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안용준 교수 (Young-Joon Ahn, Ph. D.)

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Presentation on theme: "안용준 교수 (Young-Joon Ahn, Ph. D.)"— Presentation transcript:

1 안용준 교수 (Young-Joon Ahn, Ph. D.)
교수실 : (02) 팩   스 : (02) (공용) Homepage : 생리활성천연물•약용곤충연구실 농업생물공학협동과정 응용곤충학 수강생 여러분 백년만의 무더위가 찾아와서 여름 지내기가 쉽지 않았을 텐데, 보람있는 여름을 보냈으리라고 생각합니다. 놀기도 좋고, 공부하기도 좋은 가을을 맞이하면서 2학기가 시작되었습니다. 3,4학년생들이 수강하는 것으로 알고 있습니다. 4학년생들에게는 재학중 대학의 마지막 과목이 되겠습니다. 이번 학기로 즐겁고 추억이 담긴 대학생활을 접자니, 만감이 교차하리라 생각되지만, 그럼으로해서 더욱더 유종의 미를 거두었으면 합니다. 3학년생들은 일반곤충학과는 달리 이론 및 응용면을 강조하고 있는 응용곤충학이라는 새로운 과목을 접하게 되었습니다. '응용곤충학'은 안용준, 이식혁, 제연호 교수 순으로 담당하고 있습니다. 저희 교수들은 일방적인 암기를 위한 강의가 아니라, 여러분들과 함께 생각하고 토론 할 수 있는 강의를 꾸려 나가고자 노력하고 있습니다. 여러분들과 새로운 관점에서 곤충학을 공부하고, 새로운 각도에서 곤충학을 바라 볼 수 있도록 저희 세 사람의 강의가 유기적으로 이어 질 수 있도록 하겠습니다. 새로운 한 학기를 여러분들과 함께 즐겁고 보람차게 보내고 싶은 것이 저희 교수들의 바램입니다.                                                                   안용준, 이시혁, 제연호

2 인 간 인간과 곤충 건 강 위 생 의 류 에너지 식 량 주 거 과 학 교 통 예 술 통 신 신 소 재 의료기구 Bio-
technology 의 류 생체공학 bioindicator 에너지 식 량 인 간 주 거 농작물 과 학 예 술 교 통 통 신 축산물 산 림 저장물 인간과 곤충

3 바이오 해충방제제 개발 및 산업화 이번 시간에는 곤충이 어떻게 자신의 기주식물을 찾아가는 지를, 고추와 담배나방, 십자화과 작물과 배추좀나방, 마늘과 고자리파리를 예를 들어 설명할 것입니다. 이 과정에는 각 기주식물이 방출하는 휘발성 화학물질 또는 기주식물의 비휘발성 화학물질들을 곤충이 감지하여 유인되는 이른바 chemical interaction이 성립되며, 이러한 과정을 거침으로서, 종이 유지되고 있습니다. 이와같이 곤충과 식물간의 관계를 구명함으로서, 생명현상의 본질을 이해 할수 있을 뿐 아니라, 이러한 화학물질을 이용하여 친환경적인 해충방제가 가능하게 됩니다. 고추와 담배나방간의 상호작용에 관한 논문을 첨부할 것이니, 참고하시기 바랍니다.

4 College of Agriculture and Life Sciences Seoul National University
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 pesticides

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

9 Botanical Insecticides
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?

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 ( FDA’s “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 Current Costs: Botanical Active Ingredients
Insecticide (source) % Active ingredients US$ per kg Pyrethrum (Australia, Kenya) 20 45 ~ 60 Neem (India) 10 125 ~ 200 Essential oils (Various) 90 ~ 98 10 ~ 25 Source: Isman (2001)

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

14 Main Allergens (%)

15 Medical Importance Asthma (哮喘) Eczema (湿疹) Atopic dermatitis (异位性皮炎)
Over 100 mites or 0.6 mg guanine per gram house dust  Allergic disorders Asthma (哮喘) Eczema (湿疹) Atopic dermatitis (异位性皮炎) Rhinitis (鼻炎) rhi ni tis [raináitis] The medical importance of the house dust mite is that they are the most important factor of allergen. These are the examples that transferred by the house dust mites. Asthma, rhinitis, atopic dermatitis and eczema And these pictures show the symptoms of the diseases. Causal association with sudden infant death syndrome

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

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

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

20 Commercialization Bio-acaricides

21 D. farinae D. pteronyssinus
Toxicity of cassia bark oil and acaricides against adults of D. farinae and D. pteronyssinus using the fabric-piece contact bioassay Material D. farinae D. pteronyssinus LD50, µg/cm2 (95% CL) RT* Cassia bark oil (4.21– 4.48) 4.6 (4.52– 4.71) 7.2 Benzyl benzoate (4.47– 5.23) 4.1 (4.33– 5.28) 6.9 Deet 24.43 (11.21–21.00) 0.8 17.22 (13.22–43.14) 1.9 Dibutyl phthalate 20.02 (17.93–22.42) 1.0 33.41 (29.57–36.84) *Relative toxicity, LD50 of dibutyl phthalate /LD50 of the material.

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 ± 1.0 100 ± 0.0 Glass 97 ± 3.3 Paper sheet 90 ± 3.5 Plastic Tin 97 ± 1.5 Wood ca mg of cassia oil was necessary to provide dust mite control in a space of 1 m2 or in a volume of 3.4 m3

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 Route of acaricidal action
gauze closed open mite filter paper 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 Material Method* Dose (µg/cm3) Mortality, % (mean ± SE) n D. farinae D. pteronyssinus Cassia bark oil A 50.96 97 100 ± 0.0 83 B 19 ± 4.5 73 11 ± 1.5 Benzyl benzoate 103 7 ± 2.9 74 8 ± 1.6 102 76 7 ± 1.5 Deet 101.92 85 14 ± 2.7 8 ± 2.1 75 5 ± 2.6 4 ± 2.0 Dibutyl phthalate 90 0 ± 0.0 66 5 ± 1.5 5 ± 0.8 5 ± 1.8 *A, vapor in close containers; and B, vapor in open containers.

29 Fumigant product Nonwoven fabric Fumigant device

30 Toxicity of cassia oil-containing fumigant to adult
D. pteronyssinus in six different spaces Method Space, m3 Mortality (%) (± SE) Hours after treatment 24 48 72 96 Direct* 0.024‡ 100 ± 0.0 0.05‡ Random† 36 ± 3.6 97 ± 3.0 41 ± 6.5 89 ± 5.6 94 ± 6.5 0.097‡ 7 ± 1.3 24 ± 3.0 77 ± 5.5 0.182‡ 16 ± 3.5 33 ± 2.9 75 ± 5.3 0.255‡ 15 ± 2.1 19 ± 2.5 25 ± 3.5 1.73‡ 0 ± 0.0 1 ± 1.0 24 ± 3.6 1.73§ 21 ± 4.3 88 ± 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 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 Nonwoven fabric

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

33 Development of Allergen denaturants
Tannic acid Atractylenolide Ⅲ

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

35 Characters of House dust mite allergens
Apolipophorin-like protein 70 Grp 14 Cytosolic fatty acid-binding proteins 10 15-17 Grp 13 Unknown 50 14 Grp 12 Paramyosin High 98 Grp 11 Melanogaster, rabbit & human alpha-tropomyosin >60 33-37 Grp 10 Colagenolytic serine protease 80 24-28 Grp 9 Rat & mouse glutathione-S-transferase 40 25-26 Grp 8 22-28 Grp 7 Chymotrypsin-like serine protease 40-60 25 Grp 6 45-60 14-15 Grp 5 Amylase 25-46 56-63 Grp 4 Trypsin-like serine protease 57-90 28-30 Grp 3 Primate epididymal or insect molting protein >90 Grp 2 Cysteine protease, e.g. papain, actinidin, Cathepsin, bromelain, frein. Grp 1 Homology Adverse Reactions [%] Molecular Weight [kDA] Allergen Group

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 Sequence Primer 5’-NNCATATGAAATTCGTTTTGGC-3’ DF1 (forward)
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 5kb 3kb 1kb D. p I D. f I M : marker 1 : PCR product
2 : pGemT-DNA with Xho I 3 : pGemT-DNA with Nde I, Xho I 4 : PCR with T7, SP6 5 : pET22b(+)-DNA with Xho I 6 : PCR with T7, T7 terminator 7 : colony PCR

43 Nitriloacetic acid-Ni2+ Nitriloacetic acid-Ni2+
Protein purification Wash Elution Target protein 6Histidine Nitriloacetic acid-Ni2+ Nitriloacetic acid-Ni2+

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

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


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