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안용준 교수 (Young-Joon Ahn, Ph. D.)
교수실 : (02)880-4702 팩 스 : (02)873-2319 ( 공용 ) E-mail : [email protected] Homepage : http://plaza.snu.ac.kr/~yjahn
생리활성천연물• 약용곤충연구실농업생물공학협동과정
응용곤충학 수강생 여러분백년만의 무더위가 찾아와서 여름 지내기가 쉽지 않았을 텐데 , 보람있는 여름을 보냈으리라고 생각합니다 .놀기도 좋고 , 공부하기도 좋은 가을을 맞이하면서 2 학기가 시작되었습니다 . 3,4 학년생들이 수강하는 것으로 알고 있습니다 . 4 학년생들에게는 재학중 대학의 마지막 과목이 되겠습니다 . 이번 학기로 즐겁고 추억이 담긴 대학생활을 접자니 , 만감이 교차하리라 생각되지만 , 그럼으로해서 더욱더 유종의 미를 거두었으면 합니다 . 3 학년생들은 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
교 통통 신
교 통통 신
건 강위 생
건 강위 생
의 류의 류
주 거주 거
식 량식 량
과 학예 술
과 학예 술
에너지에너지
Bio-technology
Bio-technology
신 소 재신 소 재
의료기구의료기구
생체공학생체공학
bioindicatorbioindicator
농작물농작물
축산물축산물
산 림산 림
저장물저장물
인간과 곤충
인 간인 간
바이오 해충방제제 개발 및 산업화바이오 해충방제제 개발 및 산업화
이번 시간에는 곤충이 어떻게 자신의 기주식물을 찾아가는 지를 , 고 추 와 담 배 나 방 , 십 자 화 과 작 물 과 배 추 좀 나 방 , 마 늘 과 고자리파리를 예를 들어 설명할 것입니다 . 이 과정에는 각 기주식물이 방출하는 휘발성 화학물질 또는 기주식물의 비휘발성 화학물질들을 곤 충 이 감 지 하 여 유 인 되 는 이 른 바 chemical interaction 이 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 .
Plant-Based Medical Arthropod Pest Control Agents: Development and Commercialization
Young-Joon AhnYoung-Joon Ahn
College of Agriculture and Life Sciences
Seoul National University
Vector controlVector control
Currently Used PesticidesCurrently Used Pesticides
Decreased persistence
Decreased application rates
Increased selectivity
Reduced riskReduced risk pesticides
““Reduced RiskReduced Risk”” Insecticides 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
Botanicals neem, essential oils (FDA GRAS list)
Insect growth regulators methoprene, diflubenzuron
Microbials Bacillus thuringiensis, B. sphaericus
New chemistries spinosyns, imidacloprid, fipronil
““Reduced RiskReduced Risk”” Insecticides 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?There are so few botanical insecticides. Why?
Botanical InsecticidesBotanical Insecticides
Barriers to the development of new botanical Barriers to the development of new botanical insecticidesinsecticides
Resource availability/sustainability
Stability, standardization, and quality control
Costs of the raw materials and refinement
Registration (toxicology) The most formidable barrier
Advantages of Plant Essential Oils as Advantages of Plant Essential Oils as Botanical InsecticidesBotanical 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
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
Current Costs: Botanical Active IngredientsCurrent Costs: Botanical Active Ingredients
Source: Isman (2001)
HOUSE DUST MITEHOUSE DUST MITE
Dermatophagoides farinae
American house dust mite
Dermatophagoides pteronyssinus
European house dust mite
0
5
10
15
20
25
30
35
40
45
Main AllergensMain Allergens
(%)
Medical ImportanceMedical Importance
Over 100 mites or 0.6 mg guanine per gram house dustOver 100 mites or 0.6 mg guanine per gram house dust
Allergic disordersAllergic disorders
Asthma (哮喘 )
Rhinitis (鼻炎 )Atopic dermatitis (异位性皮炎 )
Eczema (湿疹 )
Causal association with sudden infant death syndromeCausal association with sudden infant death syndrome
0
5
10
15
20
25
30
35
40
45
Market of allergic diseaseMarket of allergic disease
100mill.
(US $)
Market of house dust mite control agentsMarket of house dust mite control agents
Control of House dust miteControl of House dust mite
Physical Control
• Freeze
• Dry
• Laundry
• Clean
Very complicate
Chemical Control
•Benzyl benzoate
(Acarosan)
•0.5% lindane
•1% pirimiphos-methyl
•Pyrethroids
Side effect
• Toxicity
• Resistance
• Environmental contamination
Need for development of biorational control agents
Plants as an alternative source
Botanical acaricidesBotanical acaricides
Toxicity of natural compounds to Toxicity of natural compounds to House dust mitesHouse dust mites
CH2
CH3
O
CH3OH
CH2
O
OCH3
CH3
O
CHO
OH
OH
OCH3
OH
CH3 O
OH O
O
O
H
CH3
H H
H
H
CH3
OHH
H
CH3
CH3
O
CH2
CH3
H
OH
O
CH3 O
O
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 Ⅲ Benzyl benzoate (RT : 1)
Butylidenephthalade (RT : 1.3)
桂皮 丁香 牧丹皮
川芎羌活
白朮
O
CH3
O
CommercializationCommercialization
Bio-acaricidesBio-acaricides
MaterialD. farinae D. pteronyssinus
LD50, µg/cm2 (95% CL) RT* LD50, µg/cm2 (95% CL) RT*
Cassia bark oil 4.35 (4.21– 4.48) 4.6 4.62 (4.52– 4.71) 7.2
Benzyl benzoate 4.84 (4.47– 5.23) 4.1 4.87 (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) 1.0
*Relative toxicity, LD50 of dibutyl phthalate /LD50 of the material.
Toxicity of cassia bark oil and acaricides against adults Toxicity of cassia bark oil and acaricides against adults of of D. farinaeD. farinae and and D. pteronyssinus D. pteronyssinus using the fabric-piece using the fabric-piece
contact bioassaycontact bioassay
Hosts and HabitatsHosts and Habitats
Hosts
• Mammals
(especially, human)
• Nests
• Occasionally, bee hives
Habitats
• Carpets
• Sofas
• Beds
• Fabric materials
• Soft toys
Formulations Formulations
Spray productSpray product
Lethal activity of 2 and 5% cassia bark oil-containing Lethal activity of 2 and 5% cassia bark oil-containing spray products to adult spray products to adult D. pteronyssinus D. pteronyssinus when applied when applied
to various materialsto various materials
SubstrateMortality (%) (± SE)
2% oil product 5% oil product
Fabric 99 ± 1.0 100 ± 0.0
Glass 97 ± 3.3 100 ± 0.0
Paper sheet 90 ± 3.5 100 ± 0.0
Plastic 100 ± 0.0 100 ± 0.0
Tin 97 ± 1.5 100 ± 0.0
Wood 100 ± 0.0 100 ± 0.0
ca 50-60 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
Spray productSpray 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
closed open
gauze
mitefilter paper
Route of acaricidal actionRoute of acaricidal action
Vapor phase toxicity bioassayVapor phase toxicity bioassay
Fumigant activity of cassia bark oil and acaricides against Fumigant activity of cassia bark oil and acaricides against adults of adults of D. farinaeD. farinae and and D. pteronyssinus D. pteronyssinus using the vapor using the vapor
phase toxicity bioassayphase toxicity bioassay
Material Method*Dose
(µg/cm3)
Mortality, % (mean ± SE)
n D. farinae n D. pteronyssinus
Cassia bark oil A 50.96 97 100 ± 0.0 83 100 ± 0.0
B 50.96 83 19 ± 4.5 73 11 ± 1.5
Benzyl benzoate A 50.96 103 7 ± 2.9 74 8 ± 1.6
B 50.96 102 8 ± 1.6 76 7 ± 1.5
Deet A 101.92 85 14 ± 2.7 83 8 ± 2.1
B 101.92 75 5 ± 2.6 74 4 ± 2.0
Dibutyl phthalate A 101.92 90 0 ± 0.0 66 5 ± 1.5
B 101.92 102 5 ± 0.8 83 5 ± 1.8
*A, vapor in close containers; and B, vapor in open containers.
Nonwoven fabric
Fumigant productFumigant product
Fumigant device
Toxicity of cassia oil-containing fumigant to adultToxicity of cassia oil-containing fumigant to adultD. pteronyssinusD. pteronyssinus in six different spaces in six different spaces
Method Space, m3
Mortality (%) (± SE)
Hours after treatment
24 48 72 96
Direct* 0.024‡ 100 ± 0.0
0.05‡ 100 ± 0.0
Random† 0.024‡ 36 ± 3.6 97 ± 3.0 100 ± 0.0
0.05‡ 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§ 0 ± 0.0 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.
Nonwoven fabric
Fumigant productFumigant 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
Route of acaricidal actionRoute of acaricidal actionCH2
CH3
O
CH3OH
CH2
O
OCH3
CH3
O
CHO
OH
OH
OCH3
OH
CH3 O OH O
O
O
H
CH3
H H
H
H
CH3
OHH
H
CH3
CH3
O
CH2
CH3
H
OH
O
CH3
O
O
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 Ⅲ
Benzyl benzoate (RT : 1)
Butylidenephthalade (RT : 1.3)
桂皮 丁香 牧丹皮 川芎
羌活 白朮
O
CH3
O
Fumigant action
Contact action
Development of Allergen denaturantsDevelopment of Allergen denaturants
Tannic acid
O
CH2
CH3
H
OH
O
CH3
Atractylenolide Ⅲ
Diagram Diagram
DENATURING
AGENT
Antibody
SKIN
Fecal pellets
allergen
Denatured protein
or
Apolipophorin-like protein70177-190Grp 14
Cytosolic fatty acid-binding proteins1015-17Grp 13
Unknown5014Grp 12
ParamyosinHigh98Grp 11
Melanogaster, rabbit & human alpha-tropomyosin>6033-37Grp 10
Colagenolytic serine protease8024-28Grp 9
Rat & mouse glutathione-S-transferase4025-26Grp 8
Unknown5022-28Grp 7
Chymotrypsin-like serine protease40-6025Grp 6
Unknown45-6014-15Grp 5
Amylase25-4656-63Grp 4
Trypsin-like serine protease57-9028-30Grp 3
Primate epididymal or insect molting protein>9014Grp 2
Cysteine protease, e.g. papain, actinidin,Cathepsin, bromelain, frein.
>9025Grp 1
HomologyAdverse Reactions
[%]
Molecular Weight[kDA]
Allergen Group
Characters of House dust mite allergensCharacters of House dust mite allergens
Protein denaturing activity using SDS-PAGEProtein denaturing activity using SDS-PAGE
AllergenicAllergenic activity using Dot blot activity using Dot blot
activeactive
moderatemoderate
Expression of allergen geneExpression 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)
PrimerPrimer
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’
Gene cloningGene cloning
Vector
- pGEM®-T Vector (Promega)
- pET22b(+) (Novagen)
E-coli
- JM109 (Promega)
- Top10 (Invitrogen)
- BL21(DE3) (Novagen)
group I gene cloninggroup I gene cloning
Recombinant DNA Recombinant DNA
D. f I D. p 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
1kb
3kb
5kb
Protein purificationProtein purification
Nitriloacetic acid-Ni2+
6Histidine
Target protein
Nitriloacetic acid-Ni2+
Wash Elution
Purification of house dust mite allergens Purification of house dust mite allergens
M: marker; C: recombinant E-coli; W: wash with buffer; E: elution protein
25 kDa
Allergen denaturing activity with active Allergen denaturing activity with active compoundscompounds
2 μg/0.5 μg
4 μg /0.1 μg