Gop Phan Nghien Cuu Phuong Phap Chiet Xuat benzen

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PHN I

t vn

Vng ng l mt cy thuc qu him, mc hoang ch yu cc nc

vng nhit i, nc ta cy mc ph bin min ng nam b, Nam trung

b, Ty nguyn. Ngoi ra cn thy mc nhiu Trung v H Lo,

Campuchia. Vng ng thu hi hu nh quanh nm, thu hoch v thi mng,

phi hay sy kh hoc khng ch bin g khc. Ngi ta tm thy trong r

v thn vng ng rt giu Berberin.

Berberin l hot cht ca nhiu cy nh : Hong lin gai (Berberis sp.),

Hong lin -r (Mahonia sp.), nhiu cy thuc chi Coptis, Hong b

(Phellodendron amurense)... Trong thn v r cy vng ng hm lng

Berberin rt cao, nc ta y l ngun nguyn liu rt qu chit xut

Berberin.

Trong nhn dn, ngi ta dng vng ng cha l, au mt,vim rut .

Trong y hc dng Berberin lm thuc cha l, au mt, vim rut, cn c th

dng lm thuc cha ung th [7]. Ngoi ra, ngi ta cn dng lm thuc cm

mu trong ph khoa sau khi sinh n, cha vim ti mt, c bit l tc dng

tt vi bnh si mt [20]. Trong k ngh ngi ta cn dng Berberin to

cht mu lm thuc nhum, hoc lm cht to mu trong gia v v trong

ung c cn [18] v trong cc lnh vc khc.

c nhiu ti liu nghin cu v phng php chit xut Berberin t

cy vng ng nh phng php chit ca Phm Vit Trang [11], phng

php chit Berberin bng p lc nng ca Nguyn Lim [8] v c rt nhiu

quy trnh khc na. Nhng cc quy trnh chit vn cn mt s nhc im

nh : ko di thi gian, tn nhiu dung mi, ho cht...


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Vi mong mun ng gp mt phn nh trong vic cung cp nhng

thng s v phng php chit xut Berberin ni chung v chit t cy vng

ng ni ring . Do vy chng ti thc hin ti : Gp phn nghin cuphng php chit xut Berberin t cy vng ng Coscinium usitatum

Pierre. Vi ni dung ti nhm gii quyt nhng vn sau :

-Kho st mt s iu kin chit xut Berberin t nng sut, cht lng cao.

-D kin quy trnh chit xut Berberin trong iu kin hin ti Vit

Nam.Trn c s nhng kt qu ny s hon thin quy trnh chit xut

Berberin a vo sn xut.

PHN II

TNG QUAN TI LIU


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1. Ngun nguyn liu chit xut berberin

Berberin l mt alcaloid c pht hin trong khong 150 loi thucnhiu h thc vt khc nhau: H Mao lng (Ranunculaceae), h Hong lin

(Berberidaceae), h Tit d (Menispermaceae), h Cam (Rutaceae)...

Theo Muraviova, Berberin thng c trong nhng cy thuc chi

Berberis :

- Berberis aristata DC.

- B. buxifolia Lam. - B. buxifolia Lam.

- B. canadonsis Mill.

- B. glauca H.B.K

- B. licium Roile

- B. noranensis Schult

- B. sinosis Desf

- B. amurensis Rupr.

C hm lng Berberin trong l 0.15% - 3% , v r n 1% [21]. C

hm lng Berberin trong l 0.15% - 3% , v r n 1% [21].

Karbovic v Bespalova cho rng berberin c trongBerberis vulgaris

L. v B.amurensis Rupr. vi hm lng alcaloid khng di 0.6% [20].

Cn theo Orekhop th trong cyBerberis heteropoda c cha alcaloid

trong c berberin [23].

Trong T in cy thuc ca V Vn Chi cho bit trong thn v r

ca Hong lin gai (Berberis wallichiana. D .C) c cha berberin [2].

Bn cnh nhng cy thuc chi Berberis cho berberin cn c nhng cy

khc cng cho berberin nh cc cy thuc chi Coptis:

- Coptis teeta Wall.

- C chinensis Franch.


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- C . teetoides C.Y. Cheng

- C. deltoidea C.Y Cheng et Hsiao [1].

vi hm lng khng t hn 4% [3].Ngoi ra berberin cng c trong cc cy thuc cy chi Mahonia vi

hm lng cao :

- M. annamica. Gagnep [19].

- M. nepalensis D.C.[2]

- M. bealei Carr. [2]

Ngoi nhng cy trn Berberin cn c tm thy trong v cc cy

nh: Hong b (Phellondendron amurense Rupr. v P. chinense Schneider.),

vi t l 1,6% [2], Th hong lin ( Thalictrum foliosolum D.C.) c chng

0,35% [1], Vng ng (Coscinium fenestratum Colebr.) vi hm lng

khng t hn 1,5% [3].

Tuy nhin hin nay ngi ta thng dng vng ng a vo sn

xut Berberin, l do hm lng Berberin trong thn v r cy cao, ngun

nguyn liu nhiu, d thu hi, c nhiu vng Vit Nam, bin gii Vit

Lo, Campuchia. Do ngi ta coi vng ng l ngun nguyn liu s mt

chit xut Berberin.

2. Chi Coscinium:

Chi Coscinium l mt chi nh trong h Tit d ( Menispermaceae)

phn b ch yu vng ni min ng Nam B, Nam Trung B, Ty

Nguyn, ngoi ra cn thy mc hoang nhiu trung v h Lo, Campuchia.

Theo Phm Hong H trong Cy c Vit Nam m t 3 loi

Coscinium l:[4]

- C. fenestratum Colebr.

- C.blumeanum Miers.

- C. colaniae Gagn.


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phn b vng Ty nguyn, Lc tnh.

Theo Bi Ging Dc liu tp II m t 2 loi:

- C. fenestratum Colebr.- C. usitatum Pierre.

phn b vng ni min ng Nam B, Ty Nguyn.

3. Cy Vng ng:

3.1. Nhng nghin cu v thc vt:

Theo Trn Cng Khnh m t: cy vng ng hay cn gi l vang

ng, vng giang, t-rn.[6]

Cy vng ng l mt loi dy leo, thn g to, sng lu nm c th di

ti 10 m hoc hn na, ng knh thn t 5-10cm hoc hn. Thn v cnh

hnh tr, mt ngoi mu vng xm, c nhng np nhn m hay nhn. Cnh

nh mi u c lng mu hi nu, sau khng c lng, i thnh hi trng,

mang cc vt so ca cung l hnh a. Mt ct ca thn v cnh c mu

vng ti; mt ct ca r c mu vng sm, nhn r nhng dy mch g to

trn nh hnh nan hoa bnh xe, cht nc chy ra t mt ct cng c mu

vng.

L n nguyn , mc so le, phin l mng di, thng hnh trng rng

hoc hnh trng di11-33cm, rng 8-23cm, gc phin l trn, thng hay hnh

tim nng, ngn l thun nhn, mt trn l khng c lng, khi kh thng

nhn, gn gia v cc gn chnh lm xung; mt di l c lng hi trng,

nhn r 5-7 gn xo ra nh hnh chn vt v 1-2 i gn mp ngoi cng,

cng nh h gn hnh mng li nh. Cung l mnh, di 3-16 cm, mi u

c lng hi nu, thng phng ra 2 u, u trn nh cch mp phin l t

0,2-0,8 cm, u di cong gp li nh u gi.

Cm hoa: hoa nh trn cc u hnh cu; ng knh 6-7mm c cung

nh di 10-30mm, c sp xp trong mt chm di 5-11cm trn nch l


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hay on thn gi khng mang l; cc cm hoa ny ng n c hay t

hp vi ci mt, trc chnh v cc nhnh ca cm hoa mnh, c lng t mu

nu, cc l bc dng di, di 4-5mm. Hoa c gn nh khng cung, bao hoac 6-9 b phn hnh bu dc gn bng nhau, mt ngoi c lng, mt trong

nhn; 3-6 b phn pha trong di 1,5-2mm, cc b phn ngoi cng nh hn

di1-1,5mm, nh thp hn. B nh c 6 nh xp thnh 2 vng; vng ngoi

gm 3 nh ri, di 1mm, 3 nh vng trong dnh lin nhau pha di. Hoa

ci c bao hoa ging nh hoa c, 6 nh lp hnh chu di 1mm, b nhy gm

3 l non di 2mm, c lng dy, vi hnh si, un cong.

Chm qu di 10-15cm, mang 20-30 qu hch hnh cu, ng knh

2,5-3cm, mt ngoi c lng mu nu n vng cam, tht qu mu vng, v

ng; v qu trong to thnh hch cng, dy ti 3mm.

Nm 1999, Tt Li [10] m t cy vng ng khc vi Trn

Cng Khnh: L mt cy leo to, c phn nhnh, mc b trn mt t hoc leo

ln nhng cy g cao. Thn non mu trng bc, thn gi mu ng, x x, c

vt tch ca l rng. Ct ngang thn c hnh bnh xe, gia c vng li tu xp.

L mt trn xanh, mt di mu trng nht, c 5 gn ( 3 gn ni r).

Hoa mu trng pht tm, mc thnh xim k l. R hnh tr, u thun

hnh nn, mt ngoi mu trng nht, mt trong mu vng.

Theo V Vn Chi m t [2]: Vng ng l mt loi dy leo to, thn

rng 5-7cm c th ti 15-20cm nhng gc gi; g mu vng, v thn nt n

mu xm trng; cc nhnh, mt di l, cm hoa v qu c lng mu trng

bc. L mc so le, phin to n 25cm, gn gc 5; mu trng mc mt di;

cung phnh v cong gc, hi dnh vo trong phin l. Hoa nh mc thnh

chm tn trn thn gi, hoa c c 5 nh, hoa ci c nh lp, 3 l non c lng.

Qu hch trn, ng knh c 2,5cm.


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Ring Phm Hong H [4] li m t cy vng ng vi nhng loi khc

nhau:

- Coscinium fenestratum Colebr. ( Dy vng ng) Dy leo to, thn rng 5-7cm, g vng; nhnh, mt di l, pht hoa v tri c lng the qun, trng,

phin l to n 25cm, c 5 gn xut pht t y, trng mc mt di, cung

ph v cong y. Chm mang tn trn thn gi; hoa c c 6 tiu nhu, hoa

ci c tiu nhu lp, 3 tm b c lng cho ra tri trn, to 2,5cm.

- C. blumeanum Miers. ( Vng ng Blume) Dy leo, thn to. L c phin xoan

hay xoan trn di, to 12-35cm, rng 6-20cm, khng lng. Lc nh nh mt

bt, mt di mc trng, c lng dy, gn y 7, gn ph 2-5 cp, Cung

di 6-20cm, gn cch ba 1,5-2cm. Hoa u to 10-13cm; hoa c c 3-6 l

i, 6 tiu nhu. Hoa ci c 6 l di, 3 tm b. Qu nhn cng xoan, c lng,

to 2,5cm.

- C. colaniae Gagn.

3.2. Nhng nghin cu v thnh phn ho hc:

*.Theo nhng nghin cu ca nc ngoi:

Trong r cy vng ng C.fenestratum Colebr. c mt s alcaloid th

yu nh Berlambine, noroxyhydrastinine v dihydroberlambine [14].

Ngoi ra trong r cy C. fenestratum Colebr. c mt alcaloid mi,

l: 12, 13-dihydro-8-oxo-berberin (I), cng c tc dng tt nh berberin.

O

O

N O

OMe

OMe

H

(I)


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Bn cnh cn c oxyberberin, tetrahydroberberin ( canadin),

sitosterol, v stigmasterol [15].Cc tc gi khc cng cho rng: Berberin c tng hp trong thn

v r ca cy vng ng C.fenestratum Colebr. [16].

*. Theo nhng nghin cu trong nc:

Trong thn v r vng ng c cha Berberin vi t l t 1,5-3%. Ngoi

ra cn c t palmatin, jatrorrhizin [1].

Theo Tt Li[10]: Trong vng ng c nhiu alcaloid dn xut ca

isoquinolein, ch yu l Berberin, t l Berberin chim t 1,5-2%, c khi n 3%.

V Vn Chi[2] li cho rng: thnh phn ho hc ch yu ca vng ng

l Berberin, saponin; thn cha Berberin c t l ti 3,5%. Ngoi ra cn c

Ceryl alcohol, hentriacontane, sitosterol, acid palmitic v acid oleic, glycosid

sitosterol, saponin v vi cht nha. nc ta trong thn v r cy vng ng

c alcaloid berberin vi t l 1,5-3%.

Nguyn Lim [9] xc nh thnh phn ho hc ca cy vng ng l

Berberin chim t nht l 82% so vi alcaloid ton phn, phn cn li l

palmatin, jatrorrhizin...

Ngoi ra, trong lun n tin s [9], Nguyn Lim kho st k cy

vng ng v hm lng alcaloid protoberberin trong cc b phn khc nhau

ca cy. Kt qu l thn cy gi c 2-3%, cnh nh 1-1,5%, l v qu 0,1-

0,7%, v thn trn 5%, v phn thn g 0,85%.

iu kin bo qun c nh hng ln n cht lng nguyn liu.

m cao lm gim nhanh hm lng alcaloid v c th mt ht hoc gim t

3% xung cn 0,13%.

3.3. Nhng nghin cu v ng dng cy vng ng trong y hc:

*. Theo Tt Li (1999) [10]:


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-Nhn dn thng dng thn v r cy lm thuc h nhit, cha st rt,

cha l, a chy, au mt. Dng dng thuc bt hay thuc vin, ngy ung 4-6(g)

-Dng lm nguyn liu chit xut Berberin. Berberin hydrocloriddng cha l, a chy, au mt. Ngy ung 0,02-0,20 g di dng thuc vin.

Ngi ta cn dng cha bnh v gan, mt, vng da, n ung kh

tiu; hoc pha dung dch 0,5-1% dng nh mt, cha mt au, hay ra mt.

*. Theo V Vn Chi [2]: Vn Chi [2]:

Vng ng l ngun nguyn liu chit xut Berberin. Thng dng

cha a chy, l trc khun, vim rut, vng da, st, st rt, km tiu ho.

Ngy dng 6-10g dng thuc sc, thuc bt hay vin. Hoc dng Berberin

chlorid dng vin nn 0,05 g, ngy 6-10 vin chia lm 2 ln.

Dng dung dch 0,5-1% Berberin chlorid cha au mt. n

ngi ta dng r tr l v dng ngoi ra mn nht, vt thng. Nc sc v

dng cha st gin cch, nc sc thn dng tr rn cn.

Ngoi ra Berberin rt ng nn c tc dng khng virus v c dng

trong mt s trng hp nhim trng nh: Bnh kit l do amip, bnh

Leishmania ngoi da v bnh dch t. Dng di dng Berberin sulfat,

hydrochlorid v dng chlorid [18].

Cc tc gi Nht chng minh: Berberin c nhiu trong cc h thc vt v

c tc dng c ch mt s ung th. Theo Phan Quc Kinh: Berberin l mt

alcaloid c tc dng thng mt, co bp t cung, c tc dng iu tr bnh

Leishmania, Trichomonas, bnh l, t, st rt v au mt. Gn y pht

hin c tnh cht chng ung th ca Berberin [7].

Karbovic v Bespalova cho bit[20]: Vng ng dng lm thuc cm mu

trong ph khoa sau khi , vim ti mt. Berberin sulfat lm gim huyt p,

dng tr vim ti mt v c bit l bnh si mt. Dng dng vin nn 0,005g.


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Cn Maskovski th li cho rng: Berberin sulfat dng cha vim gan,

vim ti mt, si mt, ngy 5-10mg, t iu tr t 2-4 tun. Ngoi ra cn c

th dng di dng cn[22].Berberin c chit ra t C.fenestratum c hiu qu c ch cc vi

khun gy bnh ng rut gia sc nh g, ln, ch...c th trn vo thc

n hoc nc ung [17].

Ngoi nhng ng dng trn, Berberin cn dng lm cht to mu

trong thc phm v trong cc loi ung c cn [18].

V nhng cng dng quan trng nh vy v nhng cng trnh nghin

cu cn phi b sung cho nn chng ti tin hnh ti trn.


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PHN III

THC NGHIM V KT QU.

1. Nguyn liu v phng php nghin cu.

1.1. Nguyn liu:

- Thn cy vng ng (Coscinium usitatum) c thu hoch ti trung

v h Lo (do anh Chu Vn thu hoch). em v thi mng, sy kh 600C,

sau tn thnh bt th 1mm.- Thi gian ly mu: thng 4/2000 (trc ma ma)

1.2. Phng php nghin cu.

1.2.1. nh lng Berberin trong vng ng (theo DVNII- tp 3).

1.2.2. Kho st tan ca cc mui Berberin:

- Dng cc dung dch acid: acid sulfuric 2%, 4%, acid hydrochlorid 1%

v acid acetic 1%.

- Chit bng phng php chit ngui.

1.2.3. Phng php chit xut Berberin trong cy vng ng:

- Kho st nng mui NaCl: tm nng NaCl cho nng sut

chit xut ti u.

- Tm nng H2SO4 cho hiu sut chit xut cao.

- Tm s ln chit t hiu qu cao.

- Tm t l dung mi/ nguyn liu chit t hiu qu cao.

- Cc ho cht, nguyn liu t tiu chun DVN II, tp 3.

1.2.4. Xc nh hm lng Berberin trong sn phm chit c

t vng ng (bng phng php HPLC):

Tin hnh ti trung tm gio dc v pht trin sc k Vit Nam

(EDC) trng i hc Bch khoa H Ni.


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- My sc k HPLC: my Hewlet Packard 1100 km 1 my vi tnh CR-

4AX.

- Ct Zorbax RX-SIL- Pha ng: Ethyl acetat: acid formic: methanol (15: 3: 2)

- Bc sng pht hin: 280 nm

- Lng mu bm: 20l, bm tay.

- Tc dng: 1ml/ 1pht.

- p sut: 66 bar (kg/cm3).

1.2.5. Phng php tinh ch Berberin:- Theo phng php tinh ch Berberin chit t v cy Hong b ca b

mn Cng nghip dc - Trng i hc Dc H Ni.

1.2.6. D kin xy dng v m t quy trnh chit.

1.2.7. Kho st cc quy trnh chit xut Berberin c. So snh vi

quy trnh d kin.

1.2.8. Phng php x l s liu [5]:

S liu trong thc nghim c x l theo phng php thng k ng

dng trong y dc. Xc nh gi tr trung bnh, lch chun (S) v khong

tin cy ().

- Gi tr trung bnh: x =

- lch chun: S2 =

- Khong tin cy trung bnh:

Trong : S: lch chun

n: s ln th nghim

f = n-1: s bc t do

t : Hng s student vi = 0,05.


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So snh 2 gi tr trung bnh theo gi thit trung bnh cng (test T) chp

nhn mc ngha ca (c quy c l p) 0,05.

Theo thng k dc hc:- Nu > 0.05: hai gi tr khc nhau khng c ngha thng k

- Nu < 0,05: hai gi tr khc nhau c ngha thng k.

2- Kt qu thc nghim:

2.1. nh lng Berberin trong vng ng:

Theo dc in Vit Nam II - tp 3 - trang 303.

- Nguyn tc nh lng: theo phng php cn- Tin hnh:

Cn chnh xc khong 10g bt dc liu cho vo dng c chit soxhlet,

chit bng methanol (TT) ti khi dch chit khng cn mu vng nht. Ct thu

hi methanol trn cch thu cho n khi cn li khong 1/10 th tch ban u.

Thm 30ml nc ct v 2 gam Magnesi oxyd (TT). Tip tc un trn cch

thu nhit 60-700

C khong 15 pht, thnh thong lc bnh. Lc ht chnkhng. Ra cn bng 50ml nc ct, chia lm nhiu ln cho n khi dch ra

khng cn mu vng. Gp cc nc ra v dch lc vo mt bnh c dung tch

200ml. Thm 5ml dung dch Kali iodid 50% v khuy u kt tu Berberin

iodat. Ly tm, gn b dch pha trn, ta cn li trong ng ly tm, thm

30ml dung dch Kali iodid 2% v khuy tht k. Dng 50ml nc ct chia ra

lm nhiu ln, chuyn ta vo bnh nn c nt mi dung tch 250ml. un trncch thu, ng thi lc cho berberin iodat phn tn u trong bnh. Khi nhit

trong bnh ln khong 700C, th va lc va thm aceton (TT) cho tan ht

berberin iodat. Thm tc khc 3ml dung dch natri hydroxyd 10% (TT), lc

cho ti khi bt u kt ta aceton - berberin. y bnh, yn ch mt qua

m, lc qua phu xp thu tinh G3 (ng knh l xp 16-40m) cn b


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trc. o th tch dch lc (V.ml). Ra ta bng 10ml nc, ri ra 2 ln na,

mi ln 5ml, sy ta 1050C trong 3h. ngui trong bnh ht m, cn ta

aceton - berberin.1g ta tng ng vi 898,2mg Berberin (C20H18NO5)

1ml dch lc tng ng vi 0,100mg Berberin.

Hm lng phn trm berberin trong dc liu kh kit c tnh theo cng thc:

a: Khi lng ta thu c (g)

v: Th tch dch lc thu c (ml)

P: Khi lng dc liu kh kit ( tr m) ly nh lng (g).

Kt qu bng 1:

Bng 1: Kt qu nh lng Berberin trong dc liu

n m (%) mta (g) Vdch lc (ml) Hm lng Berberin (%)

1

2

3

8,04

8,12

8,09

0,3154

0,2679

0,2854

119

156

138

3,21

2,79

2,94 8,94

TB 2,98(%)

2.2. Kho st tan ca cc mui Berberin:

T lu nhiu tc gi t vn nghin cu dng dung mi thay th

cn Ethylic chit Berberin trong vng ng nh Nguyn Lim, Triu Duy

it, H c Trinh ...


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Mun vy, chng ta phi tm tan ca cc mui Berbrin khc nhau

hay ni cch khc l dng cc dung dch acid khc nhau chit xut

Berberin t dc liu.Nm 1983 H c Trinh a ra h s ho tan ca cc mui Berberin

[12]:

- B. iodat: 1: 2130 : 1: 2130

- B.clohydrat: 1: 500 : 1: 500

- B. citrat: 1: 125 : 1: 125

- B. photphat: 1: 15 : 1: 15

- B. acetat: 1: 20 : 1: 20

- B. sulfat: 1: 30 : 1: 30

- B. sulfat acid: 1: 100 : 1: 100

Nh trn ta thy h s ho tan ca cc mui Berberin trong nc ln

nht l Berberin photphat, Berberin acetat ri n Berberin sulfat trung tnh.

Acid photphoric v Acid acetic l loi acid kh kim t tin nn khng c

s dng lm dung mi chit xut, cn acid sulfuric cng nghip l loi ho

cht d mua, r tin nn vic s dng lm dung mi chit xut l thch hp.

Da trn c s chng ti tin hnh kho st tan ca Berberin vi

cc dung dch acid sau: acid sulfuric 2%, 4%, acid clohydrid 1%, acid acetic 1%.

Cn 5 gam dc liu, chit vi 4 loi dung dch trn theo phng php

ngm. Mi ln chit vi 40ml dung dch acid (T l dung mi/dc liu: 8/1),

ngm t 2-3 ting, thnh thong lc, chit 3 ln. Gp cc dch chit li. Cn

0,1g than hot cho vo bnh ng dch chit, lc khong 10 pht. Lc loi b

than hot. Lng dch lc thu c l 100ml. Cn 10g NaCl cho vo mi

bnh, lc cho tan ht, lng qua m.Lc ly ta, em sy 600C n khi

lng khng i. em cn.


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Quy trnh chit theo s 1.

+ dung dch acidngm 2-3h,thnh

Bt dc

Dch B 1


Dch B 2


Dch B 3

Dch

Dch B

0,1g thanhot

+ NaCl, ta qua m

Dch Berberin clorid th

C

Sy nhit


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Kt qu thu c bng 2:

Bng 2: Khi lng Berberin thu c khi chit vi 4 dung dch acid

khc nhau.

Dung mi

S ln

H2SO4 2%

(g)

H2SO4 4%

(g)

HCl 1%

(g)

CH3COOH 1%

(g)1

2

3

4

5

0,2629

0,2927

0,2768

0,2813

0,2750

0,1783

0,1826

0,1950

0,1736

0,2036

0,1344

0,1530

0,1486

0,1366

0,1467

0,3917

0,3719

0,3820

0,3898

0,3905 1,3887 0,9331 0,7187 1,9259

TB0,2777 0,0134

0,1866 0,0153

0,1437 0,0101

0,3852 0,0126

Kt qu c tnh theo phng php thng k vi mc ngha = 0,05.Cc kt qu c biu din qua th 1.

th 1: Kho st tan ca cc mui Berberin.


18/38

Qua bng 2 v th 1 ta thy nu chit vi dung dch acid acetic 1%

s cho kt qu cao nht, sau l n dung dch acid sulfuric 2%. Nhng do

acid acetic l mt acid d bay hi, c v t tin, cn acid sulfuric r, d

mua, hu nh khng bay hi, do chng ti chn acid sulfuric chit

Berberin t vng ng vi nng l 2%.

2.3. Chit xut Berberin t cy vng ng:

*Kho st nng H2SO4:

Vi nng acid H2SO4 nm vng 2% cho hiu qu chit xut cao,

nhng chnh xc vng no th cha r, do vy chng ti tip tc th nhgim,

tm c nh ca th nghim. V th chng ti tin hnh th nghim nh trn

nhng chit vi dung dch acid sulfuric cc nng khc nhau v nng

mui NaCl l 32,4%. Nhng qua qu trnh thc nghim cho thy: sau khi sy

kh vn cn ln rt nhiu mui NaCl bt u t nng 20%. Do chng

ti tin hnh ra ta vi 5ml nc, em sy li v cn. Do Berberin clorid tan

t trong nc nn trong qu trnh ra s mt i mt lng nh Berberin

clorid nhng khng ng k.

Kt qu c ghi bng 3.


19/38

Bng 3: Khi lng Berberin thu c khi thay i nng H2SO4

H2SO4(%)

n1,5 1,6 1,7 1,8 1,9

1

2

3

4

5

0,6032

0,6139

0,6003

0,6108

0,6083

0,6304

0,6318

0,6280

0,6311

0,6258

0,6645

0,6589

0,6603

0,6680

0,6586

0,7241

0,7281

0,7192

0,7301

0,7308

0,6844

0,6903

0,6859

0,6930

0,6873 3,0365 3,1471 3,3103 3,6323 3,4409

TB0,6373 0,0068

0,6294 0,0030

0,6621 0,0050

0,7265 0,0059

0,6882 0,0042

(Tip bng 3):

H2SO4(%)

n2,0 2,1 2,2 2,3 2,4

1

2

3

4

5

0,8800

0,8763

0,8789

0,8808

0,8732

0,8613

0,8645

0,8598

0,8601

0,8585

0,8676

0,8613

0,8571

0,8601

0,8549

0,7336

0,7348

0,7402

0,7299

0,7367

0,6968

0,6950

0,7003

0,7109

0,6980 4,3892 4,3042 4,3010 3,6752 3,5010

TB0,8778 0,0038

0,8608 0,0028

0,8602 0,0059

0,7350 0,0046

0,7002 0,0077

T kt qu trn a ra th sau:

V th.


20/38

th II: Mi lin quan gia khi lng Berberin v nng H2SO4.

Nhn vo th ta thy nng dung dch sulfuric 2% v nng mui

32,4% th thu c kt qu cao nht, sau l nng acid H2SO4 2,1 -

2,2%, cn nng H2SO4 1,5% th thu c kt qu thp nht.

Kho st nng NaCl:

tin trong qu trnh lm thc nghim, chng ti chn nng dung

dch H2SO4 l 2%.

Chng ti tin hnh thay i hm lng NaCl, chit vi 40ml dung dch

H2SO4 2%, chit 3 ln.

Kt qu c ghi bng 4.


21/38

Bng 4: Khi lng Berberin thu c khi thay i nng Natri clorua

NaCl(g)

n10 15 20 25 26

1

2

3

4

5

0,2852

0,2782

0,2612

0,2518

0,2820

0,3596

0,3630

0,3542

0,3841

0,3456

0,4621

0,4632

0,4716

0,4526

0,4710

0,6343

0,6318

0,6081

0,6218

0,6280

0,6439

0,6430

0,6512

0,6632

0,6581 1,3584 1,0865 2,3205 3,1240 3,2594

TB0,2717 0,0179

0,36130,0178

0,4641 0,0096

0,6248 0,0130

0,6519 0,0109

(tip Bng 4):

NaCl(g)

n27 28 29 30 31

1

2

3

4

5

0,6509

0,6532

0,6548

0,6601

0,6492

0,6723

0,6681

0,6703

0,6678

0,6715

0,7003

0,7089

0,7102

0,7058

0,7041

0,7517

0,7508

0,7489

0,7520

0,7498

0,7652

0,7701

0,7685

0,7718

0,7690 3,2682 3,3500 3,5293 3,7532 3,8446

TB0,6536 0,0052

0,6700 0,0023

0,7059 0,0048

0,7506 0,0015

0,7689 0,0029


22/38

(Tip bng 4):

NaCl(g)

n

31,5

A1

31,6

A2

31,7

A3

31,8

A4

31,9

A51

2

3

4

5

0,7068

0,7013

0,7102

0,7078

0,7115

0,6699

0,6632

0,6708

0,6657

0,6715

0,6803

0,6815

0,6789

0,6776

0,6808

0,7173

0,7158

0,7192

0,7208

0,7165

0,7637

0,7648

0,7592

0,7719

0,7586 3,5376 3,3411 3,3991 3,5896 3,7882

TB 0,7075 0,0049 0,6682 0,0044 0,6798 0,0017 0,7179 0,0025 0,7576 0,0107

(Tip bng 4):

NaCl

n

32,0

A6

32,1

A7

32,2

A8

32,3

A9

32,4

A101

23

4

5

0,8815

0,87860,8802

0,8820

0,8791

0,8203

0,81690,8210

0,8187

0,8190

0,8943

0,89030,8918

0,8896

0,8908

0,9312

0,93080,9362

0,9296

0,9301

0,9339

0,93260,9348

0,9358

0,9316 4,4014 4,0959 4,4568 4,6579 4,6687

TB0,8803 0,0017

0,8192 0,0029

0,8914 0,0022

0,9316 0,0033

0,9337 0,0010


23/38

(Tip bng 4):

NaCl

n

32,5

A11

32,6

A12

32,7

A13

32,8

A14

32,9

A151

2

3

4

5

0,7135

0,7102

0,7206

0,7186

0,7218

0,7073

0,7166

0,7054

0,7091

0,7125

0,6467

0,6474

0,6508

0,6623

0,6457

0,6491

0,6472

0,6389

0,6596

0,6460

0,6393

0,6308

0,6412

0,6381

0,6481 3,5847 3,5509 3,2529 3,2408 3,1975

TB 0,7169 0,0061 0,7102 0,0054 0,6506 0,0085 0,6482 0,0092 0,6395 0,0077

Cc s liu trn c biu din bng th 3.

th 3: Mi lin quan gia khi lng Berberin v nng NaCl.

Nhn xt: Qua th 3 ta thy Berberin clorid ta nhiu nht nng l t

32,2-> 32,4 (%).

*Thay i s ln chit:

Sau khi tm c nng H2SO4, NaCl t nng sut chit ti u

chng ti tin hnh kho st s ln chit xut Berberin t dc liu. Chit vi


24/38

dung dch acid H2SO4 2,0%, V = 40ml, chit 3 ln, lng mui 32,4 (g). Tin

hnh nh quy trnh trn.

Kt qu bng 5.Bng 5. Khi lng Berberin thu c khi thay i s ln chit

S ln chit

n2 3 4 5

1

2

3

4

5

0,6961

0,6813

0,6938

0,7048

0,7082

0,8443

0,8550

0,8476

0,8493

0,8513

0,8445

0,8490

0,8507

0,8473

0,8586

0,8668

0,8672

0,8591

0,8732

0,8701 3,4842 4,2475 4,2501 4,3364

TB 0,6968 0,0130

0,8495 0,0049

0,8500 0,0066

0,8673 0,0064

T kt qu trn chng ti a ra th sau:

th 4: Mi lin quan gia khi lng ta berberin v s ln chit.


25/38

Vi 2 ln chit th lng dch chit khong 70-> 75ml m lng NaCl

l 32,4(g) nn NaCl khng th tan ht. Sau khi sy th ta Berberin clorid cnln rt nhiu mui v mui berberin thu c s khng kh v rt bt. Mt

khc nu chit 2 ln th khng th ly ht hot cht c trong dc liu.

Vi 4 ln chit ta thu c 0,8500 ( 0,0066) (g), vi 5 ln chit c

0,8673 ( 0,0065) (g) so vi chit 3 ln (0,8495 0,0049) th c cao hn

nhng khng ng k. Mt khc nu chit 4 ln, 5 ln th s tn thi gian, tn

dung mi chit v ln nhiu tp , xt v hiu qu kinh t th khng bc nhn cng v ho cht, do ta ch chn phng php chit 3 ln.

*Thay i t l dung mi /dc liu:

Sau khi kho st c nng H Sau khi kho st c nng H2SO4,

nng mui NaCl v s ln chit, chit c berberin t vng ng t

nng sut chit cao, chng ti tip tc tin hnh thay i t l dung mi/ dc

liu.Tin hnh tng t nh trn: dung dch H Tin hnh tng t nh trn:

dung dch H2SO4 2%, m NaCl = 32,4 (g) chit 3 ln.

Kt qu ghi bng 6.

Bng 6. Khi lng Berberin thu c sau khi thay i t l dung mi/ dc

liu.

V H2SO4 2%

(ml)

n

30 40 50 60

1

2

3

4

5

0,8770

0,8560

0,8668

0,8713

0,8692

0,8150

0,8210

0,8089

0,7960

0,8132

0,6646

0,6543

0,6610

0,6702

0,6589

0,5266

0,5248

0,5132

0,5301

0,5272 4,3403 4,0541 3,3090 2,6220


26/38

TB 0,8681 0,0062

0,8108 0,0115

0,6618 0,0074

0,5244 0,0081

th 5: Mi lin quan gia khi lng Berberin v t l dung mi/dc

liu.

Nu chit vi 30ml dung dch H2SO4 2% th sau khi chit xong lng

dch chit khong 70-80ml, tng t nh trng hp chit vi 2 ln.

Cn nu chit vi 50, 60ml dung dch H 2SO4 2% th mui s tan ht

nhng lng ta Berberin clorid thu c khng cao, li tn dung mi, ta

thu c khng ti, m bt. Do ta chn th tch dung mi chit l:

40(ml)/5g nguyn liu tc l t l dung mi /dc liu l 8/1.

2.4. nh lng berberin trong sn phm chit c t vng ng:

- Theo phng php sc k lng hiu nng cao (HPLC)

Cn 8,0mg berberin clorid chit c pha trong dung mi ng

(ethyl acetat: acid formic: metanol= 13:5:2) trong bnh nh mc 100ml.

Sau ht 1ml bng pipet chnh xc ca dung dch ny pha vo trong bnh

nh mc 100ml. Sau tin hnh o. (xem ph lc)

Kt qu:

C Berberin clorid = 6,7.10-5 (mg/ml)


27/38

T tnh c:

Hm lng Berberin clorid trong sn phm chit c l

83,75(%).

2.5. Phng php tinh ch:

Hin nay Berberin clorid c chit ra di dng th, cn ln rt nhiu

tp cht, bn ra th trng vi gi r, hiu qu cha bnh li khng cao. Mt

khc Berberin clorid th c bn cho Trung Quc vi gi r, ti ngi ta

tinh ch bn li cho Vit Nam vi gi rt cao. Vy ti sao chng ta c ngun

nguyn liu di do, dung mi d kim, sau khi chit xong li khng tinh ch

t hiu qu kinh t cao. Chnh v th chng ti a ra phng php tinh

ch Berberin clorid sau khi chit c Berberin th t vng ng nh sau:

Cn 1,5g berberin clorid th ho tan trong 30ml cn tuyt i, em un

nng cho tan ht, thm 0,1g than hot, un si cch thu khong 15 pht. Sau

lc nng qua giy lc, loi b than hot. Dch lc em c cn 1/4 th tch,

kt tinh iu kin bnh thng, lc ly tinh th. Tip tc tinh ch tip 2

ln na ta thu c berberin clorid tinh khit.

Kt qu: Ln 1: 0,5178 (g) Ln 1: 0,5178 (g)

Ln 2: 0,3806 (g)

Ln 3: 0,2789 (g)

Sau khi tinh ch chng ti tin hnh em nh lng berberin trong sn

phm theo DVN II - tp 3.

Kt qu: hm lng berberin sau khi tinh ch l 98%.

2.6. D kin xy dng, m t quy trnh chit:

Sau mt qu trnh thc nghim, chng ti d kin a ra quy trnh chit

berberin t vng ng nh sau:


28/38

S quy trnh chit:

Dch

B than Dch

+ Than hot, lc khong 10

Dch lc Berberin clorid

+ NaCl nng 32,2->32,4%lc cho tan hon ton, Berberinclorid ta

+ Cn tuyt i, un cch thun tan ht, thm than hot,un thm 15.Lc nng. Bc hi cn khong 1/4th tch. yn, kt tinh thnh

Berberin clorid

Berberinclorid

Kt tinh li 2-3 lnna

Bt dc liu vng

+Chit vi acid H2SO4 2,0-2,2% chit 3lnt l dun mi dc liu: 8 1


29/38

* M t:

Thn vng ng em v thi mng, sy kh nhit 600C, em xay

thnh bt th c 0,1mm. Bt dc liu em chit vi acid sulfuric 2,2%, t l

dung mi/ dc liu l 8/1, ngm 2-3h, thnh thong lc. Chit 3 ln, gp cc

dch chit, thm than hot, lc khong 10 pht sau lc loi b than hot.

T s ml dch lc thu c tnh xem cn khi lng NaCl l bao nhiu

dung dch c nng NaCl l 32,2 - 32,4%, lc cho mui tan thy xut hin

ta Berberin clorid, tip tc lc cho mui tan hon ton. yn qua m. Lc

ly ta c berberin clorid th. em tinh ch: ho tan Berberin clorid th thu

c trn trong cn tuyt i, un cch thu Berberin clorid tan hon

ton, thm than hot un thm khong 15 sau em lc nng. Dch lc thu

c em bc hi cn khong 1/4 th tch. yn iu kin bnh thng

Berberin clorid s kt tinh thnh tinh th hnh kim mu vng. Lc loi b

nc m ly tinh th. Tip tc kt tinh 2-3 ln na ta thu c Berberin clorid

tinh khit em sy kh nhit 50-600C. Kim tra sn phm, cn sn phm,

tnh hiu sut.

2.7. Kho st cc quy trnh chit xut c. So snh vi quy trnh chit

Berberin trn:

Quy trnh sn xut Berberin clorid t v cy Hong B VN ca BM cng

nghip dc:

Bt dc liu cho vo thit b, chit vi cn 90 0. un nng dung mi

790C, un nh th trong 5,30 gi k t khi cn si. Sau em tho kit dch

chit v em thu hi dung mi.


30/38

Ct thu hi dung mi: dch chit cho vo thit b ct thu hi dung mi (cn

khong1/3 th tch ban u), dung mi thu hi dng chit m sau. Dch

m c chuyn sang thit b kt ta. Kt ta Berberin clorid: thm t t ncmui bo ho (t l 500g/1lt) vo dch chit c c. Khuy u trong 30.

ngui iu kin bnh thng Berberin clorid s kt ta c.

Lc ta bng lc chn khng, nc ci thi b. Ra ta ngay trn phu lc

bng cn 900.

Tinh ch: Ho tan ta Berberin clorid trn vo cn, un nng, thm

than hot, un thm 30 khuy u.

Lc nng loi b b than hot. Dch lc cha trong ni kt tinh. Lc ly

tinh th bng lc chn khng. Cn c em ct thu hi. Tinh ch li 1 ln

na.

Sy: tinh th Berberin clorid thu c em sy 50-600 C trong 6gi.

S quy trnhca BM cng

B Dch

Chit bng cn0

Nc c i b Ta Berberin

+ NaCl bo

Dch lc

+ Cn 900, than

kt tinh bnh th-ng

Nc me

Bt dc liu

S d kin

B b Dchchit

Chit bng H2SO

42,0-

2,2%Chit 3 ln

B b Dchlc

+ Than hot

Ta Berberinclorid

+ NaCl 32,2-32,4%

+ Cn tuyti+ Than hot

B Dchlc

Berberin

Bt d-c liu

Dch lcb

Kt tinh bnh thngLc ht chnkhng


31/38

Theo quy trnh chit ca Nguyn Lim:

Ba quy trnh trn c so snh qua bng sau:

Ni dung Quy trnh ca BM

Cng nghip Dc

Quy trnh ca

Nguyn Lim

Quy trnh d

kinThit b Cng knh, mun sn n gin n gin

Tinh thBerberin

Vng ng ti thi lt

Chit bng acid

Dch B b

+ HCl + NaCl

Berberin Dch trong

Ra ta, sy

Berberin HCl


32/38

xut phi u t nhiuThi gian Khng tn nhiu thi

gian

Tn thi gian Tn thi gian

Nng lng Tn nhiu Khng tn Khng tn tan Berberin t tan trong

cn(1/100), do vy

hiu sut chit sut

km

Tan trong acid

sulfuric(1/30), do

vy hiu sut chit

sut cao hn

Tan trong acid

sulfuric(1/30), do

vy hiu sut chit

sut cao hnQu trnh chit - Khi chit bng cn

s ko theo nhiu tp

cht do trong qu

trnh tinh ch s mt

nhiu thi gian v

Berberin s mt mt

phn do ta tp cht

ko theo.

-Khi cho nc mui

bo ho, th B. clorid

khng kt ta ngay

m kt ta rt chm

v khng ht

Khi chit xong,

dch chit cho ta

ln vi HCl +

NaCl, khng c

giai on loi tp,

v th trong sn

phm s cn ln

nhiu tp cht.

Phi tn thm

HCl, mt khc

mui B. HCl rt

t tan(1/500)

Chng ti tin

hnh loi tp ngay

giai on u, mc

d tn t thi gian

nhng trong qu

trnh tinh ch B.

clorid s tinh khit

hn.

Tinh ch Ta B. clorid th thu c vn cn ln rt

nhiu mui do khi dng cn 90

0

vn c mt lng nh NaCl tan. V th khi

kt tinh vn cn ln mui khng loi b

c trit .

Dng cn tuyt

i s loi cmui, sn phm

tinh khit hn, t

tn thi gian tinh

ch v B. s khng

b hao ht nhiu.


33/38

2.8. Mt s nhn xt:

Phng php chit xut ca chng ti qua nghin cu v thc nghim

thy c u im:- Hiu sut chit tng ng vi cn etylic.

- Hm lng B. clorid trong sn phm 83,75%.

- Phng php n gin: khng cn my mc, khng cn nng lng

(t0), c th p dng cho bt c x nghip no.

- Dung mi d kim, r tin, tit kim 1 lng ho cht.

- Gi thnh h.

- Sn phm thu c khng ln nhiu tp cht v trong qu trnh tinh

ch khng b hao ht nhiu.

- Quy trnh c th dng cho dc liu ti, khng cn phi, do

khng tn thi gian v din tch phi.

- Gim bt s ln kt ta trnh lng Berberin hao ht theo nc t.

- C th tin hnh chit Berberin clorit t vng ng ngay sau khi thu

hoch v c bit l ngay ti ni thu hoch (ti rng ni), Berberin clorid th

c th em v cc x nghip c iu kin tinh ch.


34/38


35/38

PHN IV

KT LUN V XUT

Qua qu trnh tin hnh thc nghim tm ra phng php chit xut

Berberin t cy vng ng, chng ti rt ra nhng kt lun sau:

- Kho st c tan ca cc mui Berberin, acid acetic l acid tt

nht chit Berberin ni chung v t vng ng ni ring.

- Vi quy trnh ci tin, chng ti tm c nng H2SO4l: 2,0 n

2,2%, nng mui l: 32,2- 32,4%, t l dung mi / dc liu l 8/1, chit 3

ln t hiu qu chit xut cao vi hm lng Berberin l: 83,75%.

- D kin c quy trnh chit xut Berberin t vng ng ni ring v

Berberin ni chung. Quy trnh ny tit kim c ho cht, dung mi. Mt

khc dung mi v ho cht r tin, d kim, khng c, khng cn u t thit

b nhiu, c th p dng rng ri cho cc x nghip v ngay c trong rng.

dng phng php tinh ch tinh ch Berberin clorid th thu c

t vng ng t hm lng 98%.

xut:

- Kim tra li quy trnh trn quy m pilot c kt lun chc chn

trong sn xut.

- Vic chit xut Berberin di dng kt tinh to hng xut khu

cng cn c k thut v trang b tt mi chit c hiu sut cao.

- C th p dng chit xut ngay ti ni thu hoch nhm tn dng

ngun nguyn liu v trnh hin tng gim hm lng Berberin trong vng

ng bi m, c bit l nhng ni c iu kin bo qun kh khn .

- Khi thu hi nn thu hi c l v r; cht cy no th nn tn thu

nguyn liu cy , nhm tit kim nguyn liu v bo v ngun ti nguyn


36/38

qu gi ca chng ta. Cn bo v v ti sinh cy vng ng bo m sn

xut lu di./

Ti liu tham kho1. Bi ging dc liu- tp 2- trng HDc HN-1998-tr. 89,90,91.

2. V Vn Chi - T in cy thuc Vit Nam - NXB Y hc - 1999-Tr, 1314

3. Dc in Vit Nam II- tp 3. Tr.303; 72-73.

4. Phm Hong H - Cy c Vit Nam - Quyn I - NXB Tr,1999, Tr.333.

5. - Thng k ng dng trong cng tc dc-Trng

H Dc H Ni-T sch sau i hc -1995-trang 16-18.6. Trn Cng Khnh- cy vng ng - Tp ch Dc hc-s 4-1983-BYT

xut bn, tr.8.

7. Phan Quc Kinh-Nghin cu nhng Alcaloid chit xut t cc cy thuc

Vit Nam-H Dc khoa H ni-,1971,tr.10, tr.42.

8. Nguyn Lim- Chit sut Berberin bng p lc nng -Tp ch Dc hc s

3 - 1980 B Y t xut bn tr.10.

9. Nguyn Lim-Gp phn v nghin cu thc vt v ho hc ca cy vng

ng-Lun n Ph Tin s khoa hc-Hc vin Qun y,1981, tr.116.

10. Tt Li-Nhng cy thuc v v thuc Vit Nam - Xut bn ln th

VIII-NXB Y hc, 1999, tr.195.

11. Phm Vit Trang, Nguyn Lim-Gp phn nghin cu ci tin quy trnh

chit xut Berberin t cy vng ng-Tp ch dc liu, tp 5, s 5/2000,

tr.131.

12. H c Trinh, Vin dc liu, chit Berberin clorid trong vng ng bng

dung dch acid sulfuric long. Tp ch Dc hc, s 3,1983, BYT xut bn, tr.19.

13. Trng H Dc khoa H ni-Sn xut Berberin clorua t v cy Hong

B Vit Nam -Thc tp k thut chit xut, tr.21.

14. Chemical Abstracts (C.A) Vol 109, 1988, 79561j.

.Nguyn Tin


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15. C.A-Vol112-1990-73749s.

16. C.A-Vol116-1992-233838h.

17. C.A-Vol 118-1993-39240h.18. Martindale-The extra pharmacopoeia-31th Edition, The Royal

pharmaceutical society- 1996 p.1678.

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20.


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LI CM N

Em xin chn thnh cm n:_Thy gio TSKH Trn Vn Thanh

- C gio Th.S Nguyn Bch Thu

- KS Nguyn Th Hanh Trung tm EDC

Cng tt c cc thy c gio b mn dc liu - Trng i hc Dc

H Ni gip em hon thnh kho lun tt nghip ny.

ng thi em cng xin gi li cm n Ban Gim hiu cng ton th

cc thy c gio cc b mn, cc phng ban to iu kin gip em

trong qu trnh hc tp v rn luyn ti trng.

H Ni, thng 5 nm 2001

Sinh vin

Nguyn Hong Thu Thu

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