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INSECTICIDAL PROPERTIES OF SECONDARY METABOLITES PRODUCED BY AN ENDOLICHENIC FUNGUS, PENICILLIUM PINOPHILUM AGAINST STORED GRAIN INSECT PESTS, CALLOSOBRUCHUS MACULATUS.

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1 Insecticidal properties of secondary metabolites produced by an endolichenic fungus , Penicillium pinophilum against stored grain insect pests, Callosobruchus maculatus . Maddumage Piyath Madushara Cooray Institute of Chemistry Ceylon, Sri Lanka

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  Insecticidal properties of secondary metabolites produced by an endolichenic fungus, Penicillium pinophilum against stored grain insect pests, Callosobruchus maculatus . Maddumage Piyath Madushara Cooray Supervisors: Prof. (Mrs.) P. A. Paranagama Dr. (Mrs.) T. Gobika 2

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3 Cowpea, ( Vigna unguiculata ) is one of the most nutritious grain legumes for human consumption worldwide. It is a valuable source of dietary protein, vitamins and minerals (Singh et al., 2003). Cowpea ( Vigna unguiculata) [http://www.beanbeetles.org/handbook/, accessed in December 2015]

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4 [http://www.beanbeetles.org/handbook/, accessed in December 2015] Different types of cowpea varieties : Clockwise from the top left: blackeye peas (cowpeas) light and dark varieties, mung, adzuki, hyacinth beans and pigeon peas.

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5 Loss of seed yield in legume crops during storage due to different types of stored-grain insects, notably bruchid beetles, is a very serious problem for farmers and traders (Rees, 2004). Cowpea weevil, Callosobruchus maculatus ( Coleoptera : Bruchidae ) is one of the most destructive pest species of cowpea. [http://www.beanbeetles.org/handbook/, accessed in December 2015]

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Kingdom : Animalia. Phylum : Arthropoda. Class : Insecta. Order : Coleoptera. Family : Chrysomelidae. Genus : Callosobruchus. Cowpea weevils. Growth temperature : 32 ºC. Life time : 10 days. Humidity : 60% - 90%. [https://en.m.wikipedia.org/wiki/callosobruchus_ maculatus/, accessed in December 2015] Callosobruchus maculatus 6

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Figure : The life cycle of the C. maculatus. Eggs: Adult females oviposit their egg on the surface of the cowpea and eggs are cemented to the surface of cowpea (Figure No: 01-A). Larva and Pupa: The larvae and pupae are normally only found in cells bored within the cowpea (Figure No: 01-C). [http://www.beanbeetles.org/handbook/, accessed in December 2015] Life cycle 7

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Adult: C. maculatus  adults are 2.1-3.5 mm long. The females are maculated with four elytral spots and males are plain with no distinct spots. [http://www.beanbeetles.org/handbook/, accessed in December 2015] Appearance of male (right) and female (left) C. maculatus 8

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9 [http://www.beanbeetles.org/handbook/, accessed in December 2015] Recent estimations have revealed that about 2.5 million tons of pesticides are used on crops and the worldwide damage caused by pesticides reaches $100 billion annually ( Koul et al., 2008). The indiscriminate use and the excessive reliance on chemical pesticides ( phosphine , organophosphates ) in crop protection have resulted in serious problems linked to water and environmental contamination, phytotoxicity, and toxic hazards to human and non-target organisms (Boyer et al., 2012).

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10 Selection of resistant insect strains, including C. maculatus against chemical pesticides is also a thorny problem in crop protection ( Odeyemi et al., 2006; Kang et al., 2013). These concerns have resulted in a renewed interest being given to the search for new approaches to control insect pests without these negative draw-backs. In recent years, many research studies focused on the use of natural materials as low-risk pest control agents. [http://www.beanbeetles.org/handbook/, accessed in December 2015]

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Bio - insecticides Bio-insecticides are certain types of insecticides derived from such natural materials as animals, plants, bacteria, fungi, viruses and certain minerals. Examples: Bacillus thuringiensis Azadirachtin Bacillus popilliae Essential oils ( citrus peel oil ) Plants and plant extracts ( Aframomum melegueta , Aglaia elliptical, Annona spp. ) 11 Khani , M.; Muhamad , A.R.; Omar, D. Insecticidal effects of peppermint and black pepper essential oils against rice weevil, Sitophilus oryzae L. and rice moth, Corcyra cephalonica (St.). J. Med. Plants 2012 , 11 , 97–110.

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Advantages of bio-insecticides : Do not persist long in the environment and have shorter shelf lives. They are effective in small quantities. Safer to humans and animals compared to synthetic insecticides. They are very specific, often affecting only a single species of insect and have a very specific mode of action. Slow in action and the timing of their application is relatively critical. 13

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14 Some fungi produce many secondary metabolites with insecticidal properties. Therefore the present study was focused to deduce bio-insecticide from extract of P. pinophilum .

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The lichen host, Pseudocyphellaria sp., was collected from the Hakgala Botanical Garden situated at an elevation of about 1745 m above mean sea level within the Hakgala Strict Natural Reserve in the Nuwara Eliya District, Sri Lanka. The collected lichen samples were kept in sterilized polythene packs and transported to the laboratory at the Department of Chemistry, University of Kelaniya and processed within 24 h. Isolation of the fungus After that endolichenic fungus, Penicillium pinophilum was isolated from the lichen host, Pseudocyphellaria sp. 15 Zhang, Y.; Li, X.M.; Shang, Z.; Li, C.S.; Ji , N.Y.; Wang, B.G. Meroterpenoid and diphenyl ether derivatives from Penicillium sp. MA-37, a fungus isolated from marine mangrove rhizospheric soil. J. Nat. Prod. 2012 , 75 , 1888–1895.

Penicillium pinophilum:

Penicillium pinophilum Kingdom : Fungi. Order : Eurotiales. Family : Trichocomaceae. Genus : Penicillium. Endolichenic fungus. Reproduce asexually (conidia). Growth temperature : 32 ºC. Growth time : 7 days. Colony size : diameter 35-36 mm. Mycelium : white to orange white. [https://en.m.wikipedia.org/wiki/callosobruchus_ maculatus/, accessed in December 2015] 16

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To investigation of the insecticidal activity from the isolation of secondary metabolites in Endolichenic fungus, Penicillium pinophilum and their toxicity against cowpea weevil, Callosobruchus maculatus . Objective 17

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P.D.A medium was prepared (potato infusion, Glucose and Agar ) . Autoclaved at 120 ºC . Allowed to cool to 32-35 º C inside the laminar flow cabinet. Fermentation Large 50 plates were prepared and allowed to cooled to room temperature. Pure fungi cultures were introduced to plate and incubated for 2 weeks. After two weeks. 18

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Cut of mycelium and PDA medium were added to large conical flasks with EtOAc. Shacked for 24 hours. Filtrates was obtained. After solvent was evaporated by using rotatory evaporator. Finally 5.5 g of semisolid was obtained. Extraction process of secondary metabolites… 19

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C. maculatus infested black eye peas cowpea were collected. After one week. Transfer in to the 2 L jar and covered from holey cap and placed at 32 ºC . Residual Film method. Insecticidal bioassay Different concentrations of series was prepared and added to clean glass vials. Solvent was evaporated by slowly rotated on a horizontal plane. Three pairs of one day old C. maculatus were introduced into them and covered from holey caps and placed at 32 ºC for 3 days. Khani , M.; Muhamad , A.R.; Omar, D. Insecticidal effects of peppermint and black pepper essential oils against rice weevil, Sitophilus oryzae L. and rice moth, Corcyra cephalonica (St.). J. Med. Plants 2012 , 11 , 97–110. 20

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Figure: Percentage mortality of C. maculatus treated with the crude extract of P. pinophilum . 3 rd day showed higher toxicity than that other days. 21

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10 % of blood suspension was prepared. 200 µl of blood + saline. Centrifuged at 3000 rpm for 10 min. Death blood cell 10 % of blood suspension (1 ml of blood + 9 ml of saline). Human Red Blood Cell (HRBC) Membrane stability method. Anti-inflammatory Assay Live blood cell Blunt, J.W.; Copp , B.R.; Hu , W.P.; Munro, M.H.G.; Northcote , P.T.; Prinsep , M.R. Marine natural products. Nat. Prod. Rep. 2008 , 25 , 35–94. 22

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Different concentrations of series was prepared. These were added into centrifuge tubes. Incubated in water bath at 56 ºC for 30 min. Tubes were cooled under running tap water. Centrifuged at 2500 rpm for 5 min. Absorbance was taken at 540 nm. Blunt, J.W.; Copp , B.R.; Hu , W.P.; Munro, M.H.G.; Northcote , P.T.; Prinsep , M.R. Marine natural products. Nat. Prod. Rep. 2008 , 25 , 35–94. 23

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The Percentage inhibition of Haemolysis was calculated as follows: % Inhibition = Blunt, J.W.; Copp , B.R.; Hu , W.P.; Munro, M.H.G.; Northcote , P.T.; Prinsep , M.R. Marine natural products. Nat. Prod. Rep. 2008 , 25 , 35–94. 24

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Figure: Anti-inflammatory assay activity of the crude extract of P. pinophilum. Crude extract showed higher anti-inflammatory activity than that aspirin. 25

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Known amount of Ethylacetate extract Dissolve in 80% Methanol Shaken with distilled hexane in a separating funnel The above methanol layer was shaken with distilled Chloroform in a separating funnel Methanol Fraction Hexane Fraction Chloroform fraction Hexane Fractionation Hexane Methanol Chloroform Methanol 26

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Hexane fraction Chloroform fraction Methanol fraction CH 2 Cl 2 : MeOH = 7:3 CH 2 Cl 2 : MeOH = 3:7 solvent systems: CH 2 Cl 2 : MeOH = 1:1 27

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Figure: Percentage mortality of C. maculatus treated with the chloroform fraction of P. pinophilum . 28 3 rd day showed higher toxicity than that other days.

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Figure: Anti-inflammatory assay activity of the hexane, chloroform and methanol fractions of P. pinophilum. Chloroform fraction only showed higher anti-inflammatory activity than that aspirin. 29

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38 Fractions were collected . TLC for above fractions. 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 1 5 10 15 21 26 32 38 Silica gel Column Chromatography 16 17 18 19 20 solvent systems: CH 2 Cl 2 : MeOH = 7:3 30

These fractions were combined on the basis of their TLC profiles to give seven fractions. :

These fractions were combined on the basis of their TLC profiles to give seven fractions . F 1 F 2 F 3 F 4 F 5 F 6 F 7 solvent systems: CH 2 Cl 2 : MeOH = 7:3 31

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Twenty two fractions (10 ml each) were collected. And combined on the basis of their TLC profiles to obtain two fractions ( F 1 A and F 1B ). Fraction F 1 A was found to be active in the insecticidal assay and anti-inflammatory assay. After fraction F 1A was next chromatographed on silica gel to give three active fractions (F 1A-1 , F 1A-2 and F 1A-3 ). Sepadex Column Chromatography 32

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Fractions were dissolute with CH 2 Cl 2 . Solvent system (CH 2 Cl 2 : MeOH = 7:3) was added into TLC Jar . After placed the TLC plate (with the sample of line drawing at the bottom ) into the jar. Normal phase preparative TLC 33

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2 1 S elected s ilica layer was peeled and put into column. Dichloromethane was passed through the column and collected eluate and after that solvent was evaporated by using rotary evaporator . 34

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2 1 solvent systems: CH 2 Cl 2 : MeOH = 7:3 Under fluoresce light : Under UV light : 35

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2 3 CH 2 Cl 2 : MeOH = 7:3 solvent systems: Under UV light : 36

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Figure: Percentage mortality of C. maculatus treated with the pure compound 3 isolated from the chloroform extract of P. pinophilum. 37 3 rd day showed higher toxicity than that other days.

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Figure: LD 50 values of the crude extract, chloroform fraction and pure compound 3 of P. pinophilum . pure compound 3 has low LD 50 value. 38

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Figure: Anti-inflammatory assay activity of the pure compounds (1, 2 and 3) isolated from the chloroform extract of P. pinophilum. 39

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Results suggest pure compound 3 can be used as bio-insecticide against C. maculatus. Pure compounds 1, 2 and 3 showed higher anti-inflammatory activity than that aspirin. Conclusion 40

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Future plans will be get NMR spectrums for pure compounds 1, 2 and 3. Future plans 41

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I would like to express my sincere and immense gratitude to my supervisors Prof. P.A. Paranagama , Dr. Gobika . My special thanks for past Honorary Rector of college Prof. J.N.O Fernendo for Initiating this programme. My special thanks for Honorary Rector of College Prof. Deraniyagala , Dean of college Mr. M.R.M.Haniffa and Prof. H.D.G.Gunawarthana , Prof. S.Sotheeswaran, Prof. M.de.P .Costa for providing the facilities at the college, supports and encouragements. My sincere thanks to Mis. Anoosheya Kuganesan and all other members of Laboratory staff of the IchemC. My special gratitude and thanks to my parents and friends those who helped and encourage me in varied ways. Acknowledgement 42

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