WO1995010187A1

WO1995010187A1 - Pesticidal compounds

Info

Publication number: WO1995010187A1
Application number: PCT/IB1994/000334
Authority: WO
Inventors: Paul John Martin
Original assignee: Mallinckrodt Veterinary Inc
Current assignee: Mallinckrodt Veterinary Inc
Priority date: 1993-10-09
Filing date: 1994-10-06
Publication date: 1995-04-20
Anticipated expiration: 1996-04-09
Also published as: ZA947827B; GB9320836D0; AU7860794A

Abstract

Bark of the plant Afrostyrax lepidophyllus Mildbr., substantially free of other plant material, or a pesticidally active component obtainable by extraction thereof. A pesticidally active component from the bark of the plant Afrostyrax lepidophyllus Mildbr., obtainable by extraction in methanol or petroleum ether or combinations thereof or by extraction successively in more than one of methanol, petroleum ether and ethyl acetate, optionally followed by freeze-drying the extract. Pesticidal compositions and methods of combating pests, particularly insect ectoparasites and insect crop and public health/premise pests and nematode parasites of crops and animals are provided.

Description

PESTICIDAL COMPOUNDS

This invention relates to pesticidal compounds, and in particular plant extracts with pesticidal, particularly insecticidal and nematocidal activity.

Many insects and nematodes are serious pests which cause problems in many areas of medical and agricultural importance. Especially important are insect and nematode crop pests, and insect ectoparasites and nematodes which are carried by livestock such as sheep, cattle, poultry, pigs, dogs and cats, equidae and the like.

Plant extracts have been the source of many medicinal and pesticidal compounds, although it cannot be predicted in advance whether a particular plant extract will provide any beneficial effect, and if so what that beneficial effect will be. Of extracts from fifteen plants collected from Cameroon, only one, that is made from Afrostyrax lepidophyllus Mildbr. species (of the Class Magnoliatae, Sub class Dilleniidae, Order Ebenales, and Family Styracaceae), has been shown to be effective as a pesticide for combating pests including nematodes and insect ectoparasites and crop pests. No one has previously reported that extracts of plants from the Family Styracaceae have nematocidal and insecticidal activity.

A first aspect of the present invention provides bark of the plant Afrostyrax lepidophyllus Mildbr. , substantially free of other plant material, or a pesticidally active component obtainable by extraction thereof.

A second aspect of the present invention provides a pesticidally active component from the bark of the plant Afrostyrax lepidophyllus, obtainable by extraction in methanol or any alcohol containing 2 to 8 carbon atoms or petroleum ether or any alkane containing 5 to 8 carbon atoms or ethyl acetate or any ester of an alcohol containing 2 to 8 carbon atoms and an organic acid containing 2 to 8 carbon atoms or chloroform or water or butanol or combinations thereof or by extraction successively in more than one of the said solvents, optionally followed by freeze-drying the extract.

Suitable alcohols include ethanol, propan-1-ol, propan-2-ol, butan-1-ol, butan-2-ol, r -butyl alcohol, pentan-1-ol, pentan-2-ol, hexan-1-ol, heptan- 2-ol, octan-1-ol and the like.

It is preferred if the alcohol is methanol.

Suitable alkanes include pentane, hexane, heptane, octane and the like.

It is preferred if petroleum ether is used in the extraction.

Suitable esters include methylacetate, ethylbutanoate, propyl butanoate and the like.

It is preferred if the ester is ethyl acetate.

By "methanol" we include aqueous methanol comprising at least 80% methanol, preferably at least 90% methanol and more preferably 95% methanol.

Afrostyrax lepidophyllus Mildbr. is found in Cameroon and Ghana. The authority on the classification of this species of plant, and related plants in the Angiospermae, is Cronquist.

The extract conveniently may be made by extraction in any suitable solvent, but extraction in 95% methanol is preferred; the pesticidal component or components are obtainable in this manner. Exhaustive extraction may be performed in a Soxhlet apparatus, in which case the solvent is heated to its boiling point and then condensed before passing through the plant material. Other means of extraction, well known in the art, may be used. These include continuous extraction with petroleum ether until exhaustion, the remaining plant material being successfully extracted with ethyl acetate and methanol using a Soxhlet apparatus.

Such extracts have been shown to have pesticidal activity, particularly insecticidal and nematocidal activity, when contacted with or ingested by the pest.

Approximately 4 ml of 95% methanol may be used per 1 g of bark. Typically, 600 mg of freeze dried material is obtained per 10 g of bark.

Suitably the extract may be made by placing powdered stem bark (100 g) of Afrostyrax lepidophyllus Mildbr. in a glass percolator and continually extracting with petroleum ether until exhaustion. The remaining plant material is successively extracted with ethyl acetate and methanol using a Soxhlet apparatus. The petroleum ether extract contains about 205 mg of material, the ethyl acetate extract contains about 280 mg of material and the methanol extract contains about 230 mg of material.

It is preferred if the pesticidally active component is extracted in methanol or petroleum ether or ethyl acetate or combinations thereof or by extraction successively in more than one of methanol, petroleum ether and ethyl acetate, optionally followed by freeze-drying the extract.

It is further preferred if the extract, obtainable by extraction in ethyl acetate of bark which has first been extracted in petroleum ether, is used as the pesticide.

The extract of the present invention may be particularly useful for combating insect ectoparasites and nematodes which infest sheep, cattle and the like. Such insects include fleas of the Order Siphonaptera, lice of the Sub Orders Mallophaga and Anoplura; biting and nuisance flies of the Order Diptera; and myiasis flies of the Families Sarcophagidae, Calliphoridae and Oestridae. In particular, it may be useful in combating Ctenocephalides spp; Damalinia spp; Linognathus spp; Tabanus spp; Stomoxys spp; Haematobia spp; Hydrotaea spp; Musca spp; Glossina spp; Simulium spp; Lucilia spp; Chrysomya spp; Cochliomyia spp; Oestrus ovis, Dermatobia hominis, Hypoderma spp, and Gastrophilus spp.

In addition the extract may be useful for combating nematodes of animals and plants. Such animal nematodes are included within, but are not exclusive to, the following families: Trichostrongylidae (eg Trichostrongylus, Nematodirus, Ostertagia, Cooperia and Haemonchus), Strongylidae (eg Strongylus), Ancylostomatidae (eg Bunostomum), Dictyocaulidae (eg Dictyocaulus), Filariidae (eg Dirofilariά) and Ascarididae (eg Ascaris).

Such plant nematodes are included within, but are not exclusive to, the following families:- Tylenchidae (eg Tylodorus and Ditylenchus), Meloidogynidae (eg Meloidogyne), Heteroderoidea (eg Heterodera and Globodera), Longidoridae (eg Longidorus, Xiphinema and Paralongidorus) , and Trichodoridae (eg Trichodorus and Paratrichodorus) .

Additionally, the extract of the present invention may be used to combat crop and public health pests, particularly insect crop and public health/premise pests including Heliothis virescens; Spodoptera littoralis; Aphis gossypii; Myzus persicae; Nephotettix cincticeps; Nilaparvata lugens; Diabrotica undecimpunctata; Blattella germanica; Blatta orientalis; Periplaneta spp; Musca spp; Culex spp; Anopheles spp; Aedes spp; Monomoήum pharaonis; Sitophilus spp; Tribolium spp; Dermestes spp; Ephestia spp; and Anthrenus spp.

A third aspect of the invention provides a pesticidal composition comprising an extract according to the first aspect of the invention and one or more carriers, diluents or solvents.

A fourth aspect of the invention provides a method of combating a pest by applying to the pest or its locus a composition according to the second aspect of the invention.

The extract may be used to control pests such as insects and nematodes. Thus, the present invention provides a method for the control of such pests which comprises administering to the pest or to its environment a pesticidally effective amount of the extract. The present invention provides a method for the control of pest infestations of animals (including humans) and/or plants (including trees) and/or stored products and/or the environment eg premises, vegetation, water courses etc which comprises administering an effective amount of the extract. The present invention further provides for the extract for use in human and veterinary medicine, in public health control and in agriculture for the control of such pests. By "control of pests" we mean the amelioration of present or future deleterious effects of pests, including killing adults, larvae and eggs, repelling pests, knocking down pests, inhibiting reproduction of pests and other influence on the behaviour of pests.

The extract is of particular value in the protection of field, forage, plantation, glasshouse, orchard and vineyard crops, of ornamentals and of plantation and forest trees, for example cereals (such as maize, wheat, rice, millet, oats, barley and sorghum), cotton, tobacco, vegetables and salads (such as beans, cole crops, cucurbits, lettuce, onions, tomatoes and peppers), field crops (such as potato, sugar beet, ground nuts, soyabean, oil seed rape), linseed, sugar cane, grassland (including playing fields) and forage crops, (such as lucerne), plantations (such as of tea, coffee, cocoa, banana, oil palm, coconut, rubber, spices), orchards and groves (such as of stone and pip fruit, citrus fruits, kiwifruit, avocado, mango, olives and walnuts), vineyards, ornamental plants, flowers and shrubs under glass and in gardens and parks, forest trees (both deciduous and evergreen) in forests, plantations and nurseries, and plants grown for industrial or pharmaceutical purposes (such as the evening primrose).

It is also valuable in the protection of timber (standing, felled, converted, stored or structural) from attack by the Hymenopteran wood wasps (eg Urocerus), the Isoptera (termites) and the Coleoptera (eg scolytids, platypodids, lyctids, bostrychids, cerambycids, anobiids).

It has applications in the protection of stored products such as grains, fruits, nuts, spices and tobacco, whether whole, milled or compounded into products, from moth and beetle attack. Also protected are stored animal products such as skins, hair, wool and feathers in natural or converted form (eg as carpets or textiles) from moth and beetle attack; also stored meat and fish from beetle and fly attack.

The extract is of value in the control of pests which are injurious to, or spread or act as vectors of diseases in, man, domestic animals and plants, for example those hereinbefore mentioned, and more especially in the control of plant parasitic nematodes and parasitic nematodes of animals which may be found in the gastrointestinal tract, the air passages or blood vessels of the respiratory tract and the heart and associated blood vessels and of such anthropod parasites as lice, fleas, biting, nuisance and myiasis flies including midges and mosquitoes.

The extract may be used for treating vertebrates including mammals, birds and fishes. By mammals we include man, pigs, sheep, cattle, equines, cats and dogs; by birds we include hens, ducks, turkeys, geese, canaries and budgerigars; by fish we include salmon, trout and ornamental fish.

The extract is of value in the control of public health pests such as flies, hemipteran bugs, cockroaches and ants.

The extract may be used for such purposes by application of the compounds themselves or when formulated in known fashion as a dip, spray, lacquer, foam, dust, powder, granules, paste, gel, cream, shampoo, grease, vapourising mat, combustible solid and coil, bait, dietary supplement, granules, pressure pack, impregnated article, pour on, bolus, drench and injectable or other standard formulations well known to those skilled in the art.

Dip concentrates are not applied per se, but are diluted with water and the animals immersed in a dipping bath containing the dip wash. Spray concentrates, diluted or otherwise, may be applied by hand or by means of a spray race or arch or vehicle or aircraft-mounted apparatus. The animal, soil, plant or other surface being treated may be saturated with the spray by means of high volume application or superficially coated with the spray by means of light or ultra low volume application.

Dips and sprays may be prepared in the field, as emulsions in water from emulsifiable concentrates (otherwise known as miscible oils), as aqueous suspensions, from for example wettable powders, and as aqueous solutions. The emulsifiable concentrate preferably comprises a mixture of the active ingredient, with or without an organic solvent and one or more emulsifiers. Solvents may be present within wide limits but preferably in an amount of from 0 to 99.9% w/v of the composition and may be selected from water, aromatic and aliphatic hydrocarbons, ketones, alcohols, aromatic naphtha, aromatic and aliphatic esters and other solvents known in the formulating art. The concentration of emulsifiers may be varied within wide limits but is preferably in the range of 1 to 25% w/v and the emulsifiers are conveniently non-ionic surface active agents including polyoxyalkylene esters of alkyl phenols and polyoxyethylene derivatives of hexitol anhydrides and anionic surface active agents including Na lauryl sulphate, fatty alcohol ester sulphates, Na and Ca salts of alkyl aryl sulphonates and alkyl sulphosuccinates, soaps, lecithins or hydrolysed glues and others known in the art. Wettable powders comprise an inert solid carrier, one or more surface active agents, and optionally stabilisers and/or anti-oxidants. These concentrates will normally contain from 0.5 to 99.5% by weight of the active ingredient, and are diluted, for example with water, before use. Sprays may also comprise a solution or dispersion of the active ingredient in an organic solvent (see those listed above).

Lacquers are applied by brush, for example, to inanimate surfaces where the pests may rest or breed. They comprise a solution of the active ingredient in an organic solvent together with a resin, and optionally a plasticiser.

Foams are applied by mechanical or aerosol driven sprays to both animate and inanimate surfaces. They comprise the active ingredient with foam stabilising surfactants and a propellant which forms the foam as it evaporates.

Dusts and powders may be distributed by means of an applicator or incorporated into perforated bags, or other such devices, attached to trees or rubbing bars. They comprise the extract in intimate admixture with a solid inert carrier, for example suitable clays, kaolin, bentonite, attapulgite, adsorbent carbon black, talc, mica, chalk, gypsum, tricalcium phosphate, powdered cork, magnesium silicate, silicon dioxide, vegetable carriers, starch and diatomaceous earths. Such solid formulations may be prepared by impregnating the solid diluents with solutions of the extract in volatile solvents, evaporating the solvents and, if desired, grinding the products so as to obtain powders and, if desired, granulating, compacting or encapsulating the products.

Pastes, gels and creams may be applied manually to the predilection sites on the host. They are semi-solid preparations in which the extract may be present as a uniform dispersion in a suitable base such as soft or liquid paraffin or made on a non-greasy basis with glycerin, mucilage or a suitable soap.

Shampoos may be applied manually after which the animal is rinsed with clean water.

Greases may be applied manually or distributed over the surface of an inert material, such as that against which animals rub and thereby transfer the material to their skins. They may be prepared from vegetable oils, synthetic esters of fatty acids or wool fat together with an inert base such as soft paraffin. The extract is preferably distributed uniformly through the mixture in solution or suspension. Greases may also be made from emulsifiable concentrates by diluting them with an ointment base.

As pastes, gels, creams, shampoos and greases are usually applied without further dilution they should contain the appropriate percentage of the extract required for treatment.

Vapourising mats may be operated electrically by heaters and combustible solids and coils are ignited to deliver the active ingredient to the environment of the parasite. Combustible coils may contain the active ingredient with a combustible filler (wood flour), a water soluble gum, a fungistat and a dye.

Baits may be applied to walls or areas where flies and crawling insects come to rest, feed and breed. They comprise a solution or suspension in either water or a suitable solvent with sugars or a pheromone like muscamone.

Dietary supplements may be applied to the feed either when they are formulated by the manufacturer or by the stock owner just prior to use.

Granules may be placed in the environment of the pest to contact it when it goes to feed, breed or rest. The active ingredient is compounded with a relatively heavy material, for example attapulgite. In this form they fall to the bottom of rivers etc and disintegrate so releasing the active ingredient. Alternatively they may be bound with a sticky oil emulsion to pellets of vermiculite when they float on the surface of the water and disperse the insecticide.

Pressure packs, prepared as simple solutions of the active ingredient in the aerosol propellant and co-solvent such as halogenated alkanes, butane, propane and dimethyl ether. They are used to spray predilection sites of the pest either on or off the host or may be used as space sprays to contact aerial stages.

Impregnated articles, eg ear tags, collars, bands, sheets and strips etc, are placed on the animal to provide immediate and residual control of pests. In certain cases they may also be attached to the housing of the animals.

Pour on formulations are dispensed as a unit of liquid of small volume on to for example the backs of animals as single point applications or stripes such that all of the liquid is retained on the animal and may then act systemically throughout the body of the animal or by surface action contact the parasite directly.

Boluses, suitably weighted or with retention devices, are given orally to stock, like cattle and sheep, to form residual devices in the reticulum/rumen. Pesticide released from the boluses may effect control of the pest directly or systemically or when the pest attempts to breed on the animal's faeces. They are a solid dosage form comprising suitable diluents, disintegrants, binders, lubricants and weights or retention devices.

Drenches are applied to stock by the oral route so affecting the pest either directly or systemically or when it attempts to breed on the faeces of the animal.

Injectables, in a suitable vehicle, are administered to stock by, for example, the intramuscular or subcutaneous route to affect the pest systemically as it feeds on the host's blood, tissues or tissue fluids, or when it attempts to breed on the animal's faeces. Injectables and drenches comprise a solution or suspension in either water or a suitable solvent eg alcohol, polyethylene glycol together with solubilising agents, surfactants, antimicrobial preservatives and stabilisers.

The concentration of the extract to be applied to an animal, premises, other substrates or outdoor areas will vary according to the interval between treatments, the nature of the formulation and the likely infestation, but in general 0.001 to 65% w/v and preferably 0.005 to 10% of the extract should be present in the applied formulation. The amount of the extract deposited will vary according to the method of application, area of application, concentration of the extract in the applied formulation, factor by which the formulation is diluted, the efficacy of the extract and the nature of the formulation. Undiluted formulations such as pour-on formulations will in general be deposited at a concentration in the range from 0.1 to 65 % and preferably 0.1 to 10% . Sprays to inanimate surfaces will be applied to give an average initial concentration of 0.001 to 1 mg/m³.

The extract is also of use in the protection and treatment of plant species, in which case an effective pesticidal amount of the active ingredient is applied. The application rate will vary according to the compound chosen, the nature of the formulation, the mode of application, the plant species, the planting density and likely infestation and other like factors but in general, a suitable use rate for agricultural crops is in the range 0.001 to 3 kg/ha and preferably between 0.01 and 1 kg/ha. Typical formulations for agricultural use contain between 0.0001 % and 50% of the extract and conveniently between 0.1 and 15 % by weight of the extract.

Dusts, greases, pastes and aerosol formulations are usually applied in a random fashion as described above and concentrations of 0.001 to 20% w/v of the extract in the applied formulation may be used.

The extract of the invention may be combined with one or more other pesticidally active ingredients (for example pyrethroids, carbamates, lipid amides, bicyclooctanes and organophosphates, acyl ureas, acylated ethers (pyriproxifen)) and/or with attractants, repellants, bacteriocides, fungicides, anthelmintics and the like.

Stabilisers for preventing any chemical degradation which may occur with the active components of the extract of the invention include, for example, antioxidants (such as tocopherols, butylhydroxyanisole and butylhydroxytoluene) and scavengers (such as epichlorhydrin and epoxydised vegetable oil) and organic or inorganic bases, for example trialkylamines such as triethylamine, which can act as basic stabilisers and as scavengers.

Buffers may be used to control the pH of the formulations.

It will be understood that what we will claim may comprise:

(a) the extract and concentrated or dried forms thereof;

(b) pesticidal compositions comprising the extract in admixture with a carrier, or carriers;

(c) processes for the preparation of such pesticidal compositions; (d) methods for the control of insect and nematode pests comprising the application to the pest or its environment of the extract in such pesticidal compositions, (e) potentiating or non-potentiating mixtures of the extract and another pesticidal compound. Preferred aspects of the invention will now be described with reference to the following Examples and

Figure 1, which shows an extraction scheme.

Example 1: Preparation of methanol: water extract

The extract is prepared by taking the ground bark and extracting it by the continuous solvent extraction Soxhlet procedure using a 95:5 volume by volume methanokwater mixture. The resultant extract is then concentrated by rotary evaporator and then freeze dried.

Example 2: Preparation of further extracts

Alternatively the extract is made by placing powdered stem bark (100 g) of Afrostyrax lepidophyllus Mildbr. in a glass percolator and continually extracting with petroleum ether until exhaustion. The remaining (ie petroleum-ether-exhausted) plant material is successively extracted with ethyl acetate and methanol using a Soxhlet apparatus. The petroleum ether extract contains about 205 mg of material, the ethyl acetate extract contains about 280 mg of material and the methanol extract contains 230 mg of material. Each may be concentrated in a rotary evaporator and freeze-dried.

Example 3: Preparation of further extracts

A successive extraction is shown in Figure 1. The ground stem bark (120 g) was extracted with petroleum ether (b.p. fraction 40-60°C) by maceration (2 x 1.5 litres, each maceration for 24 hours). The resulting extract was concentrated under vacuum at 40°C to give a solid (SHM-5- 2A, 0.59 g).

The plant material was dried and then re-extracted in an identical manner using methanol (2 x 1.5 litres) as the solvent. Concentration of the extract gave an oil (SHM-5-2B, 6.726 g). This oil was shaken for 24 hours with a mixture of 500 ml of chloroform and 1.5 litres of distilled water. This process was repeated with fresh solvent for a further 24 hours. The chloroform and water layers were allowed to separate. Concentration of the chloroform extract under vacuum at 40 °C gave a solid, SHM-5-2C (1.3 g). The water layer was further extracted with n-butanol (1 part water to 3 parts butanol) by liquid-liquid extraction for 24 hours (repeated for a further 24 hours with a further batch of solvents). The two butanol extracts were bulked and concentrated under vacuum (maximum 60°C) to give a butanol-soluble fraction (SHM-5-2D, 4.28 g) and a water-soluble fraction (SHM-5-2E, 0.506 g).

Finally, the plant material was dried and then subjected to a third maceration with water (3 litres). Concentration of the resulting solution (under vacuum at 60°C) gave SHM-5-2F (1.5 g).

Each extract was tested as indicated below.

Example A: Assays of pesticidal activity of extract

Species and strains of ectoparasites used

Sheep body louse, Damalinia ovis, the IRL89 (Illabo) strain with a resistance factor to synthetic pyrethroids of approximately 8 fold.

Sheep blowfly, Lucilia cuprina, the CFS strain with a resistance factor to organophosphorus compounds of approximately 12-15 fold.

Buffalo fly, Haematobia irritans. The CSIRO Standard susceptible strain was used for the methanol extraction assays and the CERIT laboratory strain, University of New South Wales, Kensington was used for the sequential extraction assays.

Species and strain of nematode used

Stomach worm, Haemonchus contortus, McM, McMaster susceptible strain.

Assay procedures

Sheep lice contact test - a serial dilution of the freeze-dried extract was made using acetone and then pipetted on to glass micro-fibre papers placed in 5 cm petri dishes. After the papers had dried 10 lice were placed on each paper and incubated at 37°C and 75% relative humidity for 18 hours. The lice were then assessed as alive (able to move in a straight line between two points) or dead (including moribund).

Sheep lice ingestion test - the freeze-dried extract was mixed, using a pestle and mortar, with the lice diet and then weighed into wells of multi well petri dishes. 10 lice were added to each well and the test incubated at 37°C and 75% relative humidity. Mortality was assessed as for the contact test at 24, 48 and 72 hours.

Sheep blowfly larval test - the freeze-dried extract was dissolved in methanol and impregnated onto a solid cotton substrate held in a tube. To this was added new born calf serum. Approximately 100 first instar larvae were added to each tube and the test was incubated at 27 °C for 48 hours at which time mortality was assessed.

Buffalo fly contact test - the freeze-dried extract was dissolved in acetone and then a 1 ml sample was pipetted onto 5 cm Whatman filter papers held in 5 cm petri dishes and allowed to dry. Ten adult flies were placed in each dish and the test incubated for 1 hour at 28 °C and 90% relative humidity when mortality was assessed.

Buffalo fly topical test - the freeze-dried extract was dissolved in acetone and, using a microapplicator, a 0.5 μl drop was applied to the dorsal thorax of each fly. Two replicates, each of 10 flies, were treated and at 1 hour after treatment the flies were assessed as live, moribund or dead.

Stomach worm larval development test - serial dilutions of each sequential extract were prepared in dimethyl sulphoxide. Test solutions were prepared following further dilution with water. 2 μl of each solution was then added to 198 μl of 2% agar and placed in a 96 well perspex (ELISA) plate. Approximately 80 eggs were added to the surface of the agar in each well, supplemented with nutrient medium and incubated for 5 days. Development was monitored to the L3 larval stage and % inhibition of development determined.

Statistical analysis

Percentage mortalities were calculated on the total number of organisms in all replicate samples for each concentration by the following formula:

(N_d/T.) x 100 where N_d and T, are the number of dead organisms and the total number treated respectively.

Corrections for control mortality using Abbott's formula were made to the contact test data before subjecting them to probit analysis to estimate the LC^.

Abbott's formula is: P = ((P'-C)/(100-Q) x 100 where Abbott's formula calculates the percentage killed by the toxin alone (P) by adjusting the total percentage killed (P') in relation to the percentage dying in the control (C).

Results

Sheep lice contact test:

LC 50 95% Fiducial Limits

(μg/ml) (μg/ml)

Afrostyrax lepidophyllus Methanol Extract 328 218-492

Sequential extraction using: petroleum ether 15 9-24

18 10-33 ethyl acetate 3 1-15

2 1-73 methanol 8 4-15

6 3-10 Sheep lice ingestion test:

% mortality at 200,000 μg/g 24 h 48 h 72 h

Afrostyrax lepidophyllus Extract 100 100 100 Control 0 3 6

Sheep blowfly test:

% mortality at 10,000 μg/ml

24 h 48 h

Afrostyrax lepidophyllus

Extract 100 100

Control 2 3

Sequential extraction using:

petroleum ether 94 100 ethyl acetate 100 100 methanol 2 20

Buffalo fly topical and contact test:

% mortality at 10,000 μg/ml Topical Contact Afrostyrax lepidopyllus Methanol Extract 63 40

Control 0 0

Sequential extraction using:

petroleum ether 75 ethyl acetate 100 methanol 40

Stomach worm larval development test:

% inhibition of development at 50 μg/ml

Sequential extraction using: petroleum ether 0 ethyl acetate 100 methanol 0

Example B: Formulations

% w/w

1. Emulsifiable Concentrate Freeze-dried extract of Example 2 (ethyl acetate) 10.00

Ethylan KEO 20.00

Xylene 67.50

Butylated Hydroxyanisole 2.50

100.00 2. Wettable Powder

Freeze-dried extract of Example 2 (ethyl acetate) 25.00

Attapulgite 69.50

Sodium isopropylbenzene sulphonate 0.50

Sodium salt of condensed naphthalene sulphonic acid 2.50

Butylated hydroxytoluene 2.50 100.00

3. Dust

Freeze-dried extract of Example 2 (ethyl acetate) 0.50

Butylated Hydroxyanisole 0.10

Talc 99.40 100.00

4. Bait

Freeze-dried extract of Example 2 (ethyl acetate) 0.25

Icing Sugar 99.65

Butylated hydroxy toluene 0.10

100.00

5. Lacquer

Extract of Example 2 (ethyl acetate) 2.50

Resin 5.00 Butylated Hydroxyanisole 0.50

High aromatic white spirit 92.00

100.00 6. Aerosol

Extract of Example 2 (ethyl acetate) 0.30

Butylated Hydroxyanisole 0.10

1,1,1 -Trichloroethane 4.00 Odourless Kerosene 15.60

Freon 11/12. 50:50 mix 80.00

100.00

7. Spray Extract of Example 2 (ethyl acetate) 0.10

Butylated Hydroxyanisole 0.10

Xylene 10.00

Odourless Kerosene 89.80

100.00

8. Potentiated Spray

Extract of Example 2 (ethyl acetate) 0.10

Permethrin 0.10

Butylated Hydroxyanisole 0.10 Xylene 10.10

Odourless Kerosene 89.60

100.00

9. Bolus mg

Extract of Example 1 1300

Sodium starch glycollate 300

Microcrystalline cellulose 1200 Lactose 2920

Povidone 250

Magnesium stearate 30

6000

10. Injection Suspension mg

Extract of Example 1 40.0

Sodium metabisulphite 1.0

Polysorbate 80 1.0

Sodium methyl hydroxybenzoate 2.0 Water for injections - to make 1.0 ml

11. Injection Solution mg

Extract of Example 1 20.0

Sodium citrate 6.0 Citric acid 1.0

Sodium chloride 7.0

Chlorcresol 1.0

Water for injections - to make 1.0 ml

12. Oral Suspension

Extract of Example 1 100.00 g

Polysorbate 80 2.00

Xanthan gum 5.00

Colloidal silicon dioxide 10.00 Methyl hydroxybenzoate 1.50 Citric acid monohydrate 5.00

Sodium citrate 10.00

Purified water - to make 1000 ml

Claims

1. Bark of the plant Afrostyrax lepidophyllus Mildbr. , substantially free of other plant material, or a pesticidally active component obtainable by extraction thereof.

2. A pesticidally active component from the bark of the plant Afrostyrax lepidophyllus, obtainable by extraction in (i) methanol, or (ii) any alcohol containing 2 to 8 carbon atoms, or (iii) petroleum ether or any alkane containing 5 to 8 carbon atoms, or

(iv) ethyl acetate or any ester of an alcohol containing 2 to 8 carbon atoms and an organic acid containing 2 to 8 carbon atoms, or (v) chloroform, or (vi) water, or (vii) butanol, or (viii) combinations thereof or by extraction successively in more than one of the said solvents, optionally followed by freeze-drying the extract.

3. A pesticidally active component according to Claim 2 obtainable by extraction in methanol or petroleum ether or ethyl acetate or combinations thereof or by extraction successively in more than one of methanol, petroleum ether and ethyl acetate, optionally followed by freeze-drying the extract.

4. A pesticidally active component according to Claim 2 obtainable by extracting petroleum-ether-exhausted bark in ethyl acetate.

5. A pesticidally active component according to Claim 2 obtainable by extraction of plant material in petroleum ether.

6. A pesticidally active component according to Claim 2 obtainable by extraction in methanol of petroleum-ether-exhausted material.

7. A pesticidally active component according to Claim 6 obtainable by extracting the methanol-exhausted material with water.

8. A pesticidally active component according to Claim 6 obtainable by extracting the methanol fraction in water/chloroform.

9. A pesticidally active component according to Claim 8 obtainable by separating the said water and chloroform fractions and obtaining the component in the chloroform fraction.

10. A pesticidally active component according to Claim 8 obtainable by separating the said water and chloroform fractions and extracting the water fraction in water/butanol.

11. A pesticidally active component according to Claim 10 obtainable from the butanol fraction.

12. A pesticidally active component according to Claim 10 obtainable from the water fraction.

13. A pesticidal composition comprising a pesticidally active component according to any of Claims 1 to 4 and one or more carriers, diluents or solvents.

14. A pesticidal composition according to Claim 13 additionally comprising one or more other pesticidally active ingredients.

15. A method of combating a pest by applying to the pest or its locus a composition according to Claim 13 or 14.

16. A method according to Claim 15 wherein the composition is contacted with or ingested by the pest.

17. A method according to Claim 15 or 16 wherein the pest is an insect crop or public health pest.

18. A method according to Claim 17 wherein the insect is selected from Heliothis virescens; Spodoptera littoralis; Aphis gossypii;

Myzus persicae; Nephotettix cincticeps; Nilaparvata lugens; Diabrotica undecimpunctata; and Blattella germanica.

19. A method according to Claim 15 or 16 wherein the pest is an insect ectoparasite of vertebrates.

20. A method according to Claim 19 wherein the insect is selected from the group consisting of fleas of the Sub Order Siphonaptera, lice of the Sub Orders Mallophaga and Anoplura; biting and nuisance flies of the Order Diptera; and myiasis flies of the families Sarcophagidae, Calliphoridae and Oestridae.

21. A method according to Claim 18 wherein the insect is selected from the group consisting of Ctenocephalides spp; Damalinia spp; Linognathus spp; Tabanus spp; Stomoxys spp; Haematobia spp; Hydrotaea spp; Musca spp; Glossina spp; Simulium spp; Lucilia spp; Chrysomya spp; Cochliomyia spp; Oestrus ovis; Dermatobia hominis; Hypoderma spp and Gastrophilus spp.

22. A method according to Claim 15 or 16 wherein the pest is a nematode parasite of vertebrates.

23. A method according to Claim 22 wherein the nematode is selected from the group consisting of Trichostrongylidae, Strongylidae, Ancylostomatidae, Dictyocaulidae, Filariidae and

Ascarididae.

24. A method according to Claim 23 wherein the nematode is selected from the group consisting of Trichostrongylus, Nematodirus, Ostertagia, Cooperia, Haemonchus, Strongylus,

Bunostomum, Dictyocaulus, Dirofilaria and Ascaris.

25. A method according to Claim 15 or 16 wherein the pest is a nematode parasite of plants.

26. A method according to Claim 25 wherein the nematode is selected from the group consisting of Tylenchidae, Meloidogynidae, Heteroderoidea, Longidoridae and Trichodoridae.

27. A method according to Claim 26 wherein the nematode is selected from the group consisting of Tylodorus, Ditylenchus, Meloidogyne, Heterodera, Globodera, Longidorus, Xiphinema, Paralongidorus , Trichodorus and Paratrichodorus.