Phan III. Cong Nghe Cac Chat Co Hoat Tinh Sinh Hoc

PHN III
CNG NGH CC CHT C

HOT TNH SINH HC
Phn III: CNG NGH CC CHT C HOT TNH SINH HC 281
XY DNG QUY TRNH CHIT XUT, TINH SCH V
XC NH HM LNG Azadirachtin TRONG HT
XOAN CHU HN (Azadirachta indica A.Juss)
V Vn , Nguyn Tin Thng
Phng Cng ngh cc cht c hot tnh sinh hc, Vin Sinh hc Nhit i
M U
Mt trong nhng cht chnh c tc ng xua ui su b cy xoan chu hn n
(Azadirachta indica) l azadirachtin. N c tc ng xua ui i vi gn 90% loi su hi.
Ngoi tc ng xua ui, azadirachtin cn c tc ng c ch pht trin v sinh sn. Cc
nghin cu trong hn 20 nm qua cho thy azadirachtin c tc ng iu ha sinh trng v
gy ngn n. Azadirachtin c tc ng gy ngn n ln nhiu loi cn trng gy bnh cng
nh mt s loi tuyn trng (2, 3).
Azadirachtin c cho l c cu trc tng t vi hocmon su hi, kim sot qu trnh
bin thi ca cn trng, t giai on u trng n nhng v giai on trng thnh.
Azadirachtin kim sot qu trnh tit hocmon cn trng, n c ch mt s loi hc mn
chnh, ngn cn s rng lng v lm thay i bin thi bnh thng ca cn trng (3).
Azadirachtin tp trung nhiu nht ht. Trung bnh 1g nhn ( nhng cy trn 5
nm tui) cha t 2g n 4g azadirachtin. Hm lng azadirachtin trong nhn ht xoan chu
hn bin ng ty thuc vo ngun gc, xut x, iu kin canh tc v kh hu v thi im
thu hoch qu (3,4,5).
Vic chit tch, tinh sch v xc nh hm lng azadirachtin trong ht xoan chu hn
l c s nghin cu tc dng a dng ca n ln su b gy hi cng nh phc v cho
vic kho st nh hng ca cc yu t ngoi cnh n hm lng azadirachtin trong ht
xoan chu hn.
V mc ch trn, cc tc gi tin hnh nghin cu xy dng quy trnh chit xut,
tinh sch v xc nh hm lng azadirachtin trong ht cy xoan chu hn trng ti Ninh
Thun, mt ngun nguyn liu qu sn xut thuc tr su tho mc v ci to mi
trng nhng vng t kh cn Vit Nam.
VT LIU V PHNG PHP
Ht xoan chu hn (Az adirachta indica) c thu hi t nhng cy xoan 4 nm
tui trng tnh Ninh Thun, c s dng l m nguyn li u chit xut, tinh
sch v xc nh hm lng azadirachtin. Thu hoch qu xoan chu hn tt nht
khi n mi chuyn sang mu vng hay vng xanh, lc ny hm lng azadirachtin
Hi ngh KHOA HC V CNG NGH 2007 282
cao nht. Nu qu chn rng xung t mi thu hoch th hm lng
azadirachtin s b gim (3,4). Chit xut, tinh sch azadirachtin bng sc k ct
(flash chromatography) vi cht nhi l silicagel 60 (0.04 - 0.063) ca hng Merck
vi hai loi ct c kch th c: 29mm x 50cm; 15mm x 50cm. Cc tiu phn
cha azdirachtin thu c qua chy ct c pht hin bng sc k bn mng vi
cht phun hin mu: 1g vanillin pha trong 100 ml axt sulfuric m c, vt
azaditachtin c mu nu. Hm lng azadirachtin c xc nh bng sc k lng
hiu xut cao (HPLC).
KT QU
1. Chit xut, tinh sch azadirachtin t ht xoan chu hn bng sc k ct
1.1. Chit th bng dung mi v loi m
Cn 250g nhn ht xoan chu hn tch v, nghin v ry qua ry 0,5mm.
a. Chit vi dung mi n-hexan loi m: Ngm kit bt nhn xoan chu hn bng
cch lc trong n-hexan 6 ln vi lng dung mi v thi gian ngm nh sau: ln 1,2 v
3 ngm, lc trong 500ml n-hexan trong 3 gi, ln 4 v 5 ngm, lc trong 300ml n-hexan
trong 2 gi, ln cui ngm, lc trong 200ml n-hexan trong 1 gi (1).
b. Chit vi methanol: B sau khi chit vi n-hexan c chit tip vi methanol
theo quy trnh sau: ln 1 v 2 ngm, lc trong 500ml methanol trong 3 gi, ln 3 v 4
ngm, lc trong 300ml methanol trong 2 gi , ln cui ngm, lc trong 200ml methanol
trong 1 gi.
Gp cc phn dch chit methanol v gim th tch dch thu c bng c quay
trong chn khng 40
o
C cn khong 50ml dch mu vng. Chit tip vi n-hexan
loi du bo trong mu. Sau gp vi dch chit n -hexan ban u, c tip thu c
89,5g du (35,8%).
Thm 20ml nc ct vo cn methanol thu c v chit 5 ln vi 50ml ethylacetat
(EtOAc), lm khan dch chit bng Na
2
SO
4
. Loi EtOAc trong chn khng 40
o
C, thu
c 4,6g cht rn mu vng sm (1,84%).
1.2. Tinh sch trn sc k ct
Nhi 120g silicagel 60 (0.040-0.063 m) ca hng Merck vo ct 29mm, di
50cm. Dung mi l hn hp EtOAc/n-hexan tng dn t 10 n 100%. Ha tan
4,6g mu thu c trn vi khong 5ml EtOAc, thm 3-5 g silicagel, loi dung
mi v a vo ct. Dung mi thi mu theo th t t l EtOAc / n -hexan tng t 0
n 100%.
Ly mi phn on khong 100 ml. Tin hnh sc k bn mng nhn dng
cht trn giy F254 ca hng Merck. Vt azadirachtin hin mu nu trong thuc
hin hnh. Hn hp dung mi chy sc k bn mng the o t l: n-hexan/aceton =
3/2, vt c gi tr Rf l 0,16. Cn t l n-hexan/aceton = 1/1, vt c gi tr Rf
l 0,32.
Thu azadirachtin t phn on 16 n 27, gom li, c quay chn khng thu c
1,6g. Ton b cht thu c t t chy sc k trn ct 1, c a tip vo ct 2 vi
kch thc 1,5 x 50cm tng ng vi lng silicagel nhi ct l 100. Ra thi ct bng
hn hp dung mi ether petroleum/aceton, t l 3/2. S dng khong 1.800ml hn hp,
mi phn on thu 20ml. Azadirachtin c trong phn on t 16 n 38. Gom dch v tin
hnh c quay chn khng, thu c 0,8432g azadirachtin.
2. Phn tch azadirachtin trn ph IR v HPLC
Phn tch trn ph HPLC bng cch cho chy sc k tr n ct Hypercil BDS
C18 vi pha ng MeOH/H
2
O, detector o 217nm vi tc dng 0,6ml/ pht.
Thi gian lu l 1,274 pht. sch ca azadirachtin t gn 95% (Hnh 1).
Ph IR: VKBr max (cm-1):
+ Azadirachtin chun: 3442(OH), 2958(CH
2
,CH
3
), 1738(ester), 1439,1377,1269
(acetat), 1046(vng furan) - (Hnh 2).
+ Azadirachtin chit: 3454(OH), 2958(CH
2
,CH
3
), 1739(ester), 1441,1377,1268
(acetat), 1046(furan) - (Hnh 3).
Qua phn tch ph IR c th kt lun cht chit t h t xoan chu hn l azadirachtin
vi hm lng 0,32% trong ht tch v.
3. nh lng azadirachtin trn HPLC
My sc k lng hiu sut cao (HPLC): Hewlett Packard 1090, series1 -liquid
chromatography.
Hnh 1. Xc nh tinh sch azadirachtin tr n HPLC
Nha Hypercil DBS-C
18,
5 m, kch thc ct 4mm x125mm
Lng mu bm vo: 0,5 l.
Detector: = 217nm.
Tc dng: 0,5ml/ pht
Dung mi chy: MeOH/H
2
O - 80/ 20.
Dng ng chun 5 im vi cht chun azadirachtin ca h ng Sigma (loi 0,5
mg/l), nhn c hng s tng quan: R = 0,99601.
Hnh 2. Sc k ph hng ngoi ca
azadirachtin chun (hng Sigma)
Hnh 3. Sc k ph hng ngoi ca
azadirachtin chit xut t nhn ht
xoan chu hn (Azadirachta indica)
trng ti Ninh Thun, VN
4. Quy trnh chit nhanh mu phn tch azadirachtin tr n HPLC
Mu c chit nhanh bng ethanol hoc methanol theo cc bc sau: 20g nhn ht
xoan chu hn c nghin nh trn trong 200ml ethanol hoc methanol, lc 30 pht
trn my lc, sau lc qua ht chn khng, b c chit tip 5 ln vi lng dung mi
nh trn. Cc phn dch chit c gom li, em c quay chn khng cho n kh kit.
Cn c loi ht m vi n-hexan bng phu chit, khong t 3 n 5 ln, mi ln vi
100ml n-hexan v c tip trn bp cch thy. Cn trng l ng v ha cn vi methanol, lc
qua lc 0,45 m. Dch lc c dng nh lng azadirachtin trn HPLC.
Hm lng azadirachtin ca mu c tch vi ethanol l 868,12mg/kg
Hm lng azadirachtin ca mu c tch vi methanol l 969,8mg/kg
Phng php tch nhanh azadirachtin b ng methanol cho kt qu cao hn 10,5% so
vi tch bng ethanol l ph hp vi cc kt qu nghi n cu cng b nc ngoi.
Methanol c cho l dung mi chit xut azadirachtin tt nht. Hm lng
azadirachtin c trong nhn ht xoan chu hn trng Ninh Thun c n thp, c th l do
ht xoan chu hn cn non, khong 4 nm tui. Theo kt qu nghi n cu ca Vin
nghin cu thc vt n , tui khai thc ht nn bt u t cy 5 nm tui tr ln
t c nng xut v cht lng n nh. Thi gian bo qun mu cng c cho l
mt yu t c nh hng n hm lng azadirachtin c trong nhn ht xoan chu hn.
KT LUN
1. chit tch v tinh sch azadirachtin t nhn ht xoan chu hn (azadir achta indica),
thu c azadirachtin vi hiu sut chit xut l 0,32% v tinh sch l 95%.
2. hon chnh phng php xc nh hm lng azadirachtin trn h sc k lng
hiu sut cao (HPLC).
TI LIU THAM KHO
1. Dng Anh Tun v cs (2001). Azadirachtin - hot cht gy ngn n mnh i vi
su khoang c phn lp t ht neem (azadirachta indica h meliaceae) di thc
vo Vit Nam, Tuyn tp hi ngh khoa hc v cng ngh ho hu c ton quc
ln th 2.
2. Nguyn Tin Thng v cs (2003). Nghin cu v pht trin cy xoan chu hn
(Azadirachta indica) t i Vit Nam, Bo co nghim thu t i nghin cu khoa
hc cng ngh cp Nh nc theo ngh nh th.
3. Dennis Dearth IR (1992). Neem, a tree for solving global problems. National
Academy press, Washington, D.c..
4. Gunasena HPM, B. Marambe (1998). Neem in Srilanka, a monograph. A
Publication of University of Peradeniya, Oxford Forestry Institute(UK) Forestry
Research Link.
5. Otmar Schaaf, Andrew P. Jarvis, S, Andrew van de Esch, Germina Giagnacovo and
Neil J. Oldham (2000). Rapid and sensitive analysis of azadirachtin and related
triterpenoids from neem (Azadirachta indica) by high - performance liquid
chromatography - atmospheric pressure chemical ionization mass spectrometry,
Journal of Chromatography A, vol 886(1-2), pp 89-97.
SUMMARY
Extraction, purification and determination azadirachtin
,
s
content in neem seed kernel (Azadirachta indica A. Juss).
Vu Van Do, Nguyen Tien Thang
Institute of Tropical Biology
The extraction and purification of azadir achtin from 3 years olds neem seed kernel
planted at Ninh Thuan pr ovince consists of two steps. The first one was the crude
extraction azadirachtin with methanol and isolation lipid by n -hexan. The residue was
concentrated with rotary vacuum evaporator and purified on flash column
chromatography. The results on IR and high performace liquid chromatography (HPLC)
showed that the compound received was azadirachtin with 95% purity. Azadirachtin
contents was determinded on Hypercil BDS - C18, 5 m, 125 x 4mm, column, reversed
phase, eluting with MeOH/ H
2
O, flow rate: 0,5ml/minute, detected at = 217nm. The
results received as follow: Azadirachtin contents extracted with ethanol and methanol
were: 868,12 ppm and 969,8 ppm, respectively.
NGHIN CU NH HNG CA Oligoglucosamine N
SINH TRNG PHT TRIN CA CY LC
(Arachis hypogea L.).
Nguyn Anh Dng, H Ty Nguyn
Nguyn Tin Thng, Vin Sinh hc Nhit i
M U
Oligoglucosamine l mt oligomer ca -1,4-glucosamine c ch to t nguyn
liu v tm ph thi. Cc nghi n cu cng b gn y cho thy oligoglucosamine l
cht c hot tnh sinh hc rt cao, l nhm kch thch sinh trng thc vt th h mi.
Hadwiger (2002) chng minh oligoglucosamine l tc nhn hot ha promoter ca
hn 20 gen lin quan n tnh khng bnh thc vt (pathogenesis -related genes) nh
ARNase, chitinase, -glucanase v nhiu enzyme lin quan n vic tng cng tng
hp phytoalexin, lignin, v qu trnh trao i cht[1]. Nhiu kt qu thc nghim c ho
thy oligoglucosamine v chitosan c kh nng khng cc loi nm gy bnh cho thc
vt nh Pythium, Slerotium, Fusarium, [2, 3, 4]. Suwalee (2002) cng khng nh khi
phun chitosan c tc dng khng bnh chy l ng (Downy mildew) tt h n cc loi
thuc khng nm trn th trng [5].
Ngoi tng cng kh nng khng bnh, oligoglucosamine cn c hi u ng kch
thch sinh trng, tng cng quang hp ca la, u lc [6,7]. Hirano (1996) khi x l
c ging khoai ty vi oligoglucosamine lm tng nng sut t 30-50% [8]. L
Quang Lun, Nawasawa, Kume (2002) c ng khng nh chitosan chiu x l m tng
chiu di r, kch thch sinh trng thc vt trong nui cy m t b o [9]. Trong cc th
nghim ca chng ti trn ng rung cng cho thy oligoglucosamine lm tng
nng sut ca ci xanh, su ho ln 20-25%, khng bnh g st cho u t ng, tng s
lng nt sn v lm tng nng sut 36,9% [10,11].
Bi bo ny l nhng kt qu th nghim ch phm oligolucosamine tr n cy trng
do nhm chng ti ch to bng cng ngh enzyme.
VT LIU V PHNG PHP
Vt liu ha cht: - Oligoglucosamine (> 8 dp) c ch to t nguyn liu v tm ph
thi bng cng ngh enzyme theo qui tr nh ca Nguyn Anh Dng v Nguyn Tin
Thng [10]. Lc trong th nghim l ging lc S a phng, c trng kh ph bin
c thi gian sinh trng l 90 ngy. Th nghim tin hnh ti Tri thc nghim Nng
lm nghip, i hc Ty Nguyn trn t bazan c ph trung bnh.
Phng php b tr th nghim:
Th nghim 1: Nghin cu nh hng ca nng Oligoglucosamine n sinh
trng v pht trin ca cy lc - Th nghim thc hin vi 5 cng thc nng l
0ppm, 20ppm, 30ppm, 40ppm v 50ppm, vi 3 ln lp li, gn 15 t h nghim,
din tch mi l 9m
2
.
Th nghim 1: So snh hiu qu ca vic phun oligoglucosamine vi cc sn phm
Sng Gianh v Komix - Th nghim c b tr theo phng php khi ngu nhin
y gm 4 cng thc: i chng (phun n c l), Sng Gianh, Komix v
Oligoglucosamine, 3 l n lp li, din tch mi th nghim l 9m
2
. Nng phun
ca Sng Gianh v Komix theo nh hng dn in trn bao b, nng ca
oligoglucosamine l 40ppm.
Cc ch tiu theo di: - Tc tng trng (cm/ngy), s cnh hu hiu, hm lng
dip lc a-b, nng sut thc thu, s lng nt sn. Cc ch tiu theo di theo 5 im
cho gc, mi im o m 5 cy.
Hm lng dip lc trong l c phn tch theo phng php quang ph [12].
Phng php x l s liu: S liu c phn tch theo phn mm Excel 7.0 t m
ra s khc bit c ngha thng k gia cc nghim thc.
KT QU V THO LUN
1. nh hng ca ch phm Oligoglucosamine n h m lng dip lc trong l lc
Sau khi phun ln l l n th 3, chng ti tin hnh ly l v phn tch hm lng
dip lc trong l lc. Kt qu ghi nhn bng 1 ch o thy oligoglucosamine lm tng
hm lng dip lc trong l lc t 16,59-32,04% so vi i chng, nng l m gia tng
hm lng dip lc cao nht l 30ppm. Hm lng dip lc gia tng l c s tng
cng , hiu sut quang hp ca cy, t l m gia tng sinh khi v nng sut.
Bng 1: nh hng ca nng oligoglucosamine n h m lng dip lc trong l lc.
Cng thc
Ch tiu
0 ppm 20 ppm 30 ppm 40 ppm 50 ppm
Chlorophyll a (mg/g l ti) 3,325 4,164 4,429 3,950 3,866
Chlorophyll b (mg/g l t i) 1,032 1,257 1,324 1,230 1,214
Tng Chlorophyll (mg/g l) 4,357 5,421 5,753 5,180 5,080
% Gia tng 0,00 24,42 32,04 18,89 16,59
2. nh hng ca ch phm oligoglucosamine n s l ng nt sn ca lc
Nt sn l s cng sinh gia vi khun c nh N v cy h u. Theo nhiu kt qu
nghin cu gn y bng N-15 t nhin chng minh vi khun Rhizobium c kh
nng cung cp t 46-60% N cho cy lc v u tng, tng ng t 106-205kg N/ha
[13,14]. V vy, nt sn c vai tr rt ln i vi sinh trng, pht trin ca cy h u.
Kt qu v nh hng ca ch phm oligoglucosamine n s lng nt sn ca lc
c ghi nhn trong bng 2.
Bng 2: nh hng ca nng glucosamine n s l ng nt sn ca cy lc.
Cng thc
LLL
I 177 189 229 354 322
II 180 237 160 187 352
III 259 158 203 384 339
Trung bnh (nt sn/cy) 205,33 194,66 197,33 308,33 337,66
Kt qu bng 2 cho thy nng 40 -50ppm lm gia tng s lng nt sn
ca lc t 50,2-64,4% so vi i chng. Ring cc th nghi m phun nng thp t
20-30ppm th khng hi u qu. Qua x l thng k th s khc bit v s l ng nt sn
gia cc cng thc phun oligoglucosamine v i chng l c ngha vi xc sut
P=0.05 (Ft = 4,30 > Fb=3,47). Trong nghin cu c bn, Carlson R. W (1994), Madigan
(2000) khng nh cc phn t oligoglucosamine l tn hiu ho hc hot ho gen Nod
iu ho qu trnh hnh thnh nt sn cy h u [15]. Kt qu ca chng ti tr n cy
u tng cng tng t. Khi x l ht ging vi olicoglucosamine lm tng gp i
lng nt sn so vi i chng [11].
3. nh hng ca ch phm Oligoglucosamine n sinh tr ng ca lc
Cc th nghim c phun oligoglucosamine vi nng t 0 -50ppm, vi 4
ln phun, cch nhau 10 ngy. Kt qu v sinh trng chiu cao cy ca lc cc th
nghim c trnh by trong bng 3.
Bng 3: nh hng ca nng oligoglucosamine n sinh tr ng ca lc.
Cng thc
Ch tiu quan trc
Tc tng trng (cm/ngy) 0,630 0,709 0,715 0,808 0,739
Sinh khi (g kh/cy) 72,89 76,99 95,49 97,21 82,13
S cnh hu hiu/cy 7,68 8,20 8,30 8,20 7,71
Kt qu bng 3 cho thy oligoglucosamine c tc dng r rt n sinh trng ca
lc. Tt c cc th nghim phun oligoglucos amine u cho sinh trng mnh hn so
vi i chng. c bit nng 40ppm c nh hng r rt nht, tc tng trng
l 0,808cm/ngy so vi 0,630cm/ngy i chng phun nc l. S khc bit v sinh
trng chiu cao cy lc cc nng l c ngha thng k vi xc sut l P=0,05.
V sinh khi, kt qu bng 1 cng cho thy oligoglucosamine c tc dng kch
thch sinh trng, lm tng sinh khi ca lc t 5,6-33,4% so vi i chng. Trong th
nghim ca Nagasawa, Nguyn Quc Hin (2000) khi b s ung chitosan chiu x vo
mi trng thy canh lm tng sinh khi ca la, lc ln 40-60% [6,7].
Nh vy qua nh hng r rt ca oligoglucosamine n hm lng dip lc, s lng
nt sn l hai ngun cung cp dinh dng C v N ch yu cho cy th vic kch thch sinh
trng ca ch phm oligoglucosamine i vi lc l iu d hiu. Cc kt qu ny cng
c chng ti khng nh trn cy rau ci, su ho, u tng, ng [10,11]
4. nh hng ca ch phm Oligoglucosamine n nng sut ca lc
Ch phm oligogl ucosamine nh hng r nt n hm lng dip lc, s
lng nt sn do nh hng n sinh tr ng, tch lu sinh khi, s c nh hu
hiu, v vy h qu dy chuyn ko theo l vic phun ch phm lm gia t ng
ng k nng sut lc nh bng 4. Kt qu cho thy ch phm oligoglucosamine
lm tng nng sut t 19,34-40,65% so vi i chng. S khc bit n y c ngha
thng k (xc sut P=0,05) Ft=5,07 > Fb=3,47. Nng cho nng sut cao nht l
40 ppm, khi gia tng n 50 ppm th s gia tng nng sut l i gim dn cn
24,75%. Kt qu ca chng ti tr n cy u tng cng lm gia tng nng sut ti
36,9 % [11].
Bng 4: nh hng ca ch phm Oligoglucosamine n nng sut lc
Cng thc
LLL
0ppm 20 ppm 30 ppm 40 ppm 50 ppm
I 3,31 3,64 3,42 4,14 4,08
II 2,81 3,53 4,40 4,10 3,54
III 3,05 3,85 3,41 4,64 3,80
Trung bnh (kg/ TN) 3,05 3,64 3,74 4,29 3,81
% Gia tng 0,00 19,34 22,62 40,65 24,75
5. So snh hiu qu ca Oligoglucosamine v cc ch phm phn bn l
nh gi hiu qu ca ch phm, ch ng ti tin hnh th nghim so snh vi cc
ch phm thng dng trn th trng l Sng Gianh v Komix. Kt qu bng 5 cho
thy ch phm oligoglucosamine cho hiu qu r rt, tng 47,98% trong khi Komix
v Sng Gianh ch gia tng nng sut t 2,82-6,45%. S gia tng nng sut ca ch
phm oligoglucosamine l hon ton c ngha (P=0,001), Ft=14,06 > Fb=4,06. Trong
khi gia tng nng sut ca 2 ch phm Komix v Sng Giang vi i chng l khng
c ngha thng k (NS).
Bng 5: So snh hiu qu ca ch phm oligoglucosamine vi cc ch phm bn l.
Cng thc
LLL
i chng Komix Sng Gianh Oligoglucosamine
I 2,06 2,42 2,55 3,67
II 2,12 2,53 2,60 3,32
III 3,27 2,97 2,51 4,01
Trung bnh 2.48 2.64 2.55 3,67
% Gia tng 0,00 4,65 2,82 47,89
Hnh: nh hng ca oligoglucosamine n sinh tr ng v pht trin ca lc.
(t tri qua phi: 0 ppm, 20 ppm, 30 ppm, 40 ppm, 50 ppm).
KT LUN
1. Ch phm oligoglucosamine c nh h ng tch cc n hm lng dip lc, s
lng nt sn ca lc. Ch phm c tc dng kch thch sinh trng, gia tng tch
lu sinh khi v lm tng nng sut t 19,34-40,65% so vi i chng.
2. Nng thch hp ca ch phm vi cy lc l 40ppm. Ch phm oligoglucosamine
hiu qu hn hn cc ch phm phn bn l l Komix v Sng Gianh.
TI LIU THAM KHO
1. Hadwiger, Klosterman, S. J and Choi, J. J (2002). Advances in Chitin Sience, Vol.
V, pp. 452-457.
2. Kendra, D. F., Hadwiger, L. A. (1984). Experimental Mycology 8, 276 -281.
3. Flach, J., Jolles, P., and Pilet, P. E. (1993). Physiologi a Plantarum, 89: 399-404.
4. Ghaouth, A. E., Arul, J.; Grenier, J., Benhamou, N. (1994). Phytopathology, 84, 3, 313.
5. Suwalee Chandrkrachang, (2002). Advances in Chitin Sience, Vol.(V): 458-462.
6. Hien, N. Q., Nagasawa , N. (2000). Radiation Physics and Chemistr y (59): 97-101.
7. Tham, L. X., Nagasawa, N. (2001). Radiation Physics and Chemistry (61): 171-175.
8. Hirano, S. (1996). Biotechnology Annual Review, (2): 237-258.
9. Luan, L. Q, Nawasawa (2002). Advances in Chitin Sience, Vol.(V): 468-474.
10. Dzung, N. A and Thang (2001). Proceedings of Scientific Conference of Tropical
Biological Institute 1999-2000, Agriculture publisher, HCMC, Viet nam, p. 162-169.
11. Dzung, N. A, Thang, N. T (2002). Advances in Chitin Sience, Vol. V, 463 -467.
12. Yoshida, S., Forno, D (1976). Laboratory manual for Physiological studies of rice,
IRRI, Philipin, pp. 43-45.
13. T.Y. Thao, P. Lieu (2003). K yu Hi ngh Cng ngh Ht nhn ton quc ln th
5, Tp. HCM 27-28/4/2003.
14. T. T. L.Hoa, T. Y.Thao, Herridge, D. (2003). K yu Hi ngh Cng ngh Ht nhn
ton quc ln th 5, Tp. HCM 27-28/4/2003.
15. Carlson, R. W., Prince, N. J. P. (1994). Mol. Plant Microb. Interact, 7, 684.
SUMMARY
Study on effects of Oligoglucosamine
on the growth and development of peanut ( Arachis hypogea L.)
Nguyen Anh Dung(1), Nguyn Tien Thang(2)
(1)Western Highland University, (2)Institute of Tropical Biology
Oligoglucosamine was prepared by enzyme degrading chitosan with averag e degree
of polymer being of 8 - 16 monomers. The effects of oligoglucosamine on chlorophyll
contents, amount of nitrogen fixing nods of peanut were investigated. The results show
that oligoglucosamine induces and enhances dramatically chlorophyll content, amount
of nitrogen fixing nods. The results also show that oligoglucosamine promotes the
growth of peanut and increases from 19,34 to 40,65% of crop yield compared with the
control. The concentration of oligoglucosamine is 40 ppm being optimal for the growth
and development of peanut.
KHO ST HOT TNH C CH SINH TRNG MT
S LOI NM GY BNG CY CA SN PHM CHIT
XUT T NHN HT XOAN CHU HN
(Azadirachta indica A. Juss) TRNG TI VIT NAM
V ng Khnh, V Vn , Nguyn Tin Thng
M U
Vn gy nhim v tc dng gy c ca thuc bo v thc vt ha hc tng
hp i vi mi tr ng sng thc y vic nghin cu s dng cc thuc bo v
thc vt c ngun gc t thc vt [5, 7, 8]. Xoan chu hn (Azadirachta indica
A.Juss) l loi cy c ngun gc n , c du nhp vo nc ta cch y khng
lu v hin ang c trng trn din tch kh ln ti cc tnh Ninh Thun, Bnh
Thun [1,3,4]. Bn cnh hot tnh xua ui, to cm gic ngn n v gy cht i vi
nhiu loi cn trng gy hi ma mng, cc sn phm chit xut t xoan chu hn
cn c ch s sinh tr ng ca mt s loi nm gy bnh cy [6-12 ]. Mc ch ca
nghin cu ny l xc nh hiu qu c ch ca sn phm chit xut t nhn ht
xoan chu hn trng ti Vit Nam i vi 3 lo i nm gy bnh cy: Fusarium
oxysporum; Sclerotium rolfsii; Rhizoctonia solani.
VT LIU V PHNG PHP
1. Nm gy bnh cy [2]
Nm Fusarium oxysporum gy bnh ho v ng khoai ty; nm Sclerotium rolfsii
gy bnh ho r gc mc trng u phng; nm Rhizoctonia solani gy bnh l c r
bng, u, da chut. Cc loi nm ny c duy tr trn mi trng PGA.
2. Chit xut hot cht t nhn ht xoan chu hn [5,7]
Ht xoan chu hn c thu hi t cc cy xoan chu (XCH) hn 4 tui Ninh
Thun, c sy kh trong 2 gi 60
o
C. Tch v ht thu nhn ht, ra sch nhn ht
trong nc ct v trng v sy kh 60
o
C trong 24 gi. Sau khi sy kh, nhn ht c
nghin thnh bt iu kin v trng.
Tin hnh chit 50g bt nhn ht xoan chu hn trong 250ml n c ct v trng
bng phng php khuy ngm kit, lc dch chit th, loi du bng petrolium ether,
sau ng kh dch chit th thu c 4,6g sn phm chit trong nc. Tng t tin
hnh chit 50g bt nhn ht xoan chu hn trong 250ml dung mi ethanol bng phng
php khuy ngm kit, lc dch chit th, loi du bng petrolium ether, sau c quay
chn khng nhit 60
o
C loi ht dung mi thu c 5,1g cao chit trong ethanol.
Tng t nhn c 4,9g cao chit trong methanol t 50g nhn ht xoan chu hn.
Cc sn phm chit ni trn c bo qun v trng nhit 4
o
C.
3. Kho st tc dng ca sn phm chit xut ln s sinh trng ca nm [5,6,8]
S dng mi trng PGA nui cy cc loi nm gy bnh cy trn cc a petri
v trng. Cc sn phm chit xut th t nhn ht xoan chu hn c ha tan trong
nc ct v trng v b sung vo mi trng nui cy cha ng ( hp kh trng,
ngui nhit 45
o
C) t nng mong mun l 0ppm (i chng); 250ppm; 500
ppm; 1000ppm; 2000ppm; 4000ppm. Rt t ng 15ml mi trng vo cc a petri v
trng v ng. Dng nt khoan c ng knh l 5mm, c l gia cc m t nm
sinh trng trn mi trng PGA trong cc a petri (ging gc), em cy v o gia
a petri (c mi trng v cht cn xc nh hot tnh) v chng sinh trng. Th
nghim c lp li 3 ln. Theo di v ghi nhn tc sinh trng dng ta trn ca
cc loi nm theo ng knh (mm) thi im t nm l i chng mc ta ht a
thch. Cc d liu c phn tch bng phn mm thng k Statgraphics 7.0. T cc
gi tr sinh trng trung bnh c th tnh ton gi tr % c ch so vi i chng theo
cng thc:
% c ch sinh trng =
Trong :
DK1: ng knh t nm trung bnh l i chng.
DK1: ng knh t nm trung bnh l th nghim.
KT QU V THO LUN
Cc s liu th v ng knh t nm ghi nhn c cc l th nghim c x l
thng k bng phn mm Stagraphics 7.0, ly gi tr trung b nh v ng knh t nm (mm)
vi 3 ln lp li mi nng ? lch chun (SE). Sau tnh % c ch t nm theo
cng thc mc I. 3. Kt qu c trnh by cc bng 1, 2 v 3 theo tng loi nm.
Bng 1. ng knh t nm v % c ch sinh trng nm Rhizoctonia solani khi x l
bi cc sn phm chit xut t nhn ht xoan chu hn
Loi sn phm i chng 250 ppm 500 ppm 1000 ppm 2000 ppm 4000 ppm
knh t
nm (mm)
87,0 0,3 86,0 0,6 85,5 0,3 76,7 1,2 46,5 0.9 24,5 0,5 Nhn XCH
trong nc
% c ch 1,2 1,7 11,9 46,6 71,8
knh t
nm (mm)
87,0 0,3 73,7 0,9 67,3 1,8 57,8 1,1 25,2 0,7 13,5 0,9 Nhn XCH
trong
ethanol
% c ch 15,3 22,6 33,5 71,1 84,5
knh t
nm (mm)
87,0 0,3 84,8 0,4 81,2 0,6 69,8 1,0 44,5 3,3 36,3 1,2 Nhn XCH
trong
methanol
% c ch 2,5 6,7 19,7 48,9 58,2
Sn phm chit xut t nhn ht XCH trong ethanol, nng 250ppm c tc
ng c ch nm Rhizoctonia solani so vi i c hng. Dch chit nhn ht XCH trong
methanol nng 500ppm v dch chit nhn ht XCH trong nc nng 1000
ppm c tc ng c ch nm ni trn. Nng cng cao th tc ng c ch cng r rt.
Bng 2. ng knh t nm v % c ch sinh trng nm Sclerotium rolfsii khi x l
bi cc sn phm chit xut t nhn ht xoan chu hn
Loi sn phm Loi sn
phm
Nhn XCH
trong nc
86,8 0,4
85,0 0,8 75,8 0,7 73,0 0,8 65,5 0,9 45,0 0,8
Nhn XCH
trong nc
2,1 12,7 15,9 24,6 48,2
Nhn XCH
trong
ethanol
86,8 0,4
73,2 0,4 58,5 0,3 43,0 0,8 39,3 0,6 34,7 0,7
Nhn XCH
trong
ethanol
15,7 32,6 50,5 55,1 60,1
Nhn XCH
trong
methanol
86,8 0,4
83,5 0,5 75,5 0,8 72,2 0,4 56,2 0,6 39,7 0,9
Nhn XCH
trong
methanol
3,8 13,1 16,9 35,3 54,3
Sn phm chit xut t nhn ht XCH trong ethanol v methanol nng 250
ppm th hin tc ng c ch nm Sclerotium rolfsii so vi i chng. Dch chit
nhn ht XCH trong nc bt u c tc ng c ch nng 500 ppm. Nng
cng cao, tc ng c ch nm cng r nt.
Sn phm chit xut t nhn ht XCH trong ethanol v methanol nng 250
ppm th hin tc ng c ch nm Fusarium oxysporum so vi i chng. Dch chit
nhn ht XCH trong nc bt u c tc ng c ch nng 500 ppm. Nng
cng cao, tc ng c ch nm cng r rt.
Bng 3. ng knh t nm v % c ch sinh trng nm Fusarium oxysporum khi x
l bi cc sn phm chit xut t nhn ht xoan chu
Loi sn phm i
chng
knh t
nm (mm)
87,3
0,3
86,2 0,6 76,2
0,4
63,3 0,6 54,8 0,4 42,2 1,2
Nhn XCH
trong nc
% c ch
1,3 12,8 27,5 37,2 51,7
knh t
nm (mm)
86,8
0,2
77,3 0,9 73,8
0,2
62,3 0,6 54,5 0,9 40,8 0,8
Nhn XCH
trong
ethanol
% c ch
10,9 14,9 28,2 37,2 52,9
knh t
nm (mm)
86,8
0,2
65,8 1,0 62,2
0,6
56,3 0,7 52,7 0,6 46,7 0,6
Nhn XCH
trong
methanol
% c ch
24,2 28,4 35,1 39,3 46,2
oi tuong nam
Loi
sn
phm
Rhizoctonia solani Sclerotium rolfsii Fusarium oxysporum
Nhn
ht
XCH
trong
nc
Nhn
ht
XCH
trong
ethanol
Nhn
ht
XCH
trong
methan
ol
KT LUN
1. nhn c 3 dng sn phm th loi du t nhn ht XCH: sn phm chit
xut trong nc, ethanol v methanol.
2. Cc sn phm chit xut t nhn ht XCH u c tc dng c ch sinh tr ng i
vi 3 loi nm gy bnh cy l Rhizoctonia solani, Sclerotium rolfsii v Fusarium
oxysporum. Trong , sn phm chit xut t nhn ht XCH trong ethanol c tc
ng c ch tt nht, tip theo l sn phm chit xut trong methanol v cui cng
l sn phm chit trong nc.
3. Cn tip tc nghin cu tc ng c ch ca cc ch phm chit xut t nhn ht XCH
trn nhng i tng nm gy bnh cy khc, cng cn tin hnh nghin cu phi ch
cc sn phm trn vi cc cht nh ha, cht chng oxy ha, cht bo qun, v.v... tng
cng hot tnh v thi gian bo qun nhng sn phm chit xut ny.
TI LIU THAM KHO
1. Lm Cng nh (1991). Gii thiu cy xoan ch u hn (Azadirachta indica A.Juss)
nhp ni vo vng ct nng hn Phan Thit - Tuy Phong. S Nng - Lm nghip
Thun Hi.
2. V Triu Mn, L Lng T (1998). Gio trnh bnh cy nng nghip. NXB Nng
nghip
3. Nguyn Tin Thng, V Vn , Th Tuyn, L Th Thanh Phng, V ng
Khnh (2003). Xy dng quy trnh chit xut hot cht sinh hc t ht neem
(Azadirachta indica A. Juss) trng ti Vit Nam v kho st nh hng ca dch
chit ln s sinh trng ca vi nm gy bnh thc vt ( Fusarium oxysporum,
Alternaria sp.) v Ngi go (Corcyra cephalonica St.). Bo co ti cp c s -
Vin Sinh hc Nhit i.
4. Nguyn Tin Thng v nhng ngi khc (2003). Nghi n cu v s dng cy xoan
chu hn (Azadirachta indica A. Juss) trng ti Vit Nam. Bo co nghim thu
ti cp Nh nc theo Ngh nh th.
5. Nguyn Th Phng Tho, Nguyn Th Qunh, Nguyn Tin Thng, Akiko Hirano.
Bc u nghin cu nh hng ca du xoan chu hn (Neem) l n s k sinh ca
b h (Cylas formicarius L.) trng thnh trong c khoai lang (Ipomoea batatas
L.). Tuyn tp cng trnh nghin cu khoa hc cng ngh, 1999 - 2000, Vin Sinh
hc Nhit i.
6. Amadioha, A. C. (2000). Controlling rice blast in vitro and in vivo with axtracts of
Azadirachta indica. Crop protection 19, 287-290.
7. 7.Amadioha, A. C. (2002). Fungitoxic effects of extracts of Azadirachta indica
against Cochlibolus miyabeanus causing brown spot disease of rice.
Arch.Phytopath.Pflanz., Vol.35, pp. 37 -42.
8. Coventry, E. & Allan, E. J. (2001). Microbiological and chemical analysis of n eem
(Azadirachta indica) extracts: New data on antimicrobial activity. Phytoparasitica 29:5.
9. Govindachari, T. R., Suresh, G., Gopalakrishan Geetha, Banumathy, B. &
Masilaman, S. (1998). Identification of antifungal compounds from seed oil of
Azadirachta indica. Phytoparasitica 26:2.
10. Narasimhan et al. (1998). Efficacy of neem EC formulation of neem oil and
pungam oil for the management of sheath rot disease of rice. Phytoparasitica 26(4):
301:306.
11. Rajappan etal. (2001). Management of grain discoloration of r ice with solvent - free
EC formulation of neem and pungam oils. Phytoparasitica 29:2.
12. Suresh, G. Narasimhan, N. S., Masilaman, S., Partho, P. D. & Gopalakrishnan
Geetha (1997). Antifungal fractions and compounds from uncrushed leveas of
Azadirachta indica. Phytoparasitica 25:1.
SUMMARY
Antifungal activity of neem seed kernel extracts from neem tree
(Azadirachta indica A.Juss) grown in vietnam on some plant
pathogens
Vu ang Khanh, Vu Van Do, Nguyen Tien Thang
Three extracts: water extract, ethanolic extract and methanolic extract of deoiled
neem seed kernel (NSK) powder at 250 -4000 ppm concentrations were evaluated as
antifungal agents to plant pathogens such as Rhizoctonia solani, Sclerotium rolfsii,
Fusarium oxysporum. All extracts significantly reduced the in vitro radial growth of
these fungi at? 2000 ppm concentrations. Ethanolic extract of deoiled NSK powder
exhibited the best control of these pathogens followed by methanolic extract and
water extract. So, we should have some researches further to confirm the results in
the field test.
KHO ST TNH N NH CA HOT CHT TRONG
DU HT XOAN CHU HN (Azadirachta indica A. Juss)
THU C BNG PHNG PHP P NGUI
Dip Qunh Nh, Nguyn Tin Thng, L th Thanh Phng
M U
Vit Nam, cy neem cn c tn gi khc l xoan chu hn, c du nhp t
kh lu v trng trn din tch kh ln ti 2 tnh Bnh Thun v Ninh Thun [8]. Kh
nng phng tr nhiu loi dch hi, c bit l khng cn trng, ca cy neem
(Azadirachta indica A. Juss) c bit n l do tc ng phi hp ca mt s hot cht
thuc nhm triterpenoid, l azadirachtin, nimbin, salannin. Cc ho t cht ny c nhiu
trong nhn ht, v th sn phm hot cht chit xut t nhn ht neem l nguyn liu y
trin vng sn xut thuc tr su tho mc [2]. Hot cht chit xut t thc vt ni
chung v t nhn ht neem ni ri ng thng khng bn, d b phn hy v mt hot tnh
di tc ng ca tia UV, s c mt ca oxy, nhit cao, pH thp, Du thc vt ni
chung v du t nhn ht neem ni ri ng c tnh cn quang v chng oxy ha tt, v do
vy, c kh nng lm cht mang n nh hot cht chit xut t thc vt [3].
Mc ch ca nghin cu l kho st tnh n nh ca 3 hot cht chnh trong du
neem thu c bng phng php p ngui v s thay i c tnh ca du neem i
vi su xanh tui 2 theo thi gian bo qun.
VT LIU V PHNG PHP
1. Vt liu
Du neem thu c bng cch p ngui nhn ht neem trng ti Ninh Thun tr n
my p chuyn dng Komet do c sn xut
Cht chun: Azadirachtin hng Sigma, M; nimbin v salannin hng Trifolio, c.
Tween 80, ethano l96
0
, petroleum ether
u trng su xanh (Heliothis armigera) tui 2 nui trn thc n bn tng hp do
Phng Cng ngh sinh hc ng vt - Vin Sinh hc Nhit i cung cp.
2. Phng php nh lng cc hot cht trong du neem bng k thut HPLC
Chun b mu phn tch: Ly 10ml du neem, loi lipid nhiu ln bng petrol ium
ether, ha tan mu trong 10ml ethanol, tip tc loi b cn bng cch bm qua
mng lc c kch thc l = 0,45m v tin hnh phn tch mu trn thit b
HPLC vi cc thng s sau:
- Lng mu bm vo ct: 0,5l
- Ct bo v: Bondapak
TM
C
18
, 125A
0
, 10m, 3,9 x 20mm
- Ct phn tch: Bondapak
TM
C
18
, 125A
0
, 10m, 3,9 x 300mm
- Dung mi ra ct: CH
3
CN: H
2
O (55:45); Tc dng: 0,5ml/pht.
- Detector DAD, bc sng 220nm.
Cht chun azadirachtin, nimbin, salannin c pha long 5 nng khc nhau
v phn tch ging nh mu th. Dng ng chun tng quan tuyn tnh gia din
tch pick ca tng cht chun v nng . Da vo ng chun, nng ppm ca hot
cht trong mu th c tnh bi phn mm Chemstation h ng Aligent-Technology.
3. Phng php xc nh c tnh ca du neem thng qua ch s LC
50
trn su xanh.
Nguyn tc: S dng gi tr LD
50
hoc LC
50
ca cc cht gy c tr n cng mt i
tng sinh vt so snh c tnh ca chng.
Th c tnh: du neem c nh ha bng tween 80, t l tween/du l 1/10. Sau
pha long bng nc ct to hn hp c nng du neem nh mong mun.
xc nh gi tr LC
50
, hn hp du neem c pha long thnh dy c 5 nng
khc nhau (tng ng vi 5 nghim thc), cc nng a ra u c thm d
trc m bo t l cht ca u tr ng su xanh tui 2 sau 5 ngy nm trong khong
16-84% gip cho vic xc nh gi tr LC
50
sau 5 ngy c tin cy cao [6].
Tin hnh th nghim: Th nghim c b tr theo kiu hon ton ngu nhin, n
yu t, lp li 3 ln mi nghim thc. Mi c s gm 20 u trng su xanh tui
2 c nui ring trong cc l nh. Thc n c qut vo l, hn hp th nghim
c nng xc nh c qut ln b mt lp thc n, kh ro. Gp u tr ng su
xanh tui 2 vo l, phun nh hn hp th nghim l n thn su, y np c c l
v theo di s su cht mi ngy.
X l s liu: Tnh t l cht tch ly ca su sau 5 ngy mi nghim thc. T
tnh gi tr LC
50
sau 5 ngy ca hn hp du neem bng phng php phn tch
Probit, thao tc trn phn mm Excel [6]
KT QU V THO LUN
Hm lng azadirachtin, nimbin, salannin trong du neem thu c bng phng
php p ngui trn my p chuyn dng KOMET do c sn xut tng ng l
930,685; 262,578; 1027,478 g/ml (bng 1, hnh 1).
Bng 1: Nng hot cht trong du neem
TT Hot cht Nng (ppm)
1
2
3
Azadirachtin
Nimbin
Salannin
930,685
262,578
1027,478
Hnh 1: Sc k hot cht trong du neem
S bin thin hm lng ca 3 hot cht ca du neem bo qun trong chai mu,
nhit phng, c trnh by bng 2.
Bng 2. S bin ng hm lng hot cht v gi tr pH ca du neem theo thi gian
(%) Hot cht cn li theo thi gian bo qun
Hot cht
0 thng 1 thng 4 thng 6 thng
Azadirachtin
Nimbin
Salanin
Total
100
100
100
100
99,45
98,45
98,99
99,16
78,15
41,26
73,84
71,81
49,56
26,02
27,12
36,35
pH 5,73 5,65 4,56 3,92
Kt qu cho thy hm lng azadirachtin, nimbin, salanin kh n nh trong du
neem. Sau thng bo qun u tin hm lng ca chng hu nh khng thay i.
Trong azadirachtin c n nh trong du neem iu kin bo qun b nh thng
cao hn so vi nimbin v salannin. Chu k bn hy ca azadirachtin l 6 thng (sau 6
thng bo qun hm lng azadirachtin cn li 49,56%), ph hp vi vic sn xut
thuc tr su tho mc. Tnh n nh ca cc cht gim c ng vi thi gian bo qun ko
di. Trong qu trnh bo qun pH ca du neem gim dn t 5,73 xung c n 3,92 sau 6
thng. S gim pH nh hng n tnh n nh ca cc hot cht trong du neem.
pH cng thp hot cht cng d b phn hy. S gim pH trong du neem c th l do s
gia tng qu trnh oxy ha ha hc v sinh hc. V trong du neem c ng ha tan,
m, khong,... l ngun dinh dng thch hp cho s pht trin ca vi sinh vt [7].
Kt qu thc nghim cho thy c ng mt iu kin bo qun, azadirachtin n nh
trong du neem tt hn so vi trong methanol v nc. Chu k bn hy ca azadirachtin
trong du neem, methanol, nc tng ng l 6 thng; 50 ngy; 1,2 - 49,9 ngy [1,4].
Bn cnh vic kho st s n nh ca hot cht trong du neem, cng tin hnh kho
st gi tr LC
50
theo thi gian bo qun. Kt qu c trnh by bng 3.
Bng 3. S bin ng gi tr LC
50
ca du neem theo thi gian bo qun
Thi
gian
(thng)
PT ton hc
Y = aX + b
H s
tng quan (R)
LC
50
(%)
0
1
4
5
6
Y = 1,32 X + 3
Y = 1,72 X + 2,38
Y = 1,378 X + 2,685
Y = 1,66 X + 2,15
Y =1,50 X + 2,22
0,985
0.960
0.995
0.979
0.994
3,20
3,30
4,79
5,16
7,00
Kt qu cho thy, theo thi gian bo qun, gi tr LC
50
tng dn, cng vi s gim
dn c tnh ca du neem. Trong thng bo qun u ti n, c tnh ca du neem hu
nh khng thay i, trng hp vi s n nh hm lng hot cht, nh phn tch
trn. Nh vy s gim hm lng hot cht trong du neem nhng thng bo qun
tip theo, lm gim c tnh ca n.
KT LUN
1. Hm lng azadirachtin, nimbin, salannin trong du neem n nh trong thng bo qun
u tin, sau gim dn theo thi gian bo qun. So vi bo qun trong methanol v
nc iu kin bnh thng, th 3 hot cht ni trn trong du neem c n nh cao
hn. Chu k bn hy ca azadirachtin trong du neem ko d i ti 6 thng. S gim pH
theo thi gian bo qun lm gim hm lng hot cht trong du neem.
2. Tng hot tnh gy c ca hot cht trong du neem gim dn t thng th 2, li n
quan n s gim hm lng ca azadirachtin, nimbin, salannin. LC
50
ca du
neem i vi u trng su xanh tui 2 thay i theo thi gian bo qun trong 0, 1,
4, 5, 6 thng tng ng l: 3,2% - 3,3% - 4,79% - 5,16% - 7% .
TI LIU THAM KHO
1. Jarvis Andrew P., Johnson Shaun & Morgan E. (1998). Stability of the natural
insecticide azadirachtin in aqueous and organic solvent s. Pestc.Sci. 53, pp. 217-222.
2. Smchmutterer H., (1995). The neem tree Azadirachta indica A. Juss and other
meliaceous plants. 695p.
3. Office of Complementary Medicines, Therapeutic Goods Administration, (2002).
Evaluation of Cold-Pressed Oil from the seed kernels of Azadirachta indica A.
Juss., Meliacaea (Neem) for Use in Listable Therapeutic Goods.
4. <http://www.health.gov.au/tga/docs/pdf/cmec/cmecde27.pdf> posting November, 2002.
5. http://www.doc.govt.nz/Publications/004~Science -and-Research/Science-for-
Conservation/PDF/sfc232.pdf
6. Phm Vn Bin v cs (2000). Cm nang thuc bo v thc vt . NXB Nng nghip.
7. Nguyn Ngc King, (2000). Thng k hc ng dng Cc kiu mu th nghim.
i hc Nng Lm Tp. H Ch Minh, trang 4-134.
8. Dip Qunh Nh, (2006). Kho st thnh phn hot cht v tc ng ca du neem
(Azadirachta indica A. Juss) i vi su xanh (Heliothis armigera). Lun vn thc
s khoa hc, H Khoa hc T nhi n TPHCM, 83 trang.
9. Nguyn Tin Thng v cng s, (2003). Nghin cu v s dng cy xoan chu hn
(Azadirachta indica A. Juss) trng ti Vit Nam. Bo co nghim thu ti cp
Nh nc theo Ngh nh th.
SUMMARY
Study on stability of bioactive substances in neem
(Azadirachta indica A.Juss) seed oil produced by cold-pressing
method
Diep Quynh Nhu, Nguyen Tien Thang, Le thi Thanh Phuong
Cold-pressed oil of neem seed kernel was preserved at room temperature and
prevented from the light. Three main biosubstances of neem oil: azadirachtin, nimbin
and salannin were quantified at the time of expe rimental beginning, one month, four
months and six months later by HPLC. Toxicity of neem oil represented by LC
50
value
was screened on the 2nd instar larvae of Heliothis armigera via Probit Analysis. As a
result of test, there was no decrease in content of three above biosubstances during the
fisrt preserved month. However, significant reduction of them was determined during
following preserved months. The half-life of azadirachtin was about six months, longer
than those of nimbin and salannin. LC
50
values of neem oil at the beginning, one month,
four month and six month pres ervation were 3.2%, 3.3%, 4.79%, 5.16% and 7%,
respectively. Thus, it was supposed that these biosubstances played important role in
toxicity of neem oil agaisnt larvae of Heliothis armigera.
NGHIN CU TC NG CA neem
(Azadirachta indica A. Juss)
I VI MT S LOI CN TRNG NNG NGHI P
L Th Thanh Phng, Nguyn Tin Thng, Trn Hnh Phc,
Phng Huy Hun, Chu Tng Khanh, Trn Tun c
Vin Sinh hc Nhit i
M U
Cy neem (Azadirachta indica A. Juss) t lu c nhiu quc gia nghin cu v pht
trin do nhng u im ca n v mt kinh t v mi trng [6]. Ring ti nc ta, hng
nghn hecta neem tp trung cc tnh min Trung, c bit l Ninh Thun v Bnh Thun
ng vai tr quan trng trong vic ph xanh t trng i trc v h tr cy phi lao
phng h tt vng ven bin. Vi lng hot cht sinh hc di do v a dng tp trung
ht v l, cy neem c th tr thnh ngun thuc bo v thc vt an ton v hiu qu nu
c nghin cu v s dng hp l [1]. Trong t i ny, mt s ch phm t ht neem
c th nghim trn mt s loi cn trng nng nghip ph bin ti nc ta, c bit l
nhng loi ang khng thuc mnh nh mt bt (Tribolium castaneum), ngi go
(Corcyra cephalonica St), ry nu (Nilaparvata lugens Stal), su t (Plutella xylostella).
VT LIU V PHNG PHP
1. Vt liu
Du neem thu c t nhn ht neem bng phng php p ngui.
Dch chit bnh du: sn phm chit xut hot cht sinh hc t bnh du neem bng
ethanol.
Ch phm neem vin nn: c thnh phn chnh l bt ht nem, dng xng hi
phng tr cn trng kho.
Mt bt , ngi go, ry nu, su t c nhn nui ti Vin Sinh hc Nhit i.
2. Phng php
* Ry nu: phun du neem ln cc chu la 5 nng 0,2 - 0,5 - 1,0 - 2,0 - 3,0%. Sau
t chu la vo cc lng li c cha 50 ry nu (10 ngy tui hoc thnh trng). Hiu lc
ca cc ch phm c nh gi theo tr s LC
50
da vo t l ry cht sau th nghim. S
ry sng st vn tip tc nui dng theo di t l thnh trng v bin dng [3] [5].
* Ngi go v mt bt
Phng thc tm c thc n: Nhng thc n nhn to ca ngi go v mt bt vo
du neem v dch chit bnh du dy nng 1,5 - 3,0 - 6,0- 12,5 - 25 - 50 -100%. Th
nghim c thc hin trong cc a petri cha 10 u trng tui 3. Da vo trng lng
thc n cn trng s dng trong 14 ngy, tnh hiu lc gy ngn n ca cc ch phm [4]
Phng thc xng hi: thc hin trong cc l nha th tch 1 lt cha 300g go sch
v 30 u trng tui 3. t ch phm vi n nn ln mt go, y kn l trong 3 ngy.
Ghi nhn s u trng cht sau x l. S sng st c nui dng theo di tc
ng ca ch phm i vi s sinh tr ng, pht trin v sinh sn ca ngi go.
* Su t: Nhng cc mu l ci (ng knh 8cm) vo cc ch phm theo dy nng
1 - 2,5 - 5 - 7,5 -10%, ro thuc v t vo cc a petri. Mi a b tr 10 u tr ng
tui 3. Ghi nhn mc ti u th thc n v s lng su cht sau th nghim tnh h
s ngn n v tr s LC
50
.
KT QU V THO LUN
1. Ry nu
Kt qu cho thy du neem gy cht ry non 10 ng y tui mnh hn so vi thnh
trng, tr s LC
50
tng ng l 2,20% v 6,87%. Khi theo di qu trnh pht tri n ca s
ry 10 ngy tui cn sng nghim thc du neem 1%, ghi nhn t l ry tr ng thnh
l 59,9%; trong 29,5% r y b bin dng (Hnh 1).
a b c d e f
Hnh 1. Tc ng gy bin dng ca neem i vi ry nu
a , b : Ry nu cnh di v cnh ngn bnh thng. c, d, e, f: Cc kiu ry bin dng
Bng 1. c (LC
50
) ca du neem i vi ry nu
2. Ngi go v mt bt
* Phng thc tm c thc n
Bng 2 cho thy dy nng 12,5 - 25 - 50 - 100%, du neem gy ngn n mnh i
vi ngi go. Trong khi dch chit bnh du ch c hiu lc mnh nng 50 - 100%.
Bng 2: Tc ng gy ngn n ca du neem, dch chit bnh du i vi ngi go
Nng x l (%)
Ch phm
100,0 50,0 25,0 12,5 6,0 3,0 1,5
Du neem 188,9*** 180,6*** 174,4*** 160,3*** 146,7** 125,2** 106,8**
Dch chit BD 165,7*** 155,2*** 147,4** 130,1** 116,7** 98,3* 89,9*
Hiu lc gy ngn n: (***):mnh, (**): trung b nh, (*): yu [4]
Tui ry Phng trnh tng quan H s tng quan P LC
50
(%)
10 ngy tui Y = 3,8824 + 0,8273 X 0,9769 0,0042 2,20
Thnh trng Y = 3,8769 + 0,6103 X 0,9903 0,0011 6,87
Ngoi tc ng gy ngn n, cc ch phm c n gy cht ngi go cc mc
khc nhau. Nhn chung trong phm vi th nghim, hiu lc gy cht ca cc ch phm
gia tng theo nng x l. Kt qu phn tch Probit (Bng 3) cho thy du neem c
c tnh cao, vi gi tr LC
50
l 0,07 %. Dch chit bnh du c tc ng gy cht thp
hn vi gi tr LC
50
l 0,23%.
Bng 3. c (LC
50
) ca cc ch phm neem i vi ng i go
Cc ch phm neem c kh nng gy cht dn ng i go nhiu giai on pht trin
khc nhau. S u trng mn cm nht s cht ngay sau khi nhim thuc. S c n li c
th chuyn qua giai on nhng v v ha, nhng thnh trng thng b bin dng v
gim kh nng sinh sn. Quan st cng cho thy mt s u tr ng v nhng c biu hin
khng bnh thng (hnh 2).
a
b
c
d
e f g h
Hnh 2. Tc ng gy bin dng ca neem i vi ng i go
a, b, e: u trng, nhng, thnh trng bnh thng.
c, d: u trng, nhng d dng. f, g, h: Cc kiu bin dng ca th nh trng
* Phng thc xng hi
S dng ch phm neem vi n nn (NV) x l ngi go v mt bt theo
phng thc xng hi cho thy cc ch phm c kh nng gy cht, c ch sinh trng
v gy bin dng thnh trng.
Bng 4: Gi tr LD50 (mg/dm
3
) ca ch phm NV i vi ngi go v mt bt
Ch phm Phng trnh tng quan
H s
tng quan
P LC
50
(%)
Du neem Y = 5.6861 + 0.5999 X 0.985000926 5.24948E-05 0,07
Dch chit bnh du Y = 5,3948 + 0,6072 X 0.985180154 5.09456E-05 0,23
Ngy sau x l
i tng x l
Phng trnh
tng quan
H s
tng quan P
LD50
(mg/dm
3
)
Ngi go y = 3,8098 + 1,4838 x 0,9538 0,0118 6,34 3 ngy
Mt bt y = 3,5726 + 1,4984 x 0,9315 0,0212 8,96
Ngi go y = 4,6161 + 1,3992 x 0,9330 0,0205 1,88 7 ngy
Mt bt y = 4,5693 + 1,4878 x 0,9312 0,0213 1,95
3. Su t
Bng 5: Tc ng gy ngn n v gy cht ca ch phm neem i vi su t
Kt qu bng 5 cho thy du neem v dch chit bnh du l cht gy ngn n mnh i
vi su t vi h s ngn n tng ng l 196,2% v 173,1% (hnh 3). Hai ch phm ny
cng c kh nng gy cht su t vi tr s LC
50
tng ng l 0,7% v 0,42%.
Hnh 4 cho thy du neem v dch chit bnh du 5% gy cht 67 - 80% su v 13 -
23% nhng, kt qu c t 7% - 10% thnh trng xut hin. Trong khi , nng
10%, du neem v dch chit bnh du gy cht ton b i tng x l qua 2 giai on
su v nhng, kt qu khng xut hin thnh trng.
a b c d
e
Hnh 3. Mt s tc ng ca neem i vi su t
a, b: Tc ng gy ngn n (a l i chng, b l mu x l neem)
c, d: tc ng lm bin dng thnh trng; e: tc ng gy cht nhng
DN (5%)
7%
13%
80%
t hanh
t r ung
nhong
chet
sau chet
DN (10%)
90%
0%
10%
nhong
chet
sau chet
t hanh
t r ung
DCH (5%)
23%
67%
10%
t hanh
t r ung
nhong
chet
sau chet
DCH (10%)
16% 0%
84%
nhong
chet
t hanh
t r ung
sau chet
Hnh 4. Tc ng ca neem i vi qu tr nh pht trin ca su t
KT LUN
Kt qu th nghim cho thy cc ch phm neem tc ng l n ry nu, ngi go, mt
bt v su t theo nhiu phng thc khc nhau nh gy cht, gy ngn n, lm bin
dng; qua c th tiu dit ngay hoc c ch dn s pht trin qun th ca chng.
Loi ch phm Ch tiu
Du neem Dch chit bnh du
H s ngn n (%) 196,2 173,1
LC
50
(%) 0,42 0,61
TI LIU THAM KHO
1. Ahmed S., (1988). Potential of using neem tree ( Azadirachta indica) for pest
control and rural development. Neem Newsl. 5(4). pp. 49 - 55.
2. Gupta B. N. and Sharma K. K., (1994). Characteristics and uses of Azadirachta
indica A. Juss. In: Neem, a wonder tree. Indian Council of Forestry Research and
Education, Dehra Dun, India. pp. 1 - 7,
3. Mathur Y. K. and Nigram S., (1993). Insecticide, antifeedi ng and juvenilizing
effects of neem oil against Corcyra cephalonica St. and Epilachna
vigintioctopunctata F. In:Neem and Environment. Volume 1. World Neem
Conference, Bangalore, India, 24 - 28 Feb. 1993. (Eds. Singh R.P, Chari M.S.,
Raheja A. K. and Kraus W.). Science Publishers, Inc., USA. pp. 335 - 342.
4. Nguyn Cu Th Hng Giang, Nguyn Cng Ho v Nguyn Ngc Sng,
(1998). Hiu qu gy ngn n ca mt s cy mc Vit Nam i vi mt thc tp.
Tuyn tp cc cng trnh nghin cu khoa hc 1993-1998, Vin Sinh hc Nhit i,
Trung tm Khoa hc T nhin v Cng ngh Quc gia. NXB Nng Nghip, trang
333 - 338.
5. Saxena R. C., (1987). Neem seed derivative for management of rice insect pests - A
review of recent studies. In: Natural pesticides from the neem t ree (Azadirachta
indica A. Juss) and other tropical plants. Eschborn, pp: 81 - 93.
6. Seeni R., Nutan K., Jitendra K., Opender K. and Balraj S. P., (1993). Azadirachtin
content and Bioactivity of some neem ecotypes of India. In: Neem and
Environment. Volume 1. World Neem Conference, Bangalore, India, 24 - 28 Feb.
1993. (Eds. Singh R. P, Chari M. S., Raheja A. K. and Kraus W.). Science
Publishers, Inc., USA, pp207-217
SUMMARY
Action modes of neem ( Azadirachta indica A. Juss)
to some agricultural insects
Le thi Thanh Phuong, Nguyen Tien Thang, Tran Hanh Phuc,
Phung Huy Huan, Chu tuong Khanh, Tran Tuan Duc
Neem seed oil, neem cake extract and neem pellet were prepared and screened for
their effects on rice moth ( Corcyra cephaloni ca), red flour beetle (Tribolium
castaneum), diamondback moth (Plutella xylostella) and brown plant hopper
(Nilaparvata lugens Stal.). It was seen that all these neem-based products affected tesed
insects as antifeedant, insecticidal, metamorphosis deterri ng and fecundity inhibiting
agents. By these ways, they controlled and inhibited development of the pests safely and
effectively.

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