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B GIO DC V O TO

B Y T

TRNG I HC DC H NI

PHNG TH M HNH

NGHIN CU CHIT XUT ACID


SHIKIMIC T I HI

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

NGHIN CU CHIT XUT


ACID SHIKIMIC T I HI

LUN VN THC S DC HC
CHUYN NGNH CNG NGH DC PHM BO CH
M S: 60720402

Ngi hng dn khoa hc: TS. Nguyn Vn Hn

H NI 2013

LI CM N !

Vi lng knh trng v bit n su sc, trc tin, em xin gi li cm


n chn thnh ti thy gio TS. Nguyn Vn Hn, ngi trc tip hng
dn, ch bo tn tnh v gip em hon thnh lun vn ny.
Em xin gi li cm n su sc ti PGS.TS. Nguyn nh Luyn v
ton th cc thy c gio, cc anh ch k thut vin B mn Cng nghip
Dc to iu kin thun li, gip em hon thnh lun vn ng thi
hn.
V em cng xin chn thnh cm n cc thy c gio trng i hc
Dc H Ni, nhng ngi dy d v ch bo em tn tnh trong sut
nhng thng nm hc tp ti trng.
Cui cng, vi lng bit n v hn, em xin php c gi li cm n
ti gia nh, ngi thn, bn b ng vin v h tr em trong sut thi
gian qua.
Do thi gian c hn v trnh bn thn cn hn ch nn lun vn
khng th trnh khi nhng thiu st. V vy, em rt mong nhn c s ch
bo tn tnh ca cc thy c v s gp chn thnh ca bn b.

Em xin chn thnh cm n !

H Ni, thng 08 nm 2013


Hc vin:
Phng Th M Hnh

MC LC

DANH MC CC K HIU, CH VIT TT


T VN ............................................................................................1
CHNG 1: TNG QUAN ......................................................................3
1.1. Tng quan v cy hi ............................................................................3
1.1.1. V tr phn loi v phn b..................................................................3
1.1.2. c im thc vt...............................................................................3
1.1.3. B phn dng, thu hi v ch bin ......................................................4
1.1.4. Thnh phn ha hc v cng dng......................................................5
1.1.5. Tinh du hi .......................................................................................6
1.2. Tng quan v acid shikimic ...................................................................7
1.2.1. Cng thc ha hc v tnh cht...........................................................7
1.2.2. K thut phn tch ..............................................................................8
1.2.3. Ngun gc acid shikimic ....................................................................8
1.2.4. Vai tr acid shikimic ..........................................................................9
1.2.5. Mt s nghin cu chit xut acid shikimic t i hi ........................10
1.3. Nhn xt ................................................................................................11
CHNG 2 : I TNG V PHNG PHP NGHIN CU .......13
2.1. Nguyn vt liu .....................................................................................13
2.1.1. Nguyn liu ........................................................................................13
2.1.2. Ha cht, thit b, dng c ..................................................................14
2.2. Ni dung nghin cu .............................................................................15
2.2.1. La chn phng php chit xut .......................................................15
2.2.2. Xy dng phng php tinh ch acid shikimic t dch chit nc ......16
2..3. Phng php nghin cu ......................................................................16
2.3.1. Phng php nh lng acid shikimic...............................................16
2.3.2. Phng php chit xut acid shikimic v ct tinh du.........................18

2.3.3. Phng php tinh ch acid shikimic ...................................................19


2.3.4. Kim tra cht lng sn phm ............................................................19
CHNG 3 : KT QU NGHIN CU.................................................21
3.1. Hm lng acid shikimic trong i hi..................................................21
3.2. Kho st giai on chit xut .................................................................22
3.2.1. Chit xut theo phng php A ..........................................................22
3.2.2. Chit xut theo phng php B .........................................................25
3.2.3. nh gi v la chn phng php chit xut ....................................29
3.3. Tinh ch acid shikimic...........................................................................33
3.3.1. Kho st v la chn dung mi tinh ch .............................................34
3.3.2. Bc u kho st yu t nh hng n qu trnh kt ta acid shikimic
bng isopropanol ..........................................................................................41
3.4. xut phng php chit xut acid shikimic v kim tra cht lng sn
phm ............................................................................................................42
3.4.1. xut phng php chit xut acid shikimic....................................42
3.4.2. Kim tra cht lng sn phm ............................................................44
3.5. M rng quy m....................................................................................48
3.5.1. M t quy trnh ...................................................................................48
3.5.2. Kt qu ...............................................................................................50
CHNG 4 : BN LUN.........................................................................51
4.1. V giai on chit xut ..........................................................................51
4.2. V giai on tinh ch .............................................................................52

KT LUN V KIN NGH....................................................................55


TI LIU THAM KHO

DANH MC CC K HIU, CH VIT TT

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

ph cng hng t ht nhn proton

ph cng hng t ht nhn 13C

H-NMR

13

C-NMR

DANH MC CC BNG

Trang

Bng 1.1. V tr phn loi ca cy hi (Illicium verum Hook. f.)........3


Bng 3.1. Kt qu hiu sut chit acid shikimic theo phng php A...24
Bng 3.2. Nng acid shikimic trong dch chit sau mi gi theo PP B.27
Bng 3.3. Kt qu hiu sut 3 ln chit acid shikimic bng PP B.28
Bng 3.4. Kt qu xc nh h s chn lc ca 2 phng php.30
Bng 3.5. Kt qu ct tinh du ca hai phng php....31
Bng 3.6. Bng so snh tc cn bng, hiu sut chit, hm lng AS
trong cn (S), th tch tinh du v thi gian chit xut.......32
Bng 3.7. Kt qu qu trnh tinh ch
s dng cc dung mi tinh ch khc nhau..........37
Bng 3.8. Kt qu kho st lin quan ti hm m ca
cao trong nc em tinh ch..........42
Bng 3.9. Cc di hp th c trng trong ph hng ngoi..........45
Bng 3.10. Kt qu phn tch ph
cng hng t ht nhn proton (1H-NMR) ..........46
Bng 3.11. Kt qu phn tch ph cng hng t carbon (13C-NMR) .47
Bng 3.12. Cc thng s ca qu trnh chit tch v phn lp
acid shikimic vi c m 2kg..48
Bng 3.13. Kt qu chit xut acid shikimic vi c m 2kg i hi...50

DANH MC CC HNH V, S , TH

Trang

Hnh 3.1. Sc k ca mu dc liu .........21


Hnh 3.2. Sc k ca acid shikimic chun.....21
Hnh 3.3. th biu din nng ca acid shikimic
theo thi gian ca PP A......23
Hnh 3.4. S quy trnh chit acid shikimic v ct tinh du
theo PP B......................26
Hnh 3.5. th biu din nng acid shikimic theo thi gian ca PP B...27
Hnh 3.6. th biu din nng AS theo thi gian ca 2 phng php...29
Hnh 3.7. th biu din hiu sut chit ca 2 phng php..30
Hnh 3.8. S quy trnh tinh ch acid shikimic
s dng cc loi dung mi..35
Hnh 3.9. Hnh nh sn phm th v sn phm tinh ch....38
Hnh 3.10. S tinh ch acid shikimic.....43
Hnh 3.11. Sn phm acid shikimic........50

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].

ti: Nghin cu chit xut acid shikimic t i hi c thc


hin nhm gp phn tm ra phng php chit xut acid shikimic t i hi
t cc tiu ch: quy trnh thao tc n gin, an ton, chi ph thp v kt hp
thu tinh du; t c th trin khai p dng trong iu kin thc t ti Vit
Nam quy m ln hn. Mc tiu nghin cu t ra gm c:
1. Kho st v la chn c phng php chit xut acid
shikimic kt hp thu tinh du t i hi vi dung mi nc.
2. Xy dng c phng php tinh ch acid shikimic t dch
chit nc.

CHNG 1: TNG QUAN

1.1. Tng quan v cy hi


1.1.1. V tr phn loi v phn b
Theo h thng phn loi thc vt ca Takhtajan, cy hi (Illicium
verum Hook. f.) c v tr phn loi khoa hc c th hin trong bng di
y [1], [9].
Bng 1.1: V tr phn loi ca cy hi (Illicium verum Hook. f.)
Gii

Thc vt - Plantae

Khng phn hng

Nhm thc vt c hoa - Angiospermae

Mc lan dy - Austrobaileyales

Hi - Illiciaceae

Chi

Hi - Illicium

Loi

I. verum

Chi Hi (Illicium) c khong trn 40 loi, phn b ch yu ng


Nam , ng v Bc M. Vit Nam chi Hi c 16 loi [8]. Cy hi
(Illicium verum Hook. f.) l mt loi cy xanh quanh nm, phn ln Trung
Quc v ng Bc Vit Nam. N cn c tn gi khc l cy i hi, bt gic
hi hng, hi hng, hi sao, mc chc, mc hi (Ty), pt cc (Dao), [2].
1.1.2. c im thc vt
Cy g nh, cao 6 - 8m, c th n 10m hay hn. Cnh thng, nhn, lc
non mu lc nht, sau chuyn sang mu nu xm. L mc so le, nhng thng
t tp nhng mu trng nh mc vng, hnh mc hoc hnh trng thun, di
8-12cm, rng 3-4cm, u nhn, mt trn mu lc sm, mt di rt nht, gn
m, cung l ngn [1], [2], [3].

Hoa mc ring l hoc 2-3 ci k l; i 5 rng, d rng, mp vin


hng, cnh hoa 5-6, u nhau, mu hng sm dn v pha gia; nh tht, nhn,
ch nh rng, mp, trung i dy [1], [2].
Qu thng cu to bi 8 i u v ri nhau, c khi 9-12 i (nhng
him), cc i hnh thoi xp ta trn thnh hnh sao hay hnh nan hoa, khi non
mu lc sau chuyn sang mu nu sm, phn nh vo cung rng bn v dt,
u c mi nhn, ngn, thng, khi chn nt mt trn; ht hnh trng nhn
bng, mu nu. Ton cy, nht l qu c mi thm [1], [2].
1.1.3. B phn dng, thu hi v ch bin
1.1.3.1. B phn dng
i hi (qu) Fructus Illicium veri l qu chn phi kh ca cy
hi - Illicium verum Hook. f. (h Hi Illiciaceae).
M t: Qu phc, thng gm 8 i, i khi nhiu i hn, mu nu
n nu sm, xp thnh hnh sao xung quanh mt tr trung tm. Mi i hnh
lng thuyn di 1-2cm, rng 0,5cm, cao 0,7-1cm. 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, tn cng bi 1 chm t, 1 gc c khong nhn hn
(ni nh gia cc i). 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 [5].
1.1.3.2. Thu hi v ch bin
Hng nm cy ra hoa kt qu theo 2 v, v chnh thu hoch vo thng
8-10 (v ma), v mun thu hoch vo thng 2-4 nm sau (v t qu). V
mun cho nng sut thp hn. Cy trng sau 5-6 nm bt u cho thu hoch.
Sau 15 nm mi cy c th cho 10-20kg qu ti/nm, sau 20 nm, nng sut
tng i n nh mc 20-30kg/nm [2].
Vo v thu hoch, hi ly qu t mu lc bin thnh vng, nhng qua
nc si, sy nh hoc phi trong bng rm khong 5-6 ngy cho kh [5].

Trn th trng, sn phm qu hi kh c chia thnh 3 loi:


- Loi 1 (hi i hng): qu 8 cnh to, ng u, khng b lp, mu
nu, cung ngn (3-5 mm), khng mc. y l loi c phm cp tt nht.
- Loi 2 (hi x): qu c cnh khng u, mu cnh gin, mt s cnh
b lp, gip, gy.
- Loi 3: qu thu hi non, qu vn, lp nhiu, mu nu en. y l loi
c cht lng km.
1.1.4. Thnh phn ha hc v cng dng
1.1.4.1. Thnh phn
- Tinh du: hm lng 3-3,5 % trong qu ti, 9-10 % trong qu kh.
Cha 80-90 % trans-anethol, ngoi ra cn c linalol, estragol, terpineol,
anysaldehyd [8], [11].
- Acid hu c: acid shikimic, protocatechic, anisatinic, isoanisatinic.
Trong quan trng nht l acid shikimic [2], [8].
- Ngoi ra cn 1 s cht thuc nhm flavonoid, tannin, sesquiterpen,
sesquiterpen lacton, sesquilignan, sesqui-neoliganan; du bo ch yu cha
trong ht [4], [8].
1.1.4.2. Cng dng
i hi (qu) c v cay, tnh n, quy kinh can, thn, t, v. N c cng
dng gip tiu ha, li sa, gim au, gim co bp nhu ng rut, dng cha
a chy, n khng tiu, bng y. Khi dng ngoi, i hi c tc dng cha
au nhc, thp khp, bong gn [4], [5]. Dng dng ca loi dc liu ny l
bt, ru thuc. Ngy dng 3-6 g.
Qu hi v 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 t tht [4].

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,

tng t nh ct ru. Thi gian ct c th ko di t 18-24 gi. c hiu


sut v cht lng tinh du cao, cn s dng cc thit b chng ct lin tc
bng hi nc c hi lu vi ni hi ring. B cn li sau khi ct tinh du c
th dng lm nhin liu un hoc trn vi phn sc vt bn cho cy
trng.
1.2. Tng quan v acid shikimic
1.2.1. Cng thc ha hc v tnh cht
Acid shikimic c cng thc ho hc v cc tnh cht c th nh sau [21], [26]:
Cng thc cu to:

1
6
2
5
3

Tn khoa hc: acid (3R,4S,5R) 3,4,5 trihydroxy -1-cyclohexen -1carboxylic.


Cng thc phn t: C7H10O5.
Trng lng phn t: 174,15.
Tnh cht: Acid shikimic l mt cht kt tinh mu trng, rt d tan trong nc
(18%), tan trong ethanol tuyt i (2,25%), trong methanol, khng tan trong
cc dung mi t phn cc ethyl acetat, aceton, cloroform, benzen, ether du
ha.
Nhit nng chy: 183 - 184,50C.
Nng sut quay cc: []18= -183,8 (c = 4,03% trong nc).

Cc i hp th: Dung dch acid shikimic trong ethanol c cc i hp th


213 nm.
1.2.2. K thut phn tch
Denis V. Bochkov v cs. trnh by cc k thut phn tch acid
shikimic trong bi tng quan v hp cht ny [17]. Cc k thut c nu ra
gm c:
- Sc k giy.

- o ph cng hng t ht nhn.

- Phn ng mu.

- Sc k lng hiu nng cao pha o.

- K thut phn tch nh lng.

- Mt s k thut phn tch khc.

1.2.3. Ngun gc acid shikimic


Acid shikimic c Eykman F. v cng s phn lp ln u tin vo
nm 1885 t mt loi hi Nht Bn (Illicium anisatum). n 50 nm sau cu
trc ha hc ca acid shikimic mi c xc nh [23]. Acid shikimic c
trong nhiu loi thc vt nh i hi (Illicium verum), bch qu (Gingko
biloba), quyn b trng sinh (Selaginella tamariscina, Hoselaginellaceae),
kha t (Terminalia chebula), chui tiu (Musa sapientum), hng dng
(Heliantus annus), tiu hi (Foeniculum vulgare) [27]. Nhng hin nay i
hi vn l nguyn liu quan trng chit xut, bi hm lng acid shikimic
trong qu hi tng i cao (5%-10%) [8].
Acid shikimic cn c ngun gc t tng hp hoc bn tng hp.
Raphael (1960) v Smissman (1959) tng hp acid shikimic t 1,3butadien-1,4-diyl diacetate qua phn ng Diels Alder. Grewe (1964) v cng
s tng hp acid shikimic t 1,3-butadien. Ngoi ra, acid shikimic c th
c tng hp t benzen, bn tng hp t acid quinic v D-manose [15].
Gio s Frost, trng i hc Michigan, nghin cu cng ngh sn
xut acid shikimic bng con ng ln men vi sinh s dng chng
Escherichia coli ti t hp, sau chit xut acid shikimic t dch ln men v

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

Trong sinh hc ni chung, acid shikimic ng vai tr quan trng trong

qu trnh sinh tng hp cc acid amin thm nh phenylalanin, tryptophan,


tyrosin; cc alcaloid, hp cht phenolic, cc phenyl propanoid [28]. N l cht
trung gian ha hc quan trng ca cc qu trnh chuyn ha trong thc vt v
vi sinh vt (con ng shikimat) [22], [23].

V tc dng dc l, acid shikimic c tc dng chng vim, gim au,

c kh nng c ch ngng tp tiu cu v bnh tc nghn ng mch do tc


ng ca acid arachidonic [24]. Cc nh khoa hc chng minh acid
shikimic c tc dng gim au, chng vim, chng co git, chng oxy ha,
km hm pht trin t bo ung th [2].

Trong lnh vc tng hp ha dc, acid shikimic c bit n l

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

trung gian c c tnh cao v c kh nng gy n. Nm 2004, Harrington v


cng s nghin cu thnh cng phng php tng hp oseltamivir
phosphat v trnh c cng on to azid. Phng php ny c cc tc
gi gi l phng php th h th 2, hiu sut tng ca phng php t
61% v c xem l phng php ti u nht hin nay [14].
1.2.5. Mt s nghin cu chit xut acid shikimic t i hi
Khi i dch cm gia cm xy ra, hng Roche v th gii lo lng v
khng acid shikimic sn xut. Nhiu nhm nghin cu c gng tm
ra acid shikimic vi cc phng php v nhiu ngun nguyn liu khc nhau.
Payne R. v Edmonds M. (2005) tin hnh chit acid shikimic t i
hi bng thit b Soxhlet vi dung mi ethanol 95%. Dch chit c ct loi
ethanol. Ha tan cn vo nc nng, gn b lp du. Dung dch c loi bt
tp bng formaldehyd v lc. Acid shikimic c tinh ch bng phng php
trao i ion (dng nha Amberlit IRA-400) v kt tinh li trong ethyl actetatmethanol. Hiu sut t 2,4-7,0 % [24]. Nguyn Quyt Chin (2006) cng
tin hnh v tinh ch acid shikimic t i hi theo phng php tng t.
Hiu sut qu trnh t 5,4%. Nhc im ca phng php ny l tinh ch
bng nha trao i ion phc tp v chi ph cao [6].
Nguyn nh Luyn v cng s (2006) chit Soxhlet bng cn 950,
chit bng nc 60oC v chit hi lu trong cn 900 v 950, quy m phng
th nghim (50 v 100g qu hi kh), loi tp vi formaldehyd, tinh ch trn
ct anionit cha 50g Amberlit v thu c acid shikimic vi hiu sut t 5,06,8 % [12]. Phng php ny c cng nhc im nu trn.
Iyer Sankar V. (2007) cng b phng php chit tch acid shikimic
gm cc bc [25]:
- Chit hi lu nguyn liu i hi (400g) vi 2x2L isopropanol 95%.
- Ct thu hi dung mi di p sut gim thu c dch chit m
c.

10

- Dung dch m c c pha long vi 900mL nc.


- Tin hnh loi tp dung dch nc ln lt bng ethyl acetat
(1200mL), dung dch formaldehyd 37% v than hot (60g).
- C c dung dch nc. Thm 50mL methanol v c n cn.
- Ha cn vo 40mL methanol, un hi lu 45 pht, lnh (0-50C)
cho acid shikimic kt tinh. Hiu sut qu trnh 3,5-5%.
Phng php ca Iyer Sankar V. c u im l bc tinh ch khng s
dng nha trao i ion. Nhng qu trnh tinh ch qua nhiu cng on v kh
phc tp, tn ethyl acetat v than hot; s dng formaldehyd 37% l dung mi
c hi loi tp v hiu sut thp.
Gn y, nhm nghin cu ca Hiroki Ohira (2009) - Trng i hc
Tohoku, Trung tm Nghin cu Cng ngh chit xut bng kh ha lng siu
ti hn (Research Center of Supereritical Fluid Tecnhnology) nghin cu
chit xut nhanh acid shikimic bng nc nng nhit 120oC trong 5 pht
vi 0,5g hi c hiu sut 8% [22]. Hiu sut chit cao, thi gian chit nhanh
nhng k thut kh phc tp, tn km, i hi trang thit b hin i.
Nguyn Thng Dong v cng s (2010) chit xut c acid
shikimic t l v qu i hi thu hoch cc tnh khc nhau Vit Nam bng
dung mi nc v methanol, tinh ch bng hn hp ethyl acetat-methanol
(3:1) cho hiu sut 0,985-1,33% trong l v 4,47% trong qu [8].
Th Loan (2011) chit xut acid shikimic t i hi bng nc theo
phng php chit nng vi nc 1000C, tinh ch bng hn hp dung mi
methanol-aceton cho hiu sut c qu trnh t 6,61% [10].
1.3. Nhn xt
Cm gia cm c nguy c bng pht thnh i dch bt c lc no
nc ta v cc nc khc trn th gii. Ch ng ngun nguyn liu acid

11

shikimic bn tng hp oseltamivir (hot cht c tc ng chng cm gia


cm hiu qu hin nay) c ngha ht sc to ln. Chit xut acid shikimic t
i hi l phng php hiu qu nht tnh n thi im hin ti thu c
hot cht ny. Gi tr kinh t ca i hi b gim ng k, do c s dng
ch yu ly tinh du m b qua hot cht acid shikimic. Thc t Vit
Nam, i hi cng ch dng ct ly tinh du m cha tn thu c acid
shikimic. Cc phng php chit xut hot cht ny c cng b trc
y thng phc tp, kh tn km, 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. Chnh v vy, chng ti tip tc nghin cu
chit xut acid shikimic t i hi s dng dung mi chit xut l nc v kt
hp chng ct tinh du nhm gp phn tm ra phng php chit xut acid
shikimic t i hi c th p dng trong iu kin thc t ti Vit Nam.

12

CHNG 2 : I TNG V PHNG PHP NGHIN CU


2.1. Nguyn vt liu
2.1.1. Nguyn liu
Ngun gc
i hi thu mua t tnh Lng Sn.

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

Tiu chun, ngun gc

Ethanol 96%

Vit Nam

Aceton

P, Trung Quc

Methanol

P, Trung Quc

Methanol

HPLC, Merck

H2SO4

P, Trung Quc

Isopropanol

P, Trung Quc

NaCl cng nghip

Vit Nam

Acid shikimic chun

99,0%, Merck

2.1.2.2. Thit b, dng c


My mc

My siu m Ultrasonic LC60H (c): Cng sut P= 400W, tn s f =

35 kHz.

T sy MEMMERT (c).

Cn k thut in t Sartorius BP 20015 (c).

Cn phn tch Mettler Toledo AB204-S ( Thy S).

14

My ct quay chn khng Bchi B-490 v R-220 (Thy S).

My khuy t Heidolph MR3001 (c).

Bp m bnh cu c bo n Heating Mantle (Trung Quc).

My sc k lng hiu nng cao Shimadzu (Nht Bn), bao gm: b

phn loi kh DGU 14A, bm cao p LC 10ADVP, bung cha ct CTO


10AVP, b iu khin SCL 10AVP, detector dy diod quang SPD
M10AVP v phn mm Class vp 6.14.

My o nhit nng chy EZ Melt (M).

Phn cc k KRSS (c).

Thit b xc nh ph hng ngoi (IR) Perkin Elmer.

Thit b xc nh ph cng hng t ht nhn (NMR) Bruker AM500

FT-NMR Spectrometer.

Thit b xc nh ph khi lng Varian 320 Ms (M).

Dng c

H thng ct tinh du: Bnh cu 2L, sinh hn, bp m bnh cu c bo

n, bnh cha tinh du.

Sinh hn hi lu.

Bnh cu 250mL, 500mL, 2L.

Cc thy tinh loi 100mL, 250mL, 500mL, 1000mL.

Bnh gn 500mL.

Bnh nh mc 10mL, 25mL, 50mL, 100mL.

Pipet 1mL, 2mL, 5mL, 10mL.

ng ong 10mL, 50mL, 300mL, 500mL.

Mng lc cellulose acetat 0,45m (Satorius).

2.2. Ni dung nghin cu


2.2.1. La chn phng php chit xut

15

Phng php chit nng kt hp ct tinh du trc (phng php A) v


phng php ngm lnh kt hp ct tinh du sau (phng php B) c kho
st. Ni dung ny nhm nh gi tc chit hot cht, hiu qu chit xut
acid shikimic, tnh chn lc v lng tinh du thu c. T c d liu
so snh v la chn phng php u vit hn.
2.2.2. Xy dng phng php tinh ch acid shikimic t dch chit nc
- Kho st v la chn dung mi tinh ch. Cc dung mi nghin cu
gm c: methanol, hn hp methanol-isopropanol, hn hp methanol-aceton,
isopropanol.
- Bc u nghin cu cc yu t nh hng n qu trnh tinh ch s
dng dung mi la chn.
- xut quy trnh chit xut acid shikimic t i hi. Quy trnh ny
c p dng vi c m ln hn nhm nng cao tnh thc tin ca ti v s
b nh gi hiu qu ca quy trnh khi m rng quy m.
2.3. Phng php nghin cu
2.3.1. Phng php nh lng acid shikimic
Xc nh hm lng acid shikimic trong dc liu, trong dch chit v
sn phm bng phng php sc k lng hiu nng cao (HPLC).
2.3.1.1. iu kin sc k
Tham kho cc ti liu [19], [20] v qua thc nghim, iu kin sc k
c xc nh nh sau:
- Ct: Supelco C8 (150 x 4mm, 5m).

- Tc dng: 0,8mL/pht.

- Pha ng: dung dch acid sulfuric 0,01%. - Th tch mu tim: 5L.
- Bc sng pht hin: 210 nm.

- Nhit bung ct: 30C.

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

SC: Din tch peak mu chun.


ST: Din tch peak mu th.
fC, fT: H s pha long ca mu chun v mu th.
VC, VT: Th tch ca dung dch chun v dung dch th.
a: hm lng acid shikimic trong mu chun.
Hiu sut chit xut:

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

Trong : H%: hiu sut c qu trnh


mSP: khi lng sn phm
mT: khi lng acid shikimic c trong dc liu em chit
2.3.2. Phng php chit xut acid shikimic v ct tinh du

Phng php A: Ct ly tinh du i hi bng phng php ct ko

hi nc, hn hp cn li em chit ly acid shikimic.

Phng php B: Chit acid shikimic bng phng php ngm lnh c

khuy trn, b dc liu c ct ly tinh du bng phng php ct ko hi


nc.

S dng phng php ct ko hi nc thu tinh du hi. Mui n

c s dng tch tinh du t dch ct nc. Tin hnh phn lp trong

18

bnh gn. Khi dch ct tch lp hon ton, gn b lp nc bn di thu c


tinh du.
2.3.3. Phng php tinh ch acid shikimic
Cc giai on tinh ch gm c:
- Dch chit nc c lng, lc qua giy lc loi tp c hc.
Dch lc c c n cao c.
- Loi bt tp cht v gim hm m trong cao tinh ch bng ethanol
96%.
- S dng cc loi dung mi sau: methanol, hn hp methanol isopropanol, hn hp methanol - aceton v isopropanol kt ta hot cht
thu sn phm th.
- S dng than hot v kt tinh li trong isopropanol thu sn phm.
2.3.4. Kim tra cht lng sn phm
Sn phm ca quy trnh c kim tra cht lng theo cc phng
php sau:
- o nhit nng chy: Tin hnh o nhit nng chy trn my
my o nhit nng chy Ez-Melt (M) ti B mn Cng nghip Dc Trng i hc Dc H Ni.
- o nng sut quay cc: Tin hnh trn phn cc k KRSS (c)
vi c = 4,03% trong nc ti B mn Cng nghip Dc - Trng i hc
Dc H Ni.
- o ph hng ngoi (IR): Mu c nghin vi KBr v ph hng
ngoi (IR) ca sn phm c xc nh trn my Perkin Elmer o trong vng
bc sng 4000-400 cm-1 ti Vin Ha hc - Vin Khoa hc v Cng ngh
Vit Nam.

19

- o ph khi lng (MS): Ph khi lng ca sn phm c xc


nh bng my Varian 320 Ms (M), ngun ion ho ESI ti Vin Ha hc Vin Khoa hc v Cng ngh Vit Nam.
- o ph cng hng t ht nhn (NMR): Ph 1H-NMR (500 MHz,
DMSO) v

13

C-NMR (125 MHz, DMSO) ca sn phm c xc nh trn

my Bruker AM500 FT-NMR Spectrometer ti Vin Ha hc - Vin Khoa


hc v Cng ngh Vit Nam.

Mi th nghim c tin hnh 3 ln, ly kt qu trung bnh

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

Hnh 3.1. Sc k ca mu dc liu


PDA-210 nm
shikimic acid
chuan merk 5.dat
Retention Time

200

2.667

mAU

400

0
0

Minutes

Hnh 3.2. Sc k ca acid shikimic chun

21

Nhn xt: Hm lng acid shikimic trong dc liu kh cao (10,8%


0,1%). Trn sc k cho thy ngoi peak ca acid shikimic trong i hi
hu nh khng c peak ca cht no khc.
3.2. Kho st giai on chit xut
3.2.1. Chit xut theo phng php A
Phng php A l phng php gn lin vi thc tin chng ct tinh
du i hi. Trong giai on u nghin cu, chng ti thc hin kho st
phng php A vi cc ni dung nghin cu gm c: tc chit acid
shikimic, hiu qu ct tinh du i hi, hiu sut chit acid shikimic v tnh
chn lc.
3.2.1.1. Kho st tc chit
Tin hnh: Cn 100g bt dc liu cho vo bnh cu th tch 2L c 2 c
c nt mi. Mt c lp h thng ct thu hi dung mi, mt c c nt y
ly mu nh k. Thm 700mL nc. un si trong 5 gi. Ly mu sau khi
si, sau mi gi ly mu 1 ln.
Cc mu c pha long nh sau: Ht chnh xc 1mL dch chit cho
vo bnh nh mc 100mL, thm nc va n vch, lc k cho u. Cc
mu c lc qua mng lc 0,45m trc khi nh lng bng HPLC theo
mc 2.3.1.
Kt qu c th hin trong hnh 3.3.

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

Thi gian (gi)

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

3.2.1.3. Chit xut acid shikimic


Tin hnh: Hn hp sau khi ct tinh du c lc thu ly dch chit. B
cn li c tip tc chit thm 2 ln na, mi ln 700mL, thi gian un si
vi mi ln l 1 gi. Lc ly dch chit. Xc nh nng acid shikimic trong
tng dch chit bng HPLC. Tin hnh nh sau:
Dch chit ln 1: Ht chnh xc 1mL dch chit cho vo bnh nh mc
100mL. Thm nc va n vch, lc k cho u.
Dch chit ln 2: Ht chnh xc 2mL dch chit cho vo bnh nh mc
50mL. Thm nc va n vch, lc k cho u.
Dch chit ln 3: Ht chnh xc 2mL dch chit cho vo bnh nh mc
25mL. Thm nc va n vch, lc k cho u.
Cc mu c lc qua mng lc 0,45m trc khi em nh lng
bng phng php HPLC. Kt qu c th hin trong bng 3.1.
Bng 3.1. Kt qu hiu sut chit acid shikimic theo phng php A
Th tch

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

Hnh 3.4. S quy trnh chit acid shikimic v ct tinh du theo PP B


Kho st phng php B v tc chit, hiu sut chit acid shikimic,
tnh chn lc v th tch tinh du thu c sau khi chit acid shikimic. Tin
hnh so snh vi phng php A, t la chn phng php chit xut acid
shikimic cho hiu sut cao.
3.2.2.1. Kho st tc chit
Kho st thi im cn bng ca phng php B trong dch chit ln 1
so snh tc chit vi phng php A. Phng php no nhanh t n
thi im cn bng tc l c tc chit nhanh hn.
Tin hnh: Cn 100g bt i hi cho vo cc c m c th tch 1L.
Thm 30mL nc ngm cho dc liu trng n hon ton. Thm 670 mL
nc. y kn. t ln my khuy t, nhit phng. Kho st trong 22
gi, ly mu sau mi gi. Mu c x l trc khi em nh lng bng
HPLC nh sau:

26

Mu ly t 1 gi n 5 gi: Ht chnh xc 1mL dch chit cho vo bnh


nh mc 50mL. Thm nc va n vch, lc k cho u.
Mu ly t 6 gi n 22 gi: Ht chnh xc 1mL dch chit cho vo
bnh nh mc 100mL. Thm nc va n vch, lc k cho u
Cc mu c lc qua mng lc 0,45m trc khi nh lng bng
HPLC theo mc 2.3.1.
Kt qu: Kt qu c th hin trong bng 3.2 v hnh 3.5.
Bng 3.2. Nng acid shikimic trong dch chit sau mi gi theo PP B
Gi

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

Thi gian (gi)

Hnh 3.5. th biu din nng acid shikimic theo thi gian ca PP B

27

3.2.2.2. Xc nh hiu sut chit


Hiu sut chit cho bit sau mi ln chit, lng acid shikimic trong
dc liu c chit kit hay cha. Tin hnh xc nh hiu sut chit ca
phng php B so snh vi phng php A, t nh gi hiu qu ca
phng php.
Tin hnh: Cn 100g bt i hi cho vo cc c m c th tch 1L.
Thm 30mL nc ngm cho dc liu trng n hon ton. Thm 670 mL
nc. y kn. t ln my khuy t, nhit phng. Khuy lin tc trong
14 gi. Lc ly dch chit ln 1. Tin hnh chit thm 2 ln nh trn, mi ln
700mL nc, khuy lin tc trong 14 gi. Lc thu c dch chit ln 2 v 3.
Xc nh nng acid shikimic trong tng dch chit bng HPLC:
Dch chit ln 1: Ht chnh xc 1mL dch chit cho vo bnh nh mc
100ml. Thm nc va n vch, lc k cho u.
Dch chit ln 2: Ht chnh xc 2mL dch chit cho vo bnh nh mc
50mL. Thm nc va n vch, lc k cho u.
Dch chit ln 3: Ht chnh xc 2mL dch chit cho vo bnh nh mc
25mL. Thm nc va n vch, lc k cho u.
Cc mu c lc qua mng lc 0,45m trc khi nh lng bng
HPLC theo mc 2.3.1. Hiu sut chit c tnh theo cng thc 2 mc 2.3.1.3.
Kt qu c th hin trong bng 3.3.
Bng 3.3. Kt qu hiu sut 3 ln chit acid shikimic bng PP B
Ln
chit

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%

3.2.2.3. Kho st tnh chn lc


Tin hnh tng t theo mc 3.2.1.4, ta c hm lng acid shikimic
trong cn tng ng vi phng php B l 60,65%.
3.2.2.4. Ct tinh du
T 100g dc liu ban u, sau khi chit ht acid shikimic, b dc
liu c cho thm 1L nc, lp h thng ct ko hi nc, ct cho n khi
ht tinh du. Phng php thu c 8 0,1 mL tinh du.
3.2.3. nh gi v la chn phng php chit xut
3.2.3.1. V tc chit
Tc chit ca hai phng php c th hin trong hnh 3.6.

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

Thi gian (gi)

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

3.2.3.2. V hiu sut chit


T cc kt qu thu c, ta c th so snh hiu sut chit ca hai
phng php (hnh 3.7).

Hnh 3.7. th biu din hiu sut chit ca 2 phng php


Nhn xt: Hm lng acid shikimic thu c sau mi ln chit trong
phng php A u ln hn hm lng trong phng php B. Ln 1, phng
php A thu c 72,5%, trong khi phng php B thu c 71,5%. Ln 2,
phng php A thu c 18,2 %, phng php B thu c 17,2%. Ln 3
phng php A thu c 7,7% , phng php B thu c 5,0%. Tng hiu
sut chit ca phng php A (98,4%) ln hn phng php B (93,7%) l
4,7%. Vy hiu sut chit ca phng php A cao hn hiu sut chit ca
phng php B.
3.2.3.3. V tnh chn lc
Kt qu kho st tnh chn lc c th hin trong bng 3.4.

30

Bng 3.4. Kt qu xc nh h s chn lc ca 2 phng php


C (%)

mAS(g)*

Phng php A 2020 36

0,53 0,01

10,706

19,62 0,89 54,57

Phng php B

0,54 0,01

10,044

16,56 1,08 60,65

V (mL)

1860 9

m (g)

S (%)

mAS = V.C/100

Nhn xt: Phng php A c hm lng AS trong cn (S) thp hn,


nh vy dch chit nhiu tp hn. Tuy nhin s khc bit v gi tr S ca hai
phng php l khng nhiu.
3.2.3.4. Kt qu ct tinh du
Tinh du c coi l sn phm ph trong nghin cu ny. Tuy nhin,
y vn l mt sn phm gi tr t i hi v c ngha ng dng thc t. Kt
qu ct tinh du ca hai phng php c tng hp trong bng 3.5.
Bng 3.5. Kt qu ct tinh du ca hai phng php

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

3.2.3.5. Nhn xt v la chn phng php chit xut


Cc kt qu c tng hp trong bng 3.6.

31

Bng 3.6. Bng so snh tc cn bng, hiu sut chit, hm lng AS


trong cn (S), th tch tinh du v thi gian chit xut
PP

PP A (chit nng kt
hp ct tinh du trc)

PP B (ngm lnh kt
hp ct tinh du sau)

14

Hiu sut chit (%)

98,4

93,7

S (%)

54,57

60,65

Th tch tinh du (mL)

9,5

8,0

Thi gian chit xut v


ct tinh du (gi)

8,5

47

Ch tiu
Tc cn bng (gi)

Cc kt qu nghin cu nghin cu trn cho thy rng:


-

Tc cn bng trong phng php ct tinh du trc (phng

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).
-

Khi nhit tng th h s khuch tn cng tng, lng cht

khuch tn cng tng ln. Hn na khi nhit tng th nht ca dung mi


gim, do s to iu kin thun li cho qu trnh chit xut. V vy tc
chit ca phng php A l nhanh hn (thi gian t n cn bng khong 3
gi) v chit c hu nh ton b acid shikimic (98,4%). Tc chit ca
phng php A nhanh hn phng php B, ng thi phng php ny c th
kt hp ct tinh du trong ln chit th nht do rt ngn c thi gian
chit xut v ct tinh du 5,5 ln so vi phng php B. Tuy nhin khi tng
nhit lm tng tan ca acid shikimic cng lm tng tan ca cc tp

32

cht, dch chit s b ln nhiu cht nhy, gm Phng php ny c hm


lng acid shikimic trong cn thp hn v lng tp trong dch chit nhiu
hn phng php ngm lnh; tuy nhin, xt v tng th, lng hot cht thu
c vi phng php A (10,706 g) vn ln hn phng php B (10,044 g).
nht ca dch chit tng gy kh khn trong vic lc v loi tp. ng
thi, phng php ct tinh du trc c s dng nhit , tinh du c ct
trc ht nn khng b tht thot, do thu c lng tinh du nhiu hn
phng php B. Nh vy phng php ny c u im l: thi gian chit
nhanh, hiu sut chit acid shikimic tt, thu c lng tinh du cao. Nhc
im: tiu tn nhit lng, dch chit thu c kh lc v nhiu tp.
-

Trong phng php ngm lnh c khuy trn lin tc to ra

s chnh lch nng lin tc gia lp dung mi pha st mng t bo vi


lp dung mi pha xa, t y nhanh tc chit. Tuy nhin, thi gian t
n cn bng ca phng php B l khong 14 gi. Lng tinh du thu c
l 8 mL, t hn 1,5 mL so vi phng php ct tinh du trc. Nguyn nhn
c th do tinh du b bo ha mt phn trong dch chit nc trong qu trnh
khuy trn lin tc trong nhiu gi. Dch chit thu c cng kh nht v kh
lc.
Vi nhng u im trn, chng ti la chn phng php chit xut
acid shikimic t i hi bng nc l phng php A: Ct ly tinh du i hi
bng phng php ct ko hi nc, hn hp cn li em chit ly acid
shikimic.
3.3. Tinh ch acid shikimic
Acid shikimic thng c phn lp bng nha trao i ion. Hot cht
ny rt d tan trong nc, tan trong ethanol, methanol v kh kt tinh. I. V.
Sankar (2007) phn lp acid shikimic bng methanol, trnh c vic dng
nha trao i ion; nhng qu trnh tinh ch phc tp, tn dung mi, s dng

33

formaldehyd l dung mi c hi. Trong khi , acid shikimic v nhiu tp


cht tan kh tt trong methanol nn hiu sut phn lp cha cao. Cc nghin
cu khc cng b thng s dng hn hp methanol v mt dung mi
khc (aceton, ethyl acetat) kt ta hot cht ny, nn hiu sut thu c
cn thp, sn phm km tinh khit v vic thu hi ti s dng dung mi kh
khn. Nghin cu ny c thc hin vi mong mun tm ra c phng
php khc phc cc hn ch nu trn ca cc cng trnh cng b trc y.
3.3.1. Kho st v la chn dung mi tinh ch
Trn nguyn tc gim dn tan ca acid shikimic (bng cch thm i
dung mi hoc gim nhit ), trc ht cao c cha acid shikimic phi
c ho tan trong dung mi sao cho hot cht c chit sang dung dch.
Methanol l dung mi r tin, d kim v ho tan tt acid shikimic; trong khi
hot cht ny t tan trong isopropanol v khng tan trong aceton. Qua thc
nghim thy, nu ch s dng dung mi methanol cng c th kt tinh hot
cht. V vy chng ti tin hnh so snh hiu qu tinh ch ca 4 loi dung
mi: methanol, isopropanol, hn hp dung mi methanol aceton, hn hp
dung mi methanol isopropanol. Sn phm th thu c c kt tinh li
trong isopropanol. Cc ch tiu nh gi chnh:
- Khi lng v hm lng sn phm th.
- Cht lng sn phm (hm lng hot cht).
- Kh nng thu hi dung mi.
Trn thc t, phng php chit bng nc nhit cao thng ko
theo rt nhiu tp vo trong dch chit nh: ng, cht nhy, gm, pectin
La chn ethanol 96% loi bt cc tp cht ny. T dung dch trong
ethanol em kt tinh sn phm bng cc loi dung mi khc nhau: methanol,
hn hp dung mi methanol aceton, hn hp methanol - isopropanol v
isopropanol.

34

Qu trnh to ta th c s ha trong hnh 3.8.

Dch chit/nc
EtOH96%

C n cao c

Cao c/EtOH 96%


MeOH-AC(IPA)

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

un, khuy 30ph


Gn
Ta
Thm 20mL IPA
un, khuy 30ph
Gn
Tin hnh 2 ln

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

Tin hnh: Th nghim chit xut acid shikimic trong mc 3.2.1.3


c tin hnh 4 ln, mi th nghim 100g dc liu. Gp cc dch chit, c
cho n khi thnh cao c. Thm 400mL ethanol 96%, khuy trn v lng
2 gi nhit phng. Lc thu dch. Ta c ra li 2 ln, mi ln 60mL
ethanol 96%, un nng, khuy, ngui, lc ly dch. Gp dch lc v dch
ra, c n cao c. Cao c trong ethanol c chia lm 4 phn bng nhau
bng phng php cn (mi phn 13g cao c tng ng vi 9,7g acid
shikimic) v s dng 4 loi dung mi khc nhau tinh ch: methanol, hn
hp dung mi methanol aceton, hn hp dung mi methanol isopropanol
v isopropanol.
3.3.1.1. Phn lp bng methanol
Ha 13g cao c (tng ng vi 9,7g acid shikimic) vo 20mL
methanol, thm 1g than hot, un cch thy 15 pht. Lc b than. kt tinh
nhit phng. Lc ly ta. Ra ta 3 ln bng 5mL methanol lnh. Sy
thu c sn phm th.
3.3.1.2. Phn lp bng hn hp methanol aceton
Ha cao c (tng ng vi 9,7g acid shikimic) vo 20mL methanol,
thm 60mL aceton. Xut hin ta keo. lng, gn ly dch. Phn ta keo
tip tc tin hnh 2 ln na, mi ln 20mL hn hp methanol : aceton (1:3),
un cch thy, khuy lin tc trong 30 pht, gn ly dch. Gp dch gn, c
bt dung mi n khi cn 20mL. kt ta nhit phng. Lc ly ta.
Ra ta 3 ln, mi ln bng 5mL aceton. Sy thu c sn phm th.
3.3.1.3. Phn lp bng hn hp methanol isopropanol
Tin hnh tng t nh kt ta trong methanol aceton. kt ta
nhit phng. Lc ly ta. Ra ta 3 ln, mi ln bng 5mL isopropanol.
Sy thu c sn phm th.

36

3.3.1.4. Phn lp bng isopropanol


Ha 13g cao c (tng ng vi 9,7g acid shikimic) vo 50mL
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 20mL isopropanol,
un cch thy, khuy lin tc trong 30 pht. Gn ly dch. Gp dch gn, em
c bt dung mi n khi cn 20mL. kt tinh nhit phng. Lc ly ta.
Ra ta 3 ln, mi ln bng 5mL isopropanol. Sy thu c sn phm th.
3.3.1.5. Kt tinh li
T sn phm th, em ha tan vo 50mL isopropanol, thm 3g than
hot, un cch thy trong 15 pht. Lc ly dch lc, dch lc c bt n cn
khong 20mL, kt tinh. Sn phm kt tinh c lc v ra 3 ln bng
isopropanol, mi ln khong 5mL. Sy kh thu c sn phm kt tinh mu
trng.
Kt qu: Kt qu thu c t qu trnh tinh ch v mt s hnh nh sn
phm c th hin trong bng 3.7 v hnh 3.10.
Bng 3.7. Kt qu qu trnh tinh ch
s dng cc dung mi tinh ch khc nhau
MeOH/Aceton MeOH/IPA
(1:3)
(1:3)

Dung mi tinh ch

MeOH

Khi lng SP th (g)

7,05
0,16

8,05 0,06

Hm lng AS trong
ta th (%)

80,20

90,60

Khi lng SP sau


tinh ch (g)

5,23
0,07

6,89 0,03

Hm lng AS trong
sn phm (%)

95,16

99,51

98,05

99,18

Hiu sut tinh ch (%)

51,31

70,68

60,65

66,87

37

IPA

7,58 0,10 7,83 0,09


83,51

88,5

6,00 0,03 6,54 0,02

Loi SP
Dung mi

Sn phm th

Sn phm tinh ch

Methanol

Hn hp
methanol aceton

Hn hp
methanol isopropanol

Isopropanol

Hnh 3.9. Hnh nh sn phm th v sn phm tinh ch

38

Nhn xt v la chn dung mi tinh ch

Kh nng loi tp ca methanol khng trit nh s dng 3 loi dung

mi cn li. Khi lng sn phm th khi kt tinh bng methanol t nht


(7,05g) do acid shikimic rt d tan trong methanol. V vy sau khi kt tinh vn
cn mt lng acid shikimic tan trong methanol m khng thu c hon
ton. u im ca phng php l tin hnh kh n gin, ch cn s dng
mt loi dung mi nn thun li thu hi dung mi. Nhc im: sn phm
th c lng tp bm dnh ln, tinh khit ca sn phm khng cao
(80,2%), cc tp cht cha c loi khi mi trng kt tinh lm dch kt
tinh nht dnh gy cn tr vic lc, thi gian lc lu, dung mi s dng c
hi.

Khi lng sn phm th kt tinh bng isopropanol l 7,83g vi hm

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

S dng hn hp methanol isopropanol thu c sn phm th c

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.

Khi lng sn phm th trong hn hp dung mi methanol aceton

(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.

Hiu sut quy trnh sp xp theo th t gim dn nh sau:

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

Qua thc nghim c th thu c thm acid shikimic t dch lc tng

ng vi dung mi tinh ch l hn hp methanol/isopropanol v isopropanol,


do c th p dng thao tc ny nng cao hiu sut quy trnh.

Vi nhng nhn xt nu trn, chng ti nhn thy rng isopropanol l

dung mi c tim nng tinh ch thu acid shikimic, p ng c cc yu


cu: loi c nhiu tp, hiu sut quy trnh tt, thu hi dung mi d dng v
dung mi r tin, d kim, t c hi. Isopropanol c la chn lm dung
mi tinh ch v qu trnh kt tinh hot cht trong dung mi ny bc u
c nghin cu trong cc th nghim tip theo.
3.3.2. Bc u kho st yu t nh hng n qu trnh kt tinh acid
shikimic bng isopropanol
Cc yu t c th nh hng n qu trnh kt tinh hot cht bng
isopropanol l: hm m ca cao trong nc em tinh ch, tp cht, nhit ,
Vi iu kin nghin cu v thi gian cn hn ch, chng ti mi ch bc
u kho st yu t hm m ca cao trong nc em tinh ch v cho kt qu
cng nh nhn nh cn s b.
Tin hnh: Th nghim chit xut acid shikimic c thc hin vi 3
mu nghin cu, mi th nghim 100g dc liu. Vi mi th nghim, tin
hnh theo cc bc sau:
- Gp cc dch chit, c thnh cao, em xc nh hm m (H) ca cao
thu c. La chn cc mu cao c H l 58,93%, 19,67% v 11,04% tip
tc nghin cu.
- Vi mi mu nghin cu: Thm 100mL ethanol 96%, lng 2 gi
nhit phng, lc thu dch. Ta c ra li 2 ln, mi ln 20mL ethanol

41

96%, un nng, khuy, ngui, lc ly dch. Gp dch lc v dch ra, c


n cao c.
- Ha cao c thu c vo 50mL 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 20mL isopropanol, un cch thy, khuy lin tc trong 30
pht. Gn ly dch. Gp dch gn, em c bt dung mi n khi cn 20mL.
kt tinh nhit phng. Lc ly ta. Ra ta 3 ln, mi ln bng 5mL
isopropanol. Sy thu c sn phm th.
Kt qu: Kt qu c th hin trong bng 3.8.
Bng 3.8. Kt qu kho st lin quan ti hm m ca
cao trong nc em tinh ch
Mu

Mu 1
(H = 58,93%)

Mu 2
(H = 19,67%)

Mu 3
(H = 11,04%)

Thi gian kt tinh hon


ton (ngy)

12

Khi lng SP th (g)

3,16

5,31

7,80

Hm lng AS (%)

82,97

85,03

87,08

Ch tiu

Nhn xt: Vi cc mu nghin cu, hm m ca cao trong nc em


tinh ch cng gim th thi gian kt ta cng ngn v khi lng ta th
cng nh hm lng acid shikimic trong ta th cng cao.
3.4. xut phng php chit xut acid shikimic v kim tra cht lng
sn phm
3.4.1. xut phng php chit xut acid shikimic

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

3.4.2. Kim tra cht lng sn phm


Chng ti tin hnh kim tra cht lng sn phm theo cc phng
php mc 2.3.4. Sn phm acid shikimic c nhit nng chy, nng sut
quay cc ph hp vi ti liu tham kho, cc d liu ph ph hp vi cng
thc cu to ca acid shikimic (ph hng ngoi, ph khi lng, ph cng
hng t ht nhn proton, ph cng hng t ht nhn

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

Ph hng ngoi (IR)


+ Kt qu: Thu c ph hng ngoi ca sn phm (xem ph lc 1).

44

Bng 3.9. Cc di hp th c trng trong ph hng ngoi

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

Bng 3.10. Kt qu phn tch ph cng hng t ht nhn proton (1H-NMR)

(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

+ Nhn xt: Kt qu phn tch ph cng hng t ht nhn proton ca


sn phm cho thy ph hp vi cng thc acid shikimic; ng thi cc gi tr
ny cng gn tng ng vi kt qu phn tch 1H NMR (399,6514MHz,
DMSO-d6) theo ti liu tham kho [30].
Ph cng hng t carbon (13C-NMR)
+ Kt qu: Thu c ph 13C-NMR ca sn phm (xem ph lc 5).

46

Bng 3.11. Kt qu phn tch ph cng hng t carbon (13C-NMR)

(ppm)
V tr C
Thc nghim

Tham kho [30]

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

+ Nhn xt: Kt qu phn tch ph cng hng t carbon ca sn phm


ph hp vi cng thc acid shikimic; ng thi cc gi tr ny cng gn
tng ng vi kt qu phn tch 13C-NMR (22,53 MHz, DMSO-d6) theo ti
liu tham kho [30].
3.4.2.3. nh lng hot cht
Hm lng acid shikimic trong sn phm t 99,85%.
Nhn xt chung: T nhng kt qu kim nghim trn c th xc nh
sn phm thu c l acid shikimic v c tinh khit cao.

47

3.5. M rng quy m


Cc cng trnh nghin cu chit xut acid shikimic cng b ch yu
nghin cu trn c m kh nh (5g, 100g, 300g, 400g). Cn p dng quy trnh
c xut vi c m ln hn nh gi hiu qu ca phng php. o
, chng ti tip tc nghin cu trn c m 2kg i hi.
3.5.1. M t quy trnh
Chit xut theo quy trnh chit xut v tinh ch nu trn (hnh 3.10). Do
thi gian c hn v iu kin thit b hn ch, thc nghim ny b qua qu
trnh ct tinh du. Tin hnh vi 3 m dc liu, c m l 2kg. Qua thc
nghim, cc thng s ca qu trnh c la chn theo bng 3.12.
Bng 3.12. Cc thng s ca qu trnh chit tch v phn lp
acid shikimic vi c m 2kg
i lng
Khi lng dc liu (kg)
Th tch dung mi chit (L)
S ln chit
Thi gian chit tnh t thi im si (gi)
Th tch ethanol 96% (L)
Th tch IPA trong giai on to ta th (L)
Khi lng bt talc (g)
Th tch IPA trong giai on kt tinh li (L)
Khi lng than hot (g)

Gi tr
2
V1 = 14, V2 = V3 = 10
3
1
1,6
1
100
1
60

3.3.1.2. M t quy trnh


Giai on 1: Chun b nguyn liu
i hi c loi b tp, sy 500C trong 2 gi. Xc nh m (a);
kt qu: a = 3,26 %. Cn 2 kg dc liu th.
Giai on 2: Chit xut

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

ra 3 ln bng isopropanol, mi ln khong 100mL. Sy kh thu c sn


phm kt tinh mu trng (hnh 3.11).

Hnh 3.11. Sn phm acid shikimic


3.5.2. Kt qu
Kt qu thu c th hin bng 3.13.
Bng 3.13. Kt qu chit xut acid shikimic vi c m 2kg i hi

Sn phm th
Khi
lng
(g)

Hm
lng
acid
shikimic
(%)

170,34

2
3

Sn phm tinh khit

Hiu sut quy trnh

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

Nhn xt: Cc m dc liu cho kt qu tng i ng nht. Nh vy,


quy trnh chit xut v tinh ch acid shikimic l n nh.

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:
-

Acid shikimic chim lng ln trong qu i hi nhng rt kh

kt ta, phn lp nu trong dch chit x l cn ln tp. V vy, cn phi x


l, loi gn nh ht tp trong dch chit.
-

Trong qu trnh tinh ch cn phi hn ch s c mt ca nc.

Nc ha tan rt tt acid shikimic v vy trong dch chit x l phi loi ht


nc c th kt ta acid shikimic.
-

La chn dung mi tinh ch m bo kh nng kt ta hot cht,

mc c hi v tnh kinh t (gi c, kh nng thu hi ti s dng, tht


thot). acid shikimic kt tinh c trong dung mi tinh ch cn phi gim
dn tan ca acid shikimic (bng cch thm i dung mi hoc gim nhit
) hoc to ra dung dch qu bo ha (bng cch lm bay hi bt dung mi
tinh ch).
-

Ty mu bng than hot v kt tinh li thu c sn phm c

tinh khit cao.


Qua kho st 4 loi dung mi tinh ch (methanol, hn hp methanolisopropanol, isopropanol, hn hp methanol-aceton) cho thy hn hp
methanol-aceton v isopropanol cho hiu qu chit tch acid shikimic tt
(70,68% v 66,87%). Tuy nhin, kh khn ln nht khi s dng hn hp
methanol-aceton l vic thu hi, ti s dng dung mi; do dung mi thu hi

52

c ln nc t cao chit nc. Vi aceton thu hi c ln methanol v nc,


qu trnh kt ta acid shikimic tr nn kh khn. Trong khi , isopropanol c
th khc phc c tnh trng ny. Song, lng nc cn li trong hn hp
kt ta hot cht cng ln th kh nng mt mt acid shikimic trong dch lc
cng cao v thi gian kt ta hon ton cng lu. Isopropanol l dung mi c
tim nng tinh ch thu acid shikimic, p ng c cc yu cu: loi c
nhiu tp, hiu sut quy trnh tt, thu hi dung mi d dng v dung mi r
tin, d kim, t c hi.
Hm m ca cao trong nc em tinh ch, tp cht, nhit , l cc
yu t c th nh hng n qu trnh kt ta hot cht bng isopropanol.
Nh trnh by trn, vi iu kin nghin cu v thi gian cn hn ch,
chng ti mi ch bc u kho st cc yu t hm m ca cao trong nc
em tinh ch v cho kt qu cng nh nhn nh cn s b. Qua thc nghim
cho thy, p dng cc bin php gim ti a lng nc ln vo hn hp em
tinh ch (s dng my ct quay chn khng, c dch chit n cao c) v loi
tp (s dng ethanol 96%, bt talc) gip rt ngn thi gian to ta, gim thiu
lng hot cht mt mt trong dch lc. Tp cht ha tan lm tng nht ca
dung dch, gy cn tr qu trnh kt tinh hot cht. Qua thc nghim cho thy,
c th s dng bt talc hoc bt giy lc trong giai on to ta th v s
dng than hot trong giai on kt tinh li loi bt tp cht ny, to iu
kin cho qu trnh kt ta/kt tinh hot cht. Tp cht lm cho khi dch rt
kh lc; do , vic hn ch lng nc ln trong hn hp em tinh ch v p
dng cc bin php loi tp gip cho thao tc lc thu hot cht tr nn d
dng hn.
Dch lc c nht ln, ng thi acid shikimic tuy kh tan, nhng
vn tan mt phn trong isopropanol, do mt lng acid shikimic b mt
mt trong dch lc. Gp cc dch lc, dch ra, c thu hi dung mi, kt

53

tinh thu hi acid shikimic. Vi cc dung mi tinh ch khc, lng acid


shikimic thu hi t dch lc l khng ng k.
Phng php tinh ch acid shikimic t dch chit nc c xut c
nhng u im sau y:
- Khng s dng nha trao i ion (phng php ny tn km v ch p
dng trong phng th nghim).
- Khng s dng ho cht c hi (methanol, formaldehyd,).
- t tht thot dung mi hn khi s dng ethyl acetat hoc aceton.
- C th ti thu hi s dng dung mi.
- Quy trnh thao tc d dng, s dng thit b n gin.
Tm li: Mc d acid shikimic c quan tm nghin cu trong v
ngoi nc t nhiu nm gn y vi nhiu cng trnh c cng b nhng
mi ch c ng dng quy m nghin cu. Cc quy trnh chit xut acid
shikimic t i hi cn kh phc tp, s dng dung mi c hi, thu hi ti s
dng dung mi kh khn, lng dung mi s dng ln nn tnh kinh t khng
cao v kh p ng vo thc tin sn xut (ni dung chi tit c trnh by
trong mc 1.2.5).
Vi quy trnh chit xut acid shikimic t i hi vi dung mi nc
c chng ti xut (hnh 3.10), cc thao tc thc hin kh d dng, thit
b n gin, s dng nc lm dung mi chit v cc dung mi tinh ch d
kim, r tin, t c hi (ethanol 96%, isopropanol). Quy trnh c th
nghim vi c m 2kg, lng nc cho mi ln chit c gim i do c m
ln, dc liu xen k vo nhau nn ch cn lng nc vi t l nh hn m
100g m vn m bo chit c gn nh ton b hot cht; ng thi gip
rt ngn qu trnh c dch chit. Lng dung mi tinh ch l tng i nhiu,
tuy nhin c th thu hi ti s dng. Quy trnh chit xut acid shikimic t i
hi vi dung mi nc c xut l n nh v cho hiu sut quy trnh t
67,83 1,62 (n = 3).

54

KT LUN V KIN NGH

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 :
-

kho st hai phng php thu c acid shikimic kt hp ct

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.
-

kho st 4 loi dung mi tinh ch gm c: methanol, hn hp

methanol-isopropanol, isopropanol v hn hp methanol-aceton. Isopropanol


c la chn tinh ch thu acid shikimic do p ng c cc yu cu:
loi c nhiu tp, hiu sut quy trnh tt, thu hi dung mi d dng v dung
mi r tin, d kim, t c hi.

55

- Bc u kho st lin quan n hm m ca cao chit nc. Kim


sot lng nc ln trong hn hp to ta hot cht v p dng nhiu bin
php loi tp s to thun li cho qu trnh to ta acid shikimic s dng dung
mi tinh ch l isopropanol.
- T cc kt qu thu c, xut quy trnh chit xut acid shikimic
t i hi, p dng trn 3 m thc nghim vi c m 2kg nguyn liu cho kt
qu tng i ng nht vi hiu sut quy trnh t 67,83 1,62 (n = 3).
Phng php khng yu cu thit b phc tp, d tin hnh, ch dng nhng
dung mi an ton v d kim.

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

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TI LIU TING TRUNG QUC:
30. Th

vin

ha

cht

http://www.basechem.org/chemical/2869.

BASECHEM,

URL:

PH LC

Ph lc 1. Ph IR ca acid shikimic

Ph lc 2. Ph khi lng (Negative) ca acid shikimic

Ph lc 3. Ph khi lng (Positive) ca acid shikimic

Ph lc 4. Ph 1H-NMR ca acid shikimic

Ph lc 5: Ph 13C-NMR ca acid shikimic

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