn dy: cha au u [34], chng oxy ha [9], [13], gii
ru [23]. Thnh phn isoflavonoid trong r c cho l cht c tc dng. Trong s
cc isoflavonoid sn dy, puerarin l cht c hm lng ln v c cho l thnh
phn c tc dng ch o. Hin nay Trung Quc puerarin c chit xut v
phn lp thnh cng t r sn dy phc v cho cho vic bo ch cc dng thuc quy
c (vin nn, vin nang, thuc tim) cha bnh.
Ti Vit Nam, sn dy c trng kh ph bin nhng ch yu nhm ch
tinh bt. Vai tr ca sn dy trong chit xut isoflavonoid cha c quan tm y
. Hin nay, isoflavonoid nguyn liu v puerarin vn phi nhp ch yu t Trung
Quc vi gi thnh cao. V vy nu c th nghin cu chit xut thnh cng
isoflavonoid v puerarin t r c sn dy song song vi vic ch tinh bt c th gip
ch ng v ngun nguyn liu isoflavonoid cng nh puerarin trong sn xut v
tn dng c ph phm ca qu trnh sn xut tinh bt sn.
Nhm gp phn tm ra phng php chit xut isoflavonoid t r sn dy
ph hp vi iu kin thc tin Vit Nam, chng ti tin hnh thc hin ti:
Xy dng phng php iu ch cao kh v phn lp puerarin t sn dy
vi hai mc tiu chnh sau:
1.

Xy dng quy trnh iu ch cao kh phun sy t r c sn dy.

2.

Phn lp c puerarin t r c sn dy.

Chng 1. TNG QUAN


1.1. Vi nt v cy sn dy
1.1.1. c im thc vt v phn b ca cy sn dy
Sn dy c tn khoa hc l Pueraria thomsonii
Benth., h u Fabaceae. Mt s ti liu Trung Quc
ghi loi Pueraria lobata (Willd.) Ohwi hoc P.
pseudohirsuta Tang et Wang [3]. Dc in Trung Quc
ghi nhn c hai loi Pueraria lobata v Pueraria
thomsonii [32].
Sn dy l mt loi dy leo, di c th n 10m, l
kp gm ba l cht. Cung l cht gia di, cung l cht

nh 1.1. n dy

hai bn ngn. L cht c th phn thnh 2-3 thy. V ma


h tr hoa mu xanh tm, mc thnh chm k l. Qu loi u c nhiu lng. C
di to nng c th ti 20kg, nhiu x.
Mun trng ngi ta o cc h su 50cm, rc v mn ri lp t xp li.
n thng 1-2 gim cnh vo cc h .

hiu ni nc ta thng kt hp lm

gin ly bng mt. Cng c nhng vng chuyn trng ch tinh bt v d lng
Cao X thuc huyn Chu Thnh, tnh Ty Ninh mi nm sn xut khong 20 tn
tinh bt [3].
R c thu hoch t thng 10 n thng 3-4 nm sau. Trung Quc, ngi ta
thng thu hoch sn dy vo ma thu v ma ng [32]. ch v Ct Cn th ra
sch, bc b lp v dy bn ngoi, ct thnh khc di 10-15 cm. Nu c to th b
dc c nhng thanh dy khong 1cm, sau xng dim sinh ri phi hoc sy
kh. Loi trng t x l loi tt. Mun ch tinh bt sn dy th bc v, em gi nh
hoc mi trn tm st ty c c thng l, hoc xay bng my, cho thm nc ri
nho lc qua ry tha, loi b, sau lc li 1 ln na qua ry dy hn, lng gn
ly tinh bt ri em phi hoc sy kh [3].
1.1.2. Thnh phn ha hc ca r c sn dy
R c cc loi ueraria u cha tinh bt, t l khong 12-15% theo ti [3]
n 40% theo kh [4]. Ngoi ra cn c saponin [4], [20] v cc flavonoid thuc
2

nhm isoflavonoid. T loi Pueraria lobata ngi ta phn lp c cc


isoflavonoid sau puerarin (1), daidzin (2), daidzein (3), formonetin (4).

m 1987

cc nh nghin cu Nht pht hin thm 13 cht mi, trong c genistein v


cc glycosid ca daidzein, genistein [20]. Cc nghin cu sau ny u ch ra rng
puerarin, daidzin, daidzein, genistein v genistin l cc isoflavonoid chim t l ln
trong r c cc loi sn dy, v cho cc tc dng sinh hc ng ch [46], [11],
[37].
R3
R2O

R1

R4

R1

R2

R3

R4

Puerarin

Glu

OH

Daidzin

Glu

OH

Daidzein

OH

Genistein

OH

OH

Genistin

OH

Glu

OH

Bng 1.1. Cng thc cu to ca mt s isoflavonoid thng gp trong sn dy


Tnh n thi im hin ti, ngi ta tm ra khong 52 flavone t cc loi
Pueraria [44]. Trong Pueraria lobata l loi c hm lng flavones cao nht v
c nhiu flavones c phn lp nht (36 cht) [44].
m lng isoflavone trong r c sn dy ph thuc nhiu vo loi, vng
trng trt [44] v thi gian thu hi [11].

m lng flavone trung bnh loi

Pueraria lobata l 7,42% trong khi hm lng flavone trung bnh loi Pueraria
thomsonii ch c 0,93% [44].

m lng flavone loi Pueraria lobata trng ti

Shanxi ln ti 10,4% trong khi hm lng ny khi trng ti Jiangxi ch t 3,3%


[44]. Tui v thi gian thu hi cng l yu t nh hng ng k ti hm lng
isoflavone ca loi Pueraria lobata trng ti Huoshan. R c 3 nm tui thu hi vo
khong thng Ging s cho nng isoflavone cao nht, t 7,26%. Tui cao hn

hoc thi gian thu hi trc thng Ging u lm gim nng isoflavone ca sn
dy [11].
1.1.3. Tc dng dc l
1.1.3.1. Tc dng trn tim mch
Th nghim tc dng trn ch bng cch tim flavon ton phn ca sn dy
cho thy c tc dng gin ng mch vnh ng thi gim lng tiu th oxy ca
c tim v gim sc khng mch vnh. Trn ch dng reserpin trc lm
tiu kit ht lng catecholamin trong m tim, dng isoflavon ton phn vi liu 30
mg/kg v puerain vi liu 20 mg/kg c tc dng lm tng lu lng mch vnh v
gim sc khng mch vnh. Nhng kt qu ny ging vi kt qu th nghim trn
ch khng dng reserpin. iu ny chng t hin tng lm gin mch vnh ca
sn dy khng lin quan ti catecholamin m l do tc dng gin c trc tip [8].
1.1.3.2. Tc dng h huyt p ca cao sn dy
Tim tnh mch vi liu 5-30 mg trn ch, mo c tc dng h huyt p. Cao
sn dy vi liu 750 mg/kg tim tnh mch c kh nng i khng vi tc dng kch
thch tim ca isoprenalin, ngoi ra cn lm gim nhp tim v gy h huyt p [8].
1.1.3.3. Tc dng chng lon nhp tim
So snh tc dng chng lon nhp tim ca puerarin, daidzein v dng chit
cn t sn dy trn chut cng trng v chut nht trng cho thy tc dng ca
daidzein tng i mnh, cho hiu qu r rt. Dng chit cn c tc dng ging vi
daidzein; iu ny chng t daidzein l thnh phn ch yu c tc dng chng lon
nhp tim. Puerarin vi liu tng ng c tc dng khng r rt vi lon nhp tim
do aconitin v bari clorid gy nn, gim nh mc lon nhp do thiu mu c tim,
tc dng i khng vi rung tht khng bng daidzein [8].
1.1.3.4. Tc dng i vi tun hon no
Th nghim trn ch gy m, flavon ton phn ca sn dy bng ng tim
ng mch cnh vi liu 0,1-5,0 mg/kg lm lu lng mu qua no tng 87,7134%, nu cho thuc bng ng tim tnh mch flavon ton phn vi liu 10-30
mg/kg ch lm lu lng mu qua no tng 20%. Trn bnh nhn cao huyt p,

flavon ton phn tim bp vi liu 200 mg gip ci thin tun hon no cho 53%
bnh nhn, lm gim tr lc mch mu no [8].
1.1.3.5. Tc dng i vi h thn kinh
Dch chit sn dy vi liu 2 g/kg cho thng vo d dy trn th gy st bng
vaccin thng hn c tc dng h nhit, bt sn dy c tc dng tng t. c sn
dy vi liu 6 g/kg cho thng vo d dy trn chut nht trng, c tc dng ci thin
hin tng tr nh b tn thng do scopolamin gy nn [8].
1.1.3.6. Tc dng i vi c trn
Trong cc dch chit bng aceton, methanol v nc t sn dy th dng
PA3, PA4 c tc dng c ch co bp hi trng c lp chut lang kiu papaverin,
dng PM1, PM3 li c tc dng gy co tht. Daidzein c tc dng gii co tht i
vi rut non c lp ca chut nht trng, tc dng ny bng khong 1/3 tc dng
ca papaverin [8].
1.1.3.7. Tc dung h ng huyt v lipid huyt
c sc sn dy vi liu 6-8 g/kg cho thng vo d dy th bnh thng c
tc dng h ng huyt nhng khng th i khng vi hin tng ng huyt
tng cao do adrenalin gy nn. Puerain vi liu 500 mg/kg hoc dng liu thp phi
hp vi aspirin 100 mg/kg dng lin tc trong 9 ngy qua ng d dy, c tc
dng lm gim cholesterol huyt thanh ca chut nht trng c gy tng
cholesterol bng alloxan. Aspirin dng n c khng c tc dng h ng huyt
hay lipid huyt [8].
1.1.3.8. Tc dng chng ung th
Dch chit cn t sn dy vi liu 10 g/kg trn ng vt th nghim c tc
dng c ch nht nh s pht trin ca t bo sarcom 180, u bng Ehrlich v t bo
ung th phi Lewis. Daidzein vi nng 14g/ml c tc dng c ch s tng
trng ca t bo ung th mu HL-60 (promyelocytic leukemia) [8].
1.1.3.9. Tc dng chng oxy ha
Th nghim trn chut gy tiu ng bng cch tim phc mc
streptozotocin. Th nghim s dng cao sn dy cha khong 10,42% puerarin v
mt vi cht lin quan, vi liu 500 mg/kg, dng ng ung trong 3 tun, kt qu
5

cho thy nng malondialdehyd trong huyt tng-c s dng nh mt du


hiu ca cht bo b oxy ha, gim xung mc tng t nh chut khe mnh,
v ging nh trong nhm c iu tr tch cc hng ngy bng tocopherol acetat
vi liu 50 mg/kg [8],[9].
1.1.3.10. Cc tc dng khc:
Dung dch puerarin 0,2-1,6% vi liu 0,2 ml tim di da chut lang theo
di phn ng gic mc v da cho thy tc dng gy m cc b. Th nghim trn
chut hamster, cao sn dy c tc dng chng nghin ru, trn chut cng trng,
daidzein c tc dng h thp lng ru trong mu v rt ngn thi gian ng do
ru gy nn. i vi gan ca chut nht trng gy nhim c bng CCl4,
isoflavonoid chit t ct cn vi liu 250 mg/kg c tc dng c ch 30,7% hot
men GOT [8].
Nghin cu ca Th oa Vin chng minh rng cao chit isoflavonoid
t c sn dy Pueraria thomsonii Benth. c hot tnh ni tit kiu estrogen trn
51,5% chut nht trng ci vi liu ung 150 mg/con/ngy. iu ny cho thy hot
tnh estrogen ca hn hp isoflavonoid chit xut t c sn dy l kh r rt [7].
1.1.4. Cng dng
Theo y hc c truyn, ct cn l mt v thuc cha st nhc u, kht nc,
kit l, ban si. Ct cn c ghi vo dc in Vit Nam. Tinh bt sn dy pha
vi nc thm ng ung gii kht [3]. Ngoi ra trong y hc c truyn cn
dng hoa ca dy sn dy vi tn Ct hoa lm thuc gi ru [3].
Theo Dc in Trung Quc, sn dy c tc dng h st, kch thch tit dch
c th, cm a chy. Do vy, sn dy c s dng iu tr st, au u, kht
nc, tiu ng, ban si, kit l hoc a chy cp, cng gy v au trong tng
huyt p [32].
1.1.5. Cc nghin cu chit xut isoflavonoid t sn dy
1.1.5.1. Chit kiu truyn thng
Mc d vi s pht trin ca khoa hc k thut hin i c rt nhiu
phng php chit hiu qu c a vo ng dng nhng chit xut theo kiu

truyn thng vn c ngh n u tin khi nghin cu v cc dc liu mi.


Ch c hiu r c im chit cc dc liu ny theo phng php hi lu n
gin mi c th p dng cc bin php ci tin nng cao hiu sut cng nh
cht lng sn phm chit. Cng v th, trong rt nhiu nghin cu chit sn dy
c thc hin trn th gii, phng php chit hi lu vn c cc nh khoa
hc quan tm thc hin. Mc tiu ca cc nghin cu ny hu ht l tm ra
c loi dung mi, nhit , thi gian ti u cho vic chit isoflavonoid t
sn dy.
Ngay t nm 1998, Li Wehong v cng s xc nh c gia hai dung
mi nc v cn, dung mi cn cho hiu sut chit cao hn v d tinh ch sn
phm hn. Cng t , nhm nguyn cu tm ra c iu kin chit xut ti
u cho sn dy l chit bng ethanol 60% trong 6 gi 60C [25].
n nm 2007,

an Jian v cng s vn tip tc nghin cu chit

isoflavonoid t sn dy bng phng php truyn thng nhng s dng dung mi


chit l nc. Mc d khng phi l mt dung mi c nh gi cao trong nhiu
nghin cu song nu tin hnh theo phng php ca Han Jian vn c th chit
c lng ln puerarin t sn dy. Chit nc iu kin ti u l chit 2 ln,
mi ln vi 10 ln th tch nc, thi gian chit ln lt l 1,5 gi v 1 gi [14].
nng cao hiu sut chit ngi ta tin hnh thay i dung mi chit,
s dng h dung mi thay v cc dung mi n thng thng. ng k n l
chui nghin cu ca Hua-Neng Xu v cng s. u tin, nhm nghin cu
tm ra h dung mi hai pha n-butanol/nc t l 1:1 (v/v) khng nhng dng
c cho qu trnh chit rn lng v tinh ch lng-lng m cn gip chit c
daidzein v isoflavonoid ton phn vi hiu sut cao hn nhiu so vi dung mi
chit mt pha thng thng [15]. Vi h dung mi hai pha nh trn, daidzein d
dng c tch v tinh ch t pha n-butanol [15]. uerarin c tinh ch t phn
pha nc bng cch iu chnh pH v lc vi n-butanol. Qu trnh tinh ch tng
i n gin nhng li cho ra sn phm tinh ch sch khi phn tch sc k [16].
Vi nhng kt qu thu c, cc tc gi tin hnh tip nhng nh gi ton
din hn v cc yu t c th nh hng n qu trnh chit isoflavonoid t sn
7

dy bng h dung mi hai pha n-butanol/nc. Cui cng, rt ra nhng kt lun


nh sau thnh phn ca h dung mi hai pha v nhit chit l nhng yu t
chnh nh hng ti tc v hiu sut chit isoflavonoid ton phn; h dung
mi n-butanol/nc vi t l 1:1 l c hiu qu hn c; hiu sut chit v h s
phn b tng ln tt c cc h dung mi kho st khi tng nhit ; nhng
kt qu tnh ton c t m hnh l hon ton ph hp vi d liu thc nghim
[17].
Ti Vit

am, nm 2009 Dc s L Thy Linh nghin cu chit

isoflavonoid t sn dy bng dung mi phn cc theo phng php chit hi lu.


Kho st ba dung mi chit l ethanol 90o, methanol v ethyl acetat. Da vo t
l phn trm hm lng cn thu c v kt qu nh tnh isoflavonoid trong dch
chit tc gi chn ethanol 90o l dung mi s dng chit isoflavonoid t sn
dy [5].
1.1.5.2. Chit di p sut cao
So vi phng php chit hi lu, chit di p sut cao cho hiu sut
chit cao hn hn. Khi s dng cng dung mi chit l ethanol 95%, chit di
p sut cao gip chit c lng puerarin cao gp 1,8 2,4 ln, lng daidzin
cao gp 1,4 1,8 ln v lng daidzein cao gp 2,6 3,7 ln so vi phng php
hi lu [31].
gi ta cng nhn thy rng, hiu sut chit isoflavonoid ph thuc vo
nhit chit khi chit di p sut cao. Hiu sut chit tng khi tng nhit
[31].
1.1.5.3. Chit bng siu m
Khi so snh phng php chit bng siu m vi phng php chit
truyn thng, Yang Hu v cng s nhn thy rng s dng siu m c th rt
ngn thi gian chit xung 20 ln so vi phng php chit hi lu.

iu sut

chit ca phng php chit bng siu m t 19% sau 20 pht, trong khi hiu
sut chit ca phng php chit hi lu ch t c 14,3% v 16,5% sau 1 v 2
gi. Chit bng siu m t c hiu sut ny sau 5 pht. Mt khc, siu m
khng lm phn hy ti cc thnh phn hot cht c trong dch chit [45].
8

Cng trong nghin cu ny, Yang Hu v cng s chng minh rng,


hiu sut chit khi s dng siu m b nh hng bi dung mi chit [45], t l
dung mi v nng lng siu m [31]. Trong cc dung mi chit nc,
methanol, ethanol v ethyl acetat ngi ta nhn thy dung mi chit ti u l
ethanol nc t l 7:3. Mc d nc l dung mi chit cho hiu sut cao nhng
li ha tan nhiu tinh bt trong dch chit gy kh khn cho qu trnh lc, dch
chit thu c khng trong v cao do dnh kh thu hi [45].
Khng ch c hiu qu hn phng php chit hi lu truyn thng, MeiHwa v cng s cn chng minh c rng, chit bng siu m cn cho hiu sut
chit cao hn phng php chit di p sut cao [31]. Di y l bng so snh
ba phng php chit hi lu truyn thng, p sut cao v siu m:
Bng 1.2. o snh ba phng php chit xut sn dy thng dng
Yu t

Truyn thng

Di p sut cao

Siu m

Thi gian chit

Tn thi gian

Ngn ( 1 gi)

Trung bnh (1-2h)

Hiu sut chit

Thp

Trung bnh hoc cao

Cao

Dung mi

Tn km

Nhit

Cao hoc
nhit phng

Tn nhiu
duy tr p sut cao
Nhit cao
p sut cao

Chi ph

Thp

Cao

m 2007, Xu

Trung bnh
Nhit phng
Trung bnh

uaneng v cng s tin hnh nghin cu chit

isoflavonoid t r Pueraria lobata bng siu m (20k z, in nng u vo thay


i t 0-650W). Tc gi nhn thy in nng u vo v thi gian chit l cc
yu t chnh nh hng ti hiu sut chit khi s dng dung mi chit l nc, nbutanol, ethanol 96% v ethanol 50%. Dung mi chit l ethanol 50% v nc
cho hiu sut chit cao ethanol 95% v n-butanol. Tc chit v hiu sut chit
tng ln r rt khi phi hp thm siu m vi tt c cc dung mi chit k trn.
ng lng in u vo cng ln trong khong 0-650W th hiu sut chit t

c cng cao. ng thi, nghin cu cng ch ra rng hiu sut chit


isoflavonoid ton phn cao hn khi s dng siu m [41].
1.1.5.4. Chit bng vi sng
m 2001, Zhenku Guo v cng s tin hnh chit isoflavonoid t sn
dy bng vi sng. Kt qu gy bt ng khi thi gian chit ca phng php ch
ko di 1 pht, ngn hn rt nhiu so vi cc phng php chit truyn thng.
Nhm nghin cu cng nhn thy rng dung mi chit, thi gian chit, nhit
v p sut chit l nhng yu t nh hng ng k ti hiu sut chit ca
phng php. Trong s cc dung mi th nghim, ethanol 70% cho l dung mi
hiu sut chit isoflavonoid cao nht, song chit puerarin th nc li l dung
mi tt hn [47].
Vi sng khng ch gip rt ngn thi gian chit m cn lm cho qu trnh c
cao tr nn nhanh chng hn rt nhiu. Yang Hu v cng s chng minh c
rng, s dng vi sng c th rt ngn thi gian sy kh dch chit xung 10 ln
so vi phng php ct thu hi dung mi thng thng [45]. n na, sng viba
khng gy ra nhng tc ng no lm nh hng ti cu trc v thnh phn cc
cht trong dch chit sn dy thu c [45].
1.1.5.5. Chit bng dung mi siu ti hn
Trong nhng nm gn y chit xut bng dung mi ti hn c p dng
rng ri trong thc phm v y hc. CO2 siu ti hn l dung mi ch yu c s
dng trong phng php ny.

u im ca CO2 siu ti hn l khng c, khng

gy chy n, gi thnh r v d loi khi sn phm cui.


Nghin cu chit puerarin t r c sn dy Pueraria lobata, Lingzhao Wang
v cng s nhn thy bn yu t chnh nh hng ti hiu sut chit puerarin t
r c Pueraria lobata l p sut, nhit , thi gian chit v th tch ng dung
mi. p sut 20 Mpa hiu sut chit t c l 5,8 0,3 mg/g, p sut cao hn
v thp hn u lm gim hiu sut chit puerarin. Tng t nh vy, chit
nhit 50C s cho hiu sut chit cao nht, nhit cao hn hoc thp hn u
lm gim hiu sut. Thi gian chit cng ln, kh nng chit kit puerarin cng
cao, c bit l trong khong thi gian di 100 pht. Hiu sut chit puerarin
10

tng ln khng r rt khi ko di thi gian hn 100 pht. iu ny c th gii


thch do tan ca puerarin trong dung mi ti hn v tc chuyn khi u l
cc yu tt ph thuc vo thi gian. Do vy, thi gian chit xut cng ln s gip
ha tan c nhiu dc cht vo dung mi chit hn, v do tng hiu sut
chit ln. ng dung mi c s dng l ethanol tuyt i.

gi ta thy rng

hiu sut chit puerarin s t gi tr ti a khi thm mt lng xc nh ethanol


(120 g) [26].
T nhng kt qu k trn, cc tc gi thc hin ti u ha v tm c
iu kin chit puerarin ti u t r c Pueraria lobata bng dung mi siu ti
hn 20,04 Mpa, 50,24C v th tch ng dung mi s dng l 181,24 ml
ethanol trong tng khong thi gian chit l 90 pht [27].
1.1.5.6. Chit bng dung mi ion
Dung mi ion lng l dng mui nng chy do trong thnh ca n c c
cation v anion dng khng phn ly. Phn cation thng l cc cation hu c
[38].
Dung mi ion lng c nhit nng chy kh thp, thng l di 100C v
thng dng lng nhit phng [38].
Trong chit xut hin i, dung mi ion lng c s dng thay th cho cc
dung mi hu c thng thng nh tnh chn lc cao hn cc dung mi phn cc
nh nc, methanol; chit c cc phn t hu c phc tp nh cyclodextrins,
glycolipid, khng sinh, artermisinin v alkaloid ; d dng ra sch hoc loi i
khi sn phm chit cui [38].
Tuy nhin, dung mi ion lng c nhc im l trong qu trnh chit xut,
mt lng ln dung mi ion lng k nc c th b ln nc to thnh h hai pha
dung mi ion/nc lm thay i nht, t trng, gim dung lng chit v tnh
chn lc ca dung mi trong qu trnh chit [18], [ 38]. hn ch nhc im
ny, Jie-Ping Pan v cng s to ra cc h dung mi hai pha thn nc
chit puerarin t r Pueraria lobatae t cc dung mi ion 1-carboxymethyl-3methylimidazolium tertrafluoroborat ([COOHMIM]BF4), 1-hydroxyethyl-3methylimidaziolium

tertrafluoroborat
11

([C2OHMIM]

BF4),

1-butyl-3-

methylimidazolium hydroxid ([BMIM]OH) v 1-butyl-3-methylimidazolium


bromua ([BMIM]Br) vi cc mui v c K2CO3, Na2CO3, K2HPO4, (NH4)2SO4,
Na2SO4, NaCl, NaH2PO4. Trong cc h hai pha dung mi ion kho st, h
K2HPO4 0,30-0,42 g/mL, [BMIM]Br 0,30-0,36 g/ml v 1,0 ml dung dch gc
puerarin, cho hiu sut chit puerarin cao nht t trn hn 99% vi mt ln chit
duy nht [18].
Li dng u im ca phng php chit bng siu m v phng php
chit bng dung mi ion, Jie-Ping Pan v cng s tip tc nghin cu chit
puerarin t r c sy kh Pueraria lobata vi cc dung mi ion [BMIM]Br,
[BMIM]BF4, [C2OHMIM]Cl, [C2OHMIM]BF4, [COOHMIM]BF4 km siu m.
S dng phng php phn tch mt p nh gi cc yu t nh hng ti
hiu sut chit puerarin v ti u ha cc iu kin chit xut. Kt qu chit xut
ti u thu c vi 0,43g dc liu trong 10ml dung mi ion [BMIM]Br 1,06
mol/L, siu m trong 27,43 pht 480W [19].
1.2. Puerarin
1.2.1. Cng thc ha hc

Cng thc phn t [33]: C21H20O9

Tn khoa hc: 7-hydroxy-3-(4-hydroxyphenyl)-8-[(2S,3R,4R,5S,6R)-3,4,


5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]chromen-4-one [33]

Tn khc: Kakonein, 3681-99-0, 8-(beta-D-Glucopyranosyl-7-hydroxy-3(4-hydroxyphenyl)-4H-1-benzopyran-4-one,AC1NQZ4N,


SureCN8581666, MLS002473178 [33].

1.2.2. Tnh cht l ha

12

1.2.2.1. L tnh: Puerarin l mt isoflavonoid t nhin, c tm thy trong r


sn dy Pueraria lobata v Pueraria thomsonii. V tan, puerarin:

t tan trong nc, aceton, tan trong nc tng ln khi tng nhit
[39].

Tan c trong ethanol, tan gim khi tng nhit [39]. Khi thm mt
lng nh dung dch CO2 siu ti hn vo dung dch ethanol s lm tng
tan ca puerarin. ong khi vt qua gii hn ny, vic thm CO2 siu
ti hn li lm tan ca puerarin gim mnh [28]. tan ca puerarin
trong hn hp ethanol + CO2 siu ti hn tng ln khi tng nhit [28]

Tan c trong hn hp methanol acid acetic, tan ph thuc vo


nhit v t l ca acid acetic trong hn hp. Tng nhit lm tng
tan, ngc li tng th tch acid acetic trong hn hp lm gim tan ca
puerarin trong hn hp dung mi [12].

1.2.2.2. Ha tnh: Do cu trc phn t ca puerarin c mt nhm carbonyl


(>C=O) v 6 nhm hydroxyl (O ) trong c 2 nhm hydroxyl phenol
nn puerarin c th
To lin kt hydro vi cc hp cht c nhm carbonyl v hydroxyl
khc
Th hin tnh acid ca 2 nhm O phenol
V vy, puerarin c th tan trong cc dung dch kim v acid mnh [18].
1.2.3. K thut phn tch
m 2007, xc nh hm lng isoflavonoid ton phn trong r c sn
dy nhanh chng, chnh xc Chen Sibao v cng s tin hnh nh gi v so
snh chnh xc ca phng php o V vi phng php

LC. Qut ph dung

dch chun ca 7 loi isoflavonoid thng gp v chim hm lng ln trong r sn


dy, cc tc gi nhn thy rng ch tr genistin c cc i hp th 260nm cn 6
isoflavonoid cn li u c cc i hp th ti 250nm. Nng isoflavonoid ton
phn trong dch chit r c sn dy ti c nh lng theo phng php o

bng cch so snh vi mt quang ca dy dung dch chun puerarin c nng


t 4,77 12,72g/ml. Kt qu cho thy, h s tng quan khi nh lng
13

isoflavonoid ton phn bng phng php o quang vi khi nh lng bng sc k
lng hiu nng cao l 0,975. iu ny chng t, kt qu nh lng isoflavonoid
ton phn bng phng php o quang 250nm l tng quan vi kt qu khi nh
lng bng phng php sc k lng hiu nng cao [11].
Cng thi gian ny, Xu Huaneng v cng s cng s dng phng php o
quang 250nm nh lng hm lng isoflavonoid ton phn trong dch chit r
c sn dy nh tc dng ca siu m.

m lng isoflavonoid trong mu th c

ngoi suy t ng chun ca dung dch puerarin [41].


m 2008, trong nghin cu so snh kh nng chit isoflavonoid t sn dy
bng cch s dng siu m v bng cch dng vi sng Yang Hu v cng s cng s
dng phng php o quang 250nm nh lng isoflavonoid trong cc dch
chit thu c. Dch chit isoflavonoid ton phn c pha long n nng thch
hp ri o quang 250nm. Nng isoflavonoid trong mu th c tnh ton t
ng chun puerarin [45].
h vy, nh lng isoflavonoid ton phn t dch chit hoc trong
nguyn liu c th s dng phng php o quang 250nm, so snh vi ng
chun ca dung dch puerarin. Kt qu nh lng bng phng php ny c
chnh xc cao, phng php tin hnh nhanh chng, n gin.
nh lng ring tng hot cht hoc nh gi kt qu ca qu trnh tinh
ch, phng php

V khng cn c th s dng c, lc ny

LC l phng

php thch hp hn c.
m 2001 Chen Bin v cng s tin hnh nghin cu xy dng hng
php

LC n gin, nhanh chng v c nhy cao dng nh lng puerarin

v daidzin trong Pueraria lobata. Isoflavonoid ton phn chit c t P.lobata


bng ethanol 70%, vi sng trong 2 pht. Phn lp bng ct Symmetry 18. Thi gian
lu ca puerarin v daidzin l 4,38 pht v 8,15 pht. ng chun trong khong
0,115 ~ 1,38g/ml c R=0,9991 i vi puerarin v trong khong 0,12 ~ 1,44 g/ml

14

c R=0,9998 i vi daidzin. Phn trm tm li trung bnh khong 103,31% vi h


s phn tn CV=1,37% puerarin v 98,73% vi CV=2,43% daidzin [10].
tch v nh lng puerarin, daidzin v daidzein trong thn v l ca
Pueraria thomsonii, Zhou Hong-Ying v cng s s dng phng php sc k
pha o RP-HPLC vi ct Diamosil TM C18 (4,6 mm x 150 mm, 5m) v gradient
ra gii, pha ng l methanol 1% dung dch acid acetic bng. Tc dng
1ml/pht. Bc sng pht hin 250nm, nhit ct 25C. Cc tc gi nhn thy
rng c ba hp cht u c ng chun vi h s tng quan tt (r > 0,9995) v
phn trm tm li trong khong 99,0% - 101,6%. Hm lng puerarin, daidzin v
daidzein trong thn cao hn trong l. Nghin cu chng minh c rng phng
php sc k pha o xy dng c c nhy cao v c th s dng s dng
nh lng puerarin, daidzin v daidzein trong thn v l ca Pueraria thomsonii
[48].
m 2008,

an Chen v cng s xy dng c phng php

LC kh

n gin v chnh xc xc nh hm lng puerarin trong r Pueraria lobata. S


dng ct sc k C18, pha ng l methanol/nc (25:75), detector UV bc sng
250nm. Kt qu cho thy mi quan h tng quan cht ch gia nng puerarin
v mt quang trong khong 20 ~ 140g/ml (r=0,9999). Phn trm tm li l
97,40%, RSD = 3,0% [29].
1.2.4. Tc dng sinh hc ca puerarin
1.2.4.1.

Tc dng trn tim mch

Gn y c rt nhiu bo co xc minh tc dng gin mch ca puerarin trn


chut. Tc dng gin mch ca puerarin trn ng mch ch ngc ca chut do
hot ha in p dn ln v knh kali canxi kch hot ng vai tr quan trng trong
iu ha nhp tim. Hot ng ca knh kali canxi kch hot gy ra hin tng u
phn cc trn mng t bo.
Tc dng th gin ni mc nguyn vn ca vng ng mch ch chut b
lm hp trc bi phenylephrine (10-9 10-6) hoc KCl (10 100 mM) ca

15

puerarin (50, 150 v 450M) khng ph thuc vo nng . Tuy nhin, tc dng
th gin ny khng quan st thy vng ng mch ch ca chut b loi b lp
ni mc, iu ny ch ra rng tc dng gin mch ca puerarin ph thuc vo hot
ng ca lp ni mc. Ngoi ra, tc dng chng co mch ca puerarin i vi
phenylephrine hon ton b v hiu ha bi dung dch Ca2+ t do. iu ny c ngha
l tc dng chng co mch ca puerarin lin quan n vic kch hot dng Ca2+ t
ngoi bo i vo ni m [43].
Puerarin bo v tim mch qua knh natri v calci dng L chut lang v t bo
c tm tht chut. Tc dng bo v tim mch ca puerarin chng li cc tn
thng do thiu mu cc b v ti ti mu t bo c tm tht ,.;m/thng qua
vic hot ha cc knh kali nhy cm ATP ti th v c ch vic m knh vn
chuyn trn mng ti th. tim chut c lp c s dng puerarin (0,24
mmol/L) trc khi gy thiu mu cc b, vic sn xut formazan c tng cng
v gim gii phng lactat dehydrogenase. Tuy nhin tc dng ny b mt khi iu tr
paxilline 1mol/L (tc nhn block knh kali canxi hot ha) [43].
Puerarin c th lm phc hi s biu hin mRNA ca yu t tng trng bin
i tim 1 v

AD3 ng thi gim s biu hin mRNA SMAD7 tim trn

chut tng huyt p t pht. Cc SMAD l cc protein ni bo vn chuyn tn hiu


t yu t tng trng bin i trong t bo, SMAD3 l loi SMAD kiu receptor
cn

AD7 l

AD i khng. Cc thay i biu hin gen ny s gip ci thin

ph i v x ha c tim, c th l c ch bo v c tim ca puerarin [43].


1.2.4.2.

Gim au, h st

Tim mng bng cho chut dung dch puerarin (25-200M) lm gim nhit
c th v nng IL-1, TNF-. IL-6, prostaglandin E2 v NO, v chn s hot ha
NF-B v s phosphoryl ha MAPKs gy ra bi LPS 100 g/kg) chut, chng t
rng tc dng h st ca puerarin lin quan ti vic iu chnh hot ng tng hp
v gii phng cc cht gy st ni sinh thng qua c ch hot ng ca NF-B v
con ng MAPK [43].
1.2.4.3.

Chng vim

16

c tnh chng vim ca puerarin c xc minh trn m hnh tai chut ph


v mt vi m hnh t bo vim. Puerarin tc dng khng ph thuc liu ngn chn
qu trnh phosphoryl ha v ging ha ca inhibitor-B v chuyn v ca ht nhn
NF-B p65 subunit, lm ngn cn qu trnh biu hin protein v mRNA ca protein
phn ng C vi LPS kch thch t bo mu n nhn ngoi bin. uerarin ngn chn
s biu hin ca protein phn ng C, iNOS, v cyclooxygenase-2, c ch ng tn
hiu NF-B t bo RAW264,7 c LPS hot ng v ngn chn r rt s bin i
hnh thi ca lipopolysaccharid t bo RAW264,7. Ngoi ra, s biu hin mRNA
NF-B p65 trong t bo v nng i thc bo protein vim 2 v TNF- trong
t bo ni b c ch bi vic s dng puerarin trc [43].
1.2.4.4.

Chng oxy ha

Puerarin thu dn c cc anion superoxid to ra bi nh snh riboflavin v gc


hydroxyl sinh ra t phn ng Fenton, lm gim phn ng oxi ha tn huyt hng
cu v nng malondialdehyd to ra t H2O2 trong t bo hng cu, sa cha
nhng bin i trn LDL gy ra bi tia cc tm v ng II sulfat, cho thy tc dng
chng peroxy ha v tim nng s dng phng nga va x ng mch ca
puerarin. uerarin ngn cn qu trnh oxi ha LDL gy ra bi ion Cu2+ trn invitro,
tng sn xut prostacyclin huyt tng v DL, c ch kt t v bm dnh tiu cu,
ngn chn qu trnh hnh thnh cc nghn. Ngoi ra puerarin cn c tc dng lm
gim sn xut LDL v cholesterol ton phn, do lm tng sn xut HDL trn m
hnh th tng lipid.
1.2.4.5.

Tc dng kiu estrogen

Puerarin vn thng c bit n nh l mt phytoestrogen bi tc dng kiu


estrogen ca n trn nhiu m hnh ng vt. Tim ngn hn puerarin (0,7 mg/kg)
lm tng s lng cc tuyn t cung chut cha trng thnh v tim ko di
puerarin (7 mg/kg) lm tng r rt t l phn trm cc t bo sng ha chut ci
trng thnh, iu ny chng t puerarin c tc dng kiu estrogen. S dng
puerarin ng ung trong 3 thng lm tng c p lc bng quang v niu o, gim
bo ph v tng cng trng lng t cung chut. Cc tc dng ny ca puerarin
17

u tng ng hoc yu hn 17-estradiol (E2). Nhng kt qu ny chng t


rng puerarin c th gip ci thin tnh trng b niu, mang li nhng li ch ng
k trong iu tr cc ri lon do mn kinh. uerarin lm tng r rt cn nng, IGF-1
v biu hin C3 v nng cht chng oxy ha trong huyt thanh, ng thi lm
gim nng melondialdehyd v tn thng D A t bo lympho trn chut,
chng t rng puerarin c tc dng trn t cung v c th l mt tc nhn tr liu c
hiu qu trong vic chng tng sn ni mc t cung v hi chng tin kinh nguyt.
1.2.4.6.

c ch cc ri lon do ru

Kh nng sng ca t bo, s tch ly lipid bn trong t bo cc t bo gan


ethanol c khi phc v bnh thng sau khi iu tr bng puerarin.
Ngoi ra, puerarin cn kch thch qu trnh phosphoryl ha AMPK, v block cc
protein ribosome S6 v protein 4E-binding l nhng protein mc tiu ng vt
c v ca rapamycin (mammalia targets of rapamycin - mTOR), phc hi c ch
t tiu hy cc t bo sn xut ethanol. Nhng kt qu ny ch ra rng c ch
bo v ca puerarin c lin quan n con ng AMPK/mTOR. Trong t bo
m m ty xng gy ng c bng ru, puerarin c ch tng sinh s lng
t bo m, c ch sn xut triglycerid v tng cng hot ng ca phosphatase
kim, osteocalcin v biu hin mRNA ca osteocalcin. iu cho thy
puerarin c tc dng phng nga cc ri lon lin quan ti nghin ru [43].
1.2.4.7.

Chng tiu ng v c ch cc bin chng tiu ng

uerarin c s dng iu tr i tho ng Trung Quc t nhng


nm 1990. Tim tnh mch puerarin gip lm h ng huyt, tng mR A v
tng biu hin protein vn chuyn glucose trong c, kch thch tuyn thng
thn tng tit -endorphin, lm gim ng huyt chut b gy tiu ng bi
streptozotocin. chut c Sparague-Dawley c cho n vi ch n giu
cht bo, cc du hiu nh tng trng lng c th v gim glucose dung
np/tng khng insulin gim, s tng nng resistin v leptin huyt
tng cng c ngn chn khi iu tr bng puerarin (100 v 20 mg/kg) [43].

18

uerarin gip tng cng ng k s hp thu glucose vo cc t bo nhy


cm insulin, gim lng ng trong mu, triglycerid, insulin, cholesterol ton
phn v trng lng c th chut Zucker bo ph v chut mc tiu ng
type 2 theo di truyn. S chuyn ha glucose v insulin c ci thin ng k
nhm chut c thm puerarin sn dy vo ch n hng ngy [43].
1.2.5. Cc nghin cu tinh ch puerarin hin nay
1.2.5.1.

Tinh ch bng sc k

Sc k iu ch l phng php c s dng rt nhiu v ng vai tr


quan trng trong nghin cu cc cht t nhin. Cc k thut sc k thng dng
l sc k lp mng iu ch, sc k lng cao p iu ch, sc k ct u im
ca phng php sc k l tch c nhiu hot cht vi tinh khit cao.
m 1999, Xueli Cao v cng s s dng sc k ngc dng tc
cao vi h dung mi ethyl acetat n-butanol nc theo t l 2:1:3 (v/v/v) tch
c ngay 6 hp cht tinh khit t 80 mg dch chit trong ln chy sc k u
tin. Cc hp cht tinh ch c gm 3-hydroxyl-puerarin, puerarin, 3methoxy-puerarin, puerarin-xylosid, puerarin-xylosid, daidzin. Cc cht tinh ch
c c tinh khit cao trn 90%, ng k nht l puerarin c tinh khit trn
98% [42].
m 2004, Xiangling He v cng s s dng sc k hp ph trn
epichlorohydrin gn ligand -cyclodextrin.

ha ng l dung dch acid acetic

10%. Dung lng phn tch khong 1,2 mg dch chit. uerarin thu c c
tinh khit khong 98% [40].
C th thy, phng php sc k tch hot cht vi tinh khit cao
(>90%) t mt lng rt nh dch chit (<100mg). hng php ny thch hp
tinh ch ra cht chun cho nh lng nhng vi lng mu nh kh trin
khai trn quy m ln.
1.2.5.2.

Tinh ch bng bin php thay i dung mi

19

Mc d phng php tinh ch bng sc k cho ra cc sn phm c tinh


khit cao song ch thc hin vi c mu nh, chi ph tn km ch yu dng
nghin cu thnh phn ca dc cht hn l tinh ch sn xut trn quy m cng
nghip. V do vy, bn cnh phng php sc k ngi ta lun phi nghin cu
tm ra cc phng php tinh ch hot cht khc r tin, d trin khai trn quy m
ln vi mc tinh khit chp nhn c.
hng php n gin nht c th k n l phng php ca HuaNeng Xu v cng s. Chit r c sn dy Pueraria lobata bng hn hp dung
mi n-butanol/nc (1:1) 25C trong 6 gi. Dch chit thu c lng cho
tch pha thu ly phn nc tinh ch puerarin (daidzein phn b ch yu pha
n-butanol). Phn dch ny c lc vi n-butanol trong 1 gi, sau iu chnh
pH ca pha nc bng acid HCl v 4,0, lc ny puerarin s chuyn sang pha nbutanol. Thu ly pha n-butanol, tip tc lc vi ng th tch nc trong 1 gi,
pH ca pha nc c iu chnh v gi tr 8,0. Lc ny puerarin li chuyn sang
pha nc. Thu ly phn chit nc. Phn tch HPLC cho thy sau 2 ln chuyn
pha puerarin thu c tng i tinh khit [16].
Ti Vit Nam, tinh ch puerarin t dch chit sn dy cha c quan tm
nghin cu mc d y l thnh phn chim hm lng ln v cho tc dng sinh
hc ch o ca sn dy. Hin ti ch c duy nht nghin cu ca Dc s L Thy
Linh tin hnh tinh ch c daidzein bng phng php sc k ct vi h dung
mi CCl3 : Methanol (98:2) [5].
1.3. Cao thuc
1.3.1. Khi nim
Cao thuc l ch phm c ch bng cch c hoc sy n th cht quy nh
cc dch chit thu c t dc liu thc vt hay ng vt vi cc dung mi thch
hp[2].
1.3.2. Phn loi
Theo th cht, cao thuc c chia lm 3 loi:

20

Cao lng: L cht lng hi snh, c mi v c trng ca dc liu s dng


trong cn v nc ng vai tr dung mi chnh (hay cht bo qun hay c hai).
Nu khng c ch dn khc, quy c 1 ml cao lng tng ng vi 1 g dc liu
dng iu ch cao thuc.
Cao c: L khi c qunh.

m lng dung mi s dng cn li trong cao

khng qu 20%.
Cao kh: L khi hoc bt kh, ng nht nhng rt d ht m. Cao kh
khng c c m ln hn 5% [2].
1.3.3. K thut iu ch
Quy trnh iu ch cao kh gm nhng giai on sau:
1.3.3.1. Chun b dung mi, dc liu

Dc liu thng c sy kh, chia nh n kch thc thch hp. Mt s


dc liu c bit cn dit enzym hoc loi cht bo. Dung mi iu ch cao
thng l nc, ethanol, ete ethylic. lm tng tan ca hot cht c th acid
ha hoc kim ha dn mi bng acid hydrochloric, acid acetic, amoniac, natri
hydroxyd.
1.3.3.2. Chit xut hot cht

Ty theo bn cht dc liu v dung mi, tiu chun cht lng thnh phm
cng nh iu kin trang thit b v quy m sn xut, c th s dng cc phng
php: Ngm, ngm kit, chit xut ngc dng hay cc phng php thch hp
khc.
1.3.3.3. Loi bt tp cht
Cn phi loi tp cht v chng thng d phn hy nh hng n cht
lng cao thuc. Trng hp iu ch cao c, cao kh, nu hm lng hot cht
cha quy nh cng c th cng c th tin hnh loi bt tp cht.
1.3.3.4. C c v sy kh

iu ch cao thuc thng phi tin hnh loi dung mi. Dch chit c
c c n t l dung mi quy nh, vi cao c v cao kh th sau khi c c,

21

sy n m khng qu 20% i vi cao c v khng qu 5% i vi cao


kh.
1.3.3.5. Xc nh v iu chnh t l hot cht.

i vi cao thuc c quy nh hm lng, sau khi iu ch phi nh lng


hot cht, nu cha t phi iu chnh cao c t l hot cht ng quy nh.
1.3.3.6. Hon chnh ch phm

Thm cc cht bo qun chng nm mc nh acid boric, natri benzoat,


nipagin, nipasol. Vic thm cht bo qun vo cao thng c thc hin cui
giai on c c [1].
1.3.4. Cc ch tiu cht lng ca cao thuc
Cm quan: Cao thuc phi c th cht, mu sc, ng nht theo quy nh.
tan: Cao lng phi tan hon ton trong dung mi dng iu ch cao.
Mt khi lng do lm kh: Thng thng cao c khng qu 20%, cao kh
khng qu 5%.
Cc ch tiu khc: nhim khun, gii hn thuc bo v thc vt, kim loi
nng, cht bo qun, nh tnh, nh lng [2].

22

Chng 2. NGUYN VT LIU, TRANG THIT B V


PHNG PHP NGHIN CU
2.1. Nguyn vt liu, thit b
2.1.1. Nguyn liu
a. C sn dy ti v kh thu Ph Th, Hi Dng, inh Bnh. Dc liu ti
em ra sch, thi lt ri xay th trc khi em vo chit xut.
b. Cht i chiu: Puerarin 82,31% (Trung Quc).

c. Ha cht:
Bng 2.1. a cht s dng
STT Ha cht

Ngun gc

Tiu chun

Ethanol 96% Vit Nam

DV IV

Cloroform

Trung Quc

Tinh khit phn tch

Ethyl acetat

Trung Quc

Cng nghip

Methanol

c ct

erck (c) Phn tch; HPLC


c t ct

2.1.2. Dng c
Bnh cu 100 ml, 500 ml, 1000 ml.
Cc thy tinh loi 100 ml, 250 ml, 500 ml, 1000 ml.
Bnh nh mc 10 ml, 25 ml, 50 ml, 100 ml.
Pipet 1 ml, 2 ml, 5 ml, 10 ml.
ng ong 10 ml, 300 ml, 500 ml.
Mng lc cellulose acetat 0,45 m ( atorius).
Bn mng silicagen GF254.
Bnh ht m.
2.1.3. Phng tin v my mc

y siu m ltrasonic LC 60 (c).

My ct quay Buchi B480 (Thu S).

23

Cn phn tch Mettler Toledo AB204S (Thu S).


Cn k thut artorius B 2001 (c).

y o quang ITACHI U-1900.

n t ngoi.
T sy

ERT (c).

My xay ba.
My xay sinh t.
H thng lc ht Buchner.
Cn xc nh hm m nhanh Precisa XM 60 (Thy ).

y o t trng biu kin ERWEKA V

(c).

H thng phun sy LPG-5 Centrifugal Spray Dryer (Changzhou Jinqiao,


Trung Quc) cng sut 5 L/gi.
My sc k lng hiu nng cao himadzu ( ht Bn), gm:
+ B phn loi kh DGU 14A
+ Bm cao p LC 10ADVP
+ Bung cha ct CTO 10AVP
+ B iu khin SCL 10AVP
+ Detector dy diod quang SPD M10AVP
+ Phn mm Class vp 6.14.
2.2. Ni dung nghin cu
2.2.1. Chit xut isoflavonoid t nguyn liu kh
Xc nh hm lng isoflavonoid ton phn v puerarin trong sn dy kh
Chit xut isoflavonoid t sn dy kh
Tinh ch dch chit, kho st mt s phng php loi tp
2.2.2. Chit xut isoflavonoid t nguyn liu ti
Xc nh hm lng isoflavonoid ton phn v puerarin trong sn dy ti
Chit xut isoflavonoid t sn dy ti
Tinh ch dch chit, kho st mt s phng php loi tp: s dng nhit ,
s dng ethanol, kt hp c ethanol v nhit .

24

2.2.3. iu ch cao kh
iu ch cao kh bng phng php phun sy
nh gi mt s ch tiu cht lng cao kh
2.2.4. Phn lp puerarin v daidzin
Phn lp daidzin v chng minh cu trc sn phm
Phn lp puerarin v chng minh cu trc sn phm
2.3.

Phng php nghin cu

2.3.1. Phng php nh lng isoflavonoid ton phn v puerarin


2.3.1.1. nh lng isoflavonoid ton phn bng phng php o quang
Qua tham kho cc ti liu [11], [41], [45] chng ti xy dng phng
php nh lng isoflavonoid ton phn trong dc liu v trong dch chit
bng quang ph hp th V nh sau.
Cn chnh xc khong 12,1 mg cht i chiu (cha 82,31% puerarin)
tng ng vi khong 10,0 mg puerarin ha tan trong khong 20ml
ethanol 96%, sau chuyn vo bnh nh mc 50ml. Thm ethanol 96%
n vch, lc u c dung dch A. Ly chnh xc 1,0 ml dung dch A cho
vo bnh nh mc 25 ml, thm ethanol 96% va n vch, lc u c
dung dch cht i chiu c hm lng puerarin khong 8g/ml. Lc qua
mng 0,45m, qut ph chn ra bc sng thch hp cho nh lng.
Chun b dy dung dch chun: Ly chnh xc 20,0 ml dung dch A cho
vo bnh nh mc 100 ml, thm ethanol 96% n vch, lc u c dung
dch B. T dung dch B pha long thnh dy dung dch chun c nng ln
lt l 1,6; 3,2; 4,8; 6,4; 8 g/ml theo nh bng sau:
Nng dung dch
chun (g/ml)
Vdung dch B (ml)

1,6

3,2

4,8

6,4

8,0

2,0

4,0

6,0

8,0

10,0

Vethanol 96% (ml)

8,0

6,0

4,0

2,0

Vnc ct (v)

50,0

50,0

50,0

50,0

50,0

25

Mu trng l dung dch ethanol 19,2% (10ml dung dch ethanol 96%
pha long vi nc ct va 50ml). Mu trng v mu chun sau khi pha
c lc qua mng 0,45m, o hp th ti bc sng chn, o lp li 3
ln, ly kt qu trung bnh xy dng ng chun.
Vi mu th l sn dy ti: Cn chnh xc khong 10 g dc liu,
thm 100 ml ethanol 70%, xay mn bng my xay sinh t. Chuyn ht vo
bnh nh mc 500 ml, thm ethanol 70% n va , siu m 30 pht trong
b siu m. au lng 15 pht iu kin phng, gn ly phn dch
trong. Ly chnh xc khong 5 ml dch trong va gn vo bnh nh mc 100
ml, thm 5 ml ethanol 96% v nc ct va , lc u c dung dch th.
Vi mu th l sn dy kh: Cn chnh xc khong 0,4 g dc liu,
thm 40 ml ethanol 70%, xay mn bng my xay sinh t. Chuyn ht vo
bnh nh mc 100 ml, thm ethanol 70% n va , siu m 30 pht trong
b siu m. au lng 15 pht iu kin phng, gn ly phn dch
trong. Ly chnh xc khong 5 ml dch trong va gn vo bnh nh mc 50
ml, thm 5 ml ethanol 96% v nc ct va , lc u c dung dch th.
Mu th l cao c, cao kh: Cn chnh xc khong 0,1 g cao xc nh
hm lng, ha tan bng ethanol 96%, chuyn vo bnh nh mc 50 ml,
thm ethanol 96% n va , lc u. Ly chnh xc 1,0 ml dung dch va
pha vo bnh nh mc 50 ml, thm 9,0 ml ethanol 96% v b sung nc ct
va , lc u. Dung dch thu c em lc qua mng 0,45m trc khi o
quang.
Hm lng isoflavonoid trong nguyn liu c tnh theo cng thc
sau:

Trong

26

At, Ac : Mt quang ca dung dch th v dung dch chun


Cc

: Nng dung dch chun

Vchit : Th tch dch chit


K

: Tch cc h s pha long

mt

: Khi lng dc liu em vo chit xut.

m lng isoflavonoid trong cao c tnh theo cng thc

Trong
At, Ac : Mt quang ca dung dch th v dung dch chun
Cc

: Nng dung dch chun

mcao

: Khi lng cao.

2.3.1.2. nh lng puerarin bng HPLC


nh lng puerarin trong nguyn liu, cao kh ton phn v sn
phm tinh ch, chng ti s dng phng php sc k lng hiu nng cao.
Qua tham kho cc ti liu [29], [45], [16] v qu trnh kho st ban u,
chng ti la chn cc iu kin sc k nh sau:
Thng s my sc k:
B phn loi kh DGU 14A
Bm cao p LC 10ADVP
Bung cha ct CTO 10AVP
B iu khin SCL 10AVP
Detector dy diod quang SPD M10AVP
Phn mm Class vp 6.14.
Ct sc k Alltech C18 vi kch thc ct 250 x 4,6 mm, kch
thc ht nhi 5 m

ha ng: methanol nc = 30:70, pha ng c lc qua mng 0,45


m, siu m trong 15 pht.

27

Tc dng: 1,2 ml/pht.


Th tch tim mu: 5 l
Detector UV pht hin bc sng 250 nm.
Chun b dy dung dch chun: Cn chnh xc khong 0,125 g cht i
chiu puerarin 82,31%. Ha tan trong khong 50ml ethanol 96% sau chuyn
vo bnh nh mc 100 ml. Thm ethanol 96% va n vch c dung dch
A. Ly chnh xc khong 5,0 ml dung dch A, pha long 10 ln bng nc ct
c dung dch B.
T dung dch B pha long thnh dy dung dch chun c nng t 0,01
mg/ml n 0,2 mg/ml theo bng sau:
Nng dung dch chun
(mg/ml)
Vdd A (ml)

0,01

0,02

0,05

0,10

0,15

0,20

Vdd B (ml)

Vpha ng v (ml)

10

10

10

10

10

10

Cc dung dch chun ny u c lc qua mng 0,45 m trc khi phn


tch sc k.
Mu th l sn dy kh: Cn chnh xc khong 10 g dc liu, thm 40 ml
ethanol 70%, xay mn bng my xay sinh t. Chuyn ht vo bnh nh mc 100
ml, thm ethanol 70% n va , siu m 30 pht trong b siu m. au
lng 15 pht iu kin phng, gn ly phn dch trong. Ly chnh xc khong 5
ml dch trong va gn pha long vi pha ng thnh 25ml. Lc dung dch qua
mng 0,45m trc khi phn tch sc k.
Mu th l sn dy ti: Cn chnh xc khong 10 g dc liu, thm 40 ml
ethanol 70%, xay mn bng my xay sinh t. Chuyn ht vo bnh nh mc 100
ml, thm ethanol 70% n va , siu m 30 pht trong b siu m. au
lng 15 pht iu kin phng, gn ly phn dch trong. Ly chnh xc khong 5
ml dch trong va gn pha long vi pha ng thnh 25ml. Lc dung dch qua
mng 0,45m trc khi phn tch sc k.
Mu th l cao c, cao kh: Cn chnh xc khong 0,115g cao. Ha tan
trong khong 20ml ethanol 96% sau chuyn vo bnh nh mc 50 ml. Thm
28

ethanol 96% va n vch. Pha long dung dch ny 10 ln bng pha ng,
sau lc qua mng 0,45m trc khi phn tch sc k.
m lng puerarin trong nguyn liu, trong cao c v cao kh c tnh
theo cng thc:

Trong
St, Sc : Mt quang ca dung dch th v dung dch chun
Cc

: Nng dung dch chun (mg/ml)

: Th tch chit hoc th tch dung dch ban u pha mu th.

: H s pha long

mcn : Khi lng dc liu, khi lng cn hoc cao kh cn thc t.


2.3.2. Phng php nh tnh cc isoflavonoid bng sc k lp mng
tit kim thi gian v chi ph trong qu trnh kho st cng nh tinh ch,
chng ti s dng phng php sc k lp mng nh tnh cc thnh phn c
trong dch chit, dch tinh ch. Tham kho cc ti liu [2], [32] chng ti xy
dng phng php sc k lp mng nh sau
Bn mng: Silicagel 60F254, hot ho 110 oC trong 1 gi.
Dung mi khai trin:
H 1: Cloroform-methanol-nc (7:2,5:0,25).
H 2: Cloroform-methanol (7:3).
Dung dch th:
+ Mu th l dch chit: Ly mt th tch dch chit tng ng vi 1
mg isoflavonoid ton phn, c cch thy n cn. Ho cn trong 1 ml
ethanol.
+ Mu th l cht cha bit: Ha tan trong ethanol 96% c dung
dch c nng khong 1 mg/ml.
Dung dch i chiu: Ha tan cht i chiu trong methanol c nng
puerarin khong 1 mg/ml.
29

Sau khi khai trin sc k, ly bn mng ra, bay hi dung mi nhit


phng, soi bn mng di n t ngoi bc sng 254 nm.
2.3.3. Phng php chit xut isoflavonoid t sn dy kh
Hu ht cc isoflavonoid trong sn dy u dng glycosid, ch c mt vi
cht l aglycon nh daidzein, genistein. V vy c th la chn dung mi phn cc
chit isoflavonoid t sn dy.

u tin chn dung mi r tin, khng c, d

kim, chng ti tin hnh th nghim vi cc dung dch ethanol c nng khc
nhau v nc. Cn xc nh khi lng cao thu c vi mi dung mi, nh lng
hm lng isoflavonoid v puerarin trong dch chit v cao thu c. Tnh hiu sut
ca qu trnh chit da vo hm lng isoflavonoid chit c.
Hiu sut ca qu trnh chit c tnh theo cng thc:

Trong

misoflavonoid/dl l lng isoflavonoid c trong dc liu a vo chit


misoflavonoid/cao l lng isoflavonoid c trong cao thu c

2.3.4. Phng php chit isoflavonoid t sn dy ti


Nhm tn dng quy trnh ch tinh bt t sn dy ti, chng ti kt hp qu
trnh chit xut isoflavonoid t sn dy ti vi qu trnh thu tinh bt. Quy trnh
chit xut c tm tt theo s trong hnh 2.1.
t cc bc tin hnh
Bc 1.

Chun b nguyn liu: Sn dy ti mua v c x l trong vng 3

ngy, c c ra sch t v v en bn ngoi, ct b nhng phn b hng.


ro nc v cn xc nh khi lng. au xay mn thnh bt nho,
trc c th thi thnh lt xay d dng hn.
Bc 2.

Tin hnh chit xut: Bt nho c ha vo nc vi t l 1:2 (w/w)

ri vt loi b bng ti vi. B sn c ra li mt ln vi lng nc nh


ln mt. Gp cc dch chit, lng qua m, gn, lc qua vi ly phn
dch trong; y l dch chit cha isoflavonoid. Ly 100ml dch chit ny

30

em c gim p n cao c c khi lng m. nh lng isoflavonoid


ton phn trong cao theo mc 2.3.1.1.
Bc 3.

Thu tinh bt sn: Phn tinh bt lng xung c ra bng cch thm

nc, khuy u ri li lng; ra n khi trng (3-5 ln) th thu c tinh


bt sn dy.
Thc hin 3 mu, ly kt qu trung bnh.
C ti
- Ra sch t, v en bn ngoi
- ro nc
C ti sau ra

B kh

Cn xc nh khi lng

Sy 60 C n kh

Xay nh
Bt nho
- a vo nc
- Vt loi b x

B t

Dch sau vt
lng qua m, gn, lc.

Tinh bt

Dch chit

nh 2.1. chit isoflavonoid t sn dy ti


2.3.5. Phng php tinh ch dch chit sn dy ti
Nhm nng cao hm lng isoflavonoid trong dch chit v loi bt mt s
tp cht, chng ti tin hnh kho st ba phng php tinh ch dch chit: kt ta
bng nhit, kt ta bng ethanol v s dng kt hp c hai phng php.
T dch gn thu c nh mc 2.3.4. Ly 3 mu, mi mu 500 ml. Tin
hnh tinh ch theo 3 phng php sau

31

Phng php 1 (kt ta tp cht bng nhit): Ly 500 ml dch chit vo cc


1000 ml, un cch thy 30 pht, ngui qua m. Lc ta, ra ta 3 ln,
mi ln 20 ml nc.
Phng php 2 (kt ta tp cht bng ethanol): C p sut gim n th
cao mm, thm 500 ml ethanol 96%, qua m. Lc ta, ra ta 3 ln, mi
ln 20 ml ethanol 96%.
Phng php 3 (kt hp phng php 1 v 2) un cch thy 30 pht,
qua m, lc ta, ra ta 3 ln, mi ln 20 ml nc. Dch lc v dch ra
c gp li, c p sut gim n cao mm, thm 500 ml ethanol 96%,
qua m. Lc ta, ra ta 3 ln, mi ln 20 ml ethanol 96%.
Dch chit sau tinh ch c c di p sut gim, 80oC n th cht cao
c. Xc nh khi lng cao v hm lng isoflavonoid ton phn trong cao thu
c.
2.3.6. Phng php iu ch cao kh
Cao kh iu ch theo phng php c c, sy kh thng c th cht do
dnh, kh thu hi, kh bo qun, qu trnh iu ch tn nhiu thi gian. hn ch
nhng nhc im trn v nng cao cht lng cao kh bo ch c c th s
dng cho bo ch cc dng thuc hin i, chng ti tin hnh iu ch cao kh
bng phng php phun sy. Qu trnh phun sy c tin hnh ti cng ty c phn
ha dc phm Vit Nam (khu cng nghip Tin n, Bc Ninh).
Quy trnh iu ch cao kh gm 2 giai on c tm tt nh hnh 2.2.
Giai on 1: Thc hin ti b mn Cng nghip Dc i hc Dc H
Ni.
Sn dy ti c ra sch t v v en bn ngoi, ct b nhng phn b
hng. ro nc v cn xc nh khi lng c 11 kg. au xay mn thnh
bt nho, bt nho c ha vo 22 l nc, ri vt loi b bng ti vi. B sn c
ra li mt ln vi 22 l nc. Gp dch cc dch chit, lng qua m, gn ly
phn dch trong; thu c 42 l dch chit. Dch chit c tinh ch bng un nng

32

90oC trong 30 pht, ngui qua m, gn ly dch trong pha trn. Phn dch ln
ta c lc ht qua giy lc bng h thng lc Buchner. Ta c ra 3 ln, mi
ln 2 l nc. Dch chit sau tinh ch c th tch l 46 l. Xc nh nng
isoflavonoid trong dch tinh ch theo mc 2.3.1.1 v xc nh khi lng cn theo
mc 2.3.9.
Giai on 2: Thc hin ti cng ty c phn ha dc phm Vit Nam (khu
cng nghip Tin n, Bc Ninh).
Dch chit sau khi tinh ch c c c bng h thng c tun hon n t
trng 1,1 g/ml. Dch chit sau khi c c em lc qua giy lc ri tin hnh phun
sy. iu kin phun sy nh sau
Thit b: H thng phun sy LPG-5 Centrifugal Spray Dryer
(Changzhou Jinqiao, Trung Quc), cng sut max 5 L/gi.
Nhit gi vo: 180C.
Nhit gi ra: 85 - 90C.
Tc phun dch: 4 lt/gi.
Cao kh thu c c nh gi v mt s ch tiu cht lng sau :
2.3.6.1. Th cht ca bt: Quan st bng mt thng bt phun sy thu c v
nhn xt v th cht, mu sc, mi v v.
2.3.6.2. Khi lng v hiu sut phun sy: Cn xc nh khi lng cao thu c
v tnh hiu sut ca qu trnh phun sy theo cng thc sau:

Trong

H:hiu sut ca qu trnh phun sy (%)


mcao: khi lng cao thu c
mdl: khi lng dc liu ban u

2.3.6.3. Mt khi lng do lm kh: Tin hnh theo phng php ghi mc
2.3.10
2.3.6.4. nh lng isoflavonoid trong cao kh: Tin hnh theo phng php ghi
mc 2.3.1.1

33

Sn dy ti

Ra sch, ct b phn hng


Cn xc nh khi lng
Xay thnh bt nho
c

Bt nho
c
B

Vt

B
Vt

Dch chit nc

Dch chit

Dch gp
lng qua m
Gn

Tinh bt

Dch trong
Ta

Tinh ch
Dch tinh ch
C
Dung dch c
(d = 1,1 g/ml)
Lc
Phun sy
Cao kh
nh 2.2. Quy trnh iu ch cao kh

34

2.3.7. Phng php phn lp puerarin v daidzin


uerarin v daidzin c phn lp t dch chit bng phng php thay
i dung mi. Sn phm tinh ch c o nhit nng chy, gi tr Rf v khng
nh cu trc bng cc phng php ph nh mc 2.3.8.
2.3.8. Phng php xc nh cu trc puerarin v daidzin
Khng nh cu trc ca hot cht phn lp c bng cc kt hp cc
phng php ph:
2.3.8.1. Ph hng ngoi (IR)

Mu c nghin vi KBr v ph hng ngoi (IR) ca sn phm c xc


nh trn my erkin Elmer o trong vng bc sng 4000 400 cm-1 ti Vin Ha
hc Vin khoa hc v Cng ngh Vit Nam.
2.3.8.2. Ph khi lng (ESI-MS)

S dng my Varian 320 Ms (M), ngun ion ha ESI ti Vin Ha hc


Vin Khoa hc v Cng ngh Vit Nam xc nh ph khi lng va chm in
t ca cc mu tinh ch c, c v bin gii ph chng minh cu trc.
2.3.8.2. Ph cng hng t ht nhn proton (1H-NMR) v ph cng hng t carbon
13 (13C-NMR)

S dng my cng hng t ht nhn phn gii cao 500MHz, BRUKER


AVANCE 500 ti Vin Ha hc Vin Khoa hc v Cng ngh Vit Nam o
ph cng hng t ht nhn proton (1H-NMR) v ph cng hng t carbon 13
(13C-NMR) ca cc mu tinh ch c. c v bin gii ph chng minh cu trc
hp cht tinh ch c.
2.3.9. Phng php xc nh khi lng cn trong dch chit

Ly chnh xc 100 ml dch chit, ct quay 95oC n th cht cao mm, cn


xc nh khi lng cao c a g. Ly mt phn cao xc nh mt khi lng do
lm kh c kt qu h%, t tnh ra khi lng cn chit c theo cng thc:

35

Trong
W: l khi lng cn chit c (g).
a: l khi lng cao mm tng ng vi 100 ml dch chit (g).
h: mt khi lng do lm kh ca cao mm (%).
V: th tch dch chit thu c (ml).
2.3.10. Phng php xc nh mt khi lng do lm kh

Cho cc cn ra sch vo trong t sy, sy nhit 1050C 20C trong 1


gi. Sau chuyn vo bnh ht m lm ngui ti nhit phng, cn xc nh
c khi lng cc mc. Chuyn mu th vo cc cn, cn c khi lng mb.
Cho cc ng mu vo trong t sy, sy ti nhit 1050C 20C. Sau chuyn
vo bnh ht m lm ngui n nhit phng, cn xc nh khi lng. Cho li
mu th vo t sy v tin hnh nh trn cho ti khi mu th t khi lng khng
i a.

u th c coi l t khi lng khng i, khi chnh lch gia 2 ln cn

lin tip khng ln hn 0,1% so vi khi lng mu th cn ban u i vi mu


th c khi lng cn ban u l 50g v khng c ln hn 0,05% i vi mu
th c khi lng cn ban u l 2g. Thi gian gia hai ln sy lin tip khng
c nh hn thi gian sy ln u.
m ca mu th c tnh theo cng thc:

Trong
mb : khi lng mu ban u v cc
mc : khi lng cc lm kh
a : khi lng mu v cc sau khi sy ti khi lng khng i

36

Chng 3. KT QU NGHIN CU
3.1. nh lng isoflavonoid ton phn trong nguyn liu
3.1.1. ng chun isoflavonoid
Tin hnh qut ph UV-VIS dung dch puerarin nng 8 g/ml trong vng
bc sng 200 n 400 nm, thu c ph hp th nh hnh 3.1.

Hnh 3.1. h V-VI ca dung dch cht i chiu


Hnh dng ph ph hp vi kt qu c cng b [35]. Hnh nh ph cho
thy c 2 cc i hp th ti bc sng 250 nm v 307 nm. T kt qu trn v tham
kho cc ti liu [11], [41], [45] chng ti chn bc sng 250 nm l bc sng o
quang trong cc php nh lng isoflavonoid ton phn sau ny.
Pha dy dung dch puerarin c nng 1,6; 3,2; 4,8; 6,4 v 8,0 g/ml nh
mc 2.3.1.1 ri tin hnh o quang ti bc sng 250 nm. Gi tr mt quang ca
dy dung dch i chiu c ghi trong bng 3.1.
Bng 3.1.

t quang ca dy dung dch cht i chiu ti bc sng 250 nm.

Nng puerarin (g/ml)

1,6

3,2

4,8

6,4

8,0

Mt quang

0,133

0,289

0,427

0,566

0,715

37

Mt quang

0.9

y = 0,0901x - 0,0063
R = 0,9996

0.7
0.5
0.3
0.1
0

10

Nng (g/l)
nh 3.2. ng chun biu th mi tng quan gia nng puerarin v mt quang
H s tng quan R2 = 0,9996 ( 1) cho thy nng puerarin v mt
quang c s ph thuc tuyn tnh trong khong nng t 1,6 8,0 g/ml ti bc
sng 250nm. Cc isoflavonoid c trong r c sn dy tr genistin u c hp th
cc i ti 250nm (dung mi ethanol 30%), trong puerarin l isoflavonoid chim
ch yu trong thnh phn r sn dy. Do c th s dng puerarin lm cht chun
so snh trong nh lng isoflavonoid ton phn bng o quang 250 nm
3.1.2. nh lng isoflavonoid trong nguyn liu
m lng isoflavonoid trong sn dy ph thuc vo nhiu yu t: thi gian
v a im thu hoch, s ch, bo qun,V vy, xc nh hm lng isoflavonoid
trong nguyn liu kh trc nghin cu s gp phn nh hng tiu chun cht
lng ca nguyn liu a vo nghin cu.
m lng isoflavonoid trong sn dy c xc nh bng phng php o
quang ti bc sng 250nm nh mc 2.3.1.1. Mu i chiu c nng 4g/ml.
Nguyn liu c thu mua t ba ni khc nhau l Ninh Bnh (NL1), Hi Dng
(NL2) v Ph Th (NL3), nh lng isoflavonoid trong cc nguyn liu ny thu
c kt qu nh bng 3.2.

38

Bng 3.2. Kt qu nh lng isoflavonoid trong nguyn liu


Mu th
NL 1

Mu th
NL2

Mu th
NL3

Khi lng mu (g)

10,0

10,0

10,0

m lng isoflavonoid
trong nguyn liu (%)

0,231

0,398

0,582

Khi lng mu (g)

0,4

0,4

0,4

m lng isoflavonoid
trong nguyn liu (%)

0,623

0,812

1,054

Mu
Nguyn liu
ti
Nguyn liu
kh

Kt qu cho thy hm lng isoflavonoid trong nguyn liu khc nhau nhiu
gia cc a phng. guyn liu t Ph Th cho hm lng isoflavonoid cao nht,
t 0,582% (theo ti) v 1,054 (theo kh) gp 2,5 ln so vi hm lng
isoflavonoid trong sn dy ti thu mua Ninh Bnh (0,231 v 0,623), v gp gn
1,5 ln so vi hm lng isoflavonoid trong sn dy Hi Dng (0,398 v 0,812).
T kt qu trn, chng ti chn nguyn liu sn ti thu mua t Ph Th l nguyn
liu a vo nghin cu.
u nnh l nguyn liu chit xut cc isoflavonoid c tc dng kiu estrogen
kh ph bin hin nay, tuy nhin so vi kt qu nh lng isoflavonoid t sn dy
trn c th thy hm lng isoflavonoid trong u nnh (0,1-0,3%) [36] thp hn
rt nhiu. Do vy, nu c th xy dng c thnh cng phng php chit xut
isoflavonoid v tinh ch puerarin t sn dy, s gp phn gia tng ngun nguyn
liu v phng php chit isoflavonoid trong nc.
3.2. nh lng puerarin trong nguyn liu
Pha dy dung dch puerarin c nng 0,01; 0,02; 0,1; 0,15; 0,20 mg/ml nh
mc 2.3.1.2 v phn tch HPLC 250nm thu c kt qu nh sau
Bng 3.3. Din tch pic sc k ca dy dung dch chun
Nng (mg/ml)

0,0103

0,0206

0,0514

0,1029

0,1543

0,2058

Spic

151175

283710

763851

1451805

2138399

2968977

39

3000000

y = 14226x + 88.251
R = 0.999
2000000

1000000

50

100

150

200

250

g/ml

nh 3.3. th biu din mi tng quan gia nng puerarin v din tch pic
sc k
T th thu c c th thy, din tch pic sc k vi nng puerarin c
mi quan h tuyn tnh trong khong 0,01 0,2 mg/ml (hay 10 200 g/ml). Kt
qu ny ph hp vi kt qu nghin cu ca Man Chen v cng s [30].
nh lng puerarin trong nguyn liu kh v ti thu mua t Ph Th,
chng ti thu c kt qu nh sau.
Bng 3.4. m lng puerarin trong nguyn liu thu mua h Th
Nguyn liu
m lng puerarin (%)

Kh

Ti

0,5640

0,2130

Nhn xt: Puerarin chim khong 50% lng isoflavonoid c trong nguyn
liu. hng php

LC l ph hp nh lng puerarin trong nguyn liu, dch

chit v sn phm tinh ch.


3.3. Chit xut isoflavonoid t sn dy kh
3.3.1. Kho st cc dung mi chit
Vi dung mi ethanol, kho st cc dung mi chit l ethanol 40, ethanol
60, ethanol 80 v ethanol 96.
Cn chnh xc khong 50g bt nguyn liu kh cho vo bnh nn 250 ml.
Thm 100 ml dung mi (ethanol 40, 60, 80, 96), khuy u. au 24h

40

nhit phng, thnh thong lc u trong qu trnh chit. Lc thu dch c th


tch V1. Tip tc chit b cn li 2 ln, mi ln vi 100ml trong 24 gi thu c cc
th tch V2, V3. C gim p cc dch chit thu c ti cao c.
Xc nh khi lng cao v nh lng hm lng isoflavonoid v puerarin
trong dch chit v cao theo phng php ghi mc 2.3.1. Kt qu thu c nh
bng 3.5.
Bng 3.5. Kt qu kho st cc dung mi ethanol c nng khc nhau

Dc liu

Dung mi chit

Dch chit

Ethanol
80%

Ethanol
96%

Khi lng dc liu (g)

50,0

50,0

50,0

50,0

m lng isoflavonoid trong


dc liu (%)

1,054

1,054

1,054

1,054

m lng puerarin trong dc


liu (%)

0,564

0,564

0,564

0,564

Ln 1

232,6

270,7

185,7

149,1

Ln 2

134,9

144,8

115,2

70,2

Ln 3

55,2

62,0

49,3

28,6

Tng

422,7

477,5

350,2

247,9

Phn trm isoflavonoid chit c


(%)

80,21

90,61

66,45

47,02

Tng lng puerarin trong dch


chit (mg)

215,0

250,1

230,1

143,0

Phn trm puerarin chit c (%)

76,24

88,69

81,61

50,70

7,1

6,3

4,3

1,6

420,0

473,7

347,0

246,5

Lng isoflavonoid
chit c (mg)

Cao chit

Ethanol Ethanol
40%
60%

Khi lng cao (g)


Lng isoflavonoid/cao (mg)

41

m lng isoflavonoid/cao (%)

5,92

7,52

8,07

15,41

Lng puerarin trong cao (mg)

213,4

248,6

228,3

142,5

m lng puerarin/cao (%)

3,01

3,95

5,31

8,91

Lng isoflavonoid chit c


(mg)

Ethanol 40%

Ethanol 60%

Ethanol 80%

Ethanol 96%

600
500
400
300
200
100
0
Ln 1

Ln 2

Ln 3

Tng

nh 3.4. Biu so snh lng isoflavonoid chit c bi cc dung mi ethanol


c nng khc nhau.
Nhn xt: Ethanol 60% cho hiu sut isoflavonoid v puerarin cao hn so vi
cc dung mi ethanol khc. Ln chit u ng vai tr quan trng, chit c ti
hn 50% hm lng isoflavonoid chit c. Vi ethanol 60%, sau ba ln chit
isoflavonoid trong dc liu c chit gn nh kit hon ton. Vic c gim p
sut trong iu ch cao c t dch chit lm thay i khng ng k hm lng
isoflavonoid v puerarin.
Vi dung mi chit l nc, tham kho quy trnh chit isoflavonoid t sn
dy ti v quy trnh chit isoflavonoid t sn dy kh theo ti liu [14], chng ti
kho st hiu sut chit isoflavonoid t sn dy ti theo hai phng php sau

42

Phng php A: Cn chnh xc khong 50g dc liu kh, em nghin vi


nc theo t l 1:4 bng my xay sinh t c hn hp bt nho. Chuyn khi bt
nho ny vo bnh nn 500 ml, ra thit b nghin v dng c bng 50 ml nc v
gp vo khi bt no ny. Lc hn hp thu c qua vi v sau l qua mng lc
thu c th tch V1. Thm khong 250 ml nc vo b lc, khuy u. Tip tc lc
qua vi v qua mng lc thu c th tch V2. Gp dch chit 1 v 2. C gim p
n cao c c khi lng m1. Ly mu cao ny em nh lng isoflavonoid
theo phng php 2.3.1.1 v nh lng puerarin theo phng php 2.3.1.2
Phng php B: Cn chnh xc khong 50g bt nguyn liu kh cho vo
bnh nn 1l. Thm 500ml nc ct, khuy trong 1,5 gi. Lc thu dch lc 1 c th
tch V1. Thm tip 500 ml nc ct vo b, khuy trong 1 gi, sau lc thu dch
lc 2 c th tch V2 . Gp dch lc 1 v 2. C gim p n cao c c khi lng
m2. Ly mu cao ny em nh lng isoflavonoid theo phng php 2.3.1.1, nh
lng puerarin theo phng php 2.3.1.2.
Kt qu kho st thu c nh sau.
Bng 3.6. Kt qu kho st chit isoflavonoid t sn dy kh bng dung mi nc
Phng php

Khi lng dc liu (g)

50,15

50,05

Khi lng cao (g)

7,08

5,99

Tng lng isoflavonoid trong cao (mg)

250,48

269,06

Phn trm isoflavonoid chit c (%)

47,39

51,00

Tng lng puerarin trong cao (mg)

125,87

146,44

Phn trm puerarin chit c (%)

44,50

51,88

Nhn xt: C hai phng php chit isoflavonoid bng dung mi nc u


cho hiu sut chit tng isoflavonoid tng i thp, ch t khong 50%. hng

43

php A c hnh thnh da trn quy trnh chit isoflavonoid t sn dy ti.


hng php B c xy dng da vo nguyn cu ca Han Jian v cng s [14].
Hiu sut chit isoflavonoid t sn dy kh bng ethanol cao hn, nhng qu
trnh chit tn nhiu thi gian. hng php chit bng dung mi nc kh lc do
c nhiu tinh bt trong dch chit, khng thu hi c tinh bt nh phng php
chit nc i vi sn dy ti, khng chit kit c hot cht nh ethanol 60%.
Do vy chng ti chn ethanol 60% l dung mi chit isoflavonoid t sn dy kh.
3.3.2. Tinh ch dch chit sn dy kh
Qua qu trnh kho st, chng ti xy dng phng php tinh ch isoflavonoid
t dch chit sn dy kh vi dung mi chit ethanol nh sau:
Vi mi dung mi chit, gp dch chit ca 3 ln chit li. C loi ht
ethanol sau thm nc ct vo phn dch cn li cho 120ml (vi 50 bt
nguyn liu kh a vo chit).
lng qua m, cc tp cht tan trong ethanol s b ta v loi i trong qu
trnh lc sau . Ly mu dch lc em nh lng isoflavonoid ton phn v
puerarin.
Kt qu thu c nh bng 3.7.
Bng 3.7. Kt qu tinh ch dch chit sn dy kh
Ethanol
40%

Ethanol
60%

Ethanol
80%

Ethanol
96%

Khi lng cao tinh ch (g)

4,81

4,28

2,99

1,30

Lng isoflavonoid cn li
trong cao tinh ch (mg)

389,20

445,71

285,21

187,39

8,09

10,41

9,53

14,359

180,86

240,54

166,5

119,60

3,76

5,62

5,57

9,20

Dung mi chit

m lng isoflavonoid trong


cao tinh ch (%)
Lng puerarin cn li trong
cao tinh ch (mg)
m lng puerarin trong cao
tinh ch (%)

h vy, sau khi tinh ch, hm lng puerarin v isoflavonoid ton phn
trong cao u tng ln. Trong s cc dung mi kho st, cao thu c sau khi tinh
44

ch dch chit ca dung mi ethanol 60% c hm lng t 10,410.

m lng

puerarin trong cao t khong 5,62%. Mc d hm lng puerarin t cao nht


cao thu c t dch chit ethanol 96%, tuy nhin khi lng cao thu c rt thp,
kh nng chit isoflavonoid v puerarin t dc liu khng cao, dung mi d chy
v t tin hn ethanol 60%, kh trin khai trn quy m ln. Do vy chng ti chn
ethanol 60% l dung mi chit isoflavonoid t sn dy kh.
3.4. Chit xut isoflavonoid t sn dy ti
3.4.1. Chit isoflavonoid t sn dy ti
Tin hnh chit isoflvonoid t sn dy ti theo phng php ghi mc 2.3.4.
nh lng isoflavonoid ton phn trong dch chit theo mc 2.3.1 v xc nh khi
lng cn trong dch chit theo mc 2.3.9.
Thc hin 3 mu, kt qu nh bng 3.8.
Bng 3.8. Kt qu chit isoflavonoid t sn dy ti
Mu

Trung bnh

Khi lng dc liu (kg)

2,35

2,15

5,3

Khi lng tinh bt (kg)

0,55

0,46

1,25

Th tch dch chit (l)

9,8

8,9

22,5

Khi lng cn chit (g)

126,29 121,17 291,82

m lng isoflavonoid trong cn (%)

9,32

8,92

9,10

9,12

Hiu sut chit


(tnh theo hm lng isoflavonoid) (%)

86,06

86,38

86,09

86,18

Nhn xt:
m lng tinh bt thu c trung bnh l 22,8% cao gn gp i so vi cng
b trong cc ti liu tham kho [2], [ 6].
Hiu sut chit isoflavonoid trung bnh t 86,18%. Mc d ch chit 2 ln
nhng hiu sut tng i cao, lng isoflavonoid cn li trong b thp (< 15%).

45

hng php chit isoflavonoid t sn dy ti bng dung mi nc khng ch


gip chit kit c hot cht trong dc liu m cn c th tn dng thu c
lng ln tinh bt. So vi chit t dc liu kh, phng php chit vi dc liu
ti t ra c nhiu u im, gi thnh r, ngoi sn phm chnh l isoflavonoid cn
thu thm sn phm ph l tinh bt.
3.4.2. Tinh ch dch chit sn dy ti
m lng isoflavonoid ton phn trong cn chit trung bnh t 9,12%.
tng hm lng hot cht trong cao. Tin hnh tinh ch dch chit nh phng php
ghi mc 2.3.5. Kt qu thu c nh bng 3.9.
Bng 3.9. iu sut v hm lng cao c sau tinh ch (thu c t 500 ml dch
chit, tng ng khong 150 g dc liu)

Phng php tinh ch

Ta tp cht
bng nhit
(1)

Khi lng cao c (g)

7,97

7,35

6,09

Mt khi lng do lm kh (%)

18,24

14,55

14,1

9,19

10,61

11,5

86,67

92,30

82,88

m lng isoflavonoid trong


cao c (%)
Hiu sut giai on tinh ch tnh
theo hm lng isoflavonoid
(%)

Ta tp cht Phi hp c hai


bng ethanol phng php
(2)
(3)

Nhn xt: m lng isoflavonoid trong cao c tinh ch ca phng php 2


(10,61%) cao hn phng php 1 (9,19%); phng php 3 (kt hp phng php 1
v 2) cho hm lng cao nht (11,5). Hiu sut tinh ch cao nht l phng php 2
(s dng ethanol), thp nht l phng php 3 (2 giai on).

h vy, mc d sn

phm thu c t phng php 3 c hm lng isoflavonoid ton phn cao hn cc


phng php khc, song tnh v hiu sut th qu trnh tinh ch theo phng php 3
cng loi i kh nhiu isoflavonoid cng vi cc tp cht khc.
hng php 2 (kt ta tp cht bng ethanol) cho sn phm c hm lng
isoflavonoid cao (10,61% tng ng vi 12,41% tnh theo cao tinh ch lm

46

kh) vi hiu sut tinh ch cao, t 92,30%. Tuy nhin phng php c nhc im
l vic ct thu hi ethanol tn thi gian.
m lng isoflavonoid ton phn trong cao tinh ch thu c t phng
php 1 (kt ta tp cht bng nhit) l 9,19% tng ng vi 11,23% tnh theo cao
tinh ch lm kh. hng php tinh ch n gin, d thc hin, t tn km, c
th trin khai trn quy m ln.
3.5. iu ch cao kh t dch chit sn dy ti
Vi hai loi nguyn liu kh v ti, qua qu trnh kho st nh gi trn
chn c cc dung mi chit ph hp, cho hiu sut chit cao, m bo chit kit
hot cht t nguyn liu. Tuy nhin do thi gian nghin cu hn ch, qu trnh phun
sy thc hin ti cng ty c phn ha dc phm Vit Nam kh ch ng v thi
gian v c m thc hin cha ln nn chng ti ch tin hnh nghin cu iu
ch cao kh phun sy t dch chit sn dy ti theo phng php ghi mc 2.3.6.,
s dng phng php tinh ch kt ta tp cht bng nhit.
Dch tinh ch thu c sau giai on 1 c nng isoflavonoid v t l cn
kh nh bng 3.10.
Bng 3.10. Dch tinh ch em vo phun sy
Thng s

Gi tr

Khi lng sn dy ti

11 kg

Th tch dch tinh ch

46 lt

Nng isoflavonoid trong dch tinh ch 1,24 mg/ml


T l cn kh ca dch tinh ch

11 mg/ml

Tin hnh phun sy vi cc iu kin nh mc 2.3.6, thu c 450g cao


kh. Th cht cao thu c nh hnh 3.5.

47

nh 3.5. Bt cao kh phun sy


Cao kh thu c c nh gi v mt s ch tiu cht lng nh mc
2.3.6.a, 2.3.6.b, 2.3.6.c v 2.3.6.d. Kt qu thu c nh bng 3.11.
Bng 3.11.
STT

t s ch tiu cht lng ca cao kh phun sy.

Ch tiu

Gi tr
Dng bt, th cht ti xp, ng nht, c mu
vng kem, mi thm, v ng v hi ngt.

Hnh thc cm quan

Khi lng cao phun sy

Khi lng ring biu kin

Kch thc tiu phn

Mt khi lng do lm kh

2,7 %

m lng isoflavonoid
ton phn trong cao

10,78 %

Hiu sut phun sy

4,09 %

450 g
0,52 g/cm3
100% bt qua ry 250 m

Nhn xt: Cao kh thu c c hm lng isoflavonoid t 10,78% tng


ng vi 11,08% tnh theo cao lm kh kit.

m lng ny thp hn khng

ng k so vi hm lng trong cn kh tinh ch. Cao kh thu c c th cht ti,

48

xp, d ng gi v bo qun hn rt nhiu so vi cao kh bo ch theo phng


php c c sy kh. Cao kh phun sy khng ch c nhiu u im hn so vi cao
kh c c sy kh m phng php cn gip tit kim nhiu thi gian v chi phi,
c th trin khai trn quy m cng nghip.
3.6. Phn lp puerarin v daidzin
Tin hnh sc k bn mng dch chit sau tinh ch, thu
c kt qu nh sau hnh 3.6.
Ghi ch: T dung dch th; C dung dch chun.
Trn bn mng sc k thy c 4 vt m, chng t trong
dch tinh ch c 4 isoflavonoid chnh:
Vt 1 tng ng vi puerarin
Vt 2 d kin l daidzin
Vt 3 cha r
Vt 4 d kin l daidzein

nh 3.6. nh nh sc k
lp mng di n t ngoi
254 nm ca dch chit

Tham kho ti liu [16] v rt nhiu qu trnh


kho st trc chng ti xy dng c quy
trnh phn lp puerarin v daidzin c tm tt nh s hnh 3.7.
3.6.1. Phn lp daidzin
3.6.1.1.

Tin hnh

Ly 1L dch tinh ch, c gim p n cao c.

a cao c ny vo 100 ml

ethyl acetat bo ha nc, lng, gn ly phn dch trong. Thm tip 50 ml ethyl
acetat bo ha nc vo phn cn cn li, lc u. au lng v gn ly phn
dch trong. Tip tc chit phn cn cn li vi 50ml ethyl acetat bo ha nc nh
trn, lng v lc ly dch. Gp cc dch lc, c gim p 50C n 50ml.
Chuyn phn dch ny vo bnh gn 250ml. Lc 2 ln, mi ln vi 100ml
nc m, trong 15 pht, sau gn ly phn nc lp di. Gp cc dch chit
nc, c gim p 70C n th tch khong 120 ml thy xut hin ta th dng li,
qua m iu kin phng, lc thu ta. Phn dch cn li dng phn lp
puerarin. Ta thu c em kt tinh li trong 10ml ethanol 50% c daidzin tinh
khit trn sc k lp mng. Cn khi lng daidzin thu c, th tinh khit bng
49

sc k lp mng theo phng php ghi mc 2.3.2 v khng nh cu trc theo


phng php ghi mc 2.3.8.

Dch tinh ch
C gim p
Ethylacetate
bo ha nc

Cao c
Chit 2-3 ln
Lc

Ta

c m
(70-80C)

Dch chit ethyl acetat


Dung dch
ethylacetat

Lc 2-3 ln, gn.


Dch chit nc m
ngui

Dch chit nc

Ta daidzin

C gim th tch
Lm lnh
c

Ethanol 50%

Kt tinh li
Daidzin

Ta puerarin
Kt tinh li
Puerarin

nh 3.7. Quy trnh phn lp daidzin v puerarin


3.6.1.2. Kt qu

Khi lng daidzin thu c: 262,5mg/m. Thc hin 5 m, thu c tng


lng daidzin l 1,31 g
Kt qu phn tch sc k lp mng thu c nh hnh 3.8. Trong A l mu
th daidzin, C l mu chun puerarin 82,31%. T hnh nh sc k lp mng ny c

50

th thy, daidzin thu c kh tinh khit, trn hnh nh sc k khng thy c xut
hin vt l, vt sc k gn, sch.

Hnh 3.8. nh nh sc k lp mng di n t ngoi 254nm ca daidzin


Xc nh cc thng s vt l ca ta daidzin thu c kt qu nh sau
Cm quan: Ta daidzin thu c c dng bt trng, hi vng.
Nhit nng chy: 238,8C (ti liu [21]: 238-240C)
Khng nh li cu trc ca daidzin bng cc phng php ph thu c kt qu
nh sau.
Ph hng ngoi - IR
Tin hnh o mu theo phng php ghi mc 2.3.8.1 thu c kt qu nh
Ph lc 13. Quan st trn hnh nh ph thu c nhn thy c cc di ph hp th
c trng nh bng sau :
Bng 3.12. Kt qu o ph hng ngoi ca daidzin
Tn cht

nh hp th c trng
3425 cm-1 , pic rng v t

Daidzin

Nhm chc tng ng


O phenol

1635 cm-1

C=O

1519 cm-1

C=C thm

Ph khi lng (ESI-MS)

51

Tin hnh o mu theo phng php ghi mc 2.3.8.2 thu c kt qu nh h


lc 14-15.
Bng 3.13. Kt qu o ph khi lng (E I-MS) ca daidzin
Tn cht
Daidzin

S khi ca mnh ion phn t m/z


Ph positive xut hin pic 438,96 ng vi mnh [M+Na]+ c s khi l
438,96, tng ng vi khi lng phn t 416,38 (C21H20O19)
Ph negative xut hin pic 415,00 ng vi mnh [M-H]- c s khi l
415,00 tng ng vi khi lng phn t 416,38 (C21H20O19)

Nhn xt : Kt qu cho thy cc cht phn tch cho mnh ion phn t ph hp vi
khi lng phn t tng ng.
Ph cng hng t ht nhn (NMR)
Tin hnh o mu theo phng php ghi mc 2.3.8.3 thu c kt qu nh
Ph lc 16-19.
OH
6''

5'' O

O 7

9 O

1''
4''

HO

6
2'' OH

3''

OH

1'

4'

OH

nh 3.9. Cng thc cu to ca daidzin


Bng 3.14. Kt qu o ph
13

R ca daidzin
1

C NMR
Thc nghim
Ti liu [22]

H NMR
Thc nghim
Ti liu [22]

(500 MHz,
DMSO, ppm)

(125 MHz,
DMSO, ppm)

(500 MHz,
DMSO, ppm)

(500 MHz,
DMSO, ppm)

153,2

153,1

8,34 s

8,35 s

123,7

123,7

174,7

174,7

126,9

126,9

8,03 d (J 9,0)

115,5

115,4

7,13 dd (J 7,0; 2,0)

8,05 d (J 8,8)
7,14 dd (J 8,8;
2,4)

161,3

161,3

103,3

103,3

7,22 d (J 2,0)

52

7,22 (J 2,4)

157,2

157,2

10

118,4

118,5

122,3

122,2

130,0

129,9

7,40 d (J 8,5)

7,41 d (J 8,7)

114,9

114,9

6,81 d (J 8,5)

6,82 d (J 8,7)

157,0

157,0

114,9

115,5

6,84 d (J 8,5)

130,0

129,9

7,35 d (J 8,5)

100,0

100,1

5,09 d (J 7,0)

5,09 d (J 7,5)

73,1

73,1

76,4

76,4

69,6

69,6

77,2

77,2

3,27-3,33 m, 2H
3,44-3,48 m, 2H
3,70-3,74 m, 1H

3,20; 3,30-3,36 m
3,44-3,52 m
3,74 m; 3,46 m

60,6

60,6
4,59; 5,06-5,13;
5,42; 9,54 (OH-4)

4,58; 5,04; 5,10;


5,39; 9,48

OH

3.6.2. Phn lp puerarin


3.6.2.1. Tin hnh
Dch lc cn li sau khi loi ta daidzin nh mc 3.6.1.1
em c bt cn khong 40ml, sau lm lnh trong t lnh qua
m, thu c ta puerarin th. Ta ny c kt tinh li trong
khong 10 ml nc thu puerarin c tinh khit cao. Cn khi
lng puerarin thu c, th tinh khit bng sc k lp mng
theo phng php ghi mc 2.3.2 v khng nh cu trc theo
phng php ghi mc 2.3.8.
3.6.2.2.

Kt qu

Khi lng puerarin thu c: 432,5mg/m. Thc hin 5

Hnh 3.10. nh nh
sc k lp mng
Kt qu phn tch sc k lp mng thu c nh hnh di n t ngoi
254nm ca puerarin
3.10. Trong B l mu th puerarin, C l mu chun puerarin
m, thu c tng lng puerarin l 2,16 g

53

82,31%. T hnh nh sc k lp mng ny c th thy, puerarin thu c c tinh


khit cao, hnh nh sc k khng thy c xut hin vt l, vt sc k gn, sch.
Xc nh cc thng s vt l ca ta daidzin thu c kt qu nh sau
Cm quan: dng bt trng, hi vng.
Nhit nng chy: 210C (ti liu [24]: 208,4-212,5C)
Rf = 0,388 vi pha ng l Cloroform - methanol - nc (7:2,5:0,25).
Rf = 0,589 vi pha ng l Cloroform methanol (7:3).
Khng nh li cu trc ca puerarin bng cc phng php ph thu c kt
qu nh sau.
Ph hng ngoi - IR
Tin hnh o mu theo phng php ghi mc 2.3.8.1 thu c kt qu nh
Ph lc 5. Quan st trn hnh nh ph thu c nhn thy c cc di ph hp th
c trng nh bng sau :
Bng 3.15. Kt qu o ph hng ngoi ca puerarin
Tn cht
Puerarin

nh hp th c trng

Nhm chc tng ng

3341 cm-1, pic rng v t

O phenol

1635 cm-1

C=O

1513 cm-1

C=C thm

Ph khi lng (ESI-MS)

Tin hnh o mu theo phng php ghi mc 2.3.8.2 thu c kt qu nh h


lc 6-7..
Bng 3.16. Kt qu o ph khi lng (ESI-MS) ca puerarin
Tn cht
Puerarin

S khi ca mnh ion phn t m/z


Ph negative xut hin pic 415,08 ng vi mnh [M-H]- c s khi l
438,96, tng ng vi khi lng phn t 416,38 (C21H20O19)
Ph positive xut hin pic 417,08 ng vi mnh [M+H]+ c s khi l
417,08, tng ng vi khi lng phn t 416,38 (C21H20O19)

Nhn xt : Kt qu cho thy cht phn tch cho mnh ion phn t ph hp vi
khi lng phn t ca puerarin.
54

Ph cng hng t ht nhn (NMR)

Tin hnh o mu theo phng php ghi mc 2.3.8.3 thu c kt qu nh h


lc 8-12.
OH

OH

4"

HO
3"

6"
5"

2"

HO

1"
8

HO

10

1'

4'

OH

nh 3.11. Cng thc cu to ca puerarin


Bng 3.17. Kt qu o ph
13

C NMR v DEPT
Thc nghim
Ti liu [22]

R ca puerarin
1

H NMR
Thc nghim
Ti liu [22]

(500 MHz, CD3OD,


ppm)

(125 MHz, DMSO,


ppm)

(500 MHz, CD3OD,


ppm)

(500 MHz, DMSO,


ppm)

154,4 (CH)

152,0

8,15 s

8,26 s

125,4 (C)

122,9

178,2 (C)

174,6

128,0 (CH)

125,9

8,03 d (J 9,0)

7,96 d (J 8,8)

116,2 (CH)

114,8

6,97 d (J 9,0)

6,99 dd (J 8,8)

162,9 (C)

160,6

113,0 (C)

113,2

158,5 (C)

155,7

10

118,4 (C)

116,8

124,1 (C)

122,4

131,3 (CH)

129,6

7,35 d (J 8,5)

7,42 d (J 8,8)

116,2 (CH)

114,7

6,84 d (J 8,5)

6,82 d (J 8,8)

158,5 (C)

156,9

116,2 (CH)

114,7

6,84 d (J 8,5)

131,3 (CH)

129,6

7,35 d (J 8,5)

75,6 (CH)

73,4

5,11 (J 9,5)

4,90 d (J 10,4)

72,99 (CH)

70,9

3,51-3,54 m, 1H

3,34m; 3,54m

55

79,9 (CH)

78,5

71,6 (CH)

70,2

82,6 (CH)

81,2

62,7 (CH2)

61,1

3,56-3,58 m, 2H
3,77 dd, 1H
3,89-3,93 m, 1H
4,14 br, 1H

OH

3,55br, d
3,74br, d
3,75m; 4,0m

4,68

Nhn xt : Cc d liu ph v hng s vt l ca mu puerarin chit c l ng


nht vi d liu ph v hng s vt l t cc ti liu tham kho v ph hp vi cu
trc ca puerarin. Kt qu o ph cng cho thy puerarin v daidzin phn lp c
l tinh khit.

56

Chng 4. BN LUN
4.1. V nguyn liu chit xut isoflavonoid
ti thc hin kho st chit isoflavonoid t c hai nguyn liu sn dy
kh v sn dy ti thu mua cc tnh Hi Dng, inh Bnh v h Th. Kt qu
kho st s b cho thy, sn dy thu mua Ph Th cho hm lng isoflavonoid v
puerarin cao nht, c a vo nghin cu.
ti mi ch thc hin nhng kho st v nghin cu bc u v chit
isoflavonoid v puerarin t sn dy, do vy cha ch ng c ngun nguyn liu.
Kt qu nghin cu ph thuc nhiu vo cht lng nguyn liu u vo. Cn c
thm nghin cu chun ha ngun nguyn liu v cc tiu ch nh hm lng
isoflavonoid, thi gian thu hi, tui ca sn dy khi thu hi, quy trnh lm kh
dc liu
Vi sn kh, tin hnh kho st cc dung mi chit l cc dung dch ethanol
c nng khc nhau v nc. Dung mi ethanol cho hiu sut chit cao hn dung
mi nc v c th chit kit hot cht trong dc liu. Trong cc nng ethanol
kho st, ethanol 60% t ra l dung mi chit hiu qu: hiu sut chit isoflavonoid
t 90,61%, cao sau tinh ch c hm lng isoflavonoid t trn 10%. Kt qu ph
hp vi nhiu kt qu c cng b trong nc [5] v nc ngoi [41], [45]. Tuy
nhin, theo phng php kho st, chit isoflavonoid bng ethanol thc hin trong
thi gian ko di, ngoi sn phm chnh l isoflavonoid th khng thu thm c
tinh bt.
Vi sn ti ch cn s dng dung mi r tin, khng c l nc chit
isoflavonoid cho hiu qu chit cao t 86,18%. c bit, quy trnh chit xy dng
c hon ton c th trin khai trn quy m ln, kt hp vi quy trnh thu tinh bt
thng s dng trong dn gian. Nguyn liu c tn dng ti a va thu tinh
bt, va chit isoflavonoid. Tuy nhin, nhc im ca quy trnh l ch sn xut
c theo thi v, vo ma thu hoch sn dy.
4.2. V phng php bo ch cao kh

57

ti xy dng c quy trnh iu ch cao kh isoflavonoid bng phng


php phun sy. hng php phun sy c cc u im:
Tit kim thi gian, nng lng (in nng, nhit nng).
Trnh phn hy hot cht do tip xc vi nhit lu.
Cao kh thu c dng bt ti xp, d thu hi, bo qun v cht lng
ng u gia cc l m.
D trin khai trn quy m ln.
ti cng nh gi c mt s c im c bn ca cao kh phun sy
c. Trong khun kh ti, vic phun sy cn gp mt s kh khn nh
Mi ch thc hin c trn dch chit nc ca nguyn liu ti.
Cn mt lng ln dch chit (42 lt), do iu kin my mc v thi gian hn
ch nn ch la chn c phng php tinh ch n gin nht tin hnh tinh
ch lng dch ch thu c mc d khng phi l phng php cho hiu qu
tinh ch cao nht.
Qu trnh iu ch phi thc hin qua hai giai on hai ni khc nhau, kh
khn trong vic vn chuyn, khng ch ng c v my mc, ph thuc nhiu
vo ni hp tc (cng ty c phn a dc phm Vit Nam).
Cn tin hnh thm nghin cu iu ch cao kh t dch chit sn dy kh
m bo c th sn xut cao kh isoflavonoid lin tc trong nm khi ngun nguyn
liu ti khng cn sn c.
4.3. V phng php phn lp puerarin v daidzin
Hu ht cc phng php phn lp puerarin v daidzin c cng b [5],
[42] u s dng phng php sc k ct. hng php ny cho sn phm c
tinh khit cao song ch tin hnh c vi c mu nh, dung mi v ha cht s
dng t tin, cho hiu qu kinh t khng cao.
ti xy dng c quy trnh n gin phn lp c c puerarin v
daidzin t dch chit sn dy ti. Quy trnh thc hin n gin, nhanh chng, c
th tin hnh vi cc c mu ln.

58

Qu trnh phn lp tri qua 4 gii on.

ai giai on u nhm mc ch loi

cc tp bng cch thay i dung mi. Do puerarin tan c trong nc, t tan trong
ethyl acetat cn daidzin tan tt trong ethyl acetat nhng li tan km hn nhiu trong
nc, s dng ethyl aceta bo ha nc gip ci thin tan ca puerarin do
nng cao hiu sut phn lp so vi khi ch s dng ethyl acetat. Qua hai ln thay i
dung mi, kim tra s b bng sc k lp mng thy tp cht v cc isoflavonoid
khng cn thit c loi i ng k.
Vic phn lp c daidzin l thnh cng nm ngoi mc tiu mong i ban
u ca ti.
Thc t, ti mi thc hin phn lp thnh cng puerarin v daidzin t dch
chit sn dy ti do iu kin thi gian c hn. Cn c thm nghin cu hon thin
quy trnh phn lp puerarin v daidzin t dch chit sn dy kh c th ch ng
trong vic sn xut khng ph thuc vo ma thu hoch sn dy ti.

59

KT LUN V XUT
KT LUN
Mc d do thi gian thc hin v iu kin thc nghim cn hn ch, nghin
cu t c cc mc tiu ra:
1.

Xy dng c quy trnh iu ch cao kh phun sy t r c sn dy c


hm lng isoflavonoid trong cao trn 10%
C th, nghin cu

Kho st v la chn c dung mi chit isoflavonoid thch hp t sn dy kh


l ethanol 60%, tinh ch dch chit bng nhit, hm lng isoflavonoid trong cao
tinh ch t 10,41%
Xy dng c quy trnh chit isoflavonoid v thu tinh bt t sn dy ti bng
dung mi r tin, khng c l nc. Hiu sut chit isoflavonoid t sn dy
ti t 86,18% sau 2 ln chit. Kho st 3 phng php tinh ch dch chit l
s dng nhit, s dng ethanol v phi hp c nhit v ethanol. La chn
phng php tinh ch n gin, thun tin l s dng nhit tinh ch dch chit
em vo iu ch cao kh phun sy.
Xy dng c quy trnh iu ch cao kh bng phng php phun sy nh

hnh 2.2. Cao kh iu ch c dng bt, th cht ti xp, ng nht, c mu


vng kem, mi thm, v ng hi ngt; khi lng ring biu kin l 0,52 g/cm3;
kch thc tiu phn nh hn 250m; hm m 2,7%; hm lng isoflavonoid
trong cao t 10,78%.
2.

Phn lp c puerarin v daidzin t r c sn dy.


C th, nghin cu

Xy dng c quy trnh phn lp c puerarin v daidzin t dch chit sn


dy ti n gin, hiu qu. Sn phm phn lp c tinh khit cao trn ph.
Cu trc ca puerarin v daidzin tinh ch c c khng nh bng cc
phng php c tin cy cao: ph IR, ph ESI MS, ph NMR.

60

XUT
Nhng kt qu t c ca nghin cu ch l nhng thnh qu kho st,
thm d bc u trong vic a ra mt quy trnh chit xut isoflavonoid v phn
lp puerarin, daidzin hon thin t sn dy (kh v ti).

hm nghin cu xin

xut cn c thm nhng nghin cu c th hn nh


1. Nghin cu chun ha nguyn liu sn dy a vo nghin cu
2. Nghin cu kho st cc yu t nh hng ti hiu qu chit isoflavonoid t
sn dy ti v sn dy kh
3. Nghin cu m rng quy trnh chit xut isoflavonoid v phn lp puerarin,
daidzin trn cc quy m ln hn.

61

TI LIU THAM KHO


TING VIT
1.

B Mn Cng Nghip Dc (2007), K thut sn xut dc phm, tp 1, tr.


199-206.

2.

B Y T (2009), Dc in Vit Nam IV, NXB Y hc, tr. 880-881, PL1-1.

3.

B Y T (2006), Bi ging dc liu, NXB Y hc, tr. 38-40, 259-289.

4.

V Vn Chi (1997), T in cy thuc Vit Nam, NXB Y hc, tr. 1023-1024.

5.

L Thy Linh (2009), "Chit xut v phn lp cc Isoflavonoid t cy sn


dy", Kha lun tt nghip Dc s, Trng i hc Dc H Ni, H Ni.

6.

g Vn Thu (2004), Bi ging Dc liu, tp 1,

Dc HN, tr. 38, 259,

273-281.
7.

Th Hoa Vin (2006), "Nghin cu in vivo tc dng ni tit kiu estrogen


ca cao chit isoflavone t c sn dy Pueraria thomsonii Benth"", Tp ch
Khoa hc v Cng ngh, tp 44, s 2, tr. 55-58.

8.

Vin Dc Liu (2006), Cy thuc v ng vt lm thuc, NXB Khoa hc &


K thut, tp 2, tr. 680-686.

TING ANH
9.

Bebrevska L., et al. (2010), "Invivo antioxidative activity of a quantified


Pueraria lobata root extract", Journal of Ethnopharmacology, 127, pp. 112117.

10.

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PH LC
PH LC 1. PIC SC K CA DUNG DC
ERARI I CHIU
PH LC 2. IC C K CA DC C IT DY K
B G
ETHANOL 60%
PH LC 3. IC C K CA
ERARI TI
C
PH LC 4. IC C K CA DAIDZI TI
C
PH LC 5.
IR CA
ERARI
PH LC 6.
K I L
G ( EGATIVE) CA
ERARIN
PH LC 7.
K I L
G ( O ITIVE) CA
ERARI
1
PH LC 8.
HR CA
ERARI
1
PH LC 9.
HexR CA
ERARI
13
PH LC 10.
CR CA
ERARI
PH LC 11.
DE T CA
ERARI
13
PH LC 12.
CexR CA
ERARI
PH LC 13.
IR CA DAIDZI
PH LC 14. PH KHI L NG (NEGATIVE) CA DAIDZIN
PH LC 15.
K I L
G ( O ITIVE) CA DAIDZI
1
PH LC 16.
HR CA DAIDZI
PH LC 17. PH 1Hex-NMR CA DAIDZIN
PH LC 18.
13CR CA DAIDZI
13
PH LC 19.
CexR CA DAIDZI

PH LC 1. PIC SC K CA DUNG DC

ERARI I CHIU

PH LC 2. IC C K CA DC C IT DY K

B G ET A OL 60%

PH LC 3. IC C K CA

ERARI TI

PH LC 4. IC C K CA DAIDZI TI

PH LC 5.

IR CA

ERARI

PH LC 6.

K I L

G ( EGATIVE) CA PUERARIN

OH

OH

4"

HO
3"
2"

HO

6"
5"

O
1"
8

HO

10

1'

4'

OH

PH LC 7.

K I L

OH

OH

4"

HO
3"

6"
5"

2"

HO

G ( O ITIVE) CA

O
1"
8

HO

10

1'

4'

OH

ERARI

PH LC 8.

1H-

R CA

OH

OH

4"

HO
3"

6"
5"

2"

HO

ERARI

O
1"
8

HO

10

1'

4'

OH

PH LC 9.

1Hex-

R CA

OH

OH

4"

HO
3"

6"
5"

2"

HO

ERARI

O
1"
8

HO

10

1'

4'

OH

13C-

PH LC 10.

OH

OH

4"

HO
3"
2"

HO

6"
5"

O
1"
8

HO

10

1'

4'

OH

R CA

ERARI

PH LC 11.

DE T CA

ERARI

OH

OH

4"

HO
3"
2"

HO

6"
5"

O
1"
8

HO

10

1'

4'

OH

PH LC 12.

OH

OH

4"

HO
3"
2"

HO

6"
5"

O
1"
8

HO

10

1'

4'

OH

13Cex-

R CA

ERARI

PH LC 13.

IR CA DAIDZI

PH LC 14. PH KHI L

NG (NEGATIVE) CA DAIDZIN

OH
6''

5'' O

O 7

9 O

1''
4''

HO

3''

OH

6
2'' OH

1'

4'

OH

K I L

PH LC 15.

G ( O ITIVE) CA DAIDZI

OH
6''

5'' O

O 7

9 O

1''
4''

HO

3''

OH

6
2'' OH

1'

4'

OH

PH LC 16.

1H-

OH
6''

5'' O

O 7

9 O

1''
4''

HO

6
3''

OH

2'' OH

1'

4'

OH

R CA DAIDZI

PH LC 17. PH 1Hex-NMR CA DAIDZIN

OH
6''

5'' O

O 7

9 O

1''
4''

HO

3''

OH

6
2'' OH

1'

4'

OH

13C-

PH LC 18.

R CA DAIDZI

OH
6''

5'' O

O 7

9 O

1''
4''

HO

3''

OH

6
2'' OH

1'

4'

OH

PH LC 19.

13Cex-

R CA DAIDZI

OH
6''

5'' O

O 7

9 O

1''
4''

HO

6
3''

OH

2'' OH

1'

4'

OH