Standardisation ofneem-extracts

Transcription

1 Standardisation ofneem-extracts Hubertus Kleeberg and Beate Ruch GmbH Dr.-Hans-Wilhelmi-Weg Lahnau, Germany

2 Purposes for which different parts of the Neem tree are used Part of the tree Seeds Oil Cake Fruits Leaves Twigs Wood Bark Roots Usage Pesticide, Oil Extraction, etc. Soap, Pesticides, Cosmetics, etc. Plant Protection, Fertiliser, Animal Fodder Food, Medicine, Oil Extraction Medicine, Cosmetics, etc. Dental Hygiene Firewood, Construction Material, etc. Toothpaste, Medicine Medicine

3 Developmental cost of Biological Plant Protection Products (estimated) Years million Euro Active ingredient Chemistry Formulation Research Biology Development DEGRADATION Extraction/Fermentation Lab-scale Development Screening Lab/Greenhouse Small plot trials Process development Development of Packing Field trials Production* Production* 2 Registration 2 AND plant, animal, soil, water, air RESIDUES Registration 8 acute u. chronic toxicity, cancerogenicity, TOXICOLOGY mutagenicity, teratogenicity, reproduction ECO- algae, daphnia, fish, birds, Registration TOXICOLOGY micro-organisms, bees, beneficials MILLION EURO NUMBER OF SUBSTANCES * without cost for production plants 5-1 1

4 LC 5 -values of some important, naturally occurring limonoids in the Epilachna varivestis bioassay (values taken from Rembold and Puhlmann, 22) Compound LC 5 ppm Azadirachtin A 1.3 Azadirachtin B 1.3 Azadirachtin D 1.6 Azadirachtin E 2.8 Azadirachtin F 1.1 Azadirachtin G 7.7* Azadirachtin H.5 Azadirachtin I.3 Salannin > 1 Nimbin 5** *Growth inhibition test (Kraus 22) ** Antifeedant activity, EC 5 -value (Kraus 22)

5 Analytical lead compound Azadirachtin A (AzA) 1H,7H-Naphtho[1,8-bc:4,4a-c ]difuran-5,1a(8h)-dicarbocylic acid,1- (acetyloxy)octahydro-3,5-dihydroxy-4-methyl-8-[(2-methyl-1-oxo-2- butenyl)oxy]-4-(3a,6a,7,7a-tetrahydro-6a-hydroxy-7a-methyl-2,7- methanofuro[2,3-b]oxireno[e]oxepin-1a(2h)-yl)-dimethylester,[2ar-[2aa, 3b, 4b(1aR*, 2S*, 3aS*, 6aS*, 7S*, 7aS*), 4ab, 5a, 7aS*, 8b(E), 1b, 1aa, 1bb)] AcO MeOOC O O 3 O H O COOMe OH O OH O OH H O

6 Variation of the content of Azadirachtin A and Azadirachtin B in samples of neem seed kernels of different origin 6 5 Ghana (AzA: 3.1 mg/g; AzA:AzB = 2.3) 4 Tansania (AzA: 4.8 mg/g; AzA:AzB = 4.1) µvolts 3 Mauretania (AzA: 5.9 mg/g; AzA:AzB = 8.3) 2 1 China (AzA: 9.6 mg/g; AzA:AzB = 4.8) China (AzA: 3.5 mg/g; AzA:AzB = 6.2) Azadirachtin standard solution.2 mg AzA/ml Minutes

7 Peak height of the HPLC-peaks of AzA and AzB in a microbiologically active test solution (ph = 7.6; T = 26ºC) Peak hight of AzadirachtinA and AzadirachtinB peak hight Azadirachtin A Azadirachtin B time [hours]

8 Variation of the content of limonoids in samples of different standardised 15 Neem-products mvolt Neem oil mvolt Time (min) Time (min) Enriched Azadirachtin extract (India) mvolt mvolt NeemAzal AzH AzA AzB Time (min) DN DS N S NeemAzal technical Time (min)

9 Neem Extraction Process yields NeemAzal technical NeemAzal (,1 kg) 34% Azadirachtin A Neem fruit (1 kg) Neem seeds (2 kg) Seed kernels (1 kg) Extraction Process Neem oil (4 kg) Neem cake (5 kg)

10 Patents and registrations in more than 2 countries, Trademarks in EU and other countries by and/or EID Parry active ingredient: NeemAzal technical Extractionplant in India (EID Parry) Technology

11 Composition NeemAzal Substance av. content in NeemAzal By weight (%) Azadirachtins: Azadirachtin A 34 Azadirachtin B approx. 5.5 Azadirachtin D aprrox. 2.1 Azadirachtin E < 1 Azadirachtin F < 1 Azadirachtin G < 1 Azadirachtin H approx. 2.3 Azadirachtin I approx..8 Azadirachtin K and other Azadirachtins < 2 Azadirachtinin approx. 2 Sum of Azadirachtins: 51,7

12 Function of the formulation

13 Accelerated Storage Test (T=54 o C) 1,5 Azadirachtin content % 1,3 1,1,9,7,5,3,1 -, day AzA AzB Sum of all Az'sAZ Sum AzB-F

14 Mode of action - concentration effect Megoura viciae (Hom., Aphididae) - aphid Anzahl von Number Number of Blattläuse, of aphids Stck aphids K,1 Konzentration, Concentration, % %,3, Tage Daysnach after der Behandlung Days after application

15 Mode of action - Megoura viciae (Hom., Aphididae): topical effect vs. oral uptake K B-b, P-ub,,2 B-b, P-ub,,4 B-ub, P-b,,2 B-ub, P-b,,

16 Mode of action & target pests: NeemAzal-T/S (ec) Effects the hormonal system of insects 1. feeding inhibition 2. moulting inhibition 3. fertility reduction

17 Mode of action: 1. feeding inhibition (after a few hours) Treated with NeemAzal-T/S untreated control

18 Feeding inhibition Typical Action of NeemAzal on larvae with 2 ppm AzadirachtinA treated untreated Mortality (%) day Ø Larval Weight (mg)

19 Mode of action - feeding inhibition Thaumetopoea processionea (Lep. Thaumetopoeidae) by Breuer (2) Cumulative faecal production (mg) of 8 larvae Cumulative faecal production (mg) of 8 larvae Days after treatment Days after treatment

20 Mode of action - feeding inhibition Thaumetopoea processionea (Lep. Thaumetopoeidae) by Breuer (2) Larval weight (mg) Larval weight (mg) Days after treatment Days after treatment

21 Mode of action: 2. moulting inhibition (after a few days) Normal larva after treatment with NeemAzal-T/S

22 Mode of action: 3. Fertility reduction (next generation)

23 Mode of action: 3. Fertility reduction (next generation) Egg deposit (untreated) Egg deposit after treatment

24 K

25 Use of Neem preparations for the control of Rosy apple aphid in Spain (modified after F. Mayoral, Agrichem, Spain) Level of ínfestation (-5) in Sariego, Spain 25 2,5 2 1,5 T+ T+21 1 T+4,5 Control Unregistered product 1 Registered product 2 Unregistered product 3 Unregistered product 4 NeemAzal- T/S

26 Use of Neem preparations for the control of Rosy apple aphid in Spain (modified after F. Mayoral, Agrichem, Spain) Efficacy of infestation 1, 5,, Unregistered product 1 Registered product 2 Unregistered product 3 Unregistered product 4 NeemAzal- T/S -5, -1, -15, 1 Efficacy damage -2, 5 Unregistered product 1 Registered product 2 Unregistered product 3 Unregistered product 4 NeemAzal- T/S

27 Efficacies, examplefrom practice: comparison of 2 spraying dates of NA TS against Rosy Apple Aphid (Univ. Hohenheim, Weinsberg) 3 25 Number of colonies on 16 trees late infestation Rote Knospe Stadium Ballonstadium Kontrolle Kienzle/Schulz, Universität Hohenheim; gefördert von der Bundesstiftung Umwelt und der Volkswagen-Stiftung, Hannover

28 Dose effect of Azadirachtin on lice (after V.H. Guerrini, Vet. Med. Online Journal) Dose effect of Azadirachtin on lice 1 Number of Lice (log) 1 1, ppm Azadirachtin

29 Neem power Konzentrat advertised as insecticide Wash-away-lice advertised as insecticide Neem power Konzentrat 2 µvolts AzH AzA AzB Wash-away-lice DN DS N S Minutes

30 .

31 HPLC-Chromatogram of limonoide standard solutions and alcoholic Neem leaf extracts from: Iran (2) (top) and Brazil (23) (bottom) DN DS N S AzH AzA AzB mvolt 5 mvolt Time (min) DN DS N S AzH AzA AzB Time (min)

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