Xây Dựng Phương Pháp Điều Chế Cao Khô Và Phân Lập Puerarin Từ Sắn Dây

7/23/2019 Xy Dng Phng Php iu Ch Cao Kh V Phn Lp Puerarin T Sn Dy

1/96

B GIO DC V O TO B Y TTRNG I HC DC H NI

PHM TH PHNG DUNG


2/96

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


3/96

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


4/96

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


5/96

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


6/96

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


7/96

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


8/96

( lch chun)

TLC : Thin Layer Chromatography

(c k bn mng)

TNF : Tumor necrosis factors

(Yu t hoi t khi u)

UV : Ultraviolet(T ngoi)


9/96

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


10/96

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
http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875144http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875144http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875144http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875144http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875144http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875148http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875148http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875148http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875148http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875148http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875148http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875148http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875148http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875148http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875148http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875148http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875148http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875148http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875148http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875148http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875148http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875152http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875152http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875152http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875152http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875152http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875153http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875153http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875153http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875154http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875154http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875154http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875154http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875154http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875154http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875154http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875154http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875154http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875154http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875154http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875154http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875154http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875154http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875154http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875153http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875152http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875148http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875148http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400875144


11/96

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


12/96

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


13/96

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


14/96

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


15/96

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


16/96

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


17/96

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


18/96

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


19/96

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)


20/96

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


21/96

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].


22/96

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


23/96

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


24/96

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].


25/96

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


26/96

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].


27/96

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


28/96

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


29/96

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


30/96

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


31/96

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


32/96

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


33/96

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


34/96

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

+


35/96

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


36/96

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


37/96

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


38/96

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


39/96

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,


40/96

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


41/96

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


42/96

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.


43/96

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.


44/96

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


45/96

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


46/96

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


47/96

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.


48/96

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


49/96

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


50/96

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


51/96

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


52/96

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%


53/96

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.


54/96

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


55/96

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


56/96

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


57/96

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


58/96

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


59/96

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


60/96

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


61/96

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


62/96

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''


63/96

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


64/96

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


65/96

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


66/96

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.


67/96

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


68/96

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.


69/96

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.


70/96

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


71/96

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.


72/96

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 &


73/96

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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

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56, pp. 85-89.

16. Hua-Neng X., Chao-Hong H. (2007), "Separation and purification of

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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


74/96

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.

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medicine by isoflavonoids", Journal of Pharmacy and Pharmacology, 62,

pp. 644-650.

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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
http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835381http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835381http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835381http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835381http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835381http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835381http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835381http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835381http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835381http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835381http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835382http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835382http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835382http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835382http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835382http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835382http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835382http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835382http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835382http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835382http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835384http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835384http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835384http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835384http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835384http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835384http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835384http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835384http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835384http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835384http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835389http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835389http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835389http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835389http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835389http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835389http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835389http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835389http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835389http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835389http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835389http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835384http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835382http://e/MASTER/Extract%20Puerarin%20from%20Puerariae%20lobata/Luan%20van/Luan%20van.docx%23_Toc400835381


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PHLC 1. PIC SC K CA DUNG DC ERARI I CHIU


79/96

PHLC 2. IC C K CA DC CIT DY K BG ETAOL 60%


80/96

PHLC 3. IC C K CA ERARI TI C


81/96

PHLC 4. IC C K CA DAIDZI TI C


82/96

PHLC 5. IR CA ERARI

( )


83/96

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


84/96

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


85/96

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


86/96

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


87/96

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


88/96

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


89/96

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


90/96

PHLC 13. IR CA DAIDZI

PH LC 14 PH KHI LNG (NEGATIVE) CA DAIDZIN


91/96

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''


92/96

PH LC 16 1H-R CA DAIDZI


93/96

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


94/96

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


95/96

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


96/96

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''

Documents

Xây Dựng Phương Pháp Điều Chế Cao Khô Và Phân Lập Puerarin Từ Sắn Dây