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(Young-Joon Ahn, Ph. D.) : (02)880-4702 : (02)873-2319 ( ) Homepage :

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Presentation on theme: "(Young-Joon Ahn, Ph. D.) : (02)880-4702 : (02)873-2319 ( ) Homepage :"— Presentation transcript:

1 (Young-Joon Ahn, Ph. D.) : (02) : (02) ( ) Homepage : 2. 3,4. 4.,,. 3. ' ',,.,.,..,,

2 Bio- technology bioindicator

3 ,,,. chemical interaction,,.,,.,.

4 Plant-Based Medical Arthropod Pest Control Agents: Development and Commercialization Young-Joon Ahn College of Agriculture and Life Sciences Seoul National University

5 Vector control

6 Currently Used Pesticides Decreased persistence Decreased application rates Increased selectivity Reduced risk Reduced risk pesticides

7 Reduced Risk Insecticides Reduced Risk Insecticides Reduced impact on human health and very low mammalian toxicity Displace chemicals of concern to human health Reduced effects on non-target organisms Reduced potential for contaminating groundwater Lower potential for pest resistance (e.g. novel mode of action) High compatibility with integrated pest management

8 Botanicals neem, essential oils (FDA GRAS list) Insect growth regulators methoprene, diflubenzuron Microbials Bacillus thuringiensis, B. sphaericus New chemistries spinosyns, imidacloprid, fipronil Reduced Risk Insecticides Reduced Risk Insecticides

9 ca 100,000 or more secondary plant metabolites Hundreds (or more) with bioactivity in insects Bioactivities Behavioral Repellence Feeding deterrence Oviposition deterrence Physiological Acute toxicity Developmental disruption Growth inhibition There are so few botanical insecticides. Why? There are so few botanical insecticides. Why? Botanical Insecticides

10 Barriers to the development of new botanical insecticides Resource availability/sustainability Stability, standardization, and quality control Costs of the raw materials and refinement Registration (toxicology) The most formidable barrier

11 Advantages of Plant Essential Oils as Botanical Insecticides Simple chemistry Relatively inexpensive World-wide supply as fragrances and flavorings Some are exempt from registration or testing ( FDAs GRAS list ) These regulatory, cost, and availability lead many venture companies to develop insect control agents based on essential oils and/or their constituents

12 Insecticide (source) % Active ingredientsUS$ per kg Pyrethrum (Australia, Kenya) 2045 ~ 60 Neem (India) ~ 200 Essential oils (Various) 90 ~ 9810 ~ 25 Current Costs: Botanical Active Ingredients Source: Isman (2001)

13 HOUSE DUST MITE Dermatophagoides farinae American house dust mite Dermatophagoides pteronyssinus European house dust mite

14 Main Allergens (%)

15 Medical Importance Over 100 mites or 0.6 mg guanine per gram house dust Allergic disorders Allergic disorders Asthma ( ) Rhinitis ( ) Atopic dermatitis ( ) Eczema ( ) Causal association with sudden infant death syndrome Causal association with sudden infant death syndrome

16 Market of allergic disease 100mill. (US $) Market of house dust mite control agents

17 Control of House dust mite Physical Control Freeze Dry Laundry Clean Very complicate Chemical Control Benzyl benzoate (Acarosan ) 0.5% lindane 1% pirimiphos-methyl Pyrethroids

18 Side effect Toxicity Resistance Environmental contamination Need for development of biorational control agents Plants as an alternative source Botanical acaricides

19 Toxicity of natural compounds to House dust mites Eugenol (RT : 1.7) Methyl eugenol (RT : 9.8) Cinnamaldehyde (RT : 9.0) Salicylaldehyde (RT : 3.6) Cinnamyl alcohol (RT : 4.7) Benzoic acid (RT : 1.07) Paeonol (RT : 1.18) Bisabolangelone (RT : 5.40) Atractylenolide Benzyl benzoate (RT : 1) Butylidenephthalade (RT : 1.3)

20 CommercializationBio-acaricides

21 Material D. farinae D. pteronyssinus LD 50, µg/cm 2 (95% CL)RT* LD 50, µg/cm 2 (95% CL)RT* Cassia bark oil4.35 (4.21– 4.48) (4.52– 4.71)7.2 Benzyl benzoate4.84 (4.47– 5.23) (4.33– 5.28)6.9 Deet24.43 (11.21–21.00) (13.22–43.14)1.9 Dibutyl phthalate20.02 (17.93–22.42) (29.57–36.84)1.0 *Relative toxicity, LD 50 of dibutyl phthalate /LD 50 of the material. Toxicity of cassia bark oil and acaricides against adults of D. farinae and D. pteronyssinus using the fabric-piece contact bioassay

22 Hosts and Habitats Hosts Mammals (especially, human) Nests Occasionally, bee hives Habitats Carpets Sofas Beds Fabric materials Soft toys

23 Formulations

24 Spray product

25 Lethal activity of 2 and 5% cassia bark oil-containing spray products to adult D. pteronyssinus when applied to various materials Substrate Mortality (%) (± SE) 2% oil product 5% oil product Fabric 99 ± ± 0.0 Glass 97 ± ± 0.0 Paper sheet 90 ± ± 0.0 Plastic100 ± 0.0 Tin 97 ± ± 0.0 Wood100 ± 0.0 ca mg of cassia oil was necessary to provide dust mite control in a space of 1 m 2 or in a volume of 3.4 m 3

26 Spray product Spray product can be applied - to homes or other facilities, such as hotels, hospitals, offices, and theaters, etc., - or directly to dust mites on mattresses, carpets, sofas, or toys

27 closedopen gauze mite filter paper Route of acaricidal action Vapor phase toxicity bioassay

28 Fumigant activity of cassia bark oil and acaricides against adults of D. farinae and D. pteronyssinus using the vapor phase toxicity bioassay MaterialMethod* Dose (µg/cm 3 ) Mortality, % (mean ± SE) n D. farinaenD. pteronyssinus Cassia bark oil A ± ± 0.0 B ± ± 1.5 Benzyl benzoate A ± ± 1.6 B ± ± 1.5 Deet A ± ± 2.1 B ± ± 2.0 Dibutyl phthalate A ± ± 1.5 B ± ± 1.8 *A, vapor in close containers; and B, vapor in open containers.

29 Nonwoven fabric Fumigant product Fumigant device

30 Toxicity of cassia oil-containing fumigant to adult D. pteronyssinus in six different spaces MethodSpace, m 3 Mortality (%) (± SE) Hours after treatment Direct * ± ± 0.0 Random ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± § 0 ± ± ± 4.9 * The tubes containing house dust mites were placed directly on fumigant devices. The tubes containing house dust mites were placed randomly in the test spaces. One fumigant was used in the space. § Two fumigants were used in the space.

31 Nonwoven fabric Fumigant product Useful for managing dust mites in enclosed spaces such as wardrobes, pet house, storage bins, factories, or buildings Placed under sofa or bedclothing, or in a mattress, a pillow, or a laundry box

32 Route of acaricidal action Eugenol (RT : 1.7) Methyl eugenol (RT : 9.8) Cinnamaldehyde (RT : 9.0) Salicylaldehyde (RT : 3.6) Cinnamyl alcohol (RT : 4.7) Benzoic acid (RT : 1.07) Paeonol (RT : 1.18) Bisabolangelone (RT : 5.40) Atractylenolide Benzyl benzoate (RT : 1) Butylidenephthalade (RT : 1.3) Fumigant action Contact action

33 Development of Allergen denaturants Tannic acid Atractylenolide

34 Diagram DENATURING AGENT Antibody SKIN Fecal pellets allergen Denatured protein or

35 Apolipophorin-like protein Grp 14 Cytosolic fatty acid-binding proteins Grp 13 Unknown 5014Grp 12 Paramyosin High98Grp 11 Melanogaster, rabbit & human alpha-tropomyosin > Grp 10 Colagenolytic serine protease Grp 9 Rat & mouse glutathione-S-transferase Grp 8 Unknown Grp 7 Chymotrypsin-like serine protease Grp 6 Unknown Grp 5 Amylase Grp 4 Trypsin-like serine protease Grp 3 Primate epididymal or insect molting protein >9014 Grp 2 Cysteine protease, e.g. papain, actinidin, Cathepsin, bromelain, frein. >9025 Grp 1 Homology Adverse Reactions [%] Molecular Weight [kDA] Allergen Group Characters of House dust mite allergens

36 Protein denaturing activity using SDS-PAGE

37 Allergenic activity using Dot blot active moderate

38 Expression of allergen gene Total RNA extraction - Trizol reagent (Invitrogen) cDNA Synthesis and PCR - AccuPower RT PreMix(Bioneer) Primer - Include restriction enzyme site (Nde l, Xho l)

39 Primer Primer Sequence DF1 (forward) 5-NNCATATGAAATTCGTTTTGGC-3 DF1 (reverse) 5-CCCTCGAGCATGATTACAACAT-3 DP1 (forward) 5-NNCATATGAAAATTGTTTTGGCCATC-3 DP1 (reverse) 5-NNCTCGAGAATGACAACATGTGG-3 T7 promoter 5-TAATACGACTCACTATAGGG-3 T7 terminator 5-GCTAGTTATTGCTCAGCGGT-3 SP6 promoter 5-TATTTAGGTGACACTATAG–3

40 Gene cloning Vector - pGEM ® -T Vector (Promega) - pET22b(+) (Novagen) E-coli - JM109 (Promega) - Top10 (Invitrogen) - BL21(DE3) (Novagen)

41 group I gene cloning

42 Recombinant DNA D. f I D. p I M : marker1 : PCR product 2 : pGemT-DNA with Xho I3 : pGemT-DNA with Nde I, Xho I 4 : PCR with T7, SP65 : pET22b(+)-DNA with Xho I 6 : PCR with T7, T7 terminator7 : colony PCR 1kb 3kb 5kb

43 Protein purification Nitriloacetic acid-Ni 2+ 6 Histidine Target protein Nitriloacetic acid-Ni 2+ WashElution

44 Purification of house dust mite allergens M: marker; C: recombinant E-coli; W: wash with buffer; E: elution protein 25 kDa

45 Allergen denaturing activity with active compounds 2 μg/0.5 μg 4 μg /0.1 μg


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