Hoàn thiện quy trình ly trích hoạt chất limonoid trên quy mô pilot từ cây neem và điều chế các phụ gia thích hợp để làm nguyên liệu pha chế thuốc bảo vệ thực vật

Muc tieu : — Hoan thien quy trinh trich ly hoat chdt limonoid quy mo pilot, tit cay neem lam nguyen lieu de pha the thu6c bao ve thgc vat, RI dung ba thai sau khi trich limonoid a lam p

UY BAN NHAN DAN TP.HCM

SCA KHOA HOC VA LONG NGHe

oa.:qo

TRUNG TAM NC OA SINH ING DUNG

BAO CAO NGHIEM THU

De tai

HOAN THICN QUY TRiNH TRICH LY HOAT CHAT

DIEU CHE CAC PHV GIA THICH HOP DE LAM NGUYEN LIN PHA CHE THUOC BAO VE THVC V41-

GSTS TrAn Kim Qui

THANH 13116 116 CHI MINH THANG 10 NAM 2006

THAN, HOA, QUA VA LA CAY NEEM

TEN KHOA HQC : AZADIRACHTA INDICA

BAO CAO NGHIEM THU DE TAI

1 Ten d tai : Hoan thien quy trinh ly trich hoat chdt limonoid tren quy m6 pilot ta'

cay neem va Eau the cac phu gia thich hdp de lam nguyen lieu pha the thu6c tra' sau bao ye thuc vat

2 Chti nhiem de tai : GSTS Tr'dn Kim Qui

3 Cd quan chi tri : Trung tam nghien eau flOa Sinh ang dung

4 Thai gian thurc hien d tai : tit thang 08/2004 den 02/2006

5 Kinh phi dtigc duyet : 161.000.000 d

6 Kinh phi 65 cap :145.000.000d theo TB s6 : 137/TB-SKHCN ngay 9/8/2004 va

TB s6: 225/TB-SKHCN ngay 18/1/2005

7 Muc tieu :

— Hoan thien quy trinh trich ly hoat chdt limonoid quy mo pilot, tit cay neem lam nguyen lieu de pha the thu6c bao ve thgc vat, RI dung ba thai sau khi trich limonoid a lam phan bon diet tuye'n trong va kie'n moi trong ddt

— Dieu the chdt phu gia nhu' chdt lam ban, chdt phu trd de ph6i the vdi limonoid tao ra thu6c bao ve thtfc vat co hoat tinh manh va ban trong mai trutrng

8 Nei dung nghien cdu :

at Nghien cdu ly trich cac hoat chdt limonoid trong hat va ld cay neem trong a Binh Thuan va Ninh Than, dm dung moi CO tinh chon loc cao trich limonoid de dat du'dc hieu sudt cao nhdt Dinh tinh va dinh lurdng cac limonoid nay de lam cd sa cho viec xay dog quy trinh trich ly limonoid dang pilot

b/ Ta' cac ke't qua thu (WO a phan tren, hoan thien quy trinh ly trich hoat chdt limonoid, quy mo pilot trong hat va IA cay neem lam nguyen lieu pha the thu6c bao ve thu'c vat clang b6t rgc, nhili dau va dung dich Dinh 'dog tong azadirachtin thu6c limonoid (AZL) va azadirachtin (AZ) trong mOi loci thu6c

c/ Dinh phan cac dtrang lieu N, P, K du' lacing limonoid va hdp chdt S trong ba thai nham sit dung ba thai nay lam nguyen lieu ph6i the vao phan hitu cd co tac dung diet tuye'n trong va kie'n m6i trong ddt

d/ Nghien cdu didu the cac phu gia thich hdp nhu chdt lam ban at au dau nanh, chdt phu trd tit can (IL me dd tang hoat limonoid

e/ Ke't hdp vdi Trung tam Nghien cdu va Phat tridn Nang Du'dc, Cong ty CP Thu6c Sat Trung Viet Nam va Trung tam Nghien cdu eng dung COng nghe Sinh hoc nhiet ddi Phan vien Cong nghe sau thu hoach de sd khao tdc dung cila thu6c d6i vdi sau mot pha hai kho lifdng thu'c, nain m6c gay benh cho cay trong va sau bo pha hai vutin rau vutrn cay an trai

9 Sin phdm nghien ctYu :

Mi?c chdt hiking an dqt

SO' ItAing sin phdm

1 QT

1 Quy trinh cong nghe ly trich limonoid

di' la neem Ham liking tang AZL

trong la

2 Quy trinh cong nghe ly trich limonoid

ta' dau neem Ham lacing tang AZL

trong cidu

ttt hat Neem Ham lacing tang AZL

trong hat

4 Quy trinh cong nghe chd bieen ba thai

trich limonoid lam phan bon Ham

luring N, P, K, limonoid va SOX

1 Limonoid ?

0,01

1 QT

(Sesamin + sesamolin) trong can du

me tinh luyen Ham Wong tang

sesamin va sesamolin trong can dau

me

dau nanh dd didu chd chdt lam bdn

vernolat Diking hiring epoxid Ma dau

dau nanh

- Ham hiring tang AZL trong hip chit

Limo

- Ham Wong tang AZL trong hop chit

Limo

- Ham Wong tang AZL trong hop chdt

Limo

10 Chdt tang hoat sesam

- Ham hiring tang sesam trong the

ph'Sm

11 Chat b e'n Ma vernolat

- Ham hiring vernolat trong chdpha'm

MIJC LUC

Trang T6m tgt

Muc luc

Danh sach bang va sd a

Danh sach hinh

PHAN II : TONG QUAN

111.2 M6t s6 che'phSm tit cay neem ten thi treing the gidi 15

PHAN III : NGHIEN CITU KET QUA VA BAN LU4N

NOI DUNG 1 : QUI TRiNH CONG NGHe LY TRiCH LIMONOID

Tip LA NEEM HAM LONG TONG AZL, AZ VA S TRONG LA

I.1.1 Phudng phdp ly trich va xac dinh ham hicing limonoid 19

1.2.1 Thanh phan hoa hoc cd ban oh la neem 35 1.2.2 Xac dinh kha Wang trich ly limonoid cila mOt s6 dung moi

1.2.3 Qui trinh ly trich limonoid tit la neem Dinh lacing tong

1.2.4 Dinh lacing sulfur 39

NoI DUNG 2 : QUI TRINH CONG NGH€ LY TRICH LIMONOID

TRONG DAU NEEM, HAM WONG TONG AZL,

AZ VA STRONG DAU

2.1.1 Pinking phap tach dau va xac dinh ham lacing limonoid trong du 41

2.2.2 Trich limonoid trong dau neem, dinh Ming AZL, AZ

2.2.3 Thanh phan acid be() ciia dAu neem 46

NOI DUNG 3 : QUI TRINH CONG NG11€ LY TRiCH LIMONOID

TRONG HAT NEEM, HAM LUNG TONG AZL VA AZ TRONG HAT

3.1.1 Phadng phap ly trich va xac dinh ham lacing limonoid 49

3.2.1 Thanh phan Ma hoc cd ban ciia vo hat neem 50 3.2.2 Thanh phan Ma hoc cd ban cua nhan hat neem 50 3.2.3 Ly trich limonoid trong banh hat neem, dinh lacing AZL va AZ 51

N6I DUNG 4 : NGHIEN CCU Sit DUNG BA THAI CiJA HAT VA LA

NEEM DE PHOI Cid VAO PHAN BON DINH LUVNG CAC THANH PHAN CCi BAN VA DAC TRUNG TRONG

BA NEEM

4.1.1 Hi'e'u qua cda viec sit dung ba neem lam phan bon 54 4.1.2 Ch6 bie'n ba neem de lam ph6i lieu cho phan bon 54

4.2 Dinh liking cac chi tieu chit lu'Ong 55 4.2.1 Dinh hiking cac chi Weil dac truing ciia ba neem 55 4.2.2 Dinh liking cac chi tieu chit liking ciia ba neem

NOI DUNG 5 : QUI TRIM! CONG NGIle DIEU CHE SESAM (SESAMIN +

SESAMOL + SESAMOLIN) TRONG CAN DAU ME TINH LUYtN

5.2 Qui trinh cong nghe trich ly hip chit sesam 63

5.4 Thu' nghiem kha nang tang host cila chit phu trd sesam 68

NOI DUNG 6 : QUI TRINH CONG NGI4 EPDXID HOA DAU DAU

'' NANH DE DIEU CHE CHAT LAM BEN VERNOLAT

6.2 Quy trinh cong nghe didu ch6 chit lam bdn ESO tir d'Au dau nanh 74 6.2.1 Tinh chit va thanh pha'n ddu dau nanh 74

6.2.4 Tinh chit cila au dau nanh epoxid h6a ESO 77

6.4 Thir nghiem kha nang lam bdn ctia au Vernolat trong sin pldm

6.4.2 Pinking phap thif nghiem 79

NOI DUNG 7 : DIEU CHE THUOC BAO Vt THVC VAT Tip LA, DAU

VA HAT NEEM

7.1 Dieu ch6 thu6c bao ve thu'c vat tit la neem 82

7.1.2 Quy trinh ly trich limonoid trong Id neem de di6u ch6 thu6c

7.1.4 Gia thanh bin thanh phgm hdp chat limonoid AZL trich

7.1.5 This nghiem tic dung cila thu6c LIMO 3000 BR 85 7.2 Dieu ch6 thu6c bao ye thu'c vat ttf au neem 89

7.2.2 Qui trinh tach &Au neem bao hoa \fa bat bao hoa a dieu ch6

7.2.4 Gid thanh ban thanh pldm d'au neem Vit bao hoa 92 7.2.5 This nghiem tic dung cua thu6c LIMO 3000 ND 93 7.3 Ditu ch6 thu6c bao ye thqc vat ta' 'Dinh hat neem 95

7.3.2 Qui trinh trich limonoid trong bdnh neem a di6u the thu6c

7.3.4 Gia thanh bin thanh phSm azadirachtin 97 7.3.5 This nghiem tic dung ciia thu6c LIMO 3000 DD 98

PHAN V : PHI) LI,JC

c l = 1,775 mg/1 c2 = 3,55 mg/1 c3 = 7,10 mg/1 c4 = 14,20 mg/1

Phu luc 5 : Si bie-n thien cua ham ltfOng azadirachtin va dau beo trong

Phu luc 9 : DO hap thu A = f(c) cua cdc mau ba neem, cao EAE cua la

Phu luc 10 : Ke-t qua thtr nghiem tdc dung phu trO cua sesam d6i vdi Limo

Ke-t qua thii nghiem nemg dq azadirachtin

- M6t s6 hoat chit limonoid da dttdc trich ra tit cac b6 phan khac

nhau cua cay neem

- M6t s6 the phdm didu the tit cay neem dang luu hanh tren thi

trtteing the gi5i

- D6c tinh cua the Olin Margosan-O dam clac

- Thu6c this biet tinh dd xac dinh thanh phAn Ma hoc cd ban

- Ke't qua xac dinh thanh phan Ma hoc cd ban cua 16 neem

- Ham luring AZL trong cao trich ly cua 100g b6t IA neem kilo vdi

mot s6 dung moi thong dung

- Ham lacing azadirachtin trich ly du'dc tit hat neem vdi cac dung

moi thong dung khac nhau

- Cac chi s6 cua du neem

- Thanh pith acid brio tong dlu neem

- Ke't qua xac dinh thanh phan Ma hoc cd ban cua vo hat neem

- lat qua xac dinh thanh phan Ma hoc cd ban oh nhan hat neem

- Ke't qua phan tich cac chi tieu chdt luring cua ba neem dung lam

phan bon

- Xac dinh su' hien dien cua sesam qua cac phan eng mau

- S6 sau con song sau khi xii' ly thu6c Limo 3000ND CT1 va CT2

- S6 sau song tru'dc va sau xit ly 24 giet, 48 gier

- Hieu laic thu6c sau 24 va 48 gier

- Thanh phan acid beo cua dAu dau nanh

- Mat dO du trung loai Tribolium castaneum a cac cong thdc till

nghiem

- Hieu lttc cua thu6c Limo 3000 BR d6i vdi loai Tribolium

castaneum

- Mat d0 loai mot Sitophilus oryzae a cac cong till:1'c till nghiem

Hieu IAN cua cac loai thu6c d6i vdi du tang loai Sitophilus

oryzae

- Ket qua did' nghiem Limo 3000 ND tren na'm Sclerotium rolfsii

sau 4 ngay

S6 salt song tru'dc va sau khi )(it ly 24 gid va 48 gier

Hieu lu'c thu6c teen sau td sau 24 gier va 48 giei

DANH SACH Si DO

1.1 - Quy trinh di6u the cao phan 136 cila la, vo va nhan hat neem 21

1.2 - Ly trich hoat chit limonoid trong la neem vdi cac dung moi có

dO phan clic Ichac nhau

22

1.4 - Quy trinh dinh lu'dng S trong cac bi) phan caa cay neem 33

1.5 - Quy trinh trich ly limonoid tit la neem de dinh lacing AZL trong

la

38

2.1 - Quy trinh ki thuat ly trich AZL trong dau neem va xic dinh

thanh phan acid beo

5.2 - Quy trinh ki that dinh phan h8n hdp sesam 66

6.1 - Quy trinh ding nghe di6u the dau dau nanh epoxy hoa ESO 76

7.1 - Quy trinh ly trich limonoid trong la neem de die'u the thu6c 83

DANH SACH H1NH

- Trich ly cdc thanh phan h6a hoc cd ban cua Id neem trong binh

Soxhlet

- Thu hdi dung mai du'di dp sudt kern trong binh co quay

- Mdy UV hidu CARY 50 do da hdp thu A d X=577nm

- Du'efrig chudn bidu dien di) hdp thu A vs n8ng dO dd azadirachtin

chudn dung trong pinking phap so mau

- Mdy HPLC

- DuTing chudn cua azadirachtin bidu dien peak area (PA) vs neIng

dO c (mg/1)

- MOt s6 limonoid thuOc nhorn AZL

- Sac kyl do HPLC cua dich trich hat neem (IARI, Delhi, 1987)

- Sac ky dd HPLC cua dich trich hat neem (Gurgaon, Haryana,

1987)

- Mdy nghidn hat neem loai c6i xay

- Mdy ep hat neem loai con dOi

- Mdy nghidn ba neem de lam phan b6n

- May hdp thu nguyen tit AAS Perkin Elmer 3300 dung dd phan

tich cac nguyen to vi 'Yong

- DO ben nhu cua dd Limo 3000 ND c6 chda Sesam

- DO bdn nhii cua dd Limo 3000ND c6 chtla Vernolat

- Mdy ep dau loai khung ban

- Tdc dung cua Limo 3000 ND d6i vdi na'm Sclerotium rolfsii sau

PHAN I

DAU

6 cac ntidc tien tien tren the gidi, vdi muc dich bao ve mai tru'efng, ngutii

!tong dan sir dung chii ye-u cac chdt an du, chdt xua odu6i con trang, hay thu6c trit sau g6c thao m6c de bac) ye hoa mau Theo tieu chudn HACCP d my, EU, tieu chudn OMIC d Nhat va d Anh, cac loci rau cu qua chi du'dc phep nhap vao thi tntelng cac nuac Aay khi nao khOng co du' lu'Ong thu6c trir sau tong hdp co g6c Cl, P huu cd, pyrethrin, carbamat

Tren the gidi hien nay cac nha hoa hoc kh6ng nein nghien cdu tim ra cac loci thao du'dc mai c6 host tinh sat trang de bao ye hoa mau ngay cang hitu hieu hdn ma khong lam o nhi8m mOi tru'dng Trong cac loci thao du'dc bie't tir trade den nay co the dbng de di6u ch6 thu6c bio ye thtic vat thi cay neem thuOc loci tot nhdt va c6 san ltrdng cao nhdt, du cid san xudt ra thu6c trii sau cong nghiep

Cay neem xudt xtt tit An DO, du'dc trong dai tra thanh rung d khgp nu& nay

va la m6t ngeon loi rat ldn cho An DO TLY nam 1960, cay neem da n6i tie-ng tren khgp the gidi do tit canh, la, va hat neem cac nha hoa hoc da trich du'dc mot s6 host chdt limonoid co tac dung gay ngan an va xua du6i eon trang rat c6 hieu qua Cac loci thu6c bao ve thqc vat nhu' Neemgold, Margocide CK cli6u ch6 tit cay neem rat du'dc tia chu6ng d An DO Nhi6u nu'dc tren the gidi, chau Phi, Nam my, chau Au da mang cay neem v6 trong tren ddt nu'ac hode? sit dung Hai san phdm Neem Azal va Neem Azal F san xudt tit cay neem tai Disc du'dc ban khgp chau Au Nam 1975, BO Nong nghiep My da xay citing ke hoach trong cay neem tren khgp nu'dc M Den nam 1985, Cd quan Bio ye M6i tradrig My da cho phep ban ra tren khgp nuac My hai loci thu6c bio ve thu'c vat trich tit la va hat cay neem vdi ten thu'dng mai Margosan-Ova Izatin Tit nam 1985, BO N6ng nghiep Nhat, B6 Nang nghiep Trung Qu6c cling da du'a cay neem ve trong va den nay ho cling da san xudt ra nhi6u loci thu6c bao ye thu'c vat va thu6c tri benh cho ngticli, gia stic, gia cam Hai the phdm Yu Teng va Ku Seng chie't xudt tit hat neem de xua clu6i con trung rat du'dc•nong dan Trung Qu6c tra chuang

Tit nam 1980 den nay da c6 8 hOi nghi khoa hoc qu6c t6 y6 cay neem to chi:1'c tai CHLB Disc (Fan 2, 1983), Pakistan (1986), Kenya (Fan 3, 1987), My (1992), An

DO (1993), Philippin (IRRI, 1994), Uc (1996), Anh (1999) Ho TA" khang dinh gia tri cua cay neem trong linh bao ye a-Ric vat, diet tar sau mot, nam m6c va tri benh cho ngtidi Den nay ngutri to da )(lc dinh attdc tren 150 limonoid trong cay neem, trong d6 c6 khoang 30 chdt c6 cdu trtic va tac dung ttidng tlf nhu' azadirachtin Cac

limonoid chi Om cac nguyen to C, H, 0, N khong co Cl, P va cac nhOm gay dOc

khac ma hien nay dang bi han the sit dung

Thanh ph6 H8 Chi Minh dinh hu6ng phat trien nong nghiep trong thi gian toi vdi 2 cay 2 con trong do co chttdng trinh phat tridn rau sach Dd san xudt rau sach theo quy dinh cita thi triteing the gidi, chting ta khong dttdc sit dung cac loci thu6c true sau tong hdp vi cac loci thu6c nay co htu ba dee lau dai gay tac hailan den sac khoe ngu'efi tieu dung va lam 6 nhi8m mei tru'Ong, ma chi co the sir dung cac chdt

an du con trUng hoac cac loci thu6c tit sau g6c thao mec khong có ltru ba dec Tuy nhien viec nghien call sit dung thu6c bao ve thttc vat loci ay a nu& ta con rat it, ke't qua con han che Tru6c day cling co met s6 ding trinh nghien citu

trich ly rotenon tut day thu6c ca Derris elleptica de diet sau rau, nhttng tac dung

kheng cao do hoat chdt bi phan hay rat nhanh khi data ra sit dung Met s6 d8 tai nghien cdu thu6c triY sau gate alio mec khdc cling chi citing lai 6 giai doan nghien cdu cd ban trong phong thi nghiem

Nam 2002 met s6 nha hoa hoc thuec Vien H6a hoc Trung tam KHTN-CNQG

va Vien Bao ve thttc vat Be NN-PTNT da ly trich dude hoat chdt azadirachtin tit nhan hat cay neem lay a Thuan Hai va ding da thit nghiem thdy no co tac dung

gay ngan an manh dol vdi sau khoang Spodoptera litura (44) Nam 2004 met s6

nha h6a hoc thuec Dai hoc Nong Lam va Vien Sinh hoc Nhiet ddi TP.H8 Chi Minh cling da didu che cao ethanol trich ly tit nhan hat neem 14 6 Ninh Thuan co ham hidng azadirachtin cao de diet ngai gao Corcyra cephalonica, dat ke'tqui tot (45)

Cay neem 6 ntt6c ta dttdc GS Lam C6ng Dinh mang gioeng tit An De ve trong tren \rung ddt kilo can a Tuy Phong, Thuan Hai tit nam 1993 Dein nay Sa Ming nghiep ctia Thuan Hai va Ninh Thuan da phat tridn trong khoang 2000 ha cay neem thanh rung cao 8 - 10m

Viec nghien cell sit dung cac thanh phAn cita cay neem lam thu6c bao ve thu'c vat de? Oat tridn chu'dng trinh rau qua sach ciia TP.H8 Chi Minh va bao ye kho lu'dng thu'c thtic phdrn la vain d8rdt carp thie't oh chting ta hien nay

Trong cid tai nay chting t6i nghien citu:

- Xac dinh ham hidng tong azadirachtin thuec limonoid AZL (Total Azadirachtin Related Limonoids) va azadirachtin AZ co tac dung bac) ve thu'c vat trong la, hat va au hat neem trong a \fang Binh Thuan, Ninh Than

- Tit cac ke't qua thu dadc, xay dung quy trinh chi& xudt hoat chdt AZL tut la, hat

va du neem lam nguyen lieu de pha che thu6c bao ve thgc vat c6 tac dung COI ma khong lam 6 nhiem rad trill:1'1g

- Sit dung ba thai sau khi trich limonoid dd phol che vao phan bon diet tuyein trUng, kie'n, m6i trong ddt va ngan chan qua trinh lam thdt thoat N2 trong ddt

- Didu che cac chdt phu gia dui chdt lam bdn (stabiliser), chdt phu trd (synergist) de ph6i ch6 vdi AZL tao ra che phdm thu6c bao ve thu'c vat LIMO 3000

co hoat tinh manh va bdn trong moi trutrng

- Geri nth' den Cd quan chuyen mon de khao sat sd be tac dung ctia thu6c

PHAN II

TONG QUAN

I Ghii thieu ve cay neem

Cay neem hay con dddc goi la cay xoan An DO, xoan chiu han, niembau,

nimb, kohumba, nimba ten khoa hoc la Azadirachta indica A Juss thuOc ho Xoan

(Meliaceae)

I.1 MO ti cay (1)

Neem la cay than g6 to, cao 15 - 20m, O clidu kien thich hdp dat 110 cao khoang 35 - 40m, chiu han t6t N6 la loci cay co la xanh quanh nam, phat trien tot ngay teen da't cat ngheo kiet, khO can

Cay co nhdnh Ong, tan la hinh oval hay hinh ton du'dng kinh khoang 15-20m Than cay ngan, thang, c6 chu vi khoing 1,5 - 2,5m, vO cay cirng, xu xi, co nhidu ranh nth, ben ngoai mau xdm va do ldt ben trong, co mai tOi, vi rat dAng Dac didm ben ngoai va dO day cila vO phu thuOc vao tudi cua cay va didu kien mOi tru'dng VO than cay non thu'ang nhgn, mau xanh nhat pha d6ng, co di) day khoing 1,25 - 2,5cm

La neem thuOc loci la kepi long chim mOt lan, dang mac, bia la co khia hinh rang cda, cuo'ng la ngan Cay ra Id non vao thdng 3 - 4 va bO la thddng xanh t6t quanh ram, khong co thdi ky rung

Hoa moc thanh chum a ndch la, mau trang, hodng thdm, cu6ng ngdn va c6 5 anh Hoa lddng tinh va hoa du'c co the xudt hien tren ding mOt cay, canh hoa rdi, xe'p ldp, pha day long min Ong nhi hinh tru, cao khoang 3 - 5mm, mieng hdi loe Cj Sri Lanka, An DO va Viet Nam, cay thu'dng ra hoa tit thang 3 den thdng 5 Tuy nhien, avai vung cua An DO, cay co the ra hoa quanh nam

Trai neem CO hinh bau due, trdn lang va dai khoing 2cm Trai hinh thanh, Oat trien va chin trong Ong 1 - 2 thang Theri didm thu hoach trai t6t nhdt la ltic trai vita chuydn sang mau xanh yang va nen hdi trai khi can teen cay khong de trai rung xu6ng ddt chdt lu'dng bi earn Cay tracing thanh m6i nam co the cho 30 - 50kg trai Th6ng thong, trai chin tit thang 6 ddn thdng 8 M6i hecta c6 thd trong tit 100 -

200 cay, san lacing trai hang nam khoing 5 - 10 dn

nu'dc to co mOt s6 cay rat de nham fan vdi cay neem nhu' :

* Cay sau dau (Brucea javanica L.) c6 la kepi long chim mOt ran nhu'ng cay nhO,

chi cao 1 - 2m, trai nhO 3mm, c6 tinh clOc, khong an click

* Cay xoan ta (Melia azedarach L.) la kep long chim 2 lan co rang cira Hoa

moc thanh teng cum tan dai, mau hoa hdi tim af Viet Nam, cay xoan ta dttdc trong tit rat lau deri, ph6 bie'n tren cac nudng ray vung Tay Bgc, Tay Nguyen va cac tinh Hoa -Binh, Thanh H6a, Darlak, Lam Do'ng chti y6u dung de ldy g8

1.2 Ngudn g6c va vung 'Alan 66(2)

Cay neem A indica dtrdc xem la co nguo'n g6c tit Burma va Sri Lanka Tuy

nhien, ngu6n g6c chinh xac cila no chu'a du'dc bieet, mOt so ngutri cho rang cay neem song tu' nhien a vung Welt An DO, con nhung ngutii khdc cho rang no thuen vung kho han tren khu vu'c Nam A, DOng Nam A bao gom Thai Lan, Malaysia va Indonesia Den nay cay neem da CO mat a tren 50 qu6c gia tren the giai

Cay neem dttdc di thu'c vao Viet Nam tit nAm 1981 do GS Lam Cong Dinh,

met nha lam hoc Viet Nam, nhan dip tham du' hei than qu6c to lam nghiep tai

Senegal, chau Phi Ong dem hat gioeng cay neem v6 trong tai Phan Thie-t, sau do cay dttdc trong rOng ra a Ninh Thuan, Binh Thuan Ong da dat ten cho loaf cay nay

la "xoan chiu ban" (A indica) de" phan biet vdi xoan dia phu'dng (Melia azadirachta)

dttdc trong ph6 bie'n a Viet Nam

GAn day cay neem co gioeng tit An DO dude du'a vao trong nhi6u ndi a mien Trung de phu xanh ddt Whig doi troc, cai tao ddt bi hoang h6a

1.3 Gia tri ctia cay neem (3)

Tai Aen DO, cay neem tit lau da dttdc xem la met tai nguyen q14, gia do tinh chdt da dung ctia no va la mOt trong s6 it loai thttc vat ma tat ca be, phAn du co cong dung tot

* La : MOt cay neem cao 7,5 - 8m cho trung binh hang nam khoing 350 kg la

tu'di (khoing 50 kg la kho)

Tai mOt s6 vung a An DO, nong dan thu'ang cho gia stic An la neem sau khi

sinh con de" gia tang su' tier sea Ngoai ra, la neem con có tac dung phong tri benh giun san cho gia stic, kie'm soat nhi8u loai tac nhan gay benh cho ngutri, gia stic va cay trong do la co club nhie'u hdp chdt nhu' nimbin, nimbinene, nimbandiol, nimbolide, quercetin NhiL ndi la neem non con dttdc dung lam rau An yea 136 sung khoang chdt, vita phong ngita giun san hoac viem nhi8m duting runt (Srivastav va ctv, 1998)

* Trai : trdi neem khi chin c6 vi ngot, có thd an dtidc hoc sit dung trong cong

nghe len men Trai neem duck dung lam thu6c CS)/ giun, thu6c giim dau va thu6c tri benh dttdng tidt nieu,.benh tri Qua khO ngam nu6c c6 thd tri mot s6 benh ngoai

• da Nu'oc thit qui khi phun len cay c6 thd xua ciu6i nhidu con trUng, (lac biet rat hiru hieu doi vdi chau chdu

* Nhan : nhan hat neem chtla nhidu hdp chit c6 host tinh sinh hoc nhu'

azadirachtin, salanin, meliantriol Dau chidt of nhan hat neem du'dc dung de sin xudt thu6c giam dau, thu6c sat triing va ca thu6c nera thai Ngoai ra, dau hat neem

, • con du'dc dung trong nganh c6ng nghiep pham, xa phong va nhidu c6ng nghiep khac Tai An D6 va Sri Lanka, khoing 25% hat neem dock sit dung cho nganh chide, s6 con 14i &Arc dung lam thu6c trU sau

* Binh au neem : banh dau neem chita khoing 1,00 - 1,40% hdp chdt

sulfur, khoing 1,5 - 2,5% N., khoing 0,7 - 1,2% P2O5 va 1,2 - 1,5% K2O Banh dau neem la ngu8n phan hi-tu cd rat tot, vita cung di) chdt dinh du'ang cho cay, vU'a diet chick cac loci tuydn trUng, Icidn moi trong ddt va c6 khi nang ttc chdqud trinh nitrat hoa trong ddt lam tang hieu qui sit dung dam cua cay trong Theo Rao (1993), khi bOn phan ure trOn vdi dau neem, mitc hdp thu dam, lan va kali 6 cay co hu'dng

(Cymbopogon iwinterianus Jowitt.) tang theo thit tu' 17, 15 va 25% so vii khi chi bon

phan ure Ladng dam mat do bay hdi NH3 cting giim 31%

* VC; cay : dich chidt tit v6 cay neem cludc dung lam thu6c chin dau rang, benh sth ret, benh yang da hoc dung dd nhu6m lua VO cay c6 chtla nimbin, nimbidin, nimbinin nen ciing du'dc dung dd didu chd thu6c bac) ve thttc vat va mot s6 thu6c tri benh ngoai da •

II Tic Ong del vdi cac loci dich h4i

II.1 Phticing thdc tac &Ong vac) con trang (4)

Tit lau ngtkri dan An DO da bidt sit dung Id neem dd bio ve cac kho lUdng thu'c, kho cluta do quan vai soi Bdo cdo dau tien vd tinh sat triing caa la neem xudt hien khoing 1927 (Mann and Burus, 1927) Nam 1928; Chopra da dung dich trich

la neem.de diet du triing cita mot ngii co'c qua doing tidp xtic (Rept of Depart of Agr Punjab, 1928) Tit do co rat nhieu cong trinh nghien citu vd tac (Icing d6i vdi cac loci dich hai cita cac b6 phan trong cay neem da dudc ding b6 Thi du, dau

neem gay ngdn An cho chat' chau An la Ida Nephotettis virescens (Neem Newl 1/3,

—5—

28, 1984); dich trich trai neem gay ngdn An cho sau bu'dm can bong lila Mythimna

separata (Neem Newl 1/3, 31, 1984); ba neem gay ngdn An cho cac loai ray nau

can ltia Nilaparvat lugens (J Economic Entol 77, 502, 1984); ddu neem ngan chAn

de tang va lam thol tr(tng cua cac loai mot phd hai lua gao Corcyra cephalonica;

dich trich nhan hat neem ngan chAn de tiling cac loai sau bu'dm cdn cay thu6c la

Spodoptera litura (Phytoparasitica 7/3, 199, 1979); dich trich ld neem gay ngdn An

cho cac loai mot ngtI c6c Rhizopertha dominica (Neem Newl 1/2, 20, 1984) Den

nay cid ca tren 20 ding trinh nghien ciiu chttng to dich trich tit cay neem c6 kha nang tic ch6 hdn 200 loai dich hai bao gom con trang, nam m6c, vi khuSn, tuy6n trang theo cac cdch tdc dOng khdc nhau nhtf sau : (5)

1/ Gay ngan An (antifeedant)

Tdc dung manh nhit cua cay neem la gay ngdn An cho con tang Dich trich 0,1% nhan hat neem trong ntidc cling del de bao ve hoa mau, gay ngdn An cho cac

loai chau chiu sa mac Schistocera gregaria, chau chau lu'u dung Locusta migratoria

va mOt s6 con trang can ldkhdc (Pradhan et al, 1962) Tac dung gay ngan An cua

cay neem tay thu6c vao gioing va tu6i cua sau bo HMI het cac sau bo d6u bi gay

ngdn An do mOt lacing nhO hdn 0,5% nhan hat neem ngam trong nu'dc Dich trich

ethanol cua nhan hat c6 tdc dung manh nhit doi vdi S gregaria, k6 dein la dich

trich cua v6 hat va ld (Mishra, 1993)

2/ Xua dudi (Repellent)

Cay neem c6 chtra mOt s6 hoat chit d8 bay hai c6 mai tdc dung tit xa lam cho con trang cam thiy kh6 chiu ldnh ra xa khOi vang tdc d6ng Saxena (1993) khao sat thiy khi phun (tau neem d dung nhit vao canh ang lila thl cac loai ray nau

Nilaparvat lugens, bo nhay Sogatella furcifera, cac loai ki6n, moi Microtermes sp

de'u ldnh ra xa Ba ld va.hat neem sau khi trich hoat chit cling thick sit dung tit lau

3/ Diet sau 139 (Insectiside)

Cay neem c6 tinh sat trang qua clu'erng tie-p xdc va Ming tiou h6a nhu'ng khong manh va nhanh bang cac loai thu6c tra' sau tong hdp Dich trich nhan hat neem trong nu'dc c6 tdc dung diet clu'dc cac loai con trang than !I-16m nint cac loai rep hilt nhu'a cay (Cherian and Menon 1944; Goyal et al, 1971; Singhm et al, 1988) CAn ddu neem a n8ng dO 0,04% diet cittdc 100% au trang mtl6i Culex fatigans (Attri

and Ravi Prasad, 1980) nhu'ng khong diet cac thien Bich cua mu8i Dich trich nhan hat

neem phun tren rung Ida diet du'crc ray nau N lugens (Shukia et al, 1992)

4/ Ngan chan phat trien (Growth disruptor)

_Cac limonoid trong cay neem có tac dung pha va he than kinh rioi tie't cilia con trong lam cho cac to bac khong phat trien du'dc, con trong a moi lda tudi bi giam kich thudc, trong ltrdng va che't clan Quan the sau bo bie'n mat sau mot theli gian natl nib chdt nay rat clac trong cho cac ch6 phdm neem Mc Milian (1969) sir dung cao chloroform cda la neem d6 ngan chdn sd phat trien va tieu diet cac loci

sau an la Spodoptera frugiperda va Heliothis zea Dich trich cda nhan hat neem

ding có tac dung ngan chdn sd phat trien va tieu diet cac loci sau pha hai vtgin rau

va vu'Ocn cay an trai nha Pieris brassicae (Gill and Lewis, 1971), Epilachna

varivestis, Leptinotarsa decemlineata va Plutella xylostella (Steets,1976), Culex fatigans (Singh, 1984) va Culex pipiens (Zebitz, 1987) Dau neem aing cd tac dung

ngan chdn sir phat trien cua cac loci rep hai lua nhu' Leptocorisa oratorius (Saxena,

1987) BOt la neem, nhan hat neem va dau neem trOn vao ngu c6c trong kho bao

ye dudc ngil co'c khoi bi sau mot pha hai

5/ Ngan ch4n de truing (Oviposition deterrent)

Cay neem có chira nhfing hoa chdt tac dOng len he than kinh not tie't ctia con tang lam phan huy cac t6 bao nuoi du'dng phoi nen to bac trdng khong tao ra cIttOc lam cho con trong khong de trong

Rieng con trong dttc, du'di tac dung cita dau neem 3%, hoan toan bi bdt ltic va khong con cam nhan ddcrc mdi cua chdt clan du sex pheromon ma con cai tie't ra de goi con ddc (Saxena et al, 1989) Dung dich nhil 3% dau neem can có tac dung

ngan chdn sir de treng cila ray nau Nilaparvat lugens (Saxena et al, 1981) ctia bo canh c?ng Callosobruchus sp (Yadav, 1985) Dung dich trich nhan hat neem ngan chdn &Plc 90% sau dau Heliothis armigera de trdng

6/ Lam thin trdng(Antifecundity effect)

Cac ch6pham neem lam hdth6i trong con tang va sau bo nhu'bo nhay can la

Nephottix virescews (Abdul Kareem, 1988), rdi duc qua Dacus cucurbitae va Dacus dorsalis (Singh and Srivastava, 1983) Dau neem vdi n8ng dO nhd 50-500

ppm lam 100% tang cua cac loci mot hai dau Callosobruchus chinensis,

C maculatus va C analis bi hu' thol, khong nd sau (Yadav, 1993)

R P Singh va S Singh (1996) tom tat trong bang II.1 sau day tac dung cila mot s6 boat chat trong cay neem d6i vdi mOt s6 sau bo (6)

Bang II.1 : Tac dung cila mot s6 hoat chat limonoid trong cay neem doi vdi mot s6 sau bo

(A) HO chit limonoid gay ngan an

Ten hcrp chdt Loai sau bo Tai lieu tham khao

T6ng Azadirachtin A - L Schistocerca gregaria Butterworth and Morgan, 1968 Salannin Musca domestica Henderson et al., 1967

Meliantriol Schistocerca gregaria Lavie at al., 1967

1, 3-Deacetyl vilasinin Epilachna varivestis Kraus and Cramer, 1981

3-Deacetyl salannin Musca domestica Schwinger et al., 1984

Mythimna separata Sankaram et al., 1987

(B) HQp chdt limonoid ngan ch4n sti Oat trien

Ten hcip chdt Loai sau bo Tai lieu tham khao

Tang Azadirachtin A-L • Plutella xylostella Ruscoe, 1972

Musca domestica Siddiqui et al., 1986

Nimbolicinoic acid Callosobruchus analis Naqvi, 1987

Deacetyl azadirachtinol Heliothis virescens Kubo, 1986

Spodoptera litura Mythimna separata Chilo partellus Myllocerus sp

Musca domestica

Sankaram et al., 1987

Naqvi, 1987

it limonoid ngan ch n sinh sin

TOng Azadirachtin A-L Oncopeltus fasciatus Dorn et al., 1987

TOng Azadirachtin A-L Spodoptera litura Gujar and Mehrotra, 1984

11.2 Doi turcing tic dOng.(1)

Nhie'u nha khoa hoc chi:mg minh c6 khoang 200 loci con trung co hai da bi cac the phdm neem tac dQng, trong do co nhffng loci rat khang thuo'c va rat kh6 diet vdi nhffrig loci thu6c sat tang thong thu'dng rani sung khoai lang, rep duc qua,

mOt s6 lc* sau an la Nhin chung the phdm neem du'dc sii dung rat co hieu qua

dd diet mOt s6 Ion sau bo co hai thu0c cac b0 con trung sau day:

1/ BO canh thong (Orthoptera):

TIngic b0 nay g8m co cac gio'ng nhti chau chdu, de, bit cao, con tang co

vay Tac dOng manh nhdt cua che phdm neem doi IAN) nay la gay ngan an Xit

15, the phdm neem gay ngan an nbie'u wan cho nhi6u loci thuOc 130 canh thong nay

2/ BO canh d6u (Homoptera)

Thu(ic 130 nay c6 cac giong nhu' rep viIng, rep sap, bo nhay can la, bo nhay

can than cay, con tang co vay Tac dung chinh ctia the phdm neem dal vdi 130

nay la gay ngan an va ngan chan phat trien D6i vdi rep but nhtfa thi the phdrn

neem to ra kern hieu qua hdn vi co tac dung Wu dan ye'u

3/ BO canh td (Thysanoptera)

Thu0c 130 nay co cac loci nhu' bo tri, sau dut than Ch6 phdm neem c6 thd diet cac.gi6ng sau bo nay khi can d dang au trung trong daet Den khi sau tracing thanh len khOi mat ddt, chi co thd,diing the plight dau neem de diet cling

4/ BO canh cuing (Coleoptera)

Thu0 c bq nay co cac loci con trung canh ding an cay va la Cac the phdm

neem diet cac loci con tun nay khi con dang au trung than ram va gay ngan an

cho cac loci con trung da trung thanh

—9—

5/ Be canh vay (Lepidoptera)

ThuOc be nay có cac loai bu'dm dem, sau dem, mot dot qua, mot dot ngu

c6c Cdc chd phdm neem gay ngan an va ngan chan stt phdt tridn cua cac sau bo thuec be nay

6/ BO hai canh (Diptera)

ThuOc b0 way c6 cac loai ru6i dut qua, ru6i can la, ru6i nha, ru8i gia

Cdc chdphdm neem diet va xua duoi cac loai con trang thuec be nay

7/ BO canh nib (Heterophtera)

Thu(ic be nay c6 cac loai rep nhtt rep lua, rep rau xanh, rep cay ca

nhVa cua cay va trdi Cdc chd phdrn neem lam ngan chan sli phat trien va gay ngan

an cho met so loci con Wing thuec be nay

8/ Nam benh:

Cdc chdphdm neem c6 tdc dung diet ciao ndm benh hai cay trong Nhi6u thi nghiem cheng to dau neem c6 tdc dung bao ve cac loai dau ch6ng cac loai ndm

benh nhir Rhizocionia solani, Sclerotium rolfsii, Sclerotinia sclerotiorum, Fusarium

oxysporum Bdnh dau neem tren trong ddt diet di.tdc nalm R solani Dich trich hat

neem c6 tdc dung tri dudc cac benh ndm ld Dich trich dau neem tri dttdc cac loai m6c strdng tren cay (t6t hdn thu6c diet ndm Benlat) va tri du'dc 90% benh ri set va benh chdy la

Dich trich Id neem c6 kha nang tic chd ski phat trier' ctia ndm Fusarium

equiseti, F semitectum, Aspergillus flavus, A.niger va na'm benh gay benh chay ld lila Pyricularia oryzae (Rajeswari and Mariappan, 1993)

9/ Vi nim tren cay

Loai vi na'm rat co lay lan do nhidu loai con tiling nheem vi na'm mang tt ndi nay sang ndi khac Ddn nay van chua c6 thu6c (lac tri Cdc chdphdm neem cho kdt qua kha t6t

MOt s6 thi nghiem a Philippines cho thdy khi phun dung dich dau neem tren canh dong ltia bi nlifem vi ram hoai sinh, sau 3 ngay cac cay lila dan dan held phuc M6t s6 thi nghiem a An DO cho day dich trich la neem c6 the ngan chan ski

lay lan cua vi ndm gay benh do'm tren iitr(!ln rau qua

10/ Tuye'n trung

Chd phSm neem diet du'Oc nhieu loai tuydn trung Didu nay rat coy nghia vi

tuydn trung rat kho tri, mot s6 thu6c tong hOp tri tuydn trung ddu rat dOc nen bi cam sit dung CI nhieu ndi

Theo Siddiqui va Alam (1985) dich trich tii la, trai, va nhan hat neem tri 0gOc

nhieu loci tuye'n trung nhg Helicotylenchus indicus, Tylenchus filiformis,

Rotylenchus reniformis Trong banh du neem sau khi op &Au va trich hoat chdt con

china mot it limonoid nen khi trOn vdi phan bon nh.t phan ure có thd diet 01rOc cac tuydn trung gay benh bu'ou re, mOt loai benh lay lan nhanh va co tac hai rat ion cho cay trong

III Sri dung cay neem cle bio vg thtic vat

Hot chg limonoid trong cay neem (4)

Hoat chit du tien c6 tac dung gay ngan an cho loai chau chdu Schistocerca

gregaria' clack 2 nha hoa hoc Morgan va Butterworth ly trich tit nam 1968 va clat

ten la azadirachtin (AZ) Kdt qui this nghiem sau 06 cho thdy azadirachtin a no'ng

ci0 50 ppm gay ngan an cho loai Earias fabia (Phytoparasiteca 9/1, 27, 1981), a

n8ng dO 5 - 10 ppm ngan chan sg phat tridn cua loai Spodoptera litura (Indian J

Expt Biol, 23(3), 16, 1985) Ira a n8ng ci0 10 - 100 ppm ngan chan sti phat tridn va

ski sinh san cila loai Epilachna verivestis (Z Angew Entomol, 93, 12, 1982) LD5o cua azadirachtin d6i vdi S litura la 1,1 y/g (J Entomol Res 11/2, 166, 1987) Tdc

dung lgu dan cua azadirachtin tii re Mil la ding dirOc Saxena va ctv chirng minh (J Econ Entomol 77/2, 502, 1984)

Tif do cldn nay c6 On 100 tetranortriterpenoid (limonoid) trong do có khoing

30 dan xudt cua azadirachtin du'Oc co lap tii cdc dich trich cua cay neem, có thd

dung de bio Ve thu'c vat Thi du 'hOn hOp azadirachtin A, B, D, E, F, G, H, I, K, L

n8ng dO 1 - 10 ppm co tac dung ngan chan skr phat tridn d6i vdi Epilachna

verisestis (Insectisides of Plant Origin ACS Symposium series, 387, 150, 1989),

azadirachtin do Morgan va Butterworth trich ra trudc day nay du'Oc goi la azadirachtin A 3-Tigloylazadirachtol n8ng 00 1ppm có tac dung gay ngan an 97%

nhidu loai con trung (Tetrahedron 45, 5175, 1989) Cac hop chit khdc (Bing 11.2)

dell co it nhieu tac dung gay ngan an, diet sau bo hay xua du6i hoac ngan chan sg

phat trien va su' sinh san cita con trung (Proceeding, 3 rd International Neem Conference, 1997)

Dau, hat Lavie, D (1967)

1 Meliantriol

Nhom 1 : Tetracyclic triterpen, protolimonoid

Nhom 2 : Apo-tetracyclic triterpen, apo-

protolimonoid can xucit tit nhom 1 do sif chuyin

vi apo

Nhom 3A : Cdc limonoid &it ngudn tet nhom 2

do mat nguyen ti C4 a mach nhcinh va sit

chuyin vi coic phcin con lai di tgo thanh vOng

y-hydroxybutenolid Pentacyclic nortriterpenoid

Nhom 3B : Ccic limonoid co ngudn gdc tit nhom

2 do mat nguyen tet C4 a mach nhcinh va sy

chuyin vi cda phcin con lai de tao thanh yang

furan Pentacyclic nortriterpenoid

Bang II.2 : Mot s6 hoat chdt limonoid cla difoc trich ra tit cac b0 phan Ichac nhau cua cay neem

17 Nimbinin DAL' hat Narayanan, C.R

(1967)

18 la-Methoxy-1,2-dihydroepoxyazadiradion Hat kho Krauss, W (1981)

19 113, 23-Diepoxy-azadiradion Hat kho Krauss, W (1981)

20 7-Desacety1-7-benzoylepoxYazadiradin Hat kilo Krauss, W (1981)

21 Meldenindiol La xanh Pachapurkar, R.V

24 7-Acetylneotrichilenon Hat kilo Krauss, W (1981)

(1969)

26 Nimbolin A Than ei Ekong, D.E.0 (1969)

su oxi hoc, cdt chit yang C Ccic limonoid yang

C-seco voi y-hydroxybutenolid yang

33 Isoazadirolid La ttidi Siddiqui, S (1986)

34 Margosinolid Canh tddi Siddiqui, S (1986)

35 Isomargosinolid Canh tadi Siddiqui, S (1986) NI- im 4B : Cdc limonoid co Vic tit nhom 3B do

su oxi hoa cdt chat veng C Cdc limonoid vang

C-seco voi furan vemg Nhom furan vOng nay có

the bi oxi hoa them, nhu cdc limonoid suein

azadirachtin C-seco

36 Azadirachtin Hat Krauss, W (1986)

37 22, 23-Dihydro-23-13-methoxyazadirachtin Hat Krauss, W (1986)

38 3-Tigloylazadirachtol Hat Klenk, A (1986)

41 Nimbandiol Dau hat, la kilo Krauss, W (1981)

42 6-0-Acetylnimbandiol Dau hat Krauss, W (1981)

(1985)

44 Nimbinen Wu hat, la, vó Krauss, W (1981)

45 6-Desacetylnimbinen Ulu hat, la, vo Krauss, W (1981)

49 Nimbidic acid Wu hat Mitra, C.R (1970)

(1968)

51 3-Desacetylsalannin Dau hat, la Krauss, W (1981)

53 2', 3'-Dehydrosalannol La Garg, H.S (1985)

Nhom 5 : Cdc limonoid xudt phcit ti! nh6m 3B

do su oxi h6a yang D de tao thanh lacton

tddi

Lavie, D (1971)

55 7-Desacetyl-7-benzoylgedunin Hat kilo Krauss, W (1981)

56 Nimolicinol Trai chin, tu'di Siddiqui, S (1984)

111.2 MOt s6 the phdm tit cay neem tren thi tru'o'ng the gidi

Tit lau nhan dan An DO cla biet sit dung cac bO phan ctia cay neem de chi& xudt thu ding mot so hoat chdt dung de bao ve thyc vit Cac the phgm nay ngay cang du'dc nhan dan u'a chuOng mac au hieu qua khong cao Do do nam 1991 Chinh pha An D6 da ban hanh Quy dinh ve cac san phSm di6u the tit cay neem NBPA (Neem Based Products Act) de quan 11 va cong nghiep hoa viec san xudt cac the pha'm tlY neem dung trong nOi dia va xudt khgu Theo quy dinh nay, ngoai mot so- tieu chuSn can dat, tieu chudn ve chdt htdng co tinh cach guy& dinh la ham litdng tong azadirachtin thu6c limonoid AZL (Total Azadirachtin Related Limonoid) Ham htdrig AZL trong ch6 phdm tit dau phai cao hdn 300 ppm va tii hat phai cao hdn 1500ppm Trong s6 hdn 20 ch6 phdm tit neem clang Idu hanh tai An

DO cla có 7 the phSm dang 1(1 theo quy dinh nay tai Oy ban Trung Ming Quan 1Yr Thu6c tra' sau An DO CIB (Central Insectiside Board) Ngoai An DO ding co khoang 15 ch6 phdm tii neem trong do co 3 ch6 phdm a My clang k1 vdi Cd quan Bao ye Moi traing EPA (Environmental Protection Agency) va 2 ch.6 phdm a Dttc Bang ky theo Chu'dng trinh Quan VT Thu6c trit sau - Ndm benh IPM (Insect - Pest Management) (Bang 11.3)

Bang 11.3 : M6t s6 ch6 phdm dieu the tU' cay neem dang ltitt hanh tren thi tru'ong the gidi (5, 7, 8)

TT Ten the pha'm Hoat chdt chinh Tac dung Co sa san xudt

2 Isatin-EC (Align) 3% AZL Tat sau Agridyne Tech Inc (Salt

Lake City, UT)

(Philadelphia, DA) Trifolio-M-GmbH (Germany) Trifolio-M-GmbH (Germany)

chin phat trien va sinh sin

M/S Godreg Agrovet Ltd, Bombay 400074

7 Margocide CK AZ va 15 clan xudt Gay ngan an, ngan

chan phat tridn va sinh san

M/S Monofix Agroproducts Ltd Hubli - 580029

—15—

8 Neemgold AZL trich tit nhan

hat neem

Gay ngan An M/S Southern Petro Chem

Ind Corp Ltd Madras

M/S T Stanes and Co Ltd Coimbatore 641018

10 RD-9-Repelin AZL 3000 ppm,

Pongania, Annona, Castor products

Gay ngan An, xua dudi

M/S ITC Ltd, Rajahmundry, Andhra Pradesh

11 Nethrin Dal u neem Diet na'm M/S Amital Agrochem Pvt

Ltd Gorakhpur - 273001, U.P

12 Amitul

Ddu neem Diet mai M/S Amitul Agrochem Pvt

Ltd Gorakhpur - 273001, U.P

Cho chuOt u6ng cao methanol 75% trich tit la neem n8ng cl() 13 pg/kg thdy bung chuOt bi co giat (Sinnials et al, 1983) Cho chuOt lang u6ng dung dich la neem kho trich vdi nu'dc d ang 6;1 50 - 200 mg/kg lam cho chuOt bi is chay va sut can Cho u6ng hoac chich cao hat vao chuOt va theo doi trong thdi gian 3 thang, khOng thdy chuOt bi ung bu'du (Sardeshpande, 1976)

Gan day cac nha hda hoc my da nghien cdu dOc tinh cua the pham

"Margosan-O dam (lac 3000 ppm" va ke't qua nghien citu nay da dudc Cd quan Bao ye Moi tru'dng my (EPA) clap nhan cho clang 14 (Bang 11.4)

Bang 11.4 : DOc tinh cua the phgni Margosan-O-dam dac

1 Chim cut LD50 (u6ng dang dam dac) 1 - 16 mUkg

2 Chim cut bach LC50 (pha trong de; an) > 7000 ppm

5 ChuOt ding bat tinh ngay LC50 > 2 mUkg

6 ChuOt bach bi me qua &Ong ho hap LC50 > 43,9 m1/1/h

7 ChuOt lang, bi di ling trong 6h Khong

PHAN III NGHIEN CeU KET QUA VA BAN LUA, N

NOI DUNG 1 : QUY TR1NH CONG NGHE LY TRICH LIMONOID 're LA NEEM HAM LUVNG TONG AZL, AZ VA S TRONG LA

I.1 Phtidng phap va nguyen vat lieu

L1.1 Phu'dng phap ly trich va xac dinh ham lu'dng limonoid

a Di6u the cao phan bo (CPB)

Dung cac dung mai th6ng dung 0 dO phan clic tang clan nhu' ether au, chloroform, ethyl acetat va ethanol dd ran lu'Ot trich ly cac thanh phan hoa hoc cd ban cua la neem trong binh Soxhlet (Hinh 1.1) Sau d6 thu h6i dung moi du'di dp

suit kern trong binh co quay (Hinh 1.2) cho ra 4 loai CPB cua la neem Quy trich dieu chd CPB difoc trinh bay trong sd a 1.1

b Xac dinh cac thanh ph'in hoa hoc cd bin cua la neem

DUng cac thu6c this biet tinh dd xac dinh thanh phan hda hoc cd ban trong CPB (Bang 1.1)

Bang 1.1 : Thu6c this biet tinh dd xac dinh thanh phan h6a hoc cd ban (9,10)

1 Sterol Liebermann-Burchard Mau d6 - hang

Tram hien nau Tram hien yang nau

Dd FeC13 1% trong nil&

Mau yang nhat a lau hoa nau

Dd chuydn mau xanh

7 Chit be° Thim vao giiy loc Vet dau loang tren giiy loc

c Xac dinh dung mei cc!, khi Wang trich ly tett nhat limonoid

Nham tim mot dung mai 0 kha nang trich ly tot nhat hoat chit limonoid trong

la neem chtIng t8i Ian lttOt trich bOt la neem vdi cac dung mai tieu bidu cho cac dung mai thuting dung dd trich hoat chit trong hOp chit HI nhien nhu' ether dau, chloroform, ethyl acetat, aceton, ethanol Thu hai dung mai du'di dp suit kern cho

ra 5 loai cao trich ly (CTL) (SO de) 1.2) (11,12) Dinh li/Ong limonoid trong moi loai

CTL dd xac dinh kha nang trich ly limonoid cua moi dung mai (11,12)

—19—

HInh 1.1 : Trich ly cdc thanh phan h6a hoc cd ban

ciia ld neem trong bInh Soxhlet

Hinh 1.2 : Thu hed dung m6i dtioi dp suit kdm trong binh c6 quay

- Chie't trong Soxhlet vdi ether dau

- Thu h6i dung mai

- Chi6t tie/p trong Soxhlet vdi chloroform

- Thu h6i dung mai du'di ap sua't kern

- Dinh tinh cac thanh plign hda hoc co ban trong cao ether au,

Ke't qua dinh tinh

i- Dinh tinh thanh phAn

hoa hoc cd ban tong

cao chloroform - Chit tie'p trong Soxhlet vdi ethyl acetat

- Thu h6i dung mai difdi dp sua't kern Kel qua dinh tinh

- Chith tiep trong Soxhlet vdi ethanol

- Thu h6i dung mai dU'di dp sua't kern

- Dinh tinh cac thanh phan hoa hoc cd ban trong cao ethyl acetat

11, - Dinh tinh cac thanh phan hoa hoc cd ban trong cao ethanol

K6t qua Binh tinh

Sci coo 1.2 :

LY TRICH HOAT CHAT LIMONOID TRONG LA NEEM VOI CAC DUNG MOI CO DO PHAN CVC KHAC NHAU

B6t la neem (I) (100g)

— Chie't trong Soxhlet 12 gier vdi 400m1 dung moi (2)

— Chang cat du'di ap sua't kern thu 118i dung moi

— Trich tie'p vdi CH2C12 (3x300m1)

— Lam khan vdi Na2SO4

— Chu'ng cat du'di ap sua't kern thu h8i

CH2C12

Cao trich ly (CTL) yeti dung moi

Ghi dal :

(1) BOt la neem &roc ;dr 15, qua cac cong doan ghi trong muc I.1.2.a

(2) Trich ly Fan lticrt vdi cac dung moi : ether au, chloroform, dichlorometan,

ethyl acetat, aceton, methanol, ethanol

d Dinh liking limonoid AZL trong 16 neem

i Chgn phtidng phap

Cay neem du'oc sit dung lam thu6c bac) ve thdc vat rat co hieu qua a nhidu ndi tren the gidi do trong la, hat va dau neem c6 chda nhidu hoat chdt tetranortriterpenoid (limonoid), quan trong nhdt la azadirachtin (AZ), dinh luting bang HPLC (Yamaski et al 1986; Sundaram and Curry, 1993; Azam et al 1995) hoac bang sac ky vdi chat long sieu tdi han (Huang and Morgan 1990) Tuy nhien

AZ khong phai la hoat chdt duy nhdt trong hat va dau neem c6 tac dung bao ye thu'c vat con trong la neem mac dau khong c6 AZ nhu'ng van co hoat tinh tot bao ve thiic vat Theo Verkerk va Wright (1993) hdp chdt limonoid ly trich tit hat neem co hoat tinh manh gdp 3, 4 lan bring AZ co trong hdp chdt nay Nhi.t the trong hdp chit limonoid ly trich tii hat neem, ngoai AZ ra con co mot s6 limonoid khac co hoat tinh tildng tii nhir AZ du'Oc goi chung la nhom AZL (Azadirachtin related Limonoids) g8m khoang 30 hdp chdt limonoid (8,4) Ngoai 5 hop chdt limonoid chinh la azadirachtin, (nay goi la azadirachtin A), salannin, meliantriol, nimbin, nimbinin, AZL can Om co 10 hdp chdt limonoid khac co cdu tn.ic siRJn arcing tti vdi

azadirachtin ddcfc goi ten azadirachtin B, C, D, E, F, G, H, I, K, L va khoang 15 hdp

chat limonoid dan xuaet cua azadirachtin, salannin, meliantriol va nimbin Cac hdp chdt nay co tac dung bao ve thiic vat phu trci lAn nhau AZL tao mau xanh luc vdi

thuoec this Salkowski (vanillin/H2SO4 dd) nen nhidu nha h6a hoc da dung phddng

phap so mau de do AZL a = 577nm tren may quang ph6 tit ngoai (9) La neem khong chda AZ ma chda nhidu hdp chdt limonoid khac thu6c AZL nen cling co the

do hoat tinh bang phddng phap so mau nay Ham luting AZL trong la phan anh dung hoat ldc cua la dung de bac) ye thiic vat (11) Nhu the de do hoat ldc cua cac chee phim tit neem ngd6i to dinh lacing AZL vdi thu6c thd Salkowski thay vi chi xac dinh AZ nhd trtidc day (9, 10)

Trong thi nghiem nay chung t6i dinh lu'Ong AZL trong la neem bang pinking phap so mau ci x = 577nm tren quang ph6 ld tit ngoai UV hieu CARY 50 (Hinh 1.3) du'a vao diking chub' xay dung theo dinh luat Beer-Lambert A = elc

ii Xfiy dtfng dtieing chudn :

— Pha 6 dung dich chan azadirachtin AZ 95% trong CH2C12 co n8ng dO: 0,01 mg/ml; 0,02 mg/ml; 0,04 mg/ml; 0,06 mg/ml; 0,08 mg/ml va 0,1 mg/ml Vdi m8i dung dich, thdc hien cac cong doan nhtt sau :

— Ldy 1 ml dung dich chuin AZ 95% trong CH2C12, cho vao 0,2 ml dung dich vanillin trong Me0H (0,02 mg/ml) Lac manh htin hdp va a yen a t° phong trong 2

phtit Cho tie-p 0,3 ml H2SO4 98%, chia lam 3 Fan (m8i Fan 0,1 ml), lgc manh trong

10 sec sau moi lAn cho H2SO4 vao

— Cho them 0,5 ml Me0H de dOng nhdt 2 pha cua h8n hdp Luc nay dung dich chuyen ra mau xanh luc De yen trong 5 phut de on dinh mau

— Do 010 hdp thu A (absorbance) cUa 6 dung dich chudn thiic hien gi6ng nhau nhu' tren, a X 577nm tren quang ph6 k6 tit ngoai, so vdi mau trang chda cling thanh phan nhung khong co AZ 95%

- ve clu'ong bleu din (dttaing chudn) tren true toa do, true hoanh ghi nOng dO

c ctia dung dich chudn, truc tung ghi (10 hip thu A Wong Ong Dodng chudn la mot clu'dng thong qua tam toa do (Hinh 1.4)

Dila vao doing chudn nay de suy ra n6ng do AZL sau khi do dOcic dO ha-pthu

A ciia dung dich CTL vdi dung mai trong CH2C12 Quy trinh k9 thuat dinh liking AZL &roc truth bay trong sct do 1.3

HInh 1.3 : May UV hieu CARY 50 do 80 hap thu A a = 577nm

HIM 1.4 : Thiong chudn bidu dien dO hip thu A vs nOng CIO dd azadirachtin chudn dung trong phuccng phap so mau

1.0 0.9

Azadirachtin ming dO c (mg/ml)

—24—

Sd del 1.3 :

QUY TRINH DINH LthiNG AZL

Cao trich ly (CTL) trong dung mai

Pha CTL trong CH2C12, chon !tong di) ci thich hop

Lay 1 ml dd CTL trong CH2C12 (ming dO ci), cho vao 0,2 ml dd vanillin trong Me0H (0,02 mg/ml) LAc manh am hop va de yen of t° phang trong 2 phtit

Cho vao tiep 0,3 ml H2SO4 98%, cho mOi rAn 0,1m1 vao va lac manh sau mtii lAn cho H2SO4 vao

Dd CTL + thu6c this Salkowski

- Cho vao 0,5 ml Me0H, lgc manh Dung dich chi dm 1 pha Mau xanh luc xuSt hien

- De yen 5 phtit de On dinh mau

Dd matt CTL

- Do A tren quang ph6 ke UV so vdi mau trong

- Du'a vao ctidng chugn suy ra n6ng d6 c2 ttiOng dng vdi A cila AZL trong dd CH2C12

- DI nong dO c2 tinh ra ham hiong c3 cila AZL trong nguyen lieu

Ham lacing AZL

1

e Dinh hfccng azadirachtin (AZ)

i ChQn phticing phap Ham lu'Ong AZ trong cac b0 phan cita cay neem bi anh hu'Ong cua nhidu ydu

to nhic am dO, khi hau, thdi gian phdi nAng du'di anh sang mat trdi, thdi gian luU till, cach say kho, dO pH va pinking phap trich ly (15)

Co nhidu pinking phap du' cc dung dd clinh Ming AZ nhu'ng phu'dng phap du'dc sit dung rOng rai nhdt la dinh hidng vdi may HPLC (Hinh 1.5)

Mac du AZ kh6ng phai la chdt duy nhdt co host tinh sinh hoc trong cay neem nhu'ng do no co n6ng d6 cao hdn cac limonoid khac nen cac nha h6a hoc chon AZ lam chdt dai dien cho limonoid trong cay neem (Hinh 1.7, 1.8, 1.9)

ii Xay thing duTing chan

- Pha 4 dung dich chugn azadirachtin AZ 95% trong Me0H co ming dO

C=1,775 mg/1; 3,55mg/1; 7,10 mg/1; 14,20 mg/1

- Chay HPLC vdi cac thong so sau : Instrument : HPLC - UV Packing column : C18, 250 mm x 2.0 mm i.d

0

Eluent : ACN-H20 (40:60) Flow rate

- Peak azadirachtin xut hien 8 R, = 8,04 min (Phu luc 1)

Detection UV detector, 217 nm

: 20 Sample loop

:

;I

ae

I 0,30 ml/min

- Peak area (PA) du'dc tinh b'Ang mV

I

c1 = 1,775 mg/1 PA I = 98555.80 c2 = 3,55 mg/1 PA2 = 189544.80 c3 = 7,10 mg/1 PA3 = 350215.89 c4 = 14,20 mg/1 PA4 = 667052.40

- Ve du'dng chuSn (Hinh 1.6)

I I

mg/1 HInh 1.6 : Diking chugn cila azadirachtin bidu din

peak area (PA) vs n6ng ci0 C (mg/1)