Professional Documents
Culture Documents
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Thi gian nui cy (ngy)
Khi lng ti t bo
Khi lng kh t bo
Hnh 3.4. Sinh trng ca t bo ngh en nui cy trong bnh tam gic trn mi
trng MS c 3% sucrose; 0,5 mg/L BA v 1,5 mg/L 2,4-D, lc 120 vng/pht
59
Hnh 3.5. Nui cy t bo ngh en trong bnh tam gic 250 ml t trn my lc
Nghin cu ca Mello v cs (2001a) ngh en cho thy, khi b sung 0,5
g t bo trong 10 ml mi trng MS c 3% sucrose, 13,4 M NAA v 2,2 M
BA, tc lc 60 vng/pht s thu c 6 g sinh khi ti sau 35 ngy [101].
Trong khi Miachir v cs (2004) nui cy 1 g t bo ngh en trong 75 ml mi
trng MS c 1 mg/L NAA, tc lc 100 vng/pht, sau 20 ngy thu c 8 g
sinh khi ti [103]. Mt s nghin cu khc, thi gian t bo sinh trng
cc i di hn t bo ngh en, chng hn nh t bo cy Solanum
eleagnifolium l 26 ngy (Nigra v cs 1989), Solanum aviculare 28 ngy
(Kittipongpatana v cs 1998), Taxus willichiana 32 ngy [112], Solanum
chrysotrichum 22 ngy (Rodrguez-Monroy v cs 2004) [134], Psoralea
corylifolia 20 ngy (Shinde v cs 2009), Solanum hainanense 28 ngy (Loc v
cs 2009), Withania somniera 28 ngy (Nagella v Murthy 2010) [109].
3.3. NUI CY T BO HUYN PH TRONG H LN MEN
3.3.1. Kho st sinh trng ca t bo
Sinh khi t bo (10 ngy tui) nui cy lc trn bnh tam gic 250 ml
trong mi trng c bn MS c 3% sucrose, b sung thm BA 0,5 mg/L v 2,4-
D 1,5 mg/L c s dng lm nguyn liu kho st qu trnh sinh trng ca
t bo ngh en khi nui cy h ln men th tch 14 L, th tch lm vic 10 L
(Hnh 3.6).
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Hnh 3.6. Nui cy t bo ngh en trong h ln men 10 L
Kt qu nghin cu trnh by hnh 3.7 cho thy, sinh khi t bo t
cc i ngy th 14 vi 241,67 g ti (22,28 g kh), sau bt u gim
dn. Sinh khi ti t bo ch cn li 132,67 g (10,08 g kh) sau 18 ngy nui
cy. Lc ny, quan st thy dch huyn ph t bo t mu vng chuyn sang
nu, nguyn nhn c th lin quan n s xut hin cc sn phm phenol
trong mi trng do qu trnh chuyn ha trao i cht ca t bo to ra.
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Thi gian nui cy (ngy)
Khi lng ti t bo
Khi lng kh t bo
Hnh 3.7. Sinh trng ca t bo ngh en trong h ln men nui cy trn mi
trng MS c 3% sucrose; 0,5 mg/L BA; 1,5 mg/L 2,4-D ; khuy 120 vng/pht;
sc kh 2,0 L/pht, c mu 100 g
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Nh vy, thi gian v ng thi sinh trng ca t bo cy ngh en
nui cy huyn ph trong h ln men 10 L tng t nui cy trong bnh tam
gic 250 ml (Hnh 3.4). Thi gian sinh trng cc i ca t bo ngh en
trong h ln men l 14 ngy, kt qu ny cng tng ng vi sinh trng
ca t bo cy Gymnema sylvestre (15 ngy) (Lee v cs 2006) [78] v
Solanum chrysotrichum (16 ngy) (Rodrguez-Monroy v cs 2004) [134] khi
nui cy trong h ln men.
Hnh 3.8. Sinh khi ti (A) v sinh khi kh (B) ca t bo ngh en sau 14 ngy
nui cy trong h ln men
Da trn kt qu kho st ng cong sinh trng ca t bo ngh en,
chng ti tin hnh kho st nh hng ca mt s iu kin nui cy trong h ln
men (c mu, tc khuy, tc sc kh) ln sinh trng ca t bo sau 14 ngy.
3.3.2. nh hng ca iu kin nui cy
3.3.2.1. C mu
Lng mu a vo nui cy trong h ln men l mt trong nhng yu
t quan trng c th nh hng n s tch ly sinh khi v cc sn phm
trao i cht. Kt qu nghin cu ca chng ti cho thy, c mu nui cy
ban u nh hng r rt n kh nng sinh trng ca t bo. Sau 14
ngy nui cy, kh nng tch ly sinh khi ca t bo tng ln cng vi s
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tng lng mu nui cy t 100 n 200 g t bo v t cc i l 514,64 g
ti (47,97 g kh). Tuy nhin, khi tng lng mu ln 250 g th sinh trng
ca t bo b c ch do tng mt t bo, v th ch t 441 g ti (38,89 g
kh) (Bng 3.11).
Bng 3.11. nh hng ca c mu ln sinh trng ca t bo nui cy huyn ph trong
h ln men 10 L
C mu (g) Khi lng ti (g) Khi lng kh (g)
100 241,67
c
22,28
d
150 358,33
bc
32,68
c
200 514,67
a
47,97
a
250 441,00
b
38,89
b
Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k p<0,05
(Duncans test).
Lee v cs (2006) khi nui cy huyn ph t bo cy Gymnema sylvestre
cho thy, c mu ban u khong 60 g/L thch hp cho sinh trng t bo v tc
sinh trng s gim nu a vo nui cy 80 hoc 100 g/L [78]. Matsubara
v cs (1989) nghin cu cy Coptis japonica nhn thy lng t bo nui cy
ban u 8 g/L thch hp cho s tch ly sinh khi [97].
3.3.2.2. Tc khuy
Khuy trn c tc dng lm tng tc ha tan ca oxy v cc cht
dinh dng trong mi trng gip t bo sinh trng tt hn. Nghin cu ca
chng ti cho thy, khi tng tc khuy t 100 n 150 vng/pht th sinh
khi t bo cng tng theo v cc i t l 561 g ti (50,84 g kh). Tuy
nhin, khi khuy trn trn 150 vng/pht th sinh trng t bo bt u gim
v ch t c 437 g ti (37,42 g kh) 200 vng/pht (Bng 3.12).
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Bng 3.12. nh hng ca tc khuy ln sinh trng ca t bo nui cy
trong h ln men 10 L
Tc khuy
(vng/pht)
Khi lng ti (g)
Khi lng kh (g)
100 435,67
c
38,19
c
120 514,67
bc
47,97
b
150 561,00
a
50,84
a
180 547,33
b
50,03
a
200 437,00
c
37,42
c
Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k
p<0,05 (Duncans test).
Paek v cs (2005) cho rng, trong qu trnh nui cy huyn ph, thy lc
sinh ra do khuy trn cn phi nh khng gy nguy hi cho m hay t bo
nhng phi ln kch thch cc chc nng ca chng [116]. Nguyn nhn
ca s c ch sinh trng t bo khi nui tc khuy cao c th l do cc t
bo b ph v c hc v gy ra hin tng t phn lm cht t bo (Ziv, 2000)
[193]. Wang v cs (2010) khi nui cy t bo cy Glycyrrhiza inflata trong h
ln men nhn thy sinh khi t bo t cao nht 150 vng/pht; tc
khuy 100 v 300 vng/pht, kh nng sinh trng ca t bo km hn [171].
Nghin cu ca Choi v cs (2008) cho thy, tc khuy 120 v 225 vng/pht
tng sinh khi t bo ca cy c du ln n 200%, nhng khi khuy vi tc
335 vng/pht th sinh khi t bo ch tng 7% [33].
3.3.2.3. nh hng ca tc sc kh
Kt qu ca chng ti cho thy, tc sc kh cng nh hng ln n sinh
trng t bo ngh en. tc sc kh 1,5-2,5 L/pht, sinh trng ca t bo tng
dn v t cc i vi 603 g ti (53,25 g kh). Nu tc sc kh tip tc tng ln
t 3-3,5 L/pht th sinh trng ca t bo s gim. tc sc kh 3,5 L/pht, khi
lng ti ca t bo ch t 441 g (33,17 g kh) (Hnh 3.9 v Bng 3.13).
Hm lng oxy ha tan tan trong mi trng nh hng ln n sinh
trng ca t bo nui cy, khi tc sc kh tng, tc ha tan ca oxy s
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tng. Tuy nhin, sinh trng ca t bo khng phi lun lun t l thun vi tc
sc kh m n b gii hn trong mt mc nht nh. Vic sc kh v khuy
trn khng ch cung cp oxy cho t bo m cn ngn chn s ng cn sinh khi
t bo. Tuy nhin, nu tc sc kh v tc khuy qu cao s lm cho t bo
b bin dng v ph v thnh t bo. Nhng t bo khi v s gii phng
polysaccharide to thnh bt, kt dnh cc mnh v t bo li vi nhau v bm
ln thnh bnh nui hoc to thnh lp vng trn b mt mi trng. Lp ny
ngn cn s lu thng kh cn thit gy ra s thiu khng kh cho sinh trng t
bo (Ziv 2000) [193]. Ngoi ra, khi vng bt ni ln trn mi trng s cun
theo cc t bo lm cho cc t bo ny khng tip xc vi mi trng dinh
dng v cht do thiu ht dinh dng.
Bng 3.13. nh hng ca tc sc kh ln sinh trng ca t bo nui cy
trong h ln men 10 L
Tc sc kh (l/pht) Khi lng ti (g) Khi lng kh (g)
1,5 489,67
c
36,9
b
2,0 561,00
b
50,84
a
2,5 603,00
a
53,25
a
3,0 583,33
b
50,33
a
3,5 441,00
d
33,17
b
Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k
p<0,05 (Duncans test).
nh hng ca s sc kh trong h ln men ln tch ly sinh khi t bo
cng c nghin cu nhiu loi thc vt. Lee v cs (2006) khi nui cy t
bo cy Gymnema sylvestre trong h ln men 2 L cho thy, tc sc kh 0,1
L/L/pht l thch hp nht [78]. Thanh v cs (2005) kho st nh hng ca
nng oxy (20,8-50%) n sinh trng ca t bo cy nhn sm nui cy trong
h ln men 5 L. Kt qu cho thy, nng oxy 40% l thch hp nht cho sn
xut sinh khi v cht ginsenoside, trong khi nng oxy 20,8%, 30% v 50%
l khng thch hp cho sinh trng ca t bo [159]. Nh vy, nhu cu oxy ca
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mi loi thc vt l khng ging nhau v n ph thuc vo nhiu yu t nh
lng mu nui cy; tc sinh trng; hot tnh sinh l ca t bo; iu kin
mi trng xung quanh nh nng ng, cc cht dinh dng, v s tch ly
cc sn phm c ca qu trnh trao i cht t bo.
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Khi lng ti t bo
Khi lng kh t bo
Hnh 3.9. Sinh trng ca t bo ngh en trong h ln men nui cy trn mi
trng MS c 3% sucrose; 0,5 mg/L BA; 1,5 mg/L 2,4-D ; khuy 150 vng/pht;
sc kh 2,5 L/pht, c mu 200 g
3.4. KHO ST S TCH LY MT S HP CHT C HOT TNH
SINH HC TRONG T BO NGH EN
3.4.1. Hm lng tinh du
Hm lng tinh du ca t bo ngh en nui trong h ln men 10 L c
trnh by bng 3.14. Nhn chung, hm lng tinh du (% khi lng kh) ca
t bo tng dn t 2-14 ngy nui cy v t gi tr cc i l 2,57% cao hn ca
c ngh en t nhin (1,61%) khong 1,6 ln (p<0,05). Hm lng tinh du tch
ly trong t bo gim nhanh sau 16 n 18 ngy; c bit sau 18 ngy nui cy,
hm lng tinh du ch cn 1,09% khi lng kh. iu ny c th gii thch l
do sinh trng ca t bo ngh en sau khi t cc i vo ngy th 14, t bo
c pha sinh trng n nh rt ngn, sau t bo bc nhanh vo pha suy vong
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sau 16 n 18 ngy nui cy (Hnh 3.9); lc hin tng phn hy (t phn)
ca t bo din ra mnh m dn n kh nng tch ly tinh du trong t bo cng
gim theo rt nhanh.
Bng 3.14. Hm lng tinh du ca t bo ngh en nui cy trong h ln men 10 L
Thi gian nui cy (ngy) Hm lng tinh du (%)
2 1,41
c
4 1,49
c
6 1,57
bc
8 1,98
c
10 2,32
b
12 2,42
b
14 2,57
a
16 1,68
bc
18 1,09
d
MTN 1,61
c
Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k
p<0,05 (Duncans test).
MTN: c ca cy ngh en t nhin 01 nm tui
Nghin cu s tch ly tinh du trong t bo thc vt nui cy in vtro
cng c nhiu tc gi cng b. Chng hn, Figueiredo v cs (1995) nui
cy t bo cy Achillea millefolium L. ssp. mille thu c hm lng tinh du
0,002% cao gp 2 ln tinh du c trong cy t nhin, v thnh phn tinh du
cng nhiu hn. Nui cy callus v huyn ph t bo cy Origanum vulgare L.
cho thy, s tch ly tinh du t 0,095% (trong callus) v 0,06% (t bo nui
cy huyn ph) cao hn hm lng tinh du tch ly trong t bo ca cy t
nhin (ch t 0,042%) (Arafeh v cs 2006).
Ngh en l cy tho dc c cha tinh du bao gm cc cht thuc nhm
sesquiterpene v monosesquiterpene [82]. Hm lng tinh du trong c ca cy
ngh en t nhin l 0,22% khi lng ti [1] hoc xp x 1,5% khi lng kh
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[10]. Mau v cs (2003) nhn thy, tinh du ngh en c hot tnh tt trong vic
qut gc t do 1,1-diphenyl-2-picrylhydrazyl [100]. Nghin cu ca Trn Th
Vit Hoa v cs (2007) nhn thy, tinh du ngh en trng Lt, Vit Nam c
kh nng chng oxy ha tng i cao [3]. Vi kt qu nghin cu ny, chng ti
nhn thy nui cy huyn ph t bo ngh en l mt phng thc trin vng
trong vic thu hi ngun tinh du c gi tr cao t loi tho dc ny.
3.4.2. Hm lng polysaccharide ha tan trong nc tng s
Bng 3.15 trnh by hm lng polysaccharide ha tan trong nc tng s
(% khi lng kh) trong t bo ngh en ti cc thi im nui cy khc nhau.
Nhn chung, hm lng polysaccharide tng t 2 - 10 ngy nui cy v t cc
i l 6,55% thp hn khong 1,4 ln so vi c ngh en (9,46%) (p<0,05). Hm
lng polysaccharide ca t bo gim sau 12 ngy nui cy v gim rt nhanh
ch t khong 1,6-2,01% trng lng kh sau 16 n 18 ngy.
Bng 3.15. Hm lng polysaccharide ha tan trong nc tng s ca t bo ngh
en nui cy trong h ln men 10 L
Thi gian nui cy (ngy) Hm lng polysaccharide (%)
2 1,73
e
4 2,23
d
6 3,53
cd
8 5,15
bc
10 6,55
a
12 5,76
b
14 4,53
c
16 2,01
d
18 1,60
e
MTN 9,46
g
Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k
p<0,05 (Duncans test).
MTN: c ca cy ngh en 01 nm tui
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Trong nhng thp k gn y, cc polysaccharide c ngun t thc vt
c ch c bit trong lnh vc y sinh hc bi chng c nhiu hot tnh sinh
hc tt trong iu tr bnh v khng c tnh c (Schepetkin v cs 2006). Nui
cy t bo thc vt c xem l ngun b sung c hiu qu cc sn phm t
nhin t thc vt, bao gm polysaccharide. Cho n nay, c hn 130 loi thc
vt c s dng trong nui cy t bo sn xut cc polysaccharide c hot
tnh sinh hc (Mahmoudifar v cs 2000). Gunter v cs (2003) nghin cu nui
cy t bo cy Silene vulgaris nhm sn xut cc polysaccharide [50]; Zhang v
Zhong (2002) cng dng h ln men CIB (th tch 30 L) nui cy huyn ph
t bo cy tam tht thu cc cht hp cht nh saponin, polysaccharide vi
hm lng t c ln lt 1,7 v 2,9 g/L [186].
Nhiu nghin cu cho thy, polysaccharide ca cc loi thuc chi ngh
nh C. longa, C kwangsiensis, C. xanhthorrhiza c cc hot tnh qu: tng
cng kh nng min dch, c ch khi u, chng oxi ha(Zeng v cs 2002;
Yuea v cs 2010). cy ngh en, mt s nghin cu cho thy, cc
polysaccharide ca n c kh nng c ch sinh trng ca t bo khi u, ngn
cn t bin nhim sc th, kch thch chc nng ca i thc bo [75], [105],
[169]. Wang v cs (2004) tch chit ca c ngh en t nhin bng sc k ct
v xc nh hm lng polysaccharide tng s l 33,12% [169]. Kt qu nghin
cu ca chng ti th hm lng polysaccharide ha tan trong nc tng s ca t
bo ngh en nui cy in vitro t 6,55% sinh khi kh, sau 10 ngy nui cy l rt
c trin vng trong sn xut cc polysaccharide c hot tnh sinh hc ca ngh en
bng con ng nui cy t bo.
3.4.3. Hm lng curcumin
Da trn kt qu nghin cu sinh trng ca t bo ngh en trong h ln
men 10 L, chng ti kho st s tch ly curcumin ca chng trong qu trnh
nui cy. Kt qu phn tch HPLC c trnh by cc hnh 3.10-3.12 v bng
3.16. Ph HPLC ca curcumin chun c 1 nh (peak) duy nht thi gian lu l
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2,05 pht. Mt peak c thi gian lu tng ng cng c tm thy dch
chit ca c ngh en t nhin (2,08 pht) v t bo nui cy cc thi gian
khc nhau t 2-18 ngy (1,97-2,09 pht). Phn tch HPLC cng nhn thy, y l
peak c din tch ln nht trong ph HPLC ca t bo in vitro cng nh c ngh
t nhin. Da vo ph HPLC ca curcumin chun, c th xc nh rng, thnh
phn chnh curcuminoid trong dch chit ca t bo ngh den nui cy l
curcumin, ging trong c ngh en t nhin.
Ngoi ra, ph phn tch HPLC dch chit ca c ngh en t nhin cn c
thm 2 peak khc, mt peak c thi gian lu nh peak th hai t bo nui cy
(1,79 pht), peak cn li c thi gian lu l 1,21 pht. Dch chit t bo nui cy
c mt peak th ba rt nh vi thi gian lu khong 1,6 pht. S bin mt ca
peak th ba 1,21 pht ( dch chit c ngh en t nhin) trong t bo in vitro c
th l do s chuyn ha sinh hc ca curcuminoid xy ra trong nui cy t bo.
Bng 3.16. Hm lng curcumin ca t bo ngh en nui cy trong h ln men 10 L
Thi gian nui
cy (ngy)
Thi gian
lu (pht)
Din tch peak
(mAU)
Hm lng
curcumin (%)
2 2,09 1909 0,44
g
4 1,98 2226 0,52
e
6 2,05 2285 0,53
e
8 2,00 2442 0,56
e
10 1,97 4244 0,98
b
12 1,99 4448 1,03
b
14 2,07 5034 1,16
a
16 1,99 3631 0,84
c
18 2,07 2573 0,60
d
MTN 2,06 1868 0,43
g
Cc ch ci khc nhau trong cng mt ct ch s sai khc c ngha thng k
p<0,05 (Duncans test).
MTN: c ca cy ngh en 1 nm tui
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Kt qu nghin cu bng 3.16 cho thy, hm lng curcumin (theo %
khi lng kh) tch ly trong t bo tng dn t ngy th 2 n ngy th 14, t
cc i l 1,16%, sau gim t ngy 16-18 cng vi sinh trng ca t bo
(Hnh 3.9). Nhn chung, hm lng curcumin trong t bo cc thi gian nui
cy khc nhau u cao hn trong c ngh en t nhin (0,43%), lc cao nht gp
khong 2,7 ln.
Thng thng, cc hp cht th cp tch ly nhiu nht vo cui giai on
sinh trng cc i. c nhng bng chng r rng cho thy c mi quan h
ngc gia tc sinh trng v kh nng sn xut cc cht th cp. Khi tc
sinh trng cao, cc qu trnh s cp ca t bo l phn chia t bo v sn xut sinh
khi t bo din ra mnh m. Ngc li, trong pha tnh khi s sinh trng gim,
lc ny hot ng sn xut v tch ly cc cht th cp mi tng ln [7]. Nghin cu
qu trnh sinh trng ca t bo ngh en nui trong h ln men cho thy, pha sinh
trng n nh ca t bo rt ngn, sau khi t bo sinh trng cc i th sinh khi
gim ngay (t ngy th 14 n 16) (Hnh 3.9). V vy, hm lng curcumin c tng
hp cao nht vo cui giai on t bo sinh trng cc i (14 ngy) l hp l. Kt qu
ny ca chng ti cng tng t kt qu nghin cu kh nng sn xut jaceosidin ca
t bo S. medusa. Hm lng jaceosidin v sinh khi t bo u t cc i vo ngy
th 14 ca qu trnh nui cy [189].
Ngoi ra, qu trnh tch ly cc hp cht th cp khng ging nhau gia cc
loi, khng phi loi thc vt no cng tng tch ly cc hp cht th cp khi sinh
trng ca t bo i vo pha n nh. Khi nui cy t bo T. cordifolia, Rao v cs
(2008) nhn thy rng trong khi khi lng kh t bo t cc i (9,9 g/L) sau 36
ngy nui cy, th hm lng berberine trong t bo li thu c cao nht vo ngy
24 trong pha log ca t bo [130]. Mt khc, nhiu nghin cu cho thy, t bo
nui cy huyn ph c kh nng tch ly cc hp cht th cp cao hn so vi cy
t nhin, chng hn nh t bo in vitro ca cy Solanum chrysotrichum c hm
lng sprirostanol saponin khong 14 mg/g, cao gp 50 ln so vi hm lng
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ca n trong l cy t nhin (Villarreal v cs 1997) [168], hoc t bo cy da
cn nui cy in vitro cng c lng vincristin cao hn l t nhin [5].
Nhiu nghin cu chng minh rng, curcumin c kh nng chng
c s pht sinh khi u; ung th rut kt, ung th d dy, ung th v v ung
th bung trng [55], [66], [152]; chng ng mu v h huyt p; chng
vim nhim, khng oxy ha v bo v t bo [152]. Syu v cs (1998) nghin
cu nhn thy, dch chit ethanol ca c ngh en c cha curcumin,
demethoxycurcumin v bisdemethoxycurcumin [152]. Paramapojn v
Gritsanapan (2007) phn tch HPLC thnh phn curcuminoid trong c ngh
en ca Thi Lan cho thy, hm lng curcumin, demethoxycurcumin v
bisdemethoxycurcumin ln lut t 1,46% n 5,73% w/w (trung bnh 2,73%
w/w); t 3,15% n 10,98% w/w (7,37%) v t 0,49% n 2,99% w/w (1,40%
w/w) [119]. Kt qu nghin cu ca chng ti cho thy, curcumin tch ly
trong t bo ngh en nui cy in vitro vi hm lng kh cao (1,16%), iu
ny c kh nng gi t bo ngh en in vitro c trin vng l ngun nguyn
liu thch hp sn xut curcumin, v ch cn trong thi gian ngn khong 2
tun l c th to c nguyn liu sn xut thay v trong t nhin trng
phi mt hng nm mi c.
Hnh 3.10. Ph HPLC ca curcumin chun (0,5 mg/ml)
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Hnh 3.11. Ph HPLC curcumin ca c ngh en 01 nm tui ngoi t nhin
73
Hnh 3.12. Ph HPLC curcumin ca t
bo ngh en sau 2-18 ngy nui cy
trong h ln men 10 L
3.4.4. Xc nh sesquiterpene
Sesquiterpennoid l cc hp cht C
15
c to thnh bi lin kt ca 3 n
v isoprenoid. Chng c mt trong nhiu t chc sng bao gm thc vt bt cao.
Khong 140 hp cht sesquitertene khc nhau c tch chit t chi Curcuma v
c chia thnh 10 nhm cu trc khc nhau. Tuy nhin, hu ht cc hp cht
ny nm trong 3 nhm chnh l bisabolane, germacrane v guaiane.
74
S phn b ca sesquiterpene trong t bo ngh en c phn tch bng
HPLC. Kt qu cho thy, s hin din ca cc peak phn tch trong c ngh t
nhin nhiu hn trong t bo huyn ph (Hnh 3.13). Nhn chung, cc peak c
thi gian lu lin quan vi cc hp cht sesquiterpene (c nh s t 1-9)
trong c ngh v t bo huyn ph c chiu cao ph hp th (mAU) khc nhau,
c bit l peak s 1 v 4 (Bng 3.17). Chng hn, peak s 1 (thi gian lu 4,7
pht) c hp th l 6,05 mAU trong c ngh nhng ch t 2,55 mAU trong t
bo huyn ph, ngc li peak s 4 (thi gian lu 7,5 pht) t bo huyn ph
c hp th l 7,33 mAU cn c ngh l 2,23 mAU. Mt s peak hin din
trong c ngh (5-8) th khng c hoc ch c dng vt (< 1 mAU) trong t bo
huyn ph. Tuy nhin, t bo huyn ph li xut hin nhng peak mi (khng
nh s) vi cc thi gian lu xp x 6,8; 7,8; 8,2 v 9,2 pht tng ng vi
hp th 1,38-4,10; 5,62-7,54; 1,70-8,41 v 1,06-8,38 mAU. Kt qu ny cho
thy, trong qu trnh nui cy t bo xut hin s chuyn ha sinh hc ca cc
sesquiterpen, to ra nhng hp cht sesquiterpen khc so vi c ngh t nhin.
Bng 3.17. Chiu cao ph hp th (mAU) ca sesquiterpene trong t bo nui cy
h ln men 10 L v t bo c ngh t nhin
Hp cht sesquiterpene (s)/thi gian lu (pht)
Thi gian
nui cy
(ngy) 1/4,7 2/5,1 3/6,0 4/7,5 5/12,7 6/13,8 7/19,2 8/20,5 9/26,7
2 2,11 - 2,49 5,99 - vt vt vt -
4 1,93 - 2,23 5,76 - vt vt vt -
6 1,15 vt 2,81 3,17 - vt - - -
8 1,06 - 1,54 3,57 - - - - -
10 1,22 - 1,02 4,05 - vt vt - -
12 1,04 vt 2,14 3,29 - vt - vt -
14 2,55 1,59 - 7,33 - vt - vt -
16 1,58 - 3,82 4,04 - vt - - -
18 2,36 1,15 1,15 5,11 - vt - vt -
MTN
6,05 2,59 2,15 2,23 6,23 1,46 3,59 1,29 1,19
MTN: c ca cy ngh en 01 nm tui
75
76
Hnh 3.13. Ph HPLC ca sesquiterpene. A: C ngh en t nhin; B: t bo
ngh en nui cy trong h ln men 10 L t 2 n18 ngy
Sakui v cs (1992) nui cy t bo callus ngh en hn 2 nm kho
st s chuyn ha sinh hc ca sesquiterpene v chng minh c s chuyn ha
sesquiterpen mch vng thnh vin s 10 thuc nhm germacrone thnh
sesquiterpene thuc nhm guaiane. Chuyn ha sinh hc trong nui cy in vitro
t bo thc vt cng c thng bo nhiu loi chng hn, Sakamoto v cs
(1994) nui cy t bo ca cc cy Lonicera japonica, Bupleurum falcatum,
Polygonum tinctorium v Solidago altissima nhn thy c s chuyn ha
germecrone thnh guaiane, eudesmane v secoguaiane; Hashimoto v cs (1999)
nui cy t bo cy Heteroscyphus planus nhn thy c s chuyn ha cubenene
thnh 7-hydroxycalamene; Chuyn ha geranyl acetate v linalyl acetate thnh
geraniol, linalool, a-terpineol cng xut hin trong nui cy t bo cy
Peganum harmala [192]. Nh vy, nui cy t bo thc vt c xem l mt
phng thc hiu qu sn xut cc hp cht th cp cng nh cc cht chuyn ha
ca chng (Rao v Ravishankar, 2002). Kt qu nghin cu ca chng ti cho thy
c s tch ly mt s hp cht sesquiterpene trong t bo ngh en nui cy in vitro
ging nh trong c ngh t nhin, ng thi trong t bo cng xut hin mt s
sesquiterpene khc vi c t nhin (c th do chuyn ha sinh hc); kt qu ny c
th gi quan trng trong sn xut sesquiterpene cng nh cc hp cht th cp
mi c hot tnh sinh hc qu thng qua nui cy huyn ph t bo cy ngh en.
77
3.5. HOT TNH KHNG KHUN CA TINH DU T BO NGH EN
Kt qu trnh by bng 3.18 cho thy, tinh du tch chit t t bo v
c ngh en t nhin u c kh nng c ch sinh trng ca c 3 loi vi
khun: Escherichi coli, Staphylococcus aureus v Bacills cereus. Nhn chung,
tinh du thu c t t bo ngh en c kh nng khng khun cao nht th
hin qua ng knh vng c ch (Hnh 3.14). Tinh du t bo nui cy c
ch mnh nht i vi S. aureus (ng knh v khun 31 mm), sau n B.
cereus (22,33 mm) v km nht i vi E. coli (18,33 mm). Trong khi ,
kh nng c ch sinh trng ca tinh du c ngh en t nhin i vi B.
cereus v E. coli l tng ng nhau (16 mm v 17,67 mm), km nht l i
vi S. aureus ch t 14 mm.
Hnh 3.14. Kh nng khng khun ca tinh du ngh en. A: E. coli ATCC 25922.
B: B. cereus ATCC 11778. C: S. aureus ATCC 6538. C: i chng. TN: tinh du c
ngh en t nhin. TB: tinh du ca t bo ngh en
78
Bng 3.18. Kh nng khng khun ca tinh du t bo ngh en
ng knh vng v khun D-d (mm)
Vi khun
i chng Tinh du t bo
Tinh du c ngh
B. cereus ATCC 11778 0 22,33
b
16,00
a
E. coli ATCC 25922 0 18,33
b
17,67
a
S. aureus ATCC 6538 0 31,00
a
14,00
a
i chng: khng c tinh du ngh en
Theo nghin cu ca nhiu tc gi, vi khun Gram m
thng t nhy cm
vi hot tnh ca nhiu loi tinh du hn vi khun Gram dng
(Dorman 2000;
Ruberto v cs 2000; Senatore v cs 2000; Tsigarida v cs 2000). iu ny c th
do s khc nhau v cu trc thnh t bo gia vi khun Gram m v Gram
dng. Vi khun Gram m
c lp mng bao quanh thnh t bo, chnh lp ny
ngn cn s khuch tn ca cc hp cht k nc thng qua lp cht
lipopolysaccharide (LPS). Ngc li, cc vi khun Gram dng khng c lp
mng ny nn tinh du c th tip xc trc tip vi lp LSP thnh t bo vi
khun, do lm tng kh nng thm ca cc ion, s tht thot cc hp cht ni
bo hoc lm suy yu h thng enzyme ca vi khun (Cowan, 1999; Wendakoon
v Sakaguchi, 1995).
Mt s nghin cu ch ra rng, cc hp cht thu c t t bo thc vt
nui cy in vitro c kh nng khng cc loi vi sinh vt. Kt qu nghin cu ca
Par v cs (1991) cho thy, hp cht aurone tch chit t t bo cy
Cephalocereus senilis nui cy huyn ph c kh nng c ch sinh trng ca
cc vi khun Gram m. Robles-Zepeda v cs (2009) cng nhn thy, cc cht
khng vi sinh vt (phytoalexin) tch chit t t bo cy xng rng (Mamillaria
huitzilopochitli) nui cy huyn ph c kh nng c ch sinh trng ca 8 loi
nm gy bnh thc vt.
Trn i tng cy ngh en, c mt s cng trnh nghin cu kh
nng khng khun ca tinh du, tuy nhin vic kho st hot tnh khng khun
79
ch yu t tinh du chit rt c ngh en ngoi t nhin. Srvidya v cs (2009)
nghin cu cho thy, tinh du ca c ngh en n c tc dng c ch mt
s vi khun, trong tc dng c ch sinh trng mnh i vi vi khun B.
cereus nhng tng i vi E. coli v S. aureus [149]. Wilson v cs (2005)
nghin cu tnh khng khun ca tinh du ngh en t nhin Nam n cng
cho thy, n c ch sinh trng nhiu loi vi sinh vt nhng khng c ch S.
aureus v E. coli [176]. Kt qu nghin cu ca chng ti cho thy, tinh du thu
c t t bo ngh en nui cy in vitro c kh nng c ch sinh trng ca c
3 loi vi khun kim nh l S. aureus, B. cereus v E. coli.
80
KTLUNVNGH
KT LUN
Trn c s cc kt qu thu c, chng ti rt ra nhng kt lun sau y:
1. Mi trng c bn MS c 20 g/L sucrose, 0,8% agar, b sung thm BA
0,5 mg/L v 2,4-D 0,5 mg/L thch hp cho nui cy callus t b l cy ngh en
in vitro. Callus c to thnh c mu c mu vng, rn v ri rc c dng
lm nguyn liu thit lp nui cy huyn ph.t bo.
2. Mi trng c bn MS c 30 g/L sucrose, b sung thm BA 0,5 mg/L
v 2,4-D 1,5 mg/L, tc lc 120 vng/pht thch hp cho sinh trng ca t
bo ngh en trong bnh tam gic 250 ml. Sinh khi t cc i l 10,44 g ti
(0,66 g) sau 14 ngy khi nui cy vi c mu l 3 g.
3. Mi trng c bn MS c 30 g/L sucrose, b sung thm BA 0,5
mg/L v 2,4-D 1,5 mg/L, tc khuy 150 vng/pht, tc sc kh 2,5
L/pht thch hp cho sinh trng ca t bo ngh en trong h ln men 10 L.
Sinh khi t cc i l 603 g ti (53,25 g kh) sau 14 ngy nui cy vi
c mu l 200 g.
4. Hm lng tinh du trong t bo t cao nht l 2,57% khi lng kh
sau 14 ngy nui cy, gp khong 1,6 ln tinh du c cy ngh en t nhin 1
nm tui. Hm lng curcumin trong t bo t cao nht l 1,16% sau 14 ngy
nui cy, gp khong 2,7 ln curcumin c cy ngh en t nhin 1 nm tui.
c chuyn ha sinh hc curcuminoid trong nui cy t bo. Hm lng
polysaccharide trong t bo cao nht l 6,55% khi lng kh sau 10 ngy nui
cy, thp hn c ngh t nhin khong 1,4 ln. C s tch ly cc hp cht
sesquiterpene trong t bo nui cy. Thnh phn sesquiterpene hin din trong t
bo huyn ph t hn so vi c cy ngh 1 nm tui. Trong t bo c 3-4 peak
ca sesquiterpene ging nh t bo c ngh. c chuyn ha sinh hc
sesquiterpene trong nui cy t bo.
81
5. Tinh du t bo cy ngh en c kh nng c ch mnh sinh trng
ca 3 chng vi khun B. cereus ATCC 11778 (ng knh v khun 31 mm),
S. aureus ATCC 6538 (22,33 mm) v E. coli ATCC 25922 (18,33 mm). Nhn
chung, kh nng khng khun ca tinh du thu hi t t bo nui cy in vitro
kh cao.
NGH
1. Nghin cu ci thin kh nng tch ly cc hot cht sinh hc trong t
bo ngh en.
2. Nghin cu thnh phn v hot tnh ca curcuminoid, sesquiterpenes,
polysaccharides c sn xut t t bo ngh en.
82
NHNG CNG TRNH C CNG B
LIN QUAN N LUN N
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PH LC
Ph lc 1: Mi trng c bn MS (Murashige v Skoog, 1962)
Mui khong Nng (mg/l)
Potasium nitrate (KNO
3
) 1900
Amonium nitrate (NH
4
NO
3
) 1650
Calcium chloride (CaCl
2
.H
2
O) 440
Magnesium sulfate (MgSO
4
.7H
2
O) 370
Potassium phosphate (KH
2
PO
4
) 170
Na
2
EDTA.2H
2
O 37,2
Manganese sulfate (MnSO
4
.4H
2
O) 22,3
Ferrous sulfate (FeSO
4
.7H
2
O) 27,8
Zinc Sulfate (ZnSO
4
.7H
2
O) 8,6
Boric Acid (H
3
BO
3
) 6,2
Potassium Iodine (KI) 0.83
Sodium molybdate (Na
2
MoO
4
.2H
2
O) 0,25
Colbalt chloride (CoCl
2
.6H
2
O) 0,025
Cupric Sulfate (CuSO
4
.5H
2
O) 0,025
Vitamin Nng (mg/l)
Myo-inositol 100
Nicotinic acid 0,5
Pyridoxine HCl 0,5
Thiamine HCl 0,1
Glycine 2
Sucrose 30 g/l
Agar 8 g/l
Ph lc 2: Hnh n h th nghim
Hnh: Nui cy t bo ngh en trong bnh tam gic 250 ml, trn my lc
Hnh: T bo ngh en lng trong bnh tam gic 250 ml
Hnh: Sinh khi ti ca t bo ngh en thu c sau kh nui cy 14 ngy
trong h ln men 10 L