CA2234476A1

CA2234476A1 - Mosquito and/or flea control

Info

Publication number: CA2234476A1
Application number: CA002234476A
Authority: CA
Inventors: William Mitchell Molyneux
Original assignee: NINETEENTH MAYBARB Pty Ltd
Current assignee: NINETEENTH MAYBARB Pty Ltd
Priority date: 1996-06-21
Filing date: 1998-04-09
Publication date: 1999-10-09
Also published as: NZ329646A; US6337071B1; AUPO060596A0

Abstract

A plant of the species Leptospermum liversidgei which is able to block the carbon dioxide message to mosquitoes and/or fleas within the vicinity of its locus.
The present invention also provides a crude or refined essential oil extract from the said plant.
Another aspect of the invention provides a method for repelling mosquitoes and/or fleas comprising the cultivation of the said plant.
A further aspect of the invention provides another method for repelling mosquitoes and/or fleas comprising the use of the said essential oil extract from the said plant.

Description

TITLE: MOSQUITO AND/OR FLEA CONTROL
FIELD OF THE INVENTION
This invention is in the field of mosquito and/or flea control.
In one aspect the invention relates to a plant which has mosquito and/or flea repellent properties.
In another aspect the invention provides an oil extract from the said plant which, in various forms and by various means, may be used as a mosquito and/or flea repellent.

BACKGROUND TO THE INVENTION
-A mosquito's and/or flea's attraction to warm-blooded animals is partly by body warmth and also by carbon dioxide as expelled breath. Certain vaporous substances, such as essential oils, are able to block the carbon dioxide message thus falsely indicating to the mosquito and/or flea that the animal simply is a source of warmth like a light globe or candle.
Many species of plants, such as tea-tree (Zeptospermum SP), are known to produce a large amount of complex mixtures of oils, which are stored in their tissues.
It is an object of this invention to isolate and characterise a true-breeding clone of a plant which, by virtue of its oil content, is able to act as a mosquito and/or flea repellant.
Such mosquito and/or flea repellency can occur by oxidisation of the oils from the leaves and stems of the plant directly into the surrounding atmosphere.
Alternatively, the oils may be extracted from the plant tissue and this extract may be used in aerosol sprays or dispersants or in pharmaceutical preparations suitable for direct topical application to the skin of a human or other warm-blooded animal.

SUi~iARY OF THE INVENTION
The present invention provides:-a plant of the Genus Zeptospermum species liversidgei which is able to block the carbon dioxide message to mosquitoes and/or fleas within the vicinity of its locus.
The present invention also provides a crude or refined essential oil extract from the said plant.
Another aspect of the invention provides a method for repelling mosquitoes and/or fleas comprising the cultivation of the said plant.
A further aspect of the invention provides another method for repelling mosquitoes and/or fleas comprising the use of the said essential oil extract from the said plant.
PREFERRED ASPECTS OF THE INVENTION
It is advantageous if the plant according to the invention, is aesthetically attractive and thus is suitable as an ornamental plant. The most preferred morphological characteristics are those given in Figures 1 and 2.
The oil which is most suitable for the purpose of the invention is citronellal however the citronellal analogues isopulegol and pulegol are also effective.
A plant according to the invention will have a citronellal and/or analogue content which falls substantially within the range of at least 55 ,- 95$ of total extractable volatile oil content. (Throughout this specification, the proportions of oil content are expressed as area percentages measured as areas under peaks by gas-chromatography.) The oil content may exhibit seasonal and/or regional variation but it is preferred that it will be at least 70$
citronellal and/or analogues.
This content may be made up of amounts of citronellal, isopulegol and pulegol which fall substantially within the ranges of 59 - 68~, 9 - 14~ and 0 - 5~ respectively.
The most preferred clone of the plant which exhibits the desired morphological features and oil content is genetically characterised by the Randomly Amplified Polymorphic DNA (RAPD) fragment analysis given in Figure 3 An essential oil mixture may be extracted from the tissues of the plant according to the invention.
It is preferred that the extracted oil will contain 55 -95~ citronellal and/or the citronellal analogues isopulegol and pulegol. It is more preferred that the oil will comprise at least 705 citronellal and/or analogues.
The most preferred embodiment of this aspect of the invention is an essential oil extract from the plant identified by the RAPD fragment analysis given in Figure 3.
The plant according to the invention may be used as a mosquito and/or flea repellant by planting one or more shrubs in either outdoor or indoor situations. The said repellency preferably occurs by oxidation into the surrounding air of oils from the leaves and stems of the plants. Alternatively, the leaves may be manually crushed or bruised to promote the efflux of the oils.
An extracted oil from the plant according to the invention may be used in a method for repelling mosquitoes and/or fleas. The oil may be vaporised by any means known in the art such as a thermal vaporiser (for example an oil burner or other heating device) or by impregnation into a vapour dispersion block or gel.

Alternatively, the oil may be applied directly to the skin or clothing of a human or animal. In this case, it is preferred that the oil is mixed with appropriate pharmaceutical carriers or excipients that make it suitable for topical application. Examples of such preparations include soaps, alcohol-based creams or lotions, oil-based creams and zinc-based creams.
These methods for repelling mosquitoes and/or fleas may be used individually or in combination with each other.
BRIEF DESCRIPTION OF THE FIGURES AND TABLES
Figure 1 is a photograph of the most preferred clone of .I~eptospermum liversidgei according to the invention.
Figure 2.is a sketch of the same plant depicted in Figure 1 with colours of the various parts identified by RHS Colour Coding Standards.
Figure 3 shows the results of Randomly Amplified Polymorphic DNA (RAPD) fragment analysis of the plant shown in Figure 1.
Table 1 characterises the DNA restriction fragments that were used as standards of known size for comparison with the RAPD fragments in Figure 3 Table 2 shows the results of gas chromatographic analysis of ethanolic extracts of leaf tissue from various individual plants according to the invention.
Table 3 gives details of the analysis parameters for the gas chromatographic mass spectrometric analysis of essential oil extracts.
Table 4 shows the results of gas chromatographic mass spectrometric analysis of oils extracted from the most preferred clone of the plant according to the invention, Table 5 shows the nucleotide sequences of the primers used to generate RAPD fragments, and Table 6 sets out the cycling conditions used in the PCR
amplification of DNA for RAPD analysis.
DETAILED DESCRIPTION WITH RESPECT TO THE FIGURES AND TABLES
Morphology Lentosnermum liversidaei Baker & Smith.
See Figures 1 and 2.
Shrub, erect, compact, 0.5 - 2.5 (3) metres tall and 1-1.5 metres wide, mostly single trunked; branches numerous, erect, ends occasionally lightly weeping, sericeous, soon becoming glabrous.
Hark hard, tight, grey, flaking in patches near base.
Leaves shortly petiolate, alternate, densely arranged up the stems, seldom spreading, flexible, apex obtuse, 3-8mm long x 1.5-3.5mm wide,~glabrous, new growth RHS Green group 141C; mature leaves RHS Green group 141A, oil glands small, numerous, emitting a citrus-like scent when crushed.
Flowers solitary, ~ sessile, mainly in axils; hypanthium 3-5mm long x 3-4mm wide, glabrous or occasionally sparsely sericeous; sepals ca 1.5mm long; petals light pink RHS Red group 56D, ca 5-7mm long x 4-5mm wide; stamens numerous C.15-20; ovary five locular, domed, glabrous.

Pro~aqation of plant Following clonal selection on the basis of tissue oil content, the plant has been propagated asexually through three generations using standard nursery techniques.
These include material cleansing, hormone stimulation, and root generation on heat beds in a glasshouse situation in a medium comprised of equal parts of peat and grade 500 perlite.
Growing-on has been carried out in a soil-less potting medium, comprising milled pinebark and coarse sand, with added trace elements and NPK fertiliser regime supplied by controlled release pellets.
Extraction and Analysis of Essential Oil Content.
Leaf tissue was obtained in June from various individuals of Leptospermum Iiversidgei. Ethanolic extracts of each sample were analysed by the single leaf extract method published by Hrophy et al, the disclosure of which is incorporated herein by reference. (J J Brophy, N W Davies, I A Southwell, I A Skiff and L R Williams, Journal of Agricultural and Food Chemistry 1989, 37 1330-1335).
Analysis was by a 60mm x 0.2mmid AT35 FSOT gas chromatography column using hydrogen as a carrier gas.
Results of this analysis are shown in Table 2. The sample with the highest total citronellal and analogue level was No. HY11 with a figure of 73~.
A further sample of this most preferred individual was taken from cultivated material when the plant was vigorously growing in March. The following procedure was used to distil the essential oils from this material.

Leaf tissue was placed in a steam distillation vessel with some boiling chips. The vessel was then filled with water to cover the plant material. The distillation cover was securely attached with a wire clamp. A Dean and Stark apparatus and water cooled condenser were then attached using a stand and clamps for support. A hot plate was placed under the distillation vessel and adjusted to bring the contents to the boil.
The essential oil was removed from the plant material by the steam, which travelled upwards to the condenser where it cooled into liquid form, collecting in the Dean and Stark apparatus. The' oil was the lower layer in the apparatus and was removed via the tap. The oil was then dried for analysis.
Analysis of the oil content was by gas chromatography and mass-spectrometry, by the parameters outlined in Table 3.
As shown in Table 4, this analysis was able to identify pulegol as well as the isopulegol and citronellal identified earlier. The measurement of total citronellal/analogue content of the oil had increased by this method to 82.5%.
Genetic characterisation of the selected clone.
Genetic characterisation of HY11 was undertaken by generation of a Randomly Amplified Polymorphic DNA (RAPD) profile.
Firstly, DNA Was isolated from leaf tissue of BY11 according to the method described by Fulton et al (T M
Fulton, J Chunwongse and~S D Tanksley, Plant Molecular Biology Reporter 1995 13, 207-209) the entire disclosure of which is incorporated herein by reference. The only modification to this method was the addition of 2% soluble polyvinyl pyrrolidone (PVP) to the micropreparation buffer.

_ g _ RAPD fragments were then generated from aliquots of this DNA by the following protocol:- .
Twenty separate PCR DNA amplification reactions were carried out, each with a different primer ( 2-Ol to 2-20, the sequences of which are shown in Table 5) under the cycling conditions set out in Table 6. The reaction mixtures contained: DNA, 50ng; Primer, 0.4pM; dNTPs, 100pM;
Mg2', 3pNl; X1 buffer; Amplitaq, 1.0 Unit; in a total reaction volume of 50 ul. When the reactions were complete, 20u1 aliquots of reaction product were sampled from each 50u1 reaction mixture and added to gel loading buffer. These samples were loaded onto a 1% agarose gel in X 1 TAE buffer and the gel was electrophoretically developed at 70V which separated the DNA fragments according to size. For comparison as known size standards, two separate preparations of Hind III and SPP1 restriction fragments of ~1DNA (as shown in Table 1) were also loaded into the 1% agarose gel in an amount of 0.5 ug total DNA
per lane. The resulting ethidium bromide stained fragments were visualised with UV light and recorded photographically as shown in Figure 3.

SPP1 = Standard A (S,) Fragment No Size (Kb) Molecular mean t Weight*
SD**

1 8.51 0.07 5,62 x 106 2 7.35 t 0.06 4.85 x 106 3 6.11 t 0.06 4.03 x 106 4 4.84 0.03 3.19 x 106 5 3.59 0.01 2.37 x 106 6 2.81 0.01 1.85 x 106 8 1.86 t 0.02 1.23 x 106 9 1.51 0.01 0.99 x 106 10 1.39 0.02 0.92 x 106 11 1.16 t 0.01 0.76 x 106 12 0.98 t 0.01 0.64 x 106 13 0.72 0.00 0.47 x 106 14 ~ 0.48 0.01 0.32 x 106 15 0.36 0.01 0.24 x 106 1 Hind III = Standard B (SH) Fragment No No of Hase Pairs Molecular Weight 1 23,130 . 15.26 x 106 2 9,416 6.21 x 106 3 6,557 4.32 x 106 4 4,361 2.88 x 106 5 2,322 1.53 x 106 6 2,027 1.33 x 106 7 564 0.37 x 106 8 125 0.08 x 106 - li -s er rid ~~al~ i:o- ~.-:a~a n.-~a~a s ;a~a ~ ,p w~ n) ~ ~

BY11 Ob 1S 592 13.8 4.0 8b 1.4 4S

13 &1 2.6 48.0 12.0 25 6.1 29 4.8 16 1.2 12 505 109 29 11.2 lit 3.d BY18 09 1.6 52.2 125 22 8d 13 52 25 1.8 2.8 495 9.4 19 113 2S 33 Insdtiment Varian GC 3400/Saturo MS

Autosampler Varian 8100 Column Dual J&W fused silica capillary) 0.25 micron DBS coadng.

Coleman Size 026 mm l.d, x 30m Sample size 0.1 microlitres of 496 solution in absolute alcohol Split ratio 2(I to 1 Carries gar Helium (c~ 60 psi) 1 ml/mia Malt up gar Nitrogen ~ 40 psi In jcctor temp 250 dcg C

GC Detector FiD A

Detector temp ZSO dcg C

Oven temp initial 60 deg C

Oven temp final 240 deg C

Oven temp pnogam 3 deg/min from 60 - 240 deg rate C

Final temp hold 2 min time FID Autoza On FID Attenuation 8 FID Range 12 Plot speed 03 an/mia Zero offset 1596 Peak reject value 30 Signal t0 nOISL 5 IdtlO

Tangent peak height 5 Initial peak width 2 Amdliary tarp 240 deg C

Manifold tanp 170 deg C

Multiplies set 1700 wits voltage A/M amplitude set 35 vole voltage Low mass 41 m/z High mass 320 m/z s~tt rate loon miu~a~

Segmwt acquire 62 min time Peak threshold 1 count Fdament/multiplier 2.00 min delay Mass defect 100 m W/ 100 W

Tune file TUN DFLT

Emission current 20 microamps AGC ptrscan ionisation 100 microsecs time AGC ptrseatt storage 20.0 m/z level EI backgrouat mass 45 m/z RF dump value 650.0 m/z AGC target 32500 counts EI max ionisation ZS000 micrasecs time alpha Pineae ~ 0.796 beta Pinene 0.1 Mynxne 0.1 I3monene +

l,s Col oa IW stool 1.4 lco Pulegol 9.7 Gtmnellal 679 Pulegol 4.9 at"O Menthol 0.6 GtoneUol 1.4 No~al 0.2 Geraniol 0.2 Methyl atronellate 0.1 Gerania( 02 Gtronellyl acetate 5.7 t Methyl cinnamate +

Ger~ryl ticetate 19 beta Caryophyllene 13 alpha Humulene 0.1 Germaame D 0.1 Geimacc~ene B 1.1 Ledol 0.1 Spathulenol 0.1 Globulol OS

beta Eudes~nol 0.1 95.696 identified Sequence of primers Primers Sequence Primers Sequence ________3 5~________3 Cycle Number Temperature Time Step in ( C ) Cycle 1 94 1 min denaturation 40 30 sec annealing 72 1 min extension I

2-34 94 10 sec denaturation 40 30 sec annealing 72 1 min extension 35 94 10 sec denaturation 40 30 sec annealing 72 5 min extension 4 Hold The claims, illustrations, photographs and drawings, if any, form part of the disclosure of this specification as does the description, claims, illustrations, photographs and drawings of any associated provisional or parent specification or of any priority document, if any, all of which are imported hereinto as part of the record hereof.
Finally it is to be understood that various alterations, modifications and/or additions may be incorporated into the various constructions and arrangements or parts without departing from the spirit and ambit of the invention.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A plant of the Genus Leptospermum species liversidgei, which is able to block the carbon dioxide message to mosquitoes and/or fleas within the vicinity of its locus.

2. A plant as claimed in Claim 1, which has the morphological characteristics given in Figure 1 and Figure 2.

3. A plant as claimed in Claim 1 or Claim 2, which has a citronellal and/or analogue content which falls substantially within the range of at least 55-95%
of total extractable volatile oil content.

4. A plant as claimed in Claim 3, wherein the plant is genetically characterised by the Randomly Amplified Polymorphic DNA (RAPD) fragment analysis given in Figure 3.

5. A plant substantially as hereinbefore described with reference to any one of the accompanying drawings.

6. An essential oil extract derived from a plant as claimed in Claim 4.

7. An essential oil extract, which contains 55-95%
citronellal and/or the citronellal analogues isopulegol and pulegol.

8. An essential oil extract, which is from the plant identified by the RAPD fragment analysis given in Figure 3.

9. A mosquito and/or flea repellent comprising the essential oil of Claims 6, 7 or 14.

10. An essential oil substantially as hereinbefore described with reference to any one of the accompanying drawings.

11. An essential oil extract derived from a plant of the Genus Leptospermum species liversidgei, in which the plant is able to block the carbon dioxide message to mosquitoes and/or fleas within the vicinity of its locus.

12. An essential oil as claimed in Claim 11, in which the plant has the morphological characteristics given in Figure 1 and Figure 2.

13. An essential oil as claimed in Claim 11 or Claim 12, in which the plant has a citronellal and/or analogue content which falls substantially within the range of at least 55-95% of total extractable volatile oil content.

14. An essential oil as claimed in Claim 13, in which the plant is genetically characterised by the Randomly Amplified Polymorphic DNA (RAPD) fragment analysis given in Figure 3.

15. An essential oil extract derived from a plant substantially as hereinbefore described with reference to any one of the accompanying drawings.

16. A method for repelling mosquitoes and/or fleas comprising cultivation of a plant of the Genus Leptospermum species liversidgei which is able to block the carbon dioxide message to mosquitoes and/or fleas within the vicinity of its locus.

17. A method as claimed in Claim 16, in which the plant has the morphological characteristics given in Figure 1 and Figure 2.

18. A method as claimed in Claim 16 or 17, in which the plant has a citronellal and/or analogue content which falls substantially within the range of at least 55-95% of total extractable volatile oil content.

19. A method as claimed in Claim 18, in which the plant is genetically characterised by the Randomly Amplified Polymorphic DNA (RAPD) fragment analysis given in Figure 3.

20. A method for repelling mosquitoes and/or fleas comprising the use of an essential oil extract as claimed in Claims 9 or 14.