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B GIO DC V O TO B Y TTRNG I HC DC H NI
PHM TH PHNG DUNG
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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 CHCAO KH V PHN LP PUERARIN
T SN DY
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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 vanh 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
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MC LC
DANH MC CC K HIU, CHVIT TT
DANH MC CC BNG, BIU
DANH MC CC HNH V, TH
T V ............................................................................................................. 1
Chng 1. TNG QUAN ......................................................................................... 2
1.1. Vi nt vcy sn dy ................................................................................. 2
1.1.1. c im thc vt v phn bca cy sn dy ..................................... 2
1.1.2. Thnh phn ha hc ca rcsn 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 tsn dy ................................ 6
1.2. Puerarin ...................................................................................................... 12
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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 tnguyn liu kh .......................................... 24
2.2.2. Chit xut isoflavonoid tnguyn liu ti .......................................... 24
2.2.3. iu chcao 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 tsn dy kh ........................... 30
2 3 4 t i fl id t d 30
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3.3.1. Kho st cc dung mi chit ................................................................. 40
3.3.2. Tinh chdch chit sn dy kh ............................................................ 44
3.4. Chit xut isoflavonoid tsn dy ti ................................................... 45
3.4.1. Chit isoflavonoid tsn dy ti ........................................................ 45
3.4.2. Tinh chdch chit sn dy ti ........................................................... 46
3.5. iu chcao kh tdch 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 LN ............................................................................................ 57
4.1. Vnguyn liu chit xut isoflavonoid .................................................... 57
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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
ESIMS : ElectroSpray Ionization - Mass spectrometry
(Khi ph - Ion habng phng phpphun 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
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( lch chun)
TLC : Thin Layer Chromatography
(c k bn mng)
TNF : Tumor necrosis factors
(Yu t hoi t khi u)
UV : Ultraviolet(T ngoi)
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DANH MC CC BNG, BIU
Bng 1.1. Cng thc cu to ca mt sisoflavonoid 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 sdng ...................................................................................... 23
Bng 3.1. Mt quang ca dy dung dch cht i chiu ti bc sng 250 nm. . 37
Bng 3.2. Kt qunh 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 qukho st cc dung mi ethanol c nng khc nhau ................ 41
Bng 3.6. Kt qukho st chit isoflavonoid tsn dy kh bng dung mi nc
................................................................................................................................... 43
Bng 3.7. Kt qutinh chdch chit sn dy kh ................................................... 44
Bng 3.8. Kt quchit isoflavonoid tsn dy ti ............................................... 45
Bng 3.9. Hiu sut v hm lng cao c sau tinh ch(thu c t500 ml dch
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DANH MC CC HNH V, TH
nh 1.1. Sn dy ........................................................................................................ 2
nh 2.1. chit isoflavonoid tsn dy ti ................................................... 31
nh 2.2. Quy trnh iu chcao kh ....................................................................... 34
Hnh 3.1. PhUV-VIS ca dung dch cht i chiu ............................................... 37
nh 3.2. ng chun biu thmi tng quan gia nng puerarin v mt
quang ......................................................................................................................... 38
nh 3.3. thbiu 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 cadch chit....... 49
nh 3.7. Quy trnh phn lp daidzin v puerarin ..................................................... 50
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T VN
Sn dy tlu c sdng trong dn gian. Theo y hc ctruyn, rsn
dy l mt vthuc mt, c tc dng gii nhit, sinh tn, chng kht, lm ra mhi,
dtiu, cht, cha cc chng st nng, nhc u, mn nga, rm sy, vim rut,
au ddy... Gn y trn thgii c kh nhiu cng trnh khoa hc chng minhtc dng ca dch chit rsn dy: cha au u [34], chng oxy ha [9], [13], gii
ru [23]. Thnh phn isoflavonoid trong rc 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 cho. Hin nay Trung Quc puerarin c chit xut v
phn lp thnh cng trsn dy phc vcho cho vic bo chcc dng thuc quyc (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 chyu tTrung
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Chng 1.TNG QUAN
1.1. Vi nt vcy sn dy
1.1.1.c im thc vt vphn bca cy sn dy
Sn dy c tn khoa hc l Pueraria thomsonii
Benth., hu 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 thn 10m, l
kp gm ba l cht. Cung l cht gia di, cung l cht
hai bn ngn. L cht c thphn thnh 2-3 thy. Vma
htrhoa mu xanh tm, mc thnh chm kl. Quloi u c nhiu lng. C
di to nng c thti 20kg, nhiu x.
Mun trng ngi ta o cc hsu 50cm, rc v mn ri lp t xp li.
nh 1.1. n dy
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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 tlln
trong rccc loi sn dy, v cho cc tc dng sinh hc ng ch [46], [11],[37].
O
R4
R1
R2O
R3
O
R1 R2 R3 R4
Puerarin H H Glu OH
Daidzin H Glu H OH
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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
Thnghim 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 thoxy cac 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 quny ging vi kt quth nghim trn
ch khng dng reserpin. iu ny chng t hin tng lm gin mch vnh casn dy khng lin quan ti catecholamin m l do tc dng gin c trc tip [8].
1.1.3.2.Tc dng hhuyt p ca cao sn dy
Tim tnh mch vi liu 5-30 mg trn ch, mo c tc dng hhuyt p. Cao
sn dy vi liu 750 mg/kg tim tnh mch c khnng i khng vi tc dng kch
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flavon ton phn tim bp vi liu 200 mg gip ci thin tun hon no cho 53%
bnh nhn, lm gim trlc mch mu no [8].
1.1.3.5.Tc dng i vi hthn kinh
Dch chit sn dy vi liu 2 g/kg cho thng vo ddy trn thgy st bng
vaccin thng hn c tc dng hnhit, bt sn dy c tc dng tng t. c sn
dy vi liu 6 g/kg cho thng vo ddy trn chut nht trng, c tc dng ci thinhin tng tr nhbtn 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 ivi 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 hng huyt v lipid huyt
c sc sn dy vi liu 6-8 g/kg cho thng vo ddy thbnh thng c
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cho thy nng malondialdehyd trong huyt tng-c s dng nh mt du
hiu ca cht bo boxy ha, gim xung mc tng tnh 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 theodi 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 ngdo
ru gy nn. i vi gan ca chut nht trng gy nhim c bng CCl4,
isoflavonoid chit tct cn vi liu 250 mg/kg c tc dng c ch30,7% hot men GOT [8].
Nghin cu ca Thoa 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
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truyn thng vn c ngh n u tin khi nghin cu vcc dc liu mi.
Chc hiu r c im chit cc dc liu ny theo phng php hi lu n
gin mi c thp 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 thgii, 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 rac loi dung mi, nhit , thi gian ti u cho vic chit isoflavonoid t
sn dy.
Ngay tnm 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 chsn
phm hn. Cng t, nhm nguyn cu tm ra c iu kin chit xut tiu cho sn dy l chit bng ethanol 60% trong 6 gi60C [25].
n nm 2007, an Jian v cng s vn tip tc nghin cu chit
isoflavonoid tsn dy bng phng php truyn thng nhng sdng dung mi
chit l nc. Mc d khng phi l mt dung mi c nh gi cao trong nhiu
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dy bng hdung mi hai pha n-butanol/nc. Cui cng, rt ra nhng kt lun
nh sau thnh phn ca hdung 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 tl1:1 l c hiu quhn c; hiu sut chit v hs
phn btng ln tt ccc hdung mi kho st khi tng nhit ; nhng
kt qutnh ton c tm hnh l hon ton ph hp vi dliu thc nghim[17].
Ti Vit am, nm 2009 Dc s L Thy Linh nghin cu chit
isoflavonoid tsn 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
lphn trm hm lng cn thu c v kt qunh tnh isoflavonoid trong dchchit tc gichn ethanol 90o l dung mi sdng chit isoflavonoid tsn
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
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Cng trong nghin cu ny, Yang Hu v cng s chng minh rng,
hiu sut chit khi sdng siu m bnh hng bi dung mi chit [45], tl
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 tl7:3. 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, dchchit thu c khng trong v cao do dnh kh thu hi [45].
Khng chc hiu quhn phng php chit hi lu truyn thng, Mei-
Hwa v cng scn 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)
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c cng cao. ng thi, nghin cu cng ch ra rng hiu sut chit
isoflavonoid ton phn cao hn khi sdng siu m [41].
1.1.5.4.Chit bng vi sng
m 2001, Zhenku Guo v cng s tin hnh chit isoflavonoid tsn
dy bng vi sng. Kt qugy bt ngkhi 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
phngphp. Trong scc dung mi thnghim, 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 chgip rt ngn thi gian chit m cn lm cho qu trnh c
cao trnn nhanh chng hn rt nhiu. Yang Hu v cng s chng minh c
rng, sdng vi sng c thrt 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
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tng ln khng r rt khi ko di thi gian hn 100 pht. iu ny c thgii
thch do tan ca puerarin trong dung mi ti hn v tc chuyn khi u l
cc yu tt phthuc vo thi gian. Do vy, thi gian chit xut cng ln sgip
ha tan c nhiu dc cht vo dung mi chit hn, v do tng hiu sut
chit ln. ng dung mi c sdng l ethanol tuyt i. gi ta thy rng
hiu sut chit puerarin st gi trti a khi thm mt lng xc nh ethanol(120 g) [26].
Tnhng kt quk trn, cc tc gi thc hin ti u ha v tm c
iu kin chit puerarin ti u trcPueraria 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].
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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]BF4km siu m.
Sdng phng php phn tch mt p nh gi cc yu tnh hng tihiu sut chit puerarin v ti u ha cc iu kin chit xut. Kt quchit 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
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1.2.2.1. L tnh: Puerarin l mt isoflavonoid tnhin, c tm thy trong r
sn dyPueraria lobata vPueraria thomsonii. Vtan, 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 nhdung dch CO2 siu ti hn vo dung dch ethanol slm tngtan ca puerarin. ong khi vt qua gii hn ny, vic thm CO2siu
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 tlca acid acetic trong hn hp. Tng nhit lm tng
tan, ngc li tng thtch 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
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isoflavonoid ton phn bng phng php o quangvi khi nh lng bng sc k
lng hiu nng cao l 0,975. iu ny chng t, kt qunh lng isoflavonoid
ton phn bng phng php o quang 250nm l tng quan vi kt qukhi nh
lng bng phng php sc k lng hiu nng cao[11].
Cng thi gian ny, Xu Huaneng v cng scng sdng phng php o
quang 250nm nh lng hm lng isoflavonoid ton phn trong dch chit r
csn dy nhtc dng ca siu m. m lng isoflavonoid trong mu thc
ngoi suy tng chun ca dung dch puerarin [41].
m 2008, trong nghin cu so snh khnng chit isoflavonoid tsn dy
bng cch sdng siu m v bng cch dng vi sng Yang Hu v cng scng sdng 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 thc tnh ton t
ng chun puerarin [45].
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c R=0,9998 i vi daidzin. Phn trm tm li trung bnh khong 103,31% vi h
sphn 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 sdng 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 ginhn thy
rng cba hp cht u c ng chun vi hs 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 thsdng sdng
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
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puerarin (50, 150 v 450M) khng ph thuc vo nng . Tuy nhin, tc dng
th gin ny khng quan st thy vng ng mch chca chut bloi blp
ni mc, iu ny chra rng tc dng gin mch ca puerarin phthuc vo hot
ng ca lp ni mc. Ngoi ra, tc dng chng co mch ca puerarin i vi
phenylephrine hon ton bv hiu ha bi dung dch Ca2+tdo. 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 vtim mch qua knh natri v calci dng L chut lang v tbo
c tm tht chut. Tc dng bo v tim mch ca puerarin chng li cc tn
thng do thiu mu cc bv ti ti mu tbo c tm tht ,.;m/thng qua
vic hot ha cc knh kali nhy cm ATP ti th v c ch vic m knh vnchuyn 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 bmt khi iu tr
paxilline 1mol/L (tc nhn block knh kali canxi hot ha) [43].
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c tnh chng vim ca puerarin c xc minh trn m hnh tai chut ph
v mt vi m hnh tbo vim. Puerarin tc dng khng phthuc liu ngn chn
qu trnh phosphoryl ha v ging ha ca inhibitor-B v chuyn vca ht nhn
NF-Bp65 subunit, lm ngn cn qu trnh biu hin protein v mRNA ca protein
phn ng C vi LPS kch thch tbo mu n nhn ngoi bin. uerarin ngn chn
sbiu hin ca protein phn ng C, iNOS, v cyclooxygenase-2, c chng tnhiu NF-B tbo RAW264,7 c LPS hot ng v ngn chn r rt sbin i
hnh thi ca lipopolysaccharid tbo RAW264,7. Ngoi ra, sbiu hin mRNA
NF-B p65 trong tbo v nng i thc bo protein vim 2 v TNF-trong
tbo ni bc chbi vic sdngpuerarin 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 tphn 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
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u tng ng hoc yu hn 17-estradiol (E2). Nhng kt qu ny chng t
rng puerarin c thgip ci thin tnh trng bniu, mang li nhng li ch ng
ktrong iu trcc 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 DA t bo lympho trn chut,
chng trng puerarin c tc dng trn tcung v c thl mt tc nhn trliu chiu qutrong vic chng tng sn ni mc tcung v hi chng tin kinh nguyt.
1.2.4.6. c chcc ri lon do ru
Khnng sng ca tbo, stch ly lipid bn trong tbo cc tbo gan
ethanol c khi phc vbnh 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
ttiu hy cc tbo sn xut ethanol. Nhng kt quny chra rng c ch
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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. Schuyn ha glucose v insulin c ci thin ng k
nhm chut c thm puerarin sn dy vo chn hng ngy [43].
1.2.5.
Cc nghin cu tinh chpuerar in h in nay
1.2.5.1. Tinh chbng sc k
Sc k iu ch l phng php c sdng rt nhiu v ng vai tr
quan trng trong nghin cu cc cht tnhin. Cc kthut sc k thng dng
l sc k lp mng iu ch, sc k lng cao p iu ch, sc k ctu imca phng php sc k l tch c nhiu hot cht vi tinh khit cao.
m 1999, Xueli Cao v cng s sdng sc k ngc dng tc
cao vi hdung mi ethyl acetatn-butanolnc theo tl2:1:3 (v/v/v) tch
c ngay 6 hp cht tinh khit t80 mg dch chit trong ln chy sc k u
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Mc d phng php tinh chbng 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 chsn 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 chhot cht khc rtin, dtrin 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 Hua-
Neng 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 chpuerarin (daidzein phn bchyu pha
n-butanol). Phn dch ny c lc vi n-butanol trong 1 gi, sau iu chnh
pH ca pha nc bng acid HCl v4,0, lc ny puerarin schuyn sang pha n-
butanol. Thu ly pha n-butanol, tip tc lc vi ng th tch nc trong 1 gi,
pH ca pha nc c iu chnh vgi tr8,0. Lc ny puerarin li chuyn sang
pha nc. Thu ly phn chit nc. Phn tch HPLC cho thy sau 2 ln chuyn
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Cao lng: L cht lng hi snh, c mi vc trng ca dc liu sdng
trong cn v nc ng vai tr dung mi chnh (hay cht bo qun hay chai).
Nu khng c chdn khc, quy c 1 ml cao lng tng ng vi 1 g dc liu
dng iu chcao thuc.
Cao c:L khi c qunh. m lng dung mi sdng 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.
Kthut iu ch
Quy trnh iu chcao kh gm nhng giai on sau:
1.3.3.1.
Chun bdung mi, dc liuDc liu thng c sy kh, chia nhn 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 thacid
ha hoc kim ha dn mi bng acid hydrochloric, acid acetic, amoniac, natri
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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 tlhot cht.
i vi cao thuc c quy nh hm lng, sau khi iu chphi nh lng
hot cht, nu cha t phi iu chnh cao c tlhot cht ng quy nh.
1.3.3.6. Hon chnh chphm
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 chtiu cht lng ca cao thuc
Cm quan: Cao thuc phi c thcht, mu sc, ng nht theo quy nh.
tan:Cao lng phi tan hon ton trong dung mi dng iu chcao.
Mt khi lng do lm kh:Thng thng cao c khng qu 20%, cao kh
khng qu 5%.
Cc chtiu khc:nhim khun, gii hn thuc bo vthc vt, kim loi
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Chng 2.NGUYN VT LIU, TRANG THIT BV
PHNG PHP NGHIN CU
2.1. Nguyn vt liu, thit b
2.1.1.Nguyn liu
a. Csn dy tiv kh thu Ph Th, Hi Dng, inh Bnh. Dc liu ti
emra 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
1 Ethanol 96% Vit Nam DV IV
2 Cloroform Trung Quc Tinh khit phn tch
3 Ethyl acetat Trung Quc Cng nghip
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Cn phn tch Mettler Toledo AB204S (ThuS).
Cn kthut artorius B2001 (c).
y o quang ITACHI U-1900.
n tngoi.
Tsy EERT (c).
My xay ba. My xay sinh t.
Hthng lc ht Buchner.
Cn xc nh hm m nhanh Precisa XM 60 (Thy ).
y o ttrng 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:
+ Bphn loi kh DGU14A
+
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2.2.3.iu chcao kh
iu chcao kh bng phng php phun sy
nh gi mt schtiu 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 phm2.3. 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 phhp thV 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
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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 thti bc sng chn, o lp li 3
ln, ly kt qutrung 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 vobnh 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
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At, Ac : Mt quang ca dung dch thv dung dch chun
Cc : Nng dung dch chun
Vchit : Thtch dch chit
K : Tch cc hspha 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 thv 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
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Tc dng: 1,2 ml/pht.
Thtch 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 dchA. Ly chnh xc khong 5,0 ml dung dch A, pha long 10 ln bng nc ct
c dung dch B.
Tdung dch B pha long thnh dy dung dch chun c nng t0,01
mg/ml n 0,2 mg/ml theo bng sau:
Nng dung dch chun(mg/ml)
0,01 0,02 0,05 0,10 0,15 0,20
Vdd A (ml) - - - 1 1 2
Vdd B (ml) 1 2 5 - 5 -
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
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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 thv dung dch chun
Cc : Nng dung dch chun (mg/ml)
V : Thtch chit hoc thtch dung dch ban u pha mu th.
K : Hspha 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,
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Sau khi khai trin sc k, ly bn mng ra, bay hi dung mi nhit
phng, soi bn mng di n tngoi bc sng 254 nm.
2.3.3.Phng php chit xut isoflavonoid tsn dy kh
Hu ht cc isoflavonoid trong sn dy u dng glycosid, chc mt vi
cht l aglycon nh daidzein, genistein. V vy c thla chn dung mi phn cc
chit isoflavonoid t sn dy. u tin chn dung mi r tin, khng c, dkim, chng ti tin hnh thnghim 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/dll lng isoflavonoid c trong dc liu a vo chit
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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 qutrung bnh.
- Ra sch t, ven bn ngoi- ro nc
Xay nhCn xc nh khi lng
Sy 60oC n kh
B kh
Cti
Cti sau ra
Bt nho
- a vo nc
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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 chc c di p sut gim, 80oC n thcht cao
c. Xc nh khi lng cao v hm lng isoflavonoid ton phn trong cao thu
c.
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90oC trong 30 pht, ngui qua m, gn ly dch trong pha trn. Phn dch ln
ta c lc ht qua giy lc bng hthng 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 chtheo mc 2.3.1.1 v xc nh khi lng cn theo
mc 2.3.9.
Giai on 2:Thc hin ti cng ty cphn ha dc phm Vit Nam (khu
cng nghip Tin n, Bc Ninh).
Dch chit sau khi tinh chc 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.
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Vt
c
c
Ra sch, ct bphn hngCn xc nh khi lng
Xay thnh bt nho
Vt
Sn dy ti
Bt nho
Dch chit nc
B
c
Dch gp
Dch chit
B
lng qua mGn
Tinh bt
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2.3.7.Phng phpphn lp puerarin v daidzin
uerarin v daidzin c phn lp tdch chit bng phng php thay
i dung mi. Sn phm tinh chc o nhit nng chy, gi trRf v khng
nh cu trc bng cc phng php phnh 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 ccphng php ph:
2.3.8.1.Phhng ngoi (IR)
Mu c nghin vi KBr v phhng ngoi (IR) ca sn phm c xc
nh trn my erkin Elmer o trong vng bc sng 4000400 cm
-1
ti Vin HahcVin khoa hc v Cng nghVit Nam.
2.3.8.2.
Phkhi lng (ESI-MS)
Sdng my Varian 320 Ms (M), ngun ion ha ESI ti Vin Ha hc
Vin Khoa hc v Cng nghVit Nam xc nh phkhi lng va chm in
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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: thtch dch chit thu c (ml).
2.3.10.Phng php xc nh mt khi lng do lm kh
Cho cccn raschvo trong tsy,synhit1050C 20C trong 1
gi.Sau chuynvo bnh ht mlm nguitinhitphng, cn xc nh
ckhi lngccmc. Chuynmu thvo cccn, cn ckhi lngmb.
Cho ccngmuvo trong tsy,sytinhit1050C 20C. Sau chuyn
vo bnh ht mlm nguinnhitphng, cn xc nhkhilng.Cho limuthvo tsyv tinhnh nhtrn cho tikhi muthtkhilngkhng
ia. uthccoi l tkhilngkhng i,khi chnh lchgia2 lncn
lin tipkhng lnhn0,1% so vikhilngmuthcn ban uivimu
thc khi lngcn ban u l 50g v khng c lnhn0,05% ivimu
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Chng 3.KT QUNGHIN CU
3.1. nh lng isoflavonoid ton phn trong nguyn liu
3.1.1.ng chun isofl avonoid
Tin hnh qut phUV-VIS dung dch puerarin nng 8 g/ml trong vng
bc sng 200 n 400 nm, thu c phhp thnh hnh 3.1.
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H s tng quan R2= 0,9996 (1) cho thy nng puerarin v mt
quang c sphthuc tuyn tnh trong khong nng t1,68,0 g/ml ti bc
sng 250nm. Cc isoflavonoid c trong r c sn dy trgenistin u c hp th
y = 0,0901x - 0,0063R = 0,9996
0.1
0.3
0.5
0.7
0.9
0 2 4 6 8 10
M
tquang
Nng (g/l)nh 3.2. ng chun biu th mi tng quan gia nng puerarin v mt quang
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Bng 3.2. Kt qu nh lng isoflavonoid trong nguyn liu
MuMu th
NL 1Mu th
NL2Mu th
NL3
Nguyn liuti
Khi lng mu (g) 10,0 10,0 10,0
m lng isoflavonoidtrong nguyn liu (%)
0,231 0,398 0,582
Nguyn liukh
Khi lng mu (g) 0,4 0,4 0,4m lng isoflavonoidtrong nguyn liu (%)
0,623 0,812 1,054
Kt qucho thy hm lng isoflavonoid trong nguyn liu khc nhau nhiu
gia cc a phng. guyn liu tPh Thcho 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).
Tkt qutrn, chng ti chn nguyn liu sn ti thu mua tPh Thl nguyn
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nh 3.3. th biu din mi tng quan gia nng puerarin v din tch pic
sc k
Tththu c c ththy, din tch pic sc k vi nng puerarin c
mi quan htuyn tnh trong khong 0,010,2 mg/ml (hay 10 200 g/ml). Kt
quny ph hp vi kt qunghin cu ca Man Chen v cng s[30].
nh lng puerarin trong nguyn liu kh v ti thu mua t Ph Th,
y = 14226x + 88.251R = 0.999
0
1000000
2000000
3000000
0 50 100 150 200 250
g/ml
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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 githu c cc
thtch 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
Dung mi chit Ethanol40%
Ethanol60%
Ethanol80%
Ethanol96%
Dcliu
Khi lng dc liu (g) 50,0 50,0 50,0 50,0
m lng isoflavonoid trongdc liu (%)
1,054 1,054 1,054 1,054
m lng puerarin trong dcliu (%)
0,564 0,564 0,564 0,564
Ln 1 232,6 270,7 185,7 149,1
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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
0
100
200
300
400
500
600
Lngisoflavonoid
chitc
(mg)
Ethanol 40% Ethanol 60% Ethanol 80% Ethanol 96%
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Phng php A:Cn chnh xc khong 50g dc liu kh, em nghin vi
nc theo tl1:4 bng my xay sinh tc hn hp bt nho. Chuyn khi bt
nho ny vo bnh nn 500 ml, ra thit bnghin v dng cbng 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 thtch 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 2.3.1.1v 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 thtch 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.
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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 tsn 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 tsn dy kh.
3.3.2. Tinh chdch chit sn dy kh
Qua qu trnh kho st, chng ti xy dng phng php tinh ch isoflavonoid
tdch 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 htethanol 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 sbta v loi i trong qu
trnh lc sau . Ly mu dch lc em nh lng isoflavonoid ton phn v
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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 tdch chit ethanol 96%, tuy nhin khi lng cao thu c rt thp,
khnng chit isoflavonoid v puerarin tdc liu khng cao, dung mi dchy
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 tsn dy kh.
3.4. Chit xut isoflavonoid tsn dy ti
3.4.1. Chit isoflavonoid tsn dy ti
Tin hnh chit isoflvonoid tsn 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 qunh bng 3.8.
Bng 3.8. Kt qu chit isoflavonoid t sn dy ti
Mu 1 2 3 Trung bnh
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hng php chit isoflavonoid tsn 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 tdc liu kh, phng php chit vi dc liu
ti tra c nhiu u im, gi thnh r, ngoi sn phm chnh l isoflavonoid cn
thu thm sn phm phl tinh bt.
3.4.2. Tinh chdch 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 chdch chit nh phng php
ghi mc 2.3.5. Kt quthu c nh bng 3.9.
Bng 3.9. iu sut v hm lng cao csau tinh ch(thu c t 500 ml dch
chit, tng ng khong 150 g dc liu)
Phng php tinh chTa tp chtbng nhit
(1)
Ta tp chtbng ethanol
(2)
Phi hp chaiphng php
(3)
Khi lng cao c (g) 7,97 7,35 6,09
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kh) vi hiu sut tinh chcao, t 92,30%. Tuy nhinphng php c nhc im
l vic ct thu hi ethanol tn thi gian.
m lng isoflavonoid ton phn trong cao tinh ch thu c tphng
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 chn gin, d thc hin, t tn km, c
thtrin khai trn quy m ln.
3.5. iu chcao kh tdch 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 tnguyn liu. Tuy nhin do thi gian nghin cu hn ch, qu trnh phun
sy thc hin ti cng ty cphn ha dc phm Vit Nam kh chng vthigian v cm thc hin cha ln nn chng ti chtin hnh nghin cu iu
chcao kh phun sy tdch chit sn dy ti theo phng php ghi mc 2.3.6.,
sdng phng php tinh chkt ta tp cht bng nhit.
Dch tinh chthu c sau giai on 1 c nng isoflavonoid v tlcn
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nh 3.5. Bt cao khphun sy
Cao kh thu c c nh gi v mt s ch tiu cht lng nh mc2.3.6.a, 2.3.6.b, 2.3.6.c v 2.3.6.d. Kt quthu c nh bng 3.11.
Bng 3.11. t s ch tiu cht lng ca cao kh phun sy.
STT Chtiu Gi tr
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xp, dng 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 chc 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 thtrin khai trn quy m cng nghip.
3.6. Phn lp puerarin v daidzin
Tin hnh sc k bn mng dch chit sau tinh ch, thuc kt qunh sau hnh 3.6.
Ghi ch: Tdung dch th; Cdung dch chun.
Trn bn mng sc k thy c 4 vt m, chng ttrong
dch tinh chc 4 isoflavonoid chnh:
Vt 1 tng ng vi puerarin
Vt 2 dkin l daidzin
Vt 3 cha r
Vt 4 dkin l daidzein
Tham kho ti liu [16]v rt nhiu qu trnh
nh 3.6. nh nh sc klp mng di n t ngoi
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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.
Chit 2-3 lnLc
Dch tinh ch
Cao c
Dch chit ethyl acetat
Lc 2-3 ln, gn.
C gim p
Ethylacetatebo ha nc
Ta
c m(70-80C)
Dung dchethylacetat
Dch chit nc m
ngui
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ththy, daidzin thu c kh tinh khit, trn hnh nh sc k khng thy c xut
hin vt l, vt sc k gn, sch.
Xc nh cc thng svt l ca ta daidzin thu c kt qunh sau
Cm quan: Ta daidzin thu c c dng bt trng, hi vng.
Nhit nng chy: 238,8C (ti liu [21]: 238-240C)
Hnh 3.8. nh nh sc k lp mng di n t ngoi 254nm ca daidzin
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Tin hnh o mu theo phng php ghi mc 2.3.8.2 thu c kt qunh h
lc 14-15.Bng 3.13. Kt qu o ph khi lng (EI-MS) ca daidzin
Tn cht Skhi ca mnh ion phn tm/zDaidzin Phpositive xut hin pic 438,96 ng vi mnh [M+Na]+c skhi l
438,96, tng ng vi khi lng phn t416,38 (C21H20O19)
Phnegative xut hin pic 415,00 ng vi mnh [M-H]-c skhi l
415,00 tng ng vi khi lng phn t416,38 (C21H20O19)
Nhn xt :Kt qucho thy cc cht phn tch cho mnh ion phn tph hp vi
khi lng phn ttng ng.
Phcng hng tht nhn (NMR)
Tin hnh o mu theo phng php ghi mc 2.3.8.3 thu c kt qunh
Phlc 16-19.
OOO
OH
896''
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9 157,2 157,2
10 118,4 118,5
1 122,3 122,2
2 130,0 129,9 7,40 d (J 8,5) 7,41 d (J 8,7)
3 114,9 114,9 6,81 d (J 8,5) 6,82 d (J 8,7)
4 157,0 157,0
5 114,9 115,5 6,84 d (J 8,5)6 130,0 129,9 7,35 d (J 8,5)
1 100,0 100,1 5,09 d (J 7,0) 5,09 d (J 7,5)
2 73,1 73,1
3,27-3,33 m, 2H
3,44-3,48 m, 2H3,70-3,74 m, 1H
3,20; 3,30-3,36 m
3,44-3,52 m3,74 m; 3,46 m
3 76,4 76,4
4 69,6 69,65 77,2 77,2
6 60,6 60,6
OH4,59; 5,06-5,13;
5,42; 9,54 (OH-4)4,58; 5,04; 5,10;
5,39; 9,48
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82,31%. Thnh nh sc k lp mng ny c ththy, 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 svt l ca ta daidzin thu c kt qunh 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 Cloroformmethanol (7:3).
Khng nh li cu trc ca puerarin bng cc phng php ph thu c kt
qunh sau.
Phhng ngoi - IR
Tin hnh o mu theo phng php ghi mc 2.3.8.1 thu c kt qunhPh lc 5. Quan st trn hnh nh phthu c nhn thy c cc di phhp th
c trng nh bng sau :
Bng 3.15. Kt qu o ph hng ngoi ca puerarin
Tn cht nh hp thc trng Nhm chc tng ng
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Phcng hng tht nhn (NMR)
Tin hnh o mu theo phng php ghi mc 2.3.8.3 thu c kt qunh hlc 8-12.
nh 3.11. Cng thc cu to ca puerarin
Bng 3.17. Kt qu o ph R ca puerarin13C NMR v DEPT 1H NMR
CThc nghim
(500 MHz, CD3OD,ppm)
Ti liu [22](125 MHz, DMSO,
ppm)
Thc nghim(500 MHz, CD3OD,
ppm)
Ti liu[22](500 MHz, DMSO,
ppm)2 154,4 (CH) 152,0 8,15 s 8,26 s
O
OH
HO
O
2
3
45
6
7
8
9
10
1'
4'
1"
2"
3"
4"
5"
6"
OHO
HO
OH OH
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3 79,9 (CH) 78,5 3,56-3,58 m, 2H3,77 dd, 1H
3,89-3,93 m, 1H4,14 br, 1H
3,55br, d3,74br, d
3,75m; 4,0m4 71,6 (CH) 70,25 82,6 (CH) 81,2
6 62,7 (CH2) 61,1
OH 4,68
Nhn xt :Cc dliu phv hng svt l ca mu puerarin chit c l ng
nht vi dliu phv hng svt l tcc ti liu tham kho v ph hp vi cu
trc ca puerarin. Kt quo phcng cho thy puerarin v daidzin phn lp c
l tinh khit.
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Chng 4. BN LUN
4.1.Vnguyn liu chit xut isoflavonoid
ti thc hin kho st chit isoflavonoid t chai nguyn liu sn dy
kh v sn dy ti thumua cc tnh Hi Dng, inh Bnh v h Th. Kt qu
kho st s bcho thy, sn dy thu mua Ph Thcho 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 tsn dy, do vy cha chng c ngun nguyn liu.
Kt qunghin 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
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ti xy dng c quy trnh iu chcao 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, dthu hi, bo qun v cht lng
ng u gia cc l m.
Dtrin khai trn quy m ln.
ti cng nh gi c mt sc im c bn ca cao kh phun sy
c. Trong khun khti, vic phun sy cn gp mt skh khn nh
Mi chthc 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
chnn chla chn c phng php tinh chn gin nht tin hnh tinh
chlng dch chthu c mc d khng phi l phng php cho hiu qu
tinh chcao nht.
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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 trongethyl 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 chsdng ethyl acetat. Qua hai ln thay i
dung mi, kim tra s bbng 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 tdch
chit sn dy tido iu kin thi gian c hn. Cn c thm nghin cu hon thin
quy trnh phn lp puerarin v daidzin tdch chit sn dy kh c thchng
trong vic sn xut khng phthuc vo ma thu hoch sn dy ti.
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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 chcao kh phun sy trcsn dy c
hm lng isoflavonoid trong cao trn 10%
Cth, nghin cu
Kho st v la chn c dung mi chit isoflavonoid thch hp tsn dy kh
l ethanol 60%, tinh chdch chit bng nhit, hm lng isoflavonoid trong cao
tinh cht 10,41% Xy dng c quy trnh chit isoflavonoid v thu tinh bt tsn 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 chdch chit l
s dng nhit, s dng ethanol v phi hp c nhit v ethanol. La chn
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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 tsn dy (kh v ti). hm nghin cu xin
xut cn c thm nhng nghin cu cthhn nh
1. Nghin cu chun ha nguyn liu sn dy a vo nghin cu
2. Nghin cu kho st cc yu tnh hng ti hiu quchit isoflavonoid t
sn dy ti v sn dy kh
3. Nghin cu mrng quy trnh chit xut isoflavonoid v phn lp puerarin,
daidzin trn cc quy m ln hn.
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TI LIU THAM KHO
TING VIT
1. BMn Cng Nghip Dc (2007),Kthut sn xut dc phm, tp 1, tr.
199-206.
2. BY T(2009),Dc in Vit Nam IV, NXB Y hc, tr. 880-881, PL1-1.
3. BY T(2006),Bi ging dc liu, NXB Y hc, tr. 38-40, 259-289.4. V Vn Chi (1997), Tin 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 nghipDc 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. ThHoa Vin (2006), "Nghin cu in vivo tc dng ni tit kiu estrogen
ca cao chit isoflavone tcsn dyPueraria thomsoniiBenth"", Tp ch
Khoa hc v Cng ngh, tp 44, s2, tr. 55-58.
8. Vin Dc Liu (2006), Cy thuc v ng vt lm thuc, NXB Khoa hc &
7/23/2019 Xy Dng Phng Php iu Ch Cao Kh V Phn Lp Puerarin T Sn Dy
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properties and effects on rat liver CYP-catalysed drug metabolism", Life
Sciences, 67, pp. 2997-3006.14. Han J., et al. (2007), "Study on extraction of puerarin from Radix Puerariae
by orthogonal test", China Hospital Pharmacy Journal, 27, pp. 332-333.
15. Hua-Neng X., Chao-Hong H. (2007), "Extraction of isoflavones from stem
of Pueraria lobata (Willd.) Ohwi using n-butanol/water two-phase solvent
system and separation of daidzein", Separation and Purification Technology,
56, pp. 85-89.
16. Hua-Neng X., Chao-Hong H. (2007), "Separation and purification of
puerarin with solvent extraction", Separation and Purification Technology,
56, pp. 397400.17. Hua-Neng X., et al. (2008), "Modeling for extraction of isoflavones from
stem of Pueraria lobata (Willd.) Ohwi using n-butanol/water two-phase
solvent system", Separation and Purification Technology, 62, pp. 590-595.
18. Jie-Ping F., et al. (2013), "Extraction of Puerarin using Ionic Liquid Based
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24. Li Liu F., inventor (2013),Puerarin hydrates, preparation methods and uses
thereof, Patent No.: US20130331345 A1.25. Li W., et al. (1998), "A study on the Extractive Technology of Effective
Composition-Pueraria Flavonoid from Pueraria Lobata Ohwi", Journal of
Northwest University, 28, No.2, pp. 131-138.
26. Lingzhao W., et al. (2009), "Investigation of supercritical fluid extraction of
puerarin from Pureraia lobata", Journal of Food Process Engineering, 32,
pp. 682-691.
27. Lingzhao W., et al. (2008), "Optimisation of supercritical fluid extraction of
flavonoids fromPueraria lobata",Food Chemistry, 108, pp. 737-741.
28. Long-Hu W., Yi-Yu C. (2005), "Solubility of Puerarin in Ethanol +Supercritical Carbon Dioxide", Journal of Chemical & Engineering Data,
50, pp. 1747-1749.
29. Man C., Gan X. (2008), "Determination of Puerarin in Radix Puerariae by
HPLC", China Feed, 10.
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36. Song T., et al. (1998), "Soy isoflavone analysis: quality control and a new
internal standard", The American journal of clinical Nutrition, 68, pp.1474S1479S.
37. Tong R. C., et al. (2010), "Evaluation of quality of Radix Puerariae herbal
medicine by isoflavonoids", Journal of Pharmacy and Pharmacology, 62,
pp. 644-650.
38. Volkmar J., Ulrich M. (2011), "Alternative Solvents in Plant Extraction",
Inductrial Scale Natural Products Extraction, Hans-Jorg Bart, Pilz S.,
editors, Wiley - VCH, pp. 55-86.
39. Wang L.-H., Cheng Y.-Y. (2005), "Solubility of Puerarin in Water, Ethanol,
and Acetone from (288.2 to 328.2) K", Journal of Chemical & EngineeringData, 50, pp. 1375-1376.
40. Xiangling H., et al. (2004), "Separation and purification of puerarin using -
cyclodextrin-coupled agarose gel media", Journal of Chromatography A,
1022, pp. 77-82.
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46. Zang Z., et al. (1999), "Studies on isoflavonoid constituents of roots of Qin
Mountain Taibai Pueraria lobata", Chinese Pharmaceutical Journal, 34(5),pp. 301-302.
47. Zhenku G., et al. (2001), "Microwave-assisted extraction of effective
constituents from a Chinese herbal medicine Radix puerariae", Analytica
Chimica Acta, 436, pp. 41-47.
48. Zhou H.-Y., et al. (2007), "Separation and determination of puerarin, daidzin
and daidzein in stems and leaves of Pueraria thomsonii by RP-HPLC",
China journal of Chinese materia medica, 32(10), pp. 937-939.
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PHLC
PHLC 1. PIC SC K CA DUNG DC ERARI I CHIUPH LC 2. IC C K CA DC CIT DY K BGETHANOL 60%PHLC 3. IC C K CA ERARI TI CPHLC 4. IC C K CA DAIDZI TI CPHLC 5. IR CA ERARIPHLC 6. KI LG (EGATIVE) CA ERARINPHLC 7. KI LG (OITIVE) CA ERARIPHLC 8. 1H-R CA ERARIPHLC 9. 1Hex-R CA ERARIPHLC 10. 13C-R CA ERARIPHLC 11. DET CA ERARIPHLC 12. 13Cex-R CA ERARIPHLC 13. IR CA DAIDZIPHLC 14. PHKHI LNG (NEGATIVE) CA DAIDZINPHLC 15. KI LG (OITIVE) CA DAIDZIPHLC 16. 1H-R CA DAIDZIPHLC 17. PH1Hex-NMR CA DAIDZIN
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PHLC 1. PIC SC K CA DUNG DC ERARI I CHIU
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PHLC 2. IC C K CA DC CIT DY K BG ETAOL 60%
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PHLC 3. IC C K CA ERARI TI C
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PHLC 4. IC C K CA DAIDZI TI C
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PHLC 5. IR CA ERARI
( )
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PHLC 6. KI LG (EGATIVE) CAPUERARIN
O
OH
HO
O
2
3
45
6
7
8
9
10
1'
4'
1"
2"
3"
4"
5"
6"
OHO
HO
OH OH
PH LC 7 KI LG (OITIVE) CA ERARI
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PHLC 7. KI LG (OITIVE) CA ERARI
O
OH
HO
O
2
3
45
6
7
8
9
10
1'
4'
1"
2"
3"
4"
5"
6"
OHO
HO
OH OH
PH LC 8 1H R CA ERARI
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PHLC 8. 1H-R CA ERARI
O
OH
HO
O
2
3
45
6
7
8
9
10
1'
4'
1"
2"
3"
4"
5"
6"
OHO
HO
OH OH
PH LC 9 H R CA ERARI
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PHLC 9. Hex-R CA ERARI
O
OH
HO
O
2
3
45
6
7
8
9
10
1'
4'
1"
2"
3"
4"
5"
6"
OHO
HO
OH OH
PHLC 10. 13C-R CA ERARI
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O
OH
HO
O
2
3
45
6
7
8
9
10 1'
4'
1"
2"
3"
4"
5"
6"
OHO
HO
OH OH
PH LC 11 DET CA ERARI
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PHLC 11. DET CA ERARI
O
OH
HO
O
2
3
45
6
7
8
9
10
1'
4'
1"
2"
3"
4"
5"
6"
OHO
HO
OH OH
PHLC 12. Cex-R CA ERARI
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O
OH
HO
O
2
3
45
6
7
8
9
10
1'
4'
1"
2"
3"
4"
5"
6"
OHO
HO
OH OH
PH LC 13 IR CA DAIDZI
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PHLC 13. IR CA DAIDZI
PH LC 14 PH KHI LNG (NEGATIVE) CA DAIDZIN
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PHLC 14. PHKHI LNG (NEGATIVE) CA DAIDZIN
O
OH
O
O
O
OH
OHHO
OH
1'
2
3
45
6
7
89
4'
1''
2''3''
4''
5''6''
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PH LC 16 1H-R CA DAIDZI
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PHLC 16. H-R CA DAIDZI
O
OH
O
O
O
OH
OHHO
OH
1'
2
3
45
6
7
89
4'
1''
2''3''
4''
5''6''
PHLC 17. PH1Hex-NMR CA DAIDZIN
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O
OH
O
O
O
OH
OHHO
OH
1'
2
3
45
6
7
89
4'
1''
2''3''
4''
5''6''
PH LC 18 13C-R CA DAIDZI
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PHLC 18. C R CA DAIDZI
O
OH
O
O
O
OH
OHHO
OH
1'
2
3
45
6
7
89
4'
1''
2''3''
4''
5''6''
PH LC 19. 13Cex-R CA DAIDZI
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PHLC 19. Cex R CA DAIDZI
O
OH
O
O
O
OH
OHHO
OH
1'
2
3
45
6
7
89
4'
1''
2''3''
4''
5''6''