PHN III

CNG NGH CC CHT C HOT TNH SINH HC

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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 h a 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 b nh 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 h m 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 lm nguyn liu chit xut, tinh sch v xc nh hm lng azadirachtin. Thu hoch qu xoan chu hn tt nht khi n mi chuyn sang m u vng hay vng xan h, lc ny hm l ng azadirachtin

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cao nht. Nu qu chn rng xung t mi thu hoch th hm lng azadirachtin s b gim (3,4). Chi t xut, tinh sch azadirachtin bng sc k ct (flash chromatography) v i 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 l ng 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 200 ml 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 200 ml methanol trong 1 gi. Gp cc phn dch chit methanol v gim th tch dch thu c bng c quay trong chn khng 40oC 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 2SO4. Loi EtOAc trong chn khng 40oC, 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 h n 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 c t. 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 m u 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, v t c gi tr Rf l 0,32.

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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 1.800 ml hn hp, mi phn on thu 20ml. Azadirachtin c trong phn o n 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% (H nh 1). Ph IR: VKBr max (cm-1): + Azadirachtin chun: 3442(OH), 2958(CH 2,CH3), 1738(ester), 1439,1377,1269 (acetat), 1046(vng furan) - (Hnh 2). + Azadirachtin chit: 3454(OH), 2958(CH 2,CH3), 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-C18, 5 m, kch thc ct 4mm x125mm Lng mu bm vo: 0,5 l. Detector: = 217nm. Tc dng: 0,5ml/ pht

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Hi ngh KHOA HC V CNG NGH 2007

Dung mi chy: MeOH/H 2O - 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 lng 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 h n 10,5% so vi tch bng ethanol l ph hp vi cc kt qu nghin cu cng b nc ngoi. Methanol c cho l dung mi chit xut azadirachtin tt nht. Hm lng azadirachtin c trong nhn h t 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%.

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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. Nguyn Tin Thng v cs (2003). Nghin cu v pht trin cy xoan chu hn (Azadirachta indica) ti Vit Nam, Bo co nghim thu t i nghin cu khoa hc cng ngh cp Nh nc theo ngh nh th. Dennis Dearth IR (1992). Neem, a tree for solving global problems. National Academy press, Washington, D.c.. Gunasena HPM, B. Marambe (1998). Neem in Srilanka, a monograph. A Publication of University of Peradeniya, Oxford Forestry Institute(UK) Forestry Research Link. 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.

2.

3. 4.

5.

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 province 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 2O, 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.

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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 tr ng 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 tng, 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.

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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 th c nng l 0ppm, 20ppm, 30ppm, 40ppm v 50ppm, v i 3 ln lp li, gn 15 t h nghim, din tch mi l 9m2. Th nghim 1: So snh hiu qu c a 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 ln lp li, din tch mi th nghim l 9m2. 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 ln th 3, chng ti tin h nh 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 lm 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 Chlorophyll a (mg/g l ti) Chlorophyll b (mg/g l t i) Tng Chlorophyll (mg/g l) % Gia tng 3,325 1,032 4,357 0,00 4,164 1,257 5,421 24,42 4,429 1,324 5,753 32,04 3,950 1,230 5,180 18,89 3,866 1,214 5,080 16,59 0 ppm 20 ppm 30 ppm 40 ppm 50 ppm

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.

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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 II III Trung bnh (nt sn/cy) 177 180 259 205,33 189 237 158 194,66 229 160 203 197,33 354 187 384 308,33 322 352 339 337,66 0 ppm 20 ppm 30 ppm 40 ppm 50 ppm

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 nghim phun nng thp t 20-30ppm th khng hiu qu. Qua x l thng k th s khc bit v s lng 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) Sinh khi (g kh/cy) S cnh hu hiu/cy 0,630 72,89 7,68 0,709 76,99 8,20 0,715 95,49 8,30 0,808 97,21 8,20 0,739 82,13 7,71 0 ppm 20 ppm 30 ppm 40 ppm 50 ppm

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 40 ppm 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].

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Nh vy qua nh hng r rt ca oligoglucosamine n h m 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 oligoglucosamine nh hng r nt n hm lng dip lc, s lng nt sn do nh h ng n sinh trng, tch lu sinh khi, s c nh hu hiu, v vy h qu dy chuyn ko theo l vic phun ch phm lm gia tng ng k nng sut lc nh bng 4. Kt qu cho thy ch phm oligoglucosamine lm tng nng su t 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 su t ti 36,9 % [11].
Bng 4: nh hng ca ch phm Oligoglucosamine n nng sut lc
Cng thc LLL I II III Trung bnh (kg/ TN) % Gia tng 3,31 2,81 3,05 3,05 0,00 3,64 3,53 3,85 3,64 19,34 3,42 4,40 3,41 3,74 22,62 4,14 4,10 4,64 4,29 40,65 4,08 3,54 3,80 3,81 24,75 0ppm 20 ppm 30 ppm 40 ppm 50 ppm

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. K t 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 su t 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 II III Trung bnh % Gia tng 2,06 2,12 3,27 2.48 0,00 2,42 2,53 2,97 2.64 4,65 2,55 2,60 2,51 2.55 2,82 3,67 3,32 4,01 3,67 47,89 i chng Komix Sng Gianh Oligoglucosamine

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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. 2. 3. 4. 5. 6. 7. 8. 9. Hadwiger, Klosterman, S. J and Choi, J. J (2002). Advances in Chitin Sience, Vol. V, pp. 452-457. Kendra, D. F., Hadwiger, L. A. (1984). Experimental Mycology 8, 276 -281. Flach, J., Jolles, P., and Pilet, P. E. (1993). Physiologi a Plantarum, 89: 399-404. Ghaouth, A. E., Arul, J.; Grenier, J., Benhamou, N. (1994). Phytopathology, 84, 3, 313. Suwalee Chandrkrachang, (2002). Advances in Chitin Sience, Vol.(V): 458-462. Hien, N. Q., Nagasawa , N. (2000). Radiation Physics and Chemistr y (59): 97-101. Tham, L. X., Nagasawa, N. (2001). Radiation Physics and Chemistry (61): 171-175. Hirano, S. (1996). Biotechnology Annual Review, (2): 237-258. Luan, L. Q, Nawasawa (2002). Advances in Chitin Sience, Vol.(V): 468-474.

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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 to n 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), Nguy n 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.

292

Hi ngh KHOA HC V CNG NGH 2007

KHO ST HOT TNH C CH SINH TR NG 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 Phng Cng ngh cc cht c hot tnh sinh hc, Vin Sinh hc Nhit i

M U Vn gy nhim v tc dng gy c ca thuc bo v thc vt ha hc tng hp i vi mi trng 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 ngu n 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 trng 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 oC. Tch v ht thu nhn ht, ra sch nhn ht trong nc ct v trng v sy kh 60 oC 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 250 ml dung mi ethanol bng phng

Phn III: CNG NGH CC CHT C HOT TNH SINH HC

293

php khuy ngm kit, lc dch chit th, loi du bng petrolium ether, sau c quay chn khng nhit 60 oC 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 oC.
3. Kho st tc dng ca sn phm chit xu t ln s sinh trng ca nm [5,6,8]

S dng mi trng PGA nui cy cc lo i 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 oC) 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 d i 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 Nhn XCH trong nc Nhn XCH trong ethanol Nhn XCH trong methanol knh t nm (mm) % c ch knh t nm (mm) % c ch knh t nm (mm) % c ch 87,0 0,3 87,0 0,3 i chng 87,0 0,3 250 ppm 86,0 1,2 73,7 15,3 84,8 2,5 0,4 0,9 0,6 500 ppm 85,5 1,7 67,3 22,6 81,2 6,7 0,6 1,8 0,3 1000 ppm 76,7 11,9 57,8 33,5 69,8 19,7 1,0 1,1 1,2 2000 ppm 46,5 46,6 25,2 71,1 44,5 48,9 3,3 0,7 0.9 4000 ppm 24,5 71,8 13,5 84,5 36,3 58,2 1,2 0,9 0,5

294

Hi ngh KHOA HC V CNG NGH 2007

Sn phm chit xut t nhn ht X CH trong ethanol, nng 250ppm c tc ng c ch nm Rhizoctonia solani so vi i c hng. Dch chit nhn ht X CH trong methanol nng 500ppm v dch chit nhn ht XCH trong n c 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 Nhn XCH trong nc Nhn XCH trong nc Loi sn phm 86,8 0,4

250 ppm 85,0 2,1 0,8

500 ppm 75,8 12,7 0,7

1000 ppm 73,0 15,9 0,8

2000 ppm 65,5 24,6 0,9

4000 ppm 45,0 48,2 0,8

Nhn XCH trong ethanol

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

15,7
Nhn XCH trong methanol Nhn XCH trong methanol 86,8 0,4

32,6 0,5 75,5 0,8

50,5 72,2 0,4

55,1 56,2 0,6

60,1 39,7 0,9

83,5

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 Nhn XCH trong nc knh t nm (mm) % c ch Nhn XCH trong ethanol Nhn XCH trong methanol knh t nm (mm) % c ch knh t nm (mm) % c ch 86,8 0,2 86,8 0,2 i chng 87,3 0,3

250 ppm 86,2 1,3 77,3 10,9 65,8 24,2 1,0 0,9 0,6

500 ppm 76,2 0,4 12,8 73,8 0,2 14,9 62,2 0,6 28,4

1000 ppm 63,3 27,5 62,3 28,2 56,3 35,1 0,7 0,6 0,6

2000 ppm 54,8 37,2 54,5 37,2 52,7 39,3 0,6 0,9 0,4

4000 ppm 42,2 51,7 40,8 52,9 46,7 46,2 0,6 0,8 1,2

Phn III: CNG NGH CC CHT C HOT TNH SINH HC

295

Loi sn phm Nhn ht XCH trong nc

Rhizoctonia solani

Sclerotium rolfsii

Fusarium oxysporum

Nhn ht XCH trong ethanol

Nhn ht XCH trong methan ol

KT LUN 1. 2. nhn c 3 dng sn phm th loi du t nhn ht XCH: sn phm chit xut trong nc, ethanol v methanol. 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 , s n 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. 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 c n 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 n y.

3.

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.

296

Hi ngh KHOA HC V CNG NGH 2007

2. 3.

V Triu Mn, L Lng T (1998). Gio trnh bnh cy nng nghip. N XB Nng nghip 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. Nguyn Tin Thng v nhng ngi khc (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. 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. Amadioha, A. C. (2000). Controlling rice blast in vitro and in vivo with axtracts of Azadirachta indica. Crop protection 19, 287-290. 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. Coventry, E. & Allan, E. J. (2001). Microbiological and chemical analysis of n eem (Azadirachta indica) extracts: New data on antimicrobial activity. Phytoparasitica 29:5. 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.

4.

5.

6. 7.

8. 9.

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.

Phn III: CNG NGH CC CHT C HOT TNH SINH HC

297

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 Institute of Tropical Biology

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 s hould have some researches further to confirm the results in the field test.

298

Hi ngh KHOA HC V CNG NGH 2007

KHO ST TNH N NH CA HOT CHT TRONG DU HT XOAN CHU HN (Azadirachta indica A. Juss) THU C BNG PHNG PHP P NGU I
Dip Qunh Nh, Nguyn Tin Thng, L th Thanh Phng Phng Cng ngh cc cht c hot tnh sinh hc, Vin Sinh hc Nhit i

M U Vit Nam, cy neem c n c tn gi khc l xoan chu hn, c du nhp t kh lu v trng trn din tch kh ln ti 2 tnh B nh 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 ring thng khng bn, d b phn h y 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 h ng Sigma, M; nimbin v salannin hng Trifolio, c. Tween 80, ethano l960, 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 b m qua mng lc c kch thc l = 0,45m v tin hnh phn tch mu trn thit b HPLC vi cc thng s sau:

Phn III: CNG NGH CC CHT C HOT TNH SINH HC

299

- Lng mu bm vo ct: 0,5l - Ct bo v: Bondapak TM C18, 125A0, 10m, 3,9 x 20mm - Ct phn tch: Bondapak TM C18, 125A0, 10m, 3,9 x 300mm - Dung mi ra ct: CH 3CN: H2O (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 LC50 ca cc cht gy c trn 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 LC50 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 1 2 3 Hot cht Azadirachtin Nimbin Salannin Nng (ppm) 930,685 262,578 1027,478

300

Hi ngh KHOA HC V CNG NGH 2007

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 m u, nhit phng, c trnh by bng 2.
Bng 2. S bin ng h m lng hot cht v gi tr pH ca du neem theo thi gian
Hot cht 0 thng Azadirachtin Nimbin Salanin Total pH 100 100 100 100 5,73 (%) Hot cht cn li theo thi gian bo qun 1 thng 99,45 98,45 98,99 99,16 5,65 4 thng 78,15 41,26 73,84 71,81 4,56 6 thng 49,56 26,02 27,12 36,35 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].

Phn III: CNG NGH CC CHT C HOT TNH SINH HC

301

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 LC50 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) 0 1 4 5 6 PT ton hc Y = aX + b 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 H s tng quan (R) 0,985 0.960 0.995 0.979 0.994 3,20 3,30 4,79 5,16 7,00 LC50 (%)

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. 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% .

2.

TI LIU THAM KHO 1. 2. 3. 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. Smchmutterer H., (1995). The neem tree Azadirachta indica A. Juss and other meliaceous plants. 695p. 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.

302

Hi ngh KHOA HC V CNG NGH 2007

4. 5. 6. 7. 8.

<http://www.health.gov.au/tga/docs/pdf/cmec/cmecde27.pdf> posting November, 2002. http://www.doc.govt.nz/Publications/004~Science -and-Research/Science-forConservation/PDF/sfc232.pdf Phm Vn Bin v cs (2000). Cm nang thuc bo v thc vt . NXB Nng nghip. 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. 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. 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.

9.

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 Institute of Tropical Biology

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.

Phn III: CNG NGH CC CHT C HOT TNH SINH HC

303

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 ti 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 ph ng 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]

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Hi ngh KHOA HC V CNG NGH 2007

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 vin nn ln mt go, y kn l trong 3 ng y. 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).

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
Tui ry 10 ngy tui Thnh trng Phng trnh tng quan Y = 3,8824 + 0,8273 X Y = 3,8769 + 0,6103 X H s tng quan 0,9769 0,9903 P 0,0042 0,0011 LC50(%) 2,20 6,87

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 ng i go
Ch phm
Du neem Dch chit BD

Nng x l (%)
100,0 188,9*** 165,7*** 50,0 180,6*** 155,2*** 25,0 174,4*** 147,4** 12,5 160,3*** 130,1** 6,0 146,7** 116,7** 3,0 125,2** 98,3* 1,5 106,8** 89,9*

Hiu lc gy ngn n: (***):mnh, (**): trung b nh, (*): yu [4]

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305

Ngoi tc ng gy ngn n, cc ch phm c n gy cht ngi go cc mc khc nhau. Nhn chung trong ph m 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
Ch phm Du neem Dch chit bnh du Phng trnh tng quan Y = 5.6861 + 0.5999 X Y = 5,3948 + 0,6072 X H s tng quan 0.985000926 0.985180154 P 5.24948E-05 5.09456E-05 LC50(%) 0,07 0,23

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).

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 ) ca ch phm NV i vi ng i go v mt bt
Ngy sau x l i tng x l
3 ngy 7 ngy Ngi go Mt bt Ngi go Mt bt
3

Phng trnh tng quan

y = 3,8098 + 1,4838 x y = 3,5726 + 1,4984 x y = 4,6161 + 1,3992 x y = 4,5693 + 1,4878 x

H s tng quan
0,9538 0,9315 0,9330 0,9312

P
0,0118 0,0212 0,0205 0,0213

LD50 3 (mg/dm )
6,34 8,96 1,88 1,95

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3. Su t
Bng 5: Tc ng gy ngn n v gy cht ca ch phm neem i vi su t Ch tiu H s ngn n (%) LC50 (%) Du neem 196,2 0,42 Loi ch phm Dch chit bnh du 173,1 0,61

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 LC50 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 to n b i tng x l qua 2 giai on su v nhng, kt qu khng xut hin th nh trng.

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% ) nhon g chet than h trun g DN (10% ) than h trun g nho g n che t

7% 13%
sau chet

0%

10%

sau chet

80%

90%
DCH (10% ) thanh trung

DCH (5% )

thanh trung nhong chet

nhong chet

10%

0%

16%
sau chet

23%
sau chet

84%
67%

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.

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TI LIU THAM KHO 1. 2. Ahmed S., (1988). Potential of using neem tree ( Azadirachta indica) for pest control and rural development. Neem Newsl. 5(4). pp. 49 - 55. 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, 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. 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. 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. 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

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6.

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 Institute of Tropical Biology Neem seed oil, neem cake extract and neem pellet were prepared and screened for their effects on rice moth (Corcyra cephalonica), 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.