azadirachtin-a and tetrahydroazadirachtin-a concentrates: preparation, lc-ms characterization and...

Pest Management Science Pest Manag Sci 62:965–975 (2006)

Azadirachtin-A andtetrahydroazadirachtin-A concentrates:preparation, LC-MS characterization andinsect antifeedant/IGR activity againstHelicoverpa armigera (Hubner)Vandana Sharma,1 Suresh Walia,1∗ Swaran Dhingra,2 Jitendra Kumar1 andBalraj S Parmar1

1Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi 110 012, India2Division of Entomology, Indian Agricultural Research Institute, New Delhi 110 012, India

Abstract: A 60% azadirachtin-A concentrate has been obtained through repeated precipitation with hexane froma methanolic solution of a 20% concentrate. Azadirachtin-A (90%) has been obtained by medium-pressure liquidchromatography of the 60% concentrate with an RP-18 column and a methanol + water (1 + 1 by volume)solvent system. Catalytic hydrogenation of the 60 and 90% azadirachtin concentrates yielded the correspondingtetrahydroazadirachtin concentrates. Dihydroazadirachtin and tetrahydroazadirachtin formed during the first 5 hof hydrogenation were identified by LC-ESI-MS on the basis of their unique mass fragmentation pattern. Theefficacy of tetrahydroazadirachtin concentrates in inhibiting the feeding and growth of Helicoverpa armigera(Hubner) larvae has been compared with that of azadirachtin concentrates. They were in general more activeand deterred larvae from feeding at all concentrations. Tetrahydroazadirachtin-A (90%) and azadirachtin-A (90%) with respective IC50 values of 280 and 390 mg L−1 were effective as insect growth regulators, whiletetrahydroazadirachtin-A (90%) displayed higher antifeedant activity (AI50 14 mg L−1) against the test insect. 2006 Society of Chemical Industry

Keywords: neem; azadirachtin; dihydroazadrachtin; tetrahydroazadirachtin; LC-MS; antifeedant; insect growthregulator

1 INTRODUCTIONThe Indian neem tree, Azadirachta indica A. Juss(Meliaceae) has received special attention from theinternational scientific community because of itsexcellent pest control properties. Its constituentsmanifest acute biological activity, reflected by insectgrowth disruption, feeding and oviposition deterrence,repellence and mortality inflicted on a variety ofpests infesting agricultural and horticultural crops.1,2

Azadirachtin-A, a meliacin obtained from this tree,has been rated as a potential botanical pesticide.Its ability to deter feeding in many insect pests atvery low concentrations makes it a valuable tool ininsect pest management.1–3 Various methods havebeen reported for the isolation and purification ofazadirachtins from neem seed kernels.4–6 Recently,a medium-pressure liquid chromatographic method(MPLC) has been effectively employed to obtain threeof the major bioactive azadirachtin congeners, A, Band H, and their structures have been establishedon the basis of the unique fragmentation pattern7

in electrospray ionization mass spectroscopy (ESI-MS). On account of the limited stability of these

compounds under natural conditions of temperatureand light,8,9 efforts have been made in the past tostabilize them, either through structural modification10

or by using stabilizers, including antioxidants andUV/sun screens.11–13 Structure–activity relationship(SAR) studies have indicated that modifications tothe basic molecule dramatically alter the activity.10,14

Detailed investigations on the antifeedant and insectgrowth regulator (IGR) activities of azadirachtins andrelated products have been reported.14–16

The vulnerability of azadirachtins to various naturalenvironmental factors such as light, heat, humidity andothers has raised concerns about the viability of neemmaterials in pest control. Thermal and photolabil-ity are serious bottlenecks for its commercializationand use. Efforts are therefore needed to developazadirachtin-A-enriched products with enhanced sta-bility and efficacy. Efforts have been made in thepast to reduce azadirachtin-A to yield more stable di-or tetrahydroazadirachtin. Dihydroazadirachtin-basedproducts are registered by the Environment Protec-tion Agency (EPA) for use in the USA. Unfortunately,tetrahydroazadirachtin-based products have still not

∗ Correspondence to: Suresh Walia, Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi 110 012, IndiaE-mail: suresh [email protected](Received 11 June 2005; revised version received 4 March 2006; accepted 14 March 2006)Published online 17 July 2006; DOI: 10.1002/ps.1265

2006 Society of Chemical Industry. Pest Manag Sci 1526–498X/2006/$30.00

V Sharma et al.

been commercialized, but they hold potential in viewof their effect in reducing the problems of lability.

The present study aims at the preparationof azadirachtin and its more stable tetrahydro-azadirachtin-enriched concentrates, their LC-ESI-MScharacterization and comparative evaluation of theirIGR and antifeedant activities against the polyphagousinsect pest Helicoverpa armigera (Hubner) which affectsa large number of agricultural and horticultural crops.

2 MATERIALS AND METHODS2.1 Chemicals and plant materialsLaboratory-grade reagents and solvents were locallyprocured. The solvents were distilled and, whereverrequired, dried before use. HPLC-grade methanol and‘SQ’ methanol (Qualigens Fine Chemicals, Mumbai)were used for analytical HPLC and MPLC separationof azadirachtins. Water for chromatographic analyseswas purified using a Milli-Q water purificationsystem. Neem seed kernels were procured from NeemMission, Pune, India.

2.2 Chromatography and spectroscopy2.2.1 High-performance liquid chromatography(HPLC)A Waters HPLC system equipped with a 600 seriespump and controller, a 996 PDA detector and a Rheo-dyne injector was used for analyses of azadirachtinsand tetrahydroazadirachtin in the concentrates. Sep-aration of azadirachtins and tetrahydroazadirachtinwas achieved under isocratic conditions at a flowrate of 0.75 mL min−1 using a mobile phase ofmethanol + water (50 + 50 by volume). A 20 µL sam-ple was injected each time via a Rheodyne injector(20 µL loop) for a run time of 10–20 min. Peaks weredetected at 217 nm and the retention time (RT) wasmeasured for each compound. Calibration and quan-tification were carried out using Waters Millennium2010 Chromatography Manager GPC software ver-sion 2.0. Azadirachtin in the samples was quantifiedby employing a standard azadirachtin sample (95%pure) obtained from Sigma Aldrich:

Azadirachtin content = (A1/A2) × (m2/m1) × P

where A1 is the peak area of azadirachtin in the sample,A2 is the peak area of azadirachtin in the referencestandard, m1 is the mass (g) of the test sample, m2 isthe mass (g) of the reference standard and P is thepurity of the reference standard sample.

2.2.2 Medium-pressure liquid chromatographyMedium-pressure liquid chromatography (MPLC)of azadirachtin concentrate was performed on anMPLC pump and a glass column (tayperling type,600 × 40 mm) prefitted with a glass guard column(15 × 25 mm) and an automatic fraction collector.Both the columns were packed with RP-18 material.The pump was operated at 10–15 psi (69–103 kPa)

pressure and the column was run with methanol +water (55 + 45 by volume) for 2 h at a flow rate of2 mL min−1.

2.2.3 Nuclear magnetic resonance spectroscopy(1H NMR)1H NMR spectra of azadirachtin-A (90%) andtetrahydroazadirachtin-A (90%) were recorded on aVarian INOVA 300 MHz instrument in deuterochlo-roform using tetramethylsilane (TMS) as an internalstandard. Chemical shifts are reported in δ valuesrelative to TMS.

2.2.4 Liquid chromatography–mass spectroscopy(LC-MS)LC-MS analysis of azadirachtin-A and tetrahydro-azadirachtin-A was performed on a Shimadzuquadrapole LCMS-2010 instrument equipped withatmospheric-pressure chemical ionization (APCI) andESI probes, an SIL-10 ADvp autoinjector, a CTO-10Avp column oven, a SCL-M10 system controllerand an SPD-M10 PDA detector (190–370 nm) withanalogue output of 217 nm. LC-MS was performedon an Inertsil ODS-3, 2.1 mm ID × 250 mm, 5 µmcolumn using an acetonitrile + water gradient at0.2 mL min−1 flow rate. MS was recorded using ESIinterphase at room temperature in the mass range300–900 m/z. For qualitative analysis, 10 µL of thesample was injected each time to record LC-MS data.

2.3 Preparation of azadirachtin-A andtetrahydroazadirachtin-A concentrates2.3.1 Azadirachtin-A concentrate (20%)Ground neem seed kernel powder (1 kg) placed in aconical flask containing hexane (2 L) was agitatedwith a mechanical stirrer for 1 h. The materialwas allowed to stand overnight, after which themixture was agitated for 30 min and filtered througha Buchner funnel under vacuum. The process wasrepeated to ensure complete removal of neem oil.The combined hexane extract on concentration undervacuum yielded neem oil. The deoiled seed cake wasextracted in the same manner with methanol (3 × 1 L)and the combined methanol extract after filtrationwas concentrated under vacuum at 40oC. It wasquickly partitioned between hexane and 95% aqueousmethanol to remove the residual oil, if any. Themethanol extract after concentration under vacuumwas partitioned between water and ethyl acetateto remove water-soluble proteins and sugars. Theorganic phase was concentrated to 25 mL volume andprecipitated with hexane. The precipitate was filteredand dried to obtain azadirachtin powder concentrate(≈20% aza-A, 8.0 g). The extraction process wasrepeated to obtain a sufficient quantity of technicalazadirachtin powder concentrate.

2.3.2 Azadirachtin-A concentrates (60%)A solution of crude azadirachtin powder concentrate(≈20% aza-A, 5 g) in methanol containing charcoal

966 Pest Manag Sci 62:965–975 (2006)DOI: 10.1002/ps

Preparation and insecticidal activity of tetrahydroazadirachtin

(1 g) was stirred magnetically to remove impurities.It was filtered through a celite-charcoal bed, con-centrated under vacuum to a viscous mass, redis-solved in ethyl acetate (10 mL) and precipitated withhexane to obtain azadirachtin-A-enriched precipitatewhich was redissolved in ethyl acetate and precipi-tated. The precipitate was filtered and dried to obtainazadirachtin-A-enriched powder concentrate contain-ing approximately 60% azadirachtin-A as analysed byHPLC. A quantity of 5 g of the azadirachtin concen-trate (∼20%) furnished about 1.25 g of azadirachtin-A(60%) concentrate. The dried white powder wasstored in a refrigerator.

2.3.3 Azadirachtin-A concentrates (90%)Azadirachtin-A concentrate (60%, 750 mg) was dis-solved in minimum quantity of the eluting solvent(5 mL) and subjected to MPLC7 using methanol +water (50 + 50 by volume) at a flow rate of2.0 mL min−1. The fractions with a TLC/HPLC pat-tern similar to that of the standard reference ofazadirachtin-A were combined. Azadirachtin-A wasrecovered after evaporating the solvent under vacuumat a temperature not exceeding 40 ◦C, mp 155–56 ◦C(Rf 0.65, dichloromethane + methanol 96 + 4 by vol-ume).

1H NMR: δ 1.31 (d, 1H, 16-Hb), 1.71 (dd, 1H, 16-H), 1.75 (s, 3H, 30-CH3), 1.78 (d, 3H, 4′-CH3), 1.85(br s, 3H, 5′-CH3), 1.95 (s, 3H, 3-OAc), 2.01 (s, 3H,18-H), 2.13 (ddd, 1H, 2-Hb) 2.34 (m, 1H, 2-Ha),2.38 (d, 1H, 17-H), 2.89 (br s, 1H, 7-OH), 2.92 (brs, 1H, 20-OH), 3.33 (s, 1H, 9-H), 3.35 (d, 1H, 5-H),

3.63 (dd, 1H, 19-Ha), 3.68 (s, 3H, 12-OCH3), 3.76(d, 1H, 28-Ha), 3.80 (s, 3H, 29-OMe), 4.07 (d, 1H,28-Hb), 4.15 (d, 1H, 19-Hb), 4.60 (dd, 1H, 6-H),4.67 (d, 1H, 15-H), 4.73 (d. 1H, 7-H), 4.75 (d, 1H,1-H), 5.03 (s, 11-OH), 5.05 (d, 1H, 22-H), 5.50 (dd,1H, 3H), 5.65 (s, 1H, 21-H), 6.46 (d, 1H, 23-H),6.93 (dq, 1H, 3′-H).

LC-ESI-MS (m/z, relative intensity): 759.3(3.125%), 743.38 (45.0%), 721.26 (11.25%), 720.0(11.25%), 703 (100%), 685 (36.875%), 667(4.375%), 625 (1.875%), 603.34 (4.375%), 585.37(11.875%), 567.32 (13.75%), 507 43 (4.375%).

2.4 Tetrahydroazadirachtin-A (60%, 90%)concentrateAzadirachtin-A (60%, 2 g) and azadirachtin-A (90%,1 g) solutions in methanol were hydrogenated asdescribed elsewhere17 to yield the correspond-ing reduced tetrahydroazadirachtin-A. LC-MS ofthe partially reduced product obtained after 5 hhydrogenation is depicted in Fig. 1. Hydrogena-tion was continued for another 3 h to obtainthe tetrahydroazadirachtin-A derivative. Its puritywas checked by HPLC, mp 158–60 ◦C (Rf 0.68,dichloromethane + methanol 96 + 4 by volume).

1H NMR: δ 0.93 (t, J = 7.4 Hz, 2.25H, 4′ Me),0.95 (t, J = 7.4 Hz, 0.75H, 4′ Me), 1.16 (d, J = 6.8Hz, 2.25H, 5′ Me), 1.17 (d, J = 7.1 Hz, 0.75H, 5′Me), 1.45 (m, 2H, H-3′ ab), 1.58 (br d, J = 12.0 Hz,1H, H-16b), 1.78 (m, 1H, H-2′), 2.06 (s, 3H, 3-Ac),2.10 (m, 2H, H-22 ab), 2.22 (s, 3H, 18-Me), 2.53 (brd, J = 5.0 Hz, 1H, H-17), 3.07 (d, J = 12.5 Hz, 1H,

0 5 10 15 20 25 min

0

25

mA

bs

ID#1 217nm (1.00)

14.2

75

15.5

61

17.0

48

RT = 15.561 min

300 400 500 600 700 800 m/z

0.0e6

1.0e6Int.

703.3

685.2585.1

766.3603.3507.1 667.2 784.1625.3 721.2463.3403.1343.2303.1 841.2 897.0

300 400 500 600 700 800 m/z

0e3

100e3Int.

719.1

720.1755.1659.1 833.3

817.0485.1 857.1599.1558.6 892.8641.1430.9312.9 373.8

B

C

Aza-A

Retention time

A

Figure 1. LC-MS spectrum of azadirachtin-A: (A) LC chromatogram; (B) ESI-MS in positive ion mode; (C) ESI-MS in negative ion mode.

Pest Manag Sci 62:965–975 (2006) 967DOI: 10.1002/ps

V Sharma et al.

H-5), 3.61 (d, J = 8.9 Hz, 1H, H-28), 3.8–4.1 (m,2H, H-23ab), 5.24 (s, 1H, H-21).

LC-ESI-MS (m/z, relative intensity): 763.33(11.875%), 747.38 (100%), 725.32 (39.375%),724 (7.5%), 707 (24.375%), 689 (16.875%),671(2.815%).

2.5 Antifeedant and insect growth regulatoryactivity against Helicoverpa armigera larvae2.5.1 Stock and test solutionsAppropriate quantities of azadirachtin-A concentrates(20, 60 and 90%) and tetrahydroazadirachtin-Aconcentrates (60 and 90%) were weighed accuratelyinto 10 mL volumetric flasks and dissolved in asmall amount of distilled acetone. The volume wasthen made up to 10 mL with acetone to provide a2000 mg L−1 active ingredient stock solution. Fromthe stock solutions, 1000, 700, 500, 100 and70 mg L−1 solutions were prepared by serial dilutionwith 5 ml L−1 Tween 80 emulsifier in distilled water.

2.5.2 Rearing of Helicoverpa armigeraLarvae of H. armigera collected from the field werereared on artificial diet in sterilized specimen tubes(10 × 3.75 cm) at 27 ± 1 ◦C and 70% RH untilpupation. The freshly emerged adults were transferredto jars (20 × 15 cm) containing cotton swab dipped in10% honey solution and covered with muslin cloth.The muslin cloth containing eggs was transferred toanother jar with 70–90% RH to prevent desiccationof the eggs. Freshly hatched larvae were transferred topetri dishes containing artificial diet. After 3 days thelarvae were transferred to individual specimen tubes toavoid cannibalism. The third-instar larvae were usedin studies on antifeedant activity and insect growthinhibition.

2.5.3 Antifeedant bioassay (no-choice method)The standard no-choice bioassay method18 was usedto quantify the antifeedant activity of the test meliacins.Leaf discs (diameter 60 mm) were cut from cabbage(Brassica oleracea botrytis L.) leaves, washed withwater, dried under shade, treated with test solutions,dried and transferred to clean rearing bottles withthe bottoms lined with a circular piece of filterpaper, to the centre of which 1 mL of tap water wasapplied. This provided a minimum humidity in thebottle, preventing rapid desiccation of the leaf discafter excision. One third-instar larva of H. armigera,prestarved for 2 h, was released in each rearing bottle.The leaf area consumption was measured after 24and 48 h treatment by taking observations using graphpapers. Later, the larvae were provided with artificialdiet and kept separately in vials. Consumption data ofleaf discs treated with methanol + aqueous emulsifierserved as control. Corrected feeding inhibition wascalculated as:

Corrected feeding inhibition (%)

= [(C − T)/(C + T)] × 100

where C and T represent the leaf areas consumed fromcontrol and treated leaves respectively.

Antifeedancy activity (AI50) values were calculatedby using a basic LD50 program version 1.1.19

Analysis of variance (ANOVA) was carried out forall AI50 values by using Duncan’s multiple range test(DMRT).20

2.5.4 Insect growth regulatory activityThird-instar larvae of H. armigera weighing between30 and 60 mg each were sprayed with differentconcentrations of the test emulsions using a Potterdirect spray tower at a pressure of 340 g cm−2.Three replicates of ten insects each were takenper treatment. The sprayed dishes were dried for5 min under a fan, after which the larvae weretransferred to separate rearing bottles. Larvae sprayedwith aqueous emulsifier served as control. Duringthe post-treatment period, the larvae were providedwith artificial diet and kept separately in vials. Larvalmortality, larval–pupal intermediates, deformities inlarval, pupal and adult stages and adult emergencewere recorded daily.

Larval weight was taken at 3, 5 and 7 daysafter treatment (DAT). The raw data on differentparameters were subjected to angular transformation(arc sine percentage)1/2 and analysed statistically.Analysis of variance was done, and means wereseparated on critical difference (CD). The percentagereduction in larval weight gain compared with control(% growth inhibition) was calculated by the followingformula:

% Growth inhibition = [(Weight gain in control

− weight gain in treatment)/Weight gain

in control] × 100

IC50 values (inhibition concentration for 50% inhibi-tion of adult emergence) were calculated by using abasic LD50 program version 1.1.19

3 RESULTS3.1 Azadirachtin-A concentrates (20, 60 and90%)Technical azadirachtin powder concentrate (∼20%azadirachtin-A) was obtained from neem seedkernel.7 Azadirachtin-A-enriched concentrate (60%azadirachtin-A) was prepared by repeated purificationand precipitation. A 90% azadirachtin-A concentratewas obtained by medium-pressure liquid chromatog-raphy (MPLC) of the 60% concentrate.7 In elec-trospray ionization mass spectroscopy (ESI-MS), themolecular ion appeared as a faint peak at m/z 721(MH+) in the positive ion mode and as a prominentpeak at m/z 719.1 (M–H) in the negative ion mode(Fig. 1). In the positive ion mode, a set of three char-acteristic peaks at m/z 703, 685 and 667 appearedas a result of the successive loss of water molecules



from the protonated parent ion. Another set of threepeaks at m/z 603, 585 and 567 was indicative ofloss of tiglic acid from the three dehydrated fragmentions. The detailed ESI mass fragmentation patternof azadirachtin-A in positive ion mode was reportedearlier.7

3.2 Hydrogenation of azadirachtin-Aconcentrates: preparation oftetrahydroazadirachtin-A concentrates (60 and90%)An efficient procedure for the catalytic reduction ofazadirachtin-A has been standardized under near-ambient conditions of temperature and pressure.17

An LC-MS chromatogram of azadirachtin-A (60%)before subjection to hydrogenation is given in Fig. 1.The major peak at RT = 15.561 min, exhibiting amolecular ion peak at m/z 719.1 (negative mode)and at m/z 703.3 (M+ – H2O, positive mode), wasidentified as that of azadirachtin-A. An LC-MSchromatogram of the sample withdrawn after 5 hhydrogenation is given in Fig. 2. The disappearanceof the azadirachtin-A peak in the LC chromatogram(217 nm) after 5 h hydrogenation was marked by theappearance of two new peaks of the reduced derivativesat RT = 12.231 and 15.411 min, which, on the basisof their unique ESI mass fragmentation pattern(positive ion mode), have been identified as those of

dihydroazadirachtin-A and tetrahydroazadirachtin-Arespectively. Their structures have been corroborated(Figs 3 and 4).

The first peak (RT 12.231 min) in the positivemode exhibited a protonated molecular ion peakat m/z 723.4 (M + H)+, a base peak at m/z 705.1[(M + H)+ – H2O)] and two major quasi-molecularsodium adduct peaks at m/z 745.2 (M + Na)+ and768.4 (M + 2Na)+. These peaks were characteristic ofdihydroazadirachtin-A. Other major fragments mostlyarise as a result of elimination of water (18 amu) orcleavage of ester bonds, leading to loss of tiglate and/oracetate functions. The first set of three prominentpeaks at m/z 705.1, 687.2 and 669.1 formed as aresult of successive elimination of water (18 amu)from the protonated molecular ion was followedby a second set of less prominent peaks at m/z605.1, 587 and 569 which originated as a result ofsequential loss of tiglic acid (100 amu) from thefirst set of fragment ions. The third set of very lowintensity peaks at m/z 545.1, 527 and 509 wereattributed to the loss of acetic acid (60 amu) fromthe second set of intermediate fragment ions. Themass fragmentation pattern of dihydroazadirachtin-Ais depicted in Fig. 3.

The second peak, recorded as a single peak (RT15.411 min) in the LC chromatogram (217 nm) wasidentified as that of tetrahydroazadirachtin-A. Like

0 5 10 15 20 25 min

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ID#1 217nm (1.00)

12.2

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15.4

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RT = 12.231

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250e3

Int.

705.1 745.2

687.2

723.4768.4

669.1605.1 747.1 786.4545.1422.1 651.3483.4 693.0351.9 875.3

RT = 15.411

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707.2689.3

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725.1

671.3605.3 770.4653.4452.9 744.5346.9 865.3

Retention time

B

C

Dihydroazadirachtin

TetrahydroazadirachtinA

Figure 2. LC-MS spectrum of hydrogenated azadirachtin-A: (A) LC chromatogram; (B) ESI-MS of dihydroazadirachtin in positive ion mode;(C) ESI-MS of tetrahydroazadirachtin in positive ion mode).


V Sharma et al.

m/z 687.2

m/z 569

m/z 605.3m/z 705.1

-H2O

-Tiglic acid

-H2O

O

O

AcO

H3COOC O

COOMe

O

H

O

OO

-H2O

m/z 587

m/z 669.1

-AcOH

m/z 545

Dihydroazadirachtin-A

O

OH

O

O

H

O

COOMeOH

OHOH3COOC

AcO

O

O

OH

Quasi-molecular adduct ion peaksm/z 723.4 (M+H)+

m/z 745.2 (M+Na)+

m/z 761.0 (M+K)+

m/z 768.4 (M+2Na)+

H

O

COOMe

OH3COOC

AcO

AcO

H3COOC O

COOMe

O

H

AcO

H3COOC O

COOMe

O

H

-H2O

-H2O

OH

OHOH

OHOH

H3COOC OOH

COOMe

O

H

H3COOC O

COOMe

O

H

-AcOH

-AcOH

H3COOC O

COOMe

O

H

m/z 527(weak)

m/z 509(weak)

OH

-H2O

-H2O

O

O

AcO

H3COOC O

COOMe

O

H

O

OO

OH

OO

O

H

O

COOMe

OH3COOC

AcO

O

O

-Tiglic acid

-Tiglic acid

Figure 3. ESI mass fragmentation pattern of dihydroazadirachtin-A.

azadirachtin-A, ESI-MS of tetrahydroazadirachtin-A displayed a characteristic molecular ion peakat m/z 725 (M + H)+ along with two prominentsodium adduct peaks at m/z 747.3 (M + Na)+ and770.4 (M + 2Na)+ and a potassium adduct peakat m/z 763. As noticed in dihydroazadirachtin-A,the first set of three prominent peaks appearingat m/z 707.2, 689.3 and 671.3, the second setat m/z 605.3, 587 and 569 and the third setof faint peaks at m/z 545, 527 and 509 wereattributed to the successive loss of water (18 amu),dihydrotiglic acid (102 amu) and acetic acid (60

amu) moieties from the protonated molecular ion andother intermediate fragment ions. Loss of dihydrotiglicacid function (102 amu) from three intermediatefragment ions at m/z 707.2, 689.3 and 671.3is indicative of hydrogenation of the tiglate estermoiety in the tetrahydroazadirachtin molecule. Whenhydrogenation was continued for 6 h, the LC-ESI-MSof the resultant final product exhibited a singlepeak (RT 15.551 min at 217 nm) corresponding totetrahydroazadirachtin-A (m/z 725, MH+). The massfragmentation pattern of tertrahydroazadirachtin isdepicted in Fig. 4.



m/z 689.3

m/z 569

m/z 605.3m/z 707.2

-H2O

-Dihydrotiglic acid

-H2O

O

O

AcO

H3COOC O

COOMe

O

H

O

OO

-H2O

m/z 587

m/z 671.3

-AcOH

m/z 545

Tetrahydroazadirachtin-A

O

OH

O

O

H

O

COOMeOH

OHOH3COOC

AcO

O

O

OH

Quasi molecular adduct ion peaksm/z 725.1 (M+H)+

m/z 747.3 (M+Na)+

m/z 763.0 (M+K)+

m/z 770.4 (M+2Na)+

H

O

COOMe

OH3COOC

AcO

AcO

H3COOC O

COOMe

O

H

AcO

H3COOC O

COOMe

O

H

-H2O

-H2O

OH

OHOH

OHOH

H3COOC OOH

COOMe

O

H

OO

O

H

O

COOMe

OH3COOC

AcO

O

O

H3COOC O

COOMe

O

H

-AcOH

-AcOH

H3COOC O

COOMe

O

H

m/z 527(weak)

m/z 509(weak)

OH

-H2O

-H2O

O

O

AcO

H3COOC O

COOMe

O

H

O

OO

OH

-Dihydrotiglic acid

-Dihydrotiglic acid

Figure 4. ESI mass fragmentation pattern of tetrahydroazadirachtin-A.

3.3 Antifeedant activityThe data on antifeedant activity of azadirachtin-A and tetrahydroazadirachtin-A after 24 and 48 htreatment of Helicoverpa armigera larvae are presentedin Table 1. A dose-dependent reduction in feeding wasnoticed at 24 h after treatment. Compared with thecontrol, test larvae consumed less of the treated food.Increasing concentrations of the five test compoundsled to higher antifeedant activity. At the lowestconcentration of 70 mg L−1, test compounds caused61.4–72.5% antifeedancy after 24 h and 47.7–67.4%antifeedancy after 48 h treatment. In terms of

antifeedant activity (AI50; Fig. 5), the most activetetrahydroaza-A (90%) showed a value of 14 mg L−1

after 24 h and 15 mg L−1 after 48 h. The remainderof the concentrates exhibited AI50 values in therange 25–30 mg L−1 after 24 h. After 48 h treatment,tetrahydroazadirachtin-A concentrates (60 and 90%)were more effective than azadirachtin-A concentrates.While tetrahydroazadirachtin-A concentrates (60 and90%) showed respective AI50 values of 30 and15 mg L−1, azadirachtin-A concentrates (20, 60 and90%) with respective AI50 values of 85, 67 and49 mg L−1 were comparatively less active.


V Sharma et al.

Table 1. Percentage feeding inhibition (AI50) of Helicoverpa armigera larvae by azadirachtin-A (aza-A) and tetrahydroazadirachtin-A

(tetrahydroaza-A) concentrates (no-choice bioassay)a

Concentration(mg L−1) Aza-A 90% Aza-A 60% Aza-A 20%

Tetrahydroaza-A90%

Tetrahydroaza-A60%

After 24 h1000 94.0 93.3 90.5 94.5 94.7700 93.1 92.9 87.6 93.5 93.1500 81.9 79.8 74.7 84.1 85.0100 73.7 71.8 68.7 72.4 76.970 66.4 65.3 61.4 67.9 72.5χ2 (3 df)b 5.63 6.09 6.15 2.94 3.36AI50 (mg L−1) 25 b 27 bc 30 c 14 a 25 bAfter 48 h1000 83.4 85.5 87.4 88.2 86.1700 77.9 81.9 86.0 82.0 85.0500 70.7 74.1 75.2 76.1 79.0100 54.5 60.2 62.4 65.9 70.570 47.7 48.5 55.0 61.4 67.4χ2 (3 df)b 2.39 1.98 1.15 0.94 2.19AI50 (mg L−1) 49 b 67 c 85 d 15 a 30 b

a Ranking order ‘a’ to ‘d’, i.e. feeding inhibition activity highest in ‘a’. Values having the same letter are not significantly different according toDuncan’s multiple range test.b df: degrees of freedom.

25

49

27

67

30

85

1415

2530

0

10

20

30

40

50

60

70

80

90

AI 5

0 (m

g L-1

)

Aza(9

0%)

Aza(6

0%)

Aza(2

0%)

THA(90%

)

THA(60%

)

1 DAT

2 DAT

Figure 5. Antifeedant activity (AI50) of azadirachtin-A andtetrahydroazadirachtin-A concentrates against Helicoverpa armigeralarvae.

3.4 Insect bioregulatory activityThe IGR effects of azadirachtin-A and tetrahydro-azadirachtin-A concentrates on larval mortality andinsect metamorphosis of H. armigera are summa-rized in Table 2. Depending on the concentrationapplied, test compounds showed significant dose-dependent increase in mortality and morphogeneticdefects expressed as larval–pupal intermediates, pupaldeformities and abnormal adults with crippled wingsand malformed legs. Tetrahydroazadirachtin-A con-centrate (90%) and azadirachtin-A concentrate (90%)with respective IC50 values of 280 and 390 mg L−1

were found to be most effective. At the high-est concentration of 1000 mg L−1, azadirachtin-A(90%) caused 76.7% growth inhibition as against12.6% at the lowest concentration. Aza-A (60%)and aza-A (20%) with 63.3 and 61.1% growth

inhibition were the least active among all the treat-ments. Out of the three azadirachtin-A concen-trates, azadirachtin-A (90%) exhibited the strongesteffect, suggesting that the activity was linked to theazadirachtin-A content of the concentrates. Of thetwo tetrahydroazadirachtin-A concentrates (60 and90%), the latter, with 89.3% growth inhibition at1000 mg L−1, was the most effective. Some of thenormal-looking pupae moulted into adults with crip-pled and malformed wings and legs. The adultsderived from treated larvae were smaller in size andfewer in number. Tetrahydroazadirachtin-A (60%)was relatively less active. At 1000 mg L−1, it produced64.1% overall growth inhibition. Based on IC50 values(Table 2), the highest IGR activity was observed withtetrahydroazadirachtin-A concentrate (90%) (IC50

280 mg L−1), followed by azadirachtin-A (90%) (IC50

390 mg L−1), tetrahydroazadirachtin-A (60%) (IC50

650 mg L−1), azadirachtin-A (60%) (IC50 740 mg L−1)and azadirachtin-A (20%) (IC50 770 mg L−1). Inter-estingly, reduced azadirachtin concentrates were moreeffective than azadirachtin concentrates, indicat-ing that hydrogenation, besides providing stability,retained/improved bioactivity. The concentrates withhigher azadirachtin-A/tetrahydroazadirachtin-A con-tent were more effective than those with a lower activeingredient content.

3.5 Effect of azadirachtin-A andtetrahydroazadirachtin-A on larval weightThe data on inhibition of larval growth of H.armigera following ingestion of food treated withazadirachtin-A and tetrahydroazadirachtin-A are pre-sented in Table 3. In all the treatments the lar-vae consumed less food (treated leaves) and con-sequently gained less weight compared with the



Table 2. Growth regulatory activity of azadirachtins and tetrahydroazadirachtins against Helicoverpa armigera larvae

Concentration(mg L−1)

Larval mortality(%) (a)

Larval pupalintermediates (%) (b)

Pupaldeformity (%) (c)

Deformedadult (%) (d)

Growth inhibition(a + b + c + d) (%)

IC50

(mg L−1)

Azadirachtin-A (90%)1000 30.0 16.7 10.0 20.0 76.7 390700 10.0 20.0 20.0 10.0 60.0500 10.0 10.0 23.3 4.0 47.3100 0.0 20.2 10.0 0.0 30.270 6.7 5.9 0.0 0.0 12.6CD 5% 7.73 6.25 30.18 15.48 10.74Azadirachtin-A (60%)1000 23.3 13.3 16.7 10.0 63.3 740700 10.3 5.6 21.6 10.0 47.5500 10.3 0.0 17.2 6.9 34.4100 10.0 0.0 16.7 0.0 26.770 0.0 0.0 16.7 0.0 16.7CD 5% 6.75 4.93 16.55 9.3 9.25Azadirachtin-A (20%)1000 19.3 16.1 19.3 6.4 61.1 770700 10.3 10.3 10.5 17.2 48.3500 10.0 0.0 23.3 0.0 33.3100 0.0 0.0 10.0 5.6 15.670 0.0 0.0 10.0 0.0 10.0CD 5% 10.76 4.78 7.11 9.62 8.79Tetrahydroazadirachtin-A (90%)1000 34.5 27.6 17.2 10.0 89.3 280700 20.0 20.0 15.0 13.3 68.3500 0.0 27.6 13.2 10.0 50.8100 20.0 0.0 0.0 10.0 30.070 0.0 0.0 16.7 0.0 16.7CD 5% 3.26 3.08 3.40 6.08 11.33Tetrahydroazadirachtin-A (60%)1000 30.0 14.1 10.0 10.0 64.1 650700 10.0 0.0 20.0 20.0 50.0500 10.0 10.0 10.0 2.6 32.6100 10.0 10.0 0.0 5.7 25.770 0.0 0.0 10.0 0.0 10.0CD 5% 5.94 5.15 6.66 3.73 6.44

control. In the control the larvae attained an averageweight of 0.1295 and 0.1564 g on 3 and 5 DATrespectively. Incorporation of azadirachtin-A andtetrahydroazadirachtin-A concentrates into the insectsystem through foliar intake led to reduction in lar-val weight gain. At 3 and 5 DAT, the feeding oflarvae with leaves treated with azadirachtin-A (90%)caused 80.66 and 77.38% reduction in larval weight at1000 mg L−1 and 29.04 and 27.12% at 70 mg L−1. Asimilar trend was observed with azadirachtin-A (60%)and azadirachtin-A (20%) concentrates. The effectwas, however, less pronounced, and decreased in adose-dependent manner.

Like azadirachtin, the foliar application of tetra-hydroazadirachtin-A (90 and 60%) also causeda significant reduction in larval weight gain.While the highest concentration (1000 mg L1) oftetrahydroazadirachtin-A (90%) caused 96.51 and87.36% reductions in larval weight gain, the lower con-centration (70 mg L−1) resulted in 51.64 and 55.65%reductions at 3 and 5 DAT. Of the various testcompounds, tetrahydroazadirachtin-A (90%) was themost effective, followed by azadirachtin-A (90%),

suggesting that hydrogenation resulted in no adverseeffect on the biological activity but rather enhanced it.

4 DISCUSSIONThere is an increasing demand for technicalazadirachtin powder concentrates and commercialneem products with a higher azadirachtin-A con-tent. Since thermal, hydrolytic and photo instabilityof azadirachtin has been a serious constraint in thepromotion and use of neem biopesticides, it is nec-essary to develop more stable neem products. Inthe present investigation, not only have azadirachtinconcentrates of higher azadirachtin-A content beenprepared by enrichment of the technical materialbut also a process for their catalytic conversion tomore stable reduced azadirachtins has been devel-oped. Azadirachtin-A 60% concentrate was preparedby repeated purification of the azadirachtin-A 20%concentrate. Azadirachtin-A 90% concentrate, on theother hand, was obtained by MPLC7 of azadirachtin-A60% concentrate. In addition to comparison of itsNMR spectral data with those of the authentic sample,


V Sharma et al.

Table 3. Effect of azadirachtins and tetrahydroazadirachtins on the

reduction in larval weight of Helicoverpa armigera

Compound

Concen-tration

(mg L−1)

Larval weightreduction (%)over control

3 DAT 5 DATAzadirachtin-A (90%)

1000 80.66 77.38700 73.82 70.43500 59.04 62.95100 35.58 42.24

70 29.04 27.12Azadirachtin-A (60%)

1000 64.42 61.23700 58.94 51.49500 46.78 41.70100 45.51 39.82

70 26.62 23.13Azadirachtin-A (20%)

1000 52.74 40.90700 34.73 30.04500 30.10 27.11100 27.23 23.25

70 13.59 9.33Tetrahydroazadirachtin-A (90%)

1000 96.51 87.36700 82.59 75.73500 82.50 70.49100 74.46 62.19

70 51.64 55.65Tetrahydroazadirachtin-A (60%)

1000 70.77 64.84700 61.17 54.32500 54.17 43.40100 46.86 39.47

70 24.23 28.99

azadirachtin-A has also been characterized on the basisof its unique mass fragmentation pattern obtained inpositive and negative ion mode electrospray ioniza-tion mass spectroscopy (ESI-MS). Interestingly, inits mass spectrum the molecular ion of azadirachtinappeared as a faint peak at m/z 721 in the positive ionmode and as a prominent peak at m/z 719.1 in thenegative ion mode (Fig. 1). The present study, alongwith the one reported earlier,7 provides useful LC-MSfragmentation data to assist identify azadirachtinoidsin neem extracts/products. 1H NMR spectral data ofazadirachtin-A and tetrahydroazadirachtin-A are alsoin agreement with those reported in the literature.10,21

The compounds show typical differences in the chem-ical shifts of protons attached to carbons C-21, C-22,C-23, C-2′, C-3′, C-4′ and C-5′ that have been affectedby the hydrogenation. While the dihydrotigloyl moietyin tetrahydroazadirachtin-A was characterized by anadditional multiplet (δ 1.78) corresponding to a C-2′proton which was otherwise absent in azadirachtin-A, one proton multiplet at δ 6.93 (C-3′ proton) inazadirachtin-A was shifted upfield as a two-protonmultiplet at δ 1.45.

The instability of azadirachtin in general has beenattributed in part to the presence of unsaturation in thetiglate and enol ether moieties in the molecule. Hydro-genation of the labile olefinic moieties is therefore cru-cial to obtain more stable reduced products.10,22 Ear-lier studies have shown that azadirachtin-A is resistantto hydrogenation at ambient conditions of temperatureand pressure. At higher pressure, and depending uponthe type of catalyst used, it has been selectively reducedat its 22,23 double bond to dihydroazadirachtin22 andadditionally at the 2′, 3′ double bond of tiglic acid21,22

to tetrahydroazadirachtin-A. In these preparations,hydrogenation has been carried out in the presenceof catalysts such as platinum oxide and palladiumon alumina at 5 and 10 atmosphere hydrogen pres-sure. An efficient procedure for the catalytic reductionof azadirachtin-A has now been standardized undernear-ambient conditions of temperature and pressureto obtain more stable tetrahydroazadirachtin concen-trates (60 and 90%). Like azadirachtin, structuresof dihydroazadirachtin-A and tetrahydroazadirachtin-A produced during the course of the hydrogenationhave been confirmed by comparison of their 1H NMRspectral data with those reported in the literature.10,22

Structures of the compounds have been additionallysubstantiated for the first time by their unique massfragmentation pattern recorded in ESI-MS.

Azadirachtin and its stable derivative tetrahy-droazadirachtin have been evaluated for their insectantifeedant and insect growth regulatory activitiesagainst the polyphagous insect Helicoverpa armigera.Results given in Table 1 revealed that, in a no-choice bioassay, H. armigera is reasonably sensitivein terms of antifeedancy to all the test concentra-tions of azadirachtin and tetrahydroazadirachtin. Thelatter were, in general, more active than the corre-sponding azadirachtin A concentrates. Based on AI50

values, antifeedant activity of the test compoundsfollowed the order: tetrahydroazadirachtin (90%) >

tetrahydroazadirachtin (60%) > azadirachtin-A (90%)> azadirachtin-A (60%) > azadirachtin-A 20%. Asreported earlier, inhibition of feeding by azadirachtinstems from blockage of input receptors for phagostim-ulants or by the stimulation of deterrent receptor cellsor both.2

Treatment of H. armigera larvae with azadirachtin-A and tetrahydroazadirachtin-A concentrates causedgrowth inhibition, malformation and mortality ina dose-dependent manner. Interestingly, tetrahy-droazadirachtin concentrates were as active as theircorresponding azadirachtin concentrates, indicat-ing that, besides providing stability, hydrogenationretained bioactivity (Table 2). The highest IGRactivity was observed with tetrahydroazadirachtin-A concentrate (90%) (IC50 280 mg L−1), fol-lowed by azadirachtin-A (90%) (IC50 390 mg L−1),tetrahydroazadirachtin-A (60%) (IC50 650 mg L−1),azadirachtin-A (60%) (IC50 740 mg L−1) and azadir-achtin-A (20%) (IC50 770 mg L−1). The concentrateswith higher azadirachtin-A/tetrahydroazadirachtin-A



contents were more active than those with lower activeingredient contents. In another study, incorporationof azadirachtin-A concentrates into the insect sys-tem through foliar intake revealed reduction in larvalweight gain. However, the effect decreased in a dose-dependent manner. Like azadirachtin, the foliar appli-cation of tetrahydroazadirachtin-A (90 and 60%) alsocaused a significant reduction in larval weight gain. Ofthe various test compounds, tetrahydroazadirachtin-A(90%) was the most effective followed by azadirachtin-A (90%). The overlapping of antifeedant effects withgrowth disruption activity accounts for the consider-able reduction in larval weight of the surviving larvae.

Based on overall consideration of insecticidal effi-cacy (antifeedant and IGR), tetrahydroazadirachtin-A (90%) and azadirachtin-A (90%) concentrateshave proved more promising against the polyphagousH. armigera larvae. Since tetrahydroazadirachtin ismore stable and equally effective to, or more effectivethan, azadirachtin, it deserves attention as a commer-cial neem biopesticide of the future.

ACKNOWLEDGEMENTSThe authors are grateful to the Head of the Division ofAgricultural Chemicals, Indian Agricultural ResearchInstitute, New Delhi, for providing the necessaryfacilities. Financial assistance from the NationalAgricultural Technology Project (NATP), ICAR, NewDelhi, is gratefully acknowledged.

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3 Ascher KRS, Nonconventional insecticidal effects of pesticidesavailable from the neem tree, Azadirachta indica. Arch InsectBiochem Physiol 22:433–449 (1993).

4 Shroeder DR and Nakanishi KA, Simplified isolation procedurefor azadirachtin. J Nat Prod 50:241–244 (1987).

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11 Chowdhuri H, Effect of curcumin and turmeric oil onphotostability and efficacy of azadirachtin. PhD Thesis, PGSchool, Indian Agricultural Research Institute, New Delhi,India (1996).

12 Sundaram KMS and Curry J, Photostabilization of the botanicalinsecticide azadirachtin in the presence of lecithin as UVprotectants J Environ Sci Health B 31:1041–1060 (1996).

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