안용준 교수 (Young-Joon Ahn, Ph. D.) 

교수실  : (02)880-4702 팩 스  : (02)873-2319 ( 공용 ) E-mail : yjahn@snu.ac.kr 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