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B Y T
TRNG I HC DC H NI
PHNG TH M HNH
LUN VN THC S DC HC
H NI 2013
B GIO DC V O TO
B Y T
TRNG I HC DC H NI
PHNG TH M HNH
LUN VN THC S DC HC
CHUYN NGNH CNG NGH DC PHM BO CH
M S: 60720402
H NI 2013
LI CM N !
MC LC
AC
aceton
AS
acid shikimic
DD
dung dch
DL
dc liu
EtOH
ethanol
IPA
isopropanol
MeOH
methanol
PP
phng php
SP
sn phm
IR
ph hng ngoi
MS
ph khi lng
H-NMR
13
C-NMR
DANH MC CC BNG
Trang
DANH MC CC HNH V, S , TH
Trang
T VN
Cy hi (cn gi l cy i hi, hi hng, ), tn khoa hc Illicium
verum Hook. f. l mt loi cy phn b ch yu Trung Quc v khu vc
ng Bc Vit Nam. Qu chn phi kh ca cy hi (i hi) c s dng
rng ri trong m thc Trung Hoa, l hng liu trong sn xut ru mi v
nng bnh cc nc phng Ty, l mt gia v trong mn ph ca Vit
Nam, Trong ng y, dc liu ny ch tr au bng, nn ma, au nhc
c-khp do lnh [4], [5]. i hi cn c bit n l ngun nguyn liu sn
xut acid shikimic, thnh phn quan trng trong bn tng hp sn xut thuc
chng virus iu tr cm gia cm type A v type B oseltamivir phosphat (tn
thng mi l Tamiflu) [7], [14]. Tamiflu ang c coi l dc phm c
trin vng nht lm gim tc hi ca bnh cm gia cm loi bnh vn
ang l mi lo ngi v tim n nguy c bng pht thnh i dch cm i vi
con ngi.
Vit Nam v trn th gii, nhiu nghin cu chit xut acid shikimic
t i hi bng dung mi ethanol, methanol, nc cho hiu sut kh cao
nhng thao tc thc hin phc tp, thu hi ti s dng dung mi kh khn, s
dng dung mi tinh ch c hi (methanol, formaldehyd,) v hu ht b
qua vn khai thc tinh du [6], [8], [10], [12], [22], [24], [25]. Gi tr ca
i hi b gim ng k v c s dng ch yu ly tinh du m b qua
acid shikimic. Thc t Vit Nam, i hi cng ch dng ct ly tinh du
m cha tn thu c hot cht ny. Trong khi , acid shikimic do Vit Nam
sn xut li c gi thnh cao hn acid shikimic do n sn xut quc gia
phi nhp i hi. c nhiu nghin cu nhm to ra ngun acid shikimic t
cc phng php tng hp ha hc hay ln men vi sinh, tuy nhin hiu sut
khng cao v gi c khng th cnh tranh vi phng php chit xut t dc
liu [8], [15], [16].
Thc vt - Plantae
Mc lan dy - Austrobaileyales
Hi - Illiciaceae
Chi
Hi - Illicium
Loi
I. verum
1.1.5. Tinh du hi
Tn thng phm: Star Anis Essential Oil, Anise Star,
1.1.5.1. Tnh cht vt l
L cht lng khng mu hay vng nht , mi c bit, v ngt, kt tinh
khi lnh. T trng 200C: 0,978-0,988. im ng c khng nh hn
150C [29].
1.1.5.2. Thnh phn
Thnh phn ch yu v quan trng nht ca tinh du hi l transanethol (82,5 %). Ngoi ra cn c linalol (0,1-1,5 %), estragol (0,3-6 %),
terpineol (0,1-1,5 %), anysaldehyd (0,1-3,5 %). V cc thnh phn khc: 1,4cineol, -bisabolen, -faruesen, -copaen, caryophylen, nerolidol, methylanisoat,
trans-methyleugenol,
cadimen,
foeniculin,
-phellandren
hydroquinon, p-allylanisol, -terpineol, -bergamoten, anysyl-aceton, 1,8cineol, limonen, 1-phellandren, sabinen, -pinen [8].
1.1.5.3. Cng dng
Tinh du hi c tc dng tng t dc liu, thng c phi hp
trong nhiu thuc khc. Ngoi ra tinh du cn dng tng hp cc hormon
oestrogen (diethyl stilbestrol, diethyl stilbestrol propionat). Tinh du hi c
dng lm gia v v hng liu cho rt nhiu sn phm dng trong k ngh
thc phm: ru mi, ko gm, bnh ko, gelatin, pudding, tht, sn phm ca
tht. Hm lng tinh du ti a c php a vo thc phm l 0,07%.
Ngoi ra tinh du cn c dng trong k ngh sn xut x phng, kem nh
rng, thuc l[4].
1.1.5.4. Chit xut tinh du t i hi
C th ct tinh du khi qu cn ti hay phi kh. ng bo cc dn
tc Lng Sn thng ct tinh du hi bng cc ni ct th cng, n gin,
1
6
2
5
3
- Phn ng mu.
tinh ch. Tuy nhin hiu sut khng cao v gi thnh khng th cnh tranh vi
phng php chit xut t i hi [16].
1.2.4. Vai tr acid shikimic
nguyn liu tng hp (-)zeylenon, hot cht c hot tnh khng sinh, c tc
dng chng virus, chng ung th v c s dng cho bnh nhn ung th
trc khi tin hnh ha tr liu [18].
c bit trong nhng nm gn y acid shikimic l nguyn liu bn
tng hp oseltamivir phosphat mt cht c vai tr c ch neuraminidase,
mt enzym cn cho qu trnh gii phng v ly lan virus t cc t bo b
nhim. Oseltamivir phosphat c tc dng trn virus cm type A v type B, c
bit vi chng H5N1. Theo tng hp ca Nguyn Quyt Chin [7]: Kim v
cng s (1997) tm ra con ng tng hp oseltamivir phosphat t acid
shikimic nhng mi ch quy m phng th nghim; Sau Rohloff v cng
s xy dng c phng php tng hp c hiu qu v c th trin khai
quy m pilot, hiu sut tng th ca qu trnh l 21% vi 10 cng on phn
ng v vn qua cng on to azid, mc d c php p dng trong sn xut
nhng n lun tim n nhng nguy c do cc tc nhn v cc hp cht azid
10
11
12
M t nguyn liu
Qu phc, thng gm 8 i, mu nu , xp thnh hnh sao xung
quanh mt tr trung tm. Mi i hnh lng thuyn di 1-2cm, cao di 1cm.
B trn gn nh thng, nhn, c 1 ng nt thnh 2 mnh l ra 1 ht. B
di hi trn v sn si, hai mt bn nhn nheo, mt trong mu vng nht hn
v nhn bng. Cung qu nh v cong, nh vo tr qu. Ht hnh tri xoan,
mu vng nu, nhn bng. Qu c mi thm d chu, v ngt. c im
nguyn liu ph hp vi m t trong Dc in Vit Nam IV [5].
13
X l dc liu
- Phng php chit nng kt hp ct tinh du trc (phng php A):
i hi loi b tp, sy kh 500C.
- Phng php ngm lnh kt hp ct tinh du sau (phng php B):
i hi loi b tp, sy kh 500C, xay, ry qua c ry 2mm.
Hm m nguyn liu xc nh c l 3,26 %.
2.1.2. Ha cht, thit b, dng c
2.1.2.1. Ha cht
TT
Tn ha cht
Ethanol 96%
Vit Nam
Aceton
P, Trung Quc
Methanol
P, Trung Quc
Methanol
HPLC, Merck
H2SO4
P, Trung Quc
Isopropanol
P, Trung Quc
Vit Nam
99,0%, Merck
35 kHz.
T sy MEMMERT (c).
14
FT-NMR Spectrometer.
Dng c
Sinh hn hi lu.
Bnh gn 500mL.
15
- Tc dng: 0,8mL/pht.
- Pha ng: dung dch acid sulfuric 0,01%. - Th tch mu tim: 5L.
- Bc sng pht hin: 210 nm.
16
2.3.1.2. Chun b mu
- Dung dch chun: Cn chnh xc 25mg acid shikimic chun vo bnh
nh mc 50mL, thm nc ct, lc ha tan hon ton. Thm nc va n
vch. Ht chnh xc 5mL, cho vo bnh nh mc 25mL, thm nc n vch,
lc u. Lc qua mng 0,45m. Dung dch chun c nng 100g/mL.
- Mu th l dc liu: Cn chnh xc 250mg bt dc liu, gi vo
giy lc, cho vo bnh nn 100mL, thm 50ml nc, ngm trong 1 gi cho
trng n hon ton. Siu m trong 30 pht. Gn ly dch chit, b tin hnh
chit ln 2, ln 3, mi ln 20mL nc, tin hnh nh ln 1. Gp dch chit cho
vo bnh nh mc 100mL, thm nc n vch, lc u. Ht chnh xc 10mL
cho vo bnh nh mc 25mL, thm nc n vch, lc u. Lc qua mng
0,45m.
- Mu th l dch chit: Pha long dch chit n nng acid shikimic
trong khong 50 150g/mL, lc qua mng 0,45m.
2.3.1.3. Cng thc tnh
Hm lng acid shikimic trong i hi:
X%
m .a.V .f .S
T T T 100%
C
m .V .f .S
T C C C
Trong :
X%: hm lng acid shikimic trong i hi.
mC: Khi lng acid shikimic chun.
mT: Khi lng i hi em chit.
17
HC %
HLA/dc
HLA/dl
100%
Trong :
HC%: hiu sut chit xut.
HLA/dc: hm lng acid shikimic trong dch chit.
HLA/dl: hm lng acid shikimic trong dc liu.
Hiu sut c qu trnh:
H %
mSP
100%
mT
Phng php B: Chit acid shikimic bng phng php ngm lnh c
18
19
13
20
CHNG 3 : KT QU NGHIN CU
3.1. Hm lng acid shikimic trong i hi
Xc nh hm lng acid shikimic bng phng php HPLC nh ch
dn mc 2.3.1. Hm lng acid shikimic c trong i hi xc nh c l
10,8% 0,1%. Sc k ca mu dc liu v acid shikimic chun c
trnh by trong hnh 3.1 v 3.2.
PDA-210 nm
shikimic acid
duoc lieu.dat
Retention Time
200
2.656
mAU
400
0
0
Minutes
200
2.667
mAU
400
0
0
Minutes
21
22
PP A
1.2
Nng (%)
1
1.01
0.8
1.07
1.1
1.12
0.76
0.6
0.4
0.2
0
0
0
Hnh 3.3. th biu din nng ca acid shikimic theo thi gian ca PP A
Nhn xt: Da trn th thy, thi gian t n cn bng ca phng
php A l khong 3gi.
3.2.1.2. Ct tinh du i hi
Tin hnh: Cho 100g nguyn liu vo bnh cu 2L. Cho thm 1,5L
nc. Lp h thng ct ko hi nc. Ct cho n khi ht tinh du. Dch ct
c cho thm 50g mui NaCl, ha tan mui ri chuyn sang bnh gn
500mL. dch ct tch lp hon ton. Gn b lp nc bn di thu c
tinh du. Th tch tinh du c ong bng ng ong th tch 10mL.
Kt qu: Thu c 9,5mL tinh du.
Nhn xt: Thc nghim cho thy: thi gian ct ht tinh du l 5 gi.
Th tch tinh du thu c kh cao 9,5% (9,5mL/100g dc liu). Phng
php ct ko hi nc tin hnh kh n gin.
Hn hp sau khi ct tinh du c em x l chit acid shikimic.
23
Nng
Hiu sut
(mL)
(%)
chit (%)
Ln 1
700 26
1,12 0,07
72,5
Ln 2
680 13
0,29 0,01
18,2
Ln 3
640 5
0,13 0,01
7,7
Ln chit
Tng
hiu
sut
chit
98,4%
Nhn xt: Hiu sut chit acid shikimic trong dch chit ln 1 l 72,5 %,
ln 2 l 18,2%, ln 3 l 7,7%. Hiu sut chit acid shikimic sau 3 ln chit l
98,4%.
24
Kt qu cho thy phng php chit acid shikimic sau khi ct tinh du
c th thu c gn nh hon ton lng acid shikimic trong dc liu vi 3
ln chit. Nh vy trong qu trnh ct tinh du acid shikimic hu nh khng b
phn hy.
3.2.1.4. Kho st tnh chn lc
Tin hnh: Dch chit ca 3 ln chit theo mc 3.2.1.3 c gp li.
Ht chnh xc 1mL dch chit cho vo bnh nh mc 50mL. Thm nc va
n vch, lc k cho u. Xc nh nng acid shikimic bng phng
php HPLC. Sau c n cn, cn lng cn thu c.
Hm lng acid shikimic trong cn (S) c tnh theo cng thc:
CxV
S (%) =
m
Trong :
C : Nng acid shikimic trong dch chit (%)
V : Th tch dch chit (mL)
m : Khi lng cn (g)
nh gi: S cng ln th dch chit cng t tp, qu trnh tinh ch thun
li hn.
Kt qu: Phng php A c S = 54,57 (%).
3.2.2. Chit xut theo phng php B
Phng php ngm lnh c khuy trn tin hnh chit acid shikimic
trc, ct tinh du sau. Qu trnh c tm tt trong hnh 3.4.
25
Bt dc liu
(100g)
Nc (700ml)
Khuy lin tc 14 gi
Lc
B DL
Dch chit L1
Khuy lin tc 14 gi
H2O (700ml) Chit 2 ln
Dch chit L2,3
Gp dch chit
Dch chit
B
DL
Ct tinh du
Tinh du
B
DL
26
Nng (%)
Gi
Nng (%)
0,30
12
1,09
0,42
14
1,14
0,55
16
1,16
0,67
17
1,17
0,79
18
1,21
0,87
20
1,20
1,02
22
1,23
Nng (%)
PP B
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
12
14
16
17
18
20
22
Hnh 3.5. th biu din nng acid shikimic theo thi gian ca PP B
27
Th tch
(ml)
Nng
(%)
Hiu sut
chit (%)
Ln 1
630 9
1,23 0,06
71,5
Ln 2
620 13
0,30 0,02
17,2
Ln 3
610 10
0,09 0,01
5,0
Tng
hiu
sut
chit
28
93,7%
Nng
(%)
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
PP B
PP A
8 12 14 16 17 18 20 22
Hnh 3.6. th biu din nng AS theo thi gian ca 2 phng php
Nhn xt: Thi gian t n cn bng ca phng php A l khong
3gi, thi gian t n cn bng ca phng php B l khong 14 gi. Vy
phng php A c tc chit nhanh hn phng php B.
29
30
mAS(g)*
0,53 0,01
10,706
Phng php B
0,54 0,01
10,044
V (mL)
1860 9
m (g)
S (%)
mAS = V.C/100
Ch tiu
Phng php
Cm quan
Th tch (ml)
Mu sc
Mi v
Phng php A
9,5
Vng nht
V ngt
Phng php B
8,0
Vng nht
V ngt
31
PP A (chit nng kt
hp ct tinh du trc)
PP B (ngm lnh kt
hp ct tinh du sau)
14
98,4
93,7
S (%)
54,57
60,65
9,5
8,0
8,5
47
Ch tiu
Tc cn bng (gi)
php A) nhanh hn trong phng php ngm lnh (phng php B). Hiu
sut chit v th tch tinh du thu c cng nhiu hn. Tuy nhin tnh chn
lc ca phng php A l thp hn so vi phng php B (th hin qua hm
lng acid shikimic trong cn).
-
32
33
34
Dch chit/nc
EtOH96%
C n cao c
MeOH
MeOH (20mL)
Than hot(1g) un
DD/MeOH
IPA
MeOH(20mL)
IPA(50mL)
(50ml)
DD/MeOH
AC (IPA)
kt tinh
Gn
60mL
Lc
Ta
Ra bng MeOH
Sy
DD/hn hp dung mi
Sn phm th
DD/IPA
C bt
C bt
20mL DD/hn hp dung mi
DD/IPA (20mL)
kt tinh
Lc
Ra bng AC(IPA)
Sy
Sn phm th
kt tinh
Lc
Ra bng IPA
Sy
Sn phm th
Hnh 3.8. S quy trnh tinh ch acid shikimic s dng cc loi dung mi
35
36
Dung mi tinh ch
MeOH
7,05
0,16
8,05 0,06
Hm lng AS trong
ta th (%)
80,20
90,60
5,23
0,07
6,89 0,03
Hm lng AS trong
sn phm (%)
95,16
99,51
98,05
99,18
51,31
70,68
60,65
66,87
37
IPA
88,5
Loi SP
Dung mi
Sn phm th
Sn phm tinh ch
Methanol
Hn hp
methanol aceton
Hn hp
methanol isopropanol
Isopropanol
38
lng acid shikimic 88,5%. Acid shikimic c tan trong isopropanol khng
cao. Qua thc nghim cho thy tan ca acid shikimic trong isopropanol l
1/50 nhit thng. Qu trnh ha tan phi s dng nhit tng tan
ca acid shikimic. V tan thp nn khi gim nhit acid shikimic d kt
tinh trong isopropanol. Sn phm trong isopropanol khng ti xp nh trong
hn hp dung mi methanol aceton m cng chc hn. u im: tin hnh
kh n gin, ch cn s dng mt loi dung mi nn thun li thu hi
dung mi; ng thi loi c ng k tp cht dng keo, d lc hn dung
mi methanol, dung mi c. Nhc im: acid shikimic kh tan trong
isopropanol, lng dung mi s dng nhiu.
39
khi lng v hm lng hot cht thp hn khi ch dng isopropanol (tng
ng l 7,58g v 83,51%) do tc ng ca methanol nh nu trn. S
dng hn hp dung mi ny c th gim bt lng isopropanol s dng so
vi phng php ch dng isopropanol tinh ch. Tuy nhin, dung mi
methanol c hi, s dng hn hp 2 dung mi kh thu hi.
(1:3) ln nht (8,05g), hm lng cng cao nht (90,6%). Acid shikimic tan
tt trong methanol v khng tan trong aceton. Mt lng methanol va phi
ha tan hon ton acid shikimic cng vi aceton thm vo vi t l
methanol/aceton 1:3 lm gim tan ca acid shikimic, to iu kin cho acid
shikimic d kt tinh trong mi trng . Mt khc, aceton thm vo gip loi
bt mt lng ng k ta dng keo, lm cho sn phm sch hn. Qu trnh
ra ta s dng dung mi aceton, trnh c mt mt do ha tan tr li mt
phn v acid shikimic khng tan trong aceton. u im: khi lng sn phm
th thu c nhiu nht vi hm lng cao nht, sn phm ti xp, t b bm
dnh dng c. Nhc im: Dung mi methanol c hi, s dng hn hp 2
dung mi kh thu hi, lng dung mi s dng ln, tht thot dung mi
nhiu.
MeOH/aceton > IPA > MeOH/IPA > MeOH. Sn phm sau tinh ch tng
ng vi dung mi tinh ch l MeOH/aceton v isopropanol c cht lng tt
v khng khc bit nhiu (hm lng hot cht l 99,51 v 99,18).
40
41
Mu 1
(H = 58,93%)
Mu 2
(H = 19,67%)
Mu 3
(H = 11,04%)
12
3,16
5,31
7,80
Hm lng AS (%)
82,97
85,03
87,08
Ch tiu
42
Sau khi tin hnh kho st nhiu th nghim, chng ti xut phng
php chit xut acid shikimic t i hi bng nc va thu c acid shikimic
va thu c tinh du. T 100g nguyn liu, chng ti thu c 6,81 g sn
phm. Phng php c s ha trong hnh 3.10.
Nc
1,5L
Bt dc liu
100 g
Bt talc
Dch IPA 1
10g
Lc
Ct tinh du
Hn hp sau ct
Dung dch
loi bt tp
Lc
Nc
2 x 700mL
B dc
liu
Dch
chit L1
C
kt tinh
*Lc, ra, sy
Lc, gp
dch chit
IPA
50mL
Dch chit
Sn phm th
Khuy trn
800C
C
EtOH 96%
100mL
Cao c/nc
Tp
Lc, ra
Than hot
3g
Dch IPA 2
Dung dch
ty mu 2
2 x 20mL
B than
Lc
Dch EtOH
IPA
50mL
C
kt tinh
*Lc, ra, sy
Cao c/EtOH
Gn, ra,
gp dch
Acid shikimic
Dch IPA 1
* Gp cc dch lc, dch ra, c thu hi dung mi, kt tinh thu hi acid shikimic
Hnh 3.10. S tinh ch acid shikimic
43
13
C) v c tinh
khit cao.
Kt qu kim tra cht lng c th nh sau:
3.4.2.1. Nhit nng chy ( tnc)
+ Kt qu: tnc= 184,30C
+ Nhn xt: Sn phm thu c c nhit nng chy o c nm
trong khong nhit nng chy ca acid shikimic (183 - 184,50C The Merck
Index 13rd [21]. Nh vy, s b nhn nh sn phm thu c cha hot cht
ny, v c tinh khit cao.
3.4.2.2. Nng sut quay cc
+ Kt qu: []D20 = - 183,6 (vi c = 4,03% trong nc).
+ Nhn xt: Gi tr thu c gn vi gi tr nng sut quay cc []D18 =
- 183,8 (vi c = 4,03% trong nc) theo The Merck Index 13rd [21].
3.4.2.3. D liu ph
44
max(cm-1)
Nhm chc
Thc nghim Tham kho [30]
1
6
2
5
3
3491,16
3482
3385,16
3384
3236,74
3236
1645,06
1649
C=C (alken)
1681,72
1682
C=O
O-H
+ Nhn xt: Kt qu phn tch ph hng ngoi cho php chng ti nhn
bit cc di hp th c trng ca cc nhm chc in hnh ca acid shikimic.
Ph khi lng
+ Kt qu: Thu c ph khi lng ca sn phm (ph lc 2 v 3).
+ Nhn xt: Ph khi lng ph hp vi khi lng phn t ca acid
shikimic. C th nh sau:
- Ph Positive : Pic ion phn t m/z 197 ng vi mnh [M+Na+].
- Ph Negative : Pic ion phn t m/z 173 ng vi mnh [M-H+].
Ph cng hng t ht nhn (1H-NMR)
+ Kt qu: Thu c ph cng hng t ht nhn ca sn phm (xem
ph lc 4).
45
(ppm)
1
6
J (Hz)
V tr H
2,035
18
1H, d, H-6
2,381 2,416
2,407
17,5
1H, d, H-6
2,535 2,554
3,560
4,75
1H, t, H-4
3,827 3,837
3,847
1H, d, H-5
4,202
4,220
1H, s, H-3
6,570
6,584
1H, s, H-2
Thc
nghim
Tham
kho [30]
1,997 2,033
2
5
3
C7H10O5
acid (3R,4S,5R) 3,4,5
trihydroxy -1-cyclohexen 1- carboxylic
46
(ppm)
V tr C
Thc nghim
30,057
29,89
65,627
65,42
66,951
66,74
70,471
70,32
128,509
128,34
trihydroxy -1-cyclohexen -
138,909
138,59
1- carboxylic
168,133
167,87
-COOH
1
6
2
5
3
C7H10O5
acid (3R,4S,5R) 3,4,5
47
Gi tr
2
V1 = 14, V2 = V3 = 10
3
1
1,6
1
100
1
60
48
Tin hnh theo phng php ngm nng, dung mi l nc. Cc thng
s k thut ca qu trnh chit xut c th hin trong bng 3.12 nu trn.
Cch tin hnh: Cho dc liu cho vo b chit. Cho thm 14L nc,
un si 1 gi, lc thu ly dch chit ln 1. B cn li c tip tc chit thm
2 ln na, mi ln 10L v un si trong 1 gi. Gp cc dch chit, lc qua
giy lc loi tp c hc.
Giai on 3: Tinh ch
+ Bc 1: To sn phm th
Dch chit c c cho n khi thnh cao c. Thm 1,6L ethanol 96%,
lng 2 gi nhit phng. Lc thu dch. Ta c ra li 2 ln, mi ln
300mL ethanol 96%, lc ly dch. Gp dch lc v dch ra, c n cao c.
Ha cao c vo 1L isopropanol, un cch thy, khuy lin tc trong
30 pht. lng, gn ly dch. Phn ta keo tip tc tin hnh 2 ln na, mi
ln 400mL isopropanol, un cch thy, khuy lin tc trong 30 pht. Gn ly
dch. Gp cc dch gn.
Thm 100g bt talc, un cch thy, khy trn 10 pht, nhit
phng 2 gi, sau em lc qua giy lc trn phu c ht chn khng. Dch
lc c c bt dung mi n khi cn 400mL. kt tinh nhit phng.
Sau 5 ngy, lc ly ta. Ra ta 3 ln, mi ln bng 100mL isopropanol. Sy
thu c sn phm th.
+Bc 2: Kt tinh li
T sn phm th, em ha tan vo 1L isopropanol, thm 30g than hot,
un cch thy trong 15 pht. Lc ly dch lc, dch lc c bt n cn khong
400mL, kt tinh 5 ngy nhit phng. Sn phm kt tinh c lc v
49
Sn phm th
Khi
lng
(g)
Hm
lng
acid
shikimic
(%)
170,34
2
3
Khi
lng
(g)
Hm
lng
acid
shikimic
(%)
Nhit
nng
chy
(0C)
89,95
150,16
99,60
184
69,52
165,04
90,03
146,18
99,81
184,2
67,68
168
90,00
143,16
99,88
184,3
66,28
X + SE
67,83
1,62
50
CHNG 4: BN LUN
4.1. V giai on chit xut
Hu ht cc cng trnh nghin cu chit xut acid shikimic t i hi s
dng alcol lm dung mi chit xut, do vic thu tinh du rt kh khn.
Phng php chit xut bng nc c nhng u im sau y:
- Dung mi r tin, khng c hi.
- C th tn dng ph phm ca qu trnh sn xut tinh du hi.
Mc ch ca th nghim kho st tc chit l tm ra thi gian cn
bng gia nng acid shikimic trong dch chit v nng acid shikimic
trong dc liu. Sau thi im cn bng, nng acid shikimic trong dch
chit khng tng thm na. Thi im cn bng l thi im hp l rt
dch chit. Phng php no nhanh t n thi im cn bng tc l c tc
chit nhanh hn. Kho st thi im cn bng ca 2 phng php trong
dch chit ln 1 so snh tc chit ca 2 phng php. Kt qu nghin
cu cho thy thi gian t n cn bng ca phng php A l 3 gi, thi
gian t n cn bng ca phng php B l 14 gi. La chn phng php A
chit xut acid shikimic gip tit kim thi gian mt cch hiu qu so vi
phng php B.
- Chit acid shikimic bng phng php chit nng nhit si ca
nc c th kt hp thu tinh du ln chit th nht (s dng b dng c ct
ko hi nc); t 100g nguyn liu thu c 9,5mL tinh du. Hiu sut chit
acid shikimic trong dch chit ln 1 l 72,5 %, ln 2 l 18,2%, ln 3 l 7,7%.
Hiu sut chit acid shikimic sau 3 ln chit l 98,4%. Nh vy trong qu
trnh ct tinh du acid shikimic hu nh khng b phn hy v c th thu c
gn nh hon ton lng acid shikimic trong dc liu vi 3 ln chit. Trn
thc t sn xut, chng ti xut s dng dch chit ln 1 v 2 x l thu
acid shikimic, dch chit ln 3 lm dung mi cho m sau. Quy trnh chit xut
51
theo phng php chit nng kt hp ct tinh du trc l phng php php
n gin, hiu qu tn thu tinh du v acid shikimic, c th ng dng trn
thc t. Tuy nhin, chng ti khng cp n t l dung mi/dc liu do
thng s ny chu tc ng ln ca c mu. ng thi, phng php ny gn
vi thc tin sn xut tinh du hi ti tnh Lng Sn ca nc ta, c th tn
thu nc ci v b i hi sau khi ct tinh du tip tc x l thu acid
shikimic.
4.2. V giai on tinh ch
Nguyn tc tinh ch acid shikimic c rt ra t nghin cu ny:
-
52
53
54
Kt lun
Mc d thi gian c hn v iu kin thc nghim cn nhiu hn ch,
nghin cu t c 2 mc tiu ra:
1. kho st v la chn phng php chit xut acid shikimic
kt hp thu tinh du t i hi vi dung mi nc.
2. xy dng phng php tinh ch acid shikimic t dch chit
nc.
C th nh sau :
-
tinh du: phng php chit nng kt hp ct tinh du trc v phng php
ngm lnh ct tinh du sau; so snh v tc chit (thi im cn bng l
khong 3 gi v 14 gi), v hiu sut chit (98,4% v 93,7%), v tnh chn
lc (hm lng hot cht trong cn ln lt l 54,57% v 60,65%) v v th
tch tinh du thu c (9,5mL v 8mL). La chn phng php: Ct ly tinh
du i hi bng phng php ct ko hi nc, hn hp cn li em chit
ly acid shikimic.
-
55
Kin ngh
Trn y l nhng kt qu nghin cu bc u, lm c s cho nhng
nghin cu tip theo. Chng ti xin kin ngh:
Tip tc trin khai nh gi quy trnh chit xut acid shikimic trn quy
m ln hn khng nh tnh kh thi ca phng php.
56
in
microorganisms,
WIPO
Patent
Application
WO/2008/128076.
17. Denis V. Bochkov et al. (2012), Shikimic acid: review of its
analytical, isolation, and purification techniques from plant and
microbial sources, J. Chem. Biol., 5(1), pp. 517.
18. Amalia M. Estvez and Ramn J. Estvez (20112), A Short
Overview on the Medicinal Chemistry of ()-Shikimic Acid,
Mini-Reviews in Medicinal Chemistry, 12, pp. 1443-1454.
19. Katarna Hroboova, Jozef Lehotaya and Jozef imrik (2007),
Determination of Quinic and Shikimic Acids in Products Derived
from Bees and their Preparates by HPLC, Journal of Liquid
Chromatography & Related Technologies, 30(17), pp. 2635-2644.
20. LI Wei, CAO Yong, WEI Hua, ZHOU Dong-wu (2008),
Reversed-Phase HPLC Determination of Shikimic Acid in Illicium
vin
ha
cht
http://www.basechem.org/chemical/2869.
BASECHEM,
URL:
PH LC
Ph lc 1. Ph IR ca acid shikimic