PROCEDES D'UTILISATION D'HUILE D'EMU ET DE FRACTIONS ACTIVES DE CELLE-CI COMME REPULSIF CONTRE LES INSECTES

METHODS OF USING EMU OIL AND ACTIVE FRACTIONS THEREOF AS AN INSECT REPELLENT

Abrégé français

Procédé permettant de repousser les insectes piqueurs tels que les moustiques par l'application topique sur la peau d'un sujet d'huile d'ému, de fractions d'huile d'ému obtenues par chromatographie éclair ou d'une composition constituée d'huile d'ému et de citronnelle. Sont également décrits des procédés permettant de repousser les insectes piqueurs par une application topique de fractions diluées d'huile d'ému. L'invention porte également sur un composé servant à repousser les insectes piqueurs présentant le spectre de résonance magnétique nucléaire de la figure 3, c'est-à-dire sensibles à la lumière ultraviolette et réagissant à la vanilline et à l'acide sulfurique. Sont également décrits des composés servant à repousser les insectes piqueurs présentant le spectre de résonance magnétique nucléaire de la figure 4, c'est-à-dire non sensibles à la lumière ultraviolette et ne réagissant pas à la vanilline et à l'acide sulfurique. Une composition servant à repousser les insectes piqueurs comprenant de l'huile d'ému et de citronnelle est également décrite.

Abrégé anglais

This invention provides a method for repelling biting insects such as
mosquitoes by topically applying to the skin of a subject emu
oil, fractions of emu oil obtained by flash chromatography or a composition
comprising emu oil and citronella. Also provided are methods
for repelling biting insects by topically applying diluted fractions of emu
oil. The invention also provides a compound useful in repelling
biting insects having the NMR spectrum of Figure 3, that is ultraviolet light
sensitive and is reactive to vanillin/sulfuric acid. Also provided
are compounds useful in repelling biting insects having the NMR spectrum of
Figure 4, that is not ultraviolet light sensitive and is not
reactive to vanillin/sulfuric acid. A composition for repelling biting insects
comprising emu oil and citronella is further provided.

Revendications

CLAIMS:
1. A method for repelling biting insects comprising the step of topically
applying emu oil to the skin of a subject.
2. A composition for repelling biting insect comprising emu oil and
citronella.
3. The composition of claim 2, wherein the composition comprises about 30
drops of 100% oil of citronella per 25 mL of pure emu oil.
4. A method for repelling biting insects comprising the step of topically
applying a composition comprising emu oil and citronella to the skin of a
subject.
9

Description

CA 02248038 1998-09-02
WO 97/33594 PCT/US97/04i01
METHODS OF USING EMU OIL AND ACTIVE FRACTIONS THEREOF
AS AN INSECT REPELLENT
TRCHNICAL FIELD
This invention is in the field of topical insect repellents. More
particularly,
effective, natural and safe mosquito repellents comprising emu oil, active
fractions of emu
oil and compositions comprising emu oil and citronella are provided.
BACKGROUND ART
This invention relates to a method of repelling insects, and more particularly
to a
method for repelling mosquitoes using a natural ingredient, emu oil and active
fractions
thereof.
Known natural oils that repel insects include rotundial {from the leaves of
Vitex
rotundifolia, Watanabe K et al. (1995) Biotech Biochem 59(10):1979-1980);
citronella oil
(e.g. U.S. Patent No. 5,346,922); eucalyptus oil (Watanabe et al. (1993) J.
Agric. Food
Chem. 41:2164-2166); neem oil (Sharma VP et al. {1993) .7 American Mosquito
Control
Association 9(3):359-360); and oil of Hedeoma pulgioides, oil of anisum and
oil of
chrysanthemum (U.S. Patent No. 5,208,209).
However, the compound most widely used as a topically-applied insect repellent
is
N,N-diethyl-m-toluamide (DEET). When applied to children's skin, DEET has been
implicated in causing convulsions. DEFT is also known to react with certain
plastics and
synthetic rubber and cause skin irritation (Watanabe et al. (1993), supra). As
a result of
these problems and other side effects, New York State had banned products
comprised of
100% DEFT.
The active fractions of the naturally occurring insect repellents are also
largely
unknown. Methods of resolving heterogeneous compounds into chemical species
are well-
known in the art. For example, silica gel flash chromatography provides for
high speed
resolution of organic compounds (see, e.g., U.S. Patent No. 4,293,422). After
separation,
the eluted fractions can be recovered and tested for the activity of interest.

CA 02248038 2003-10-28
Accordingly, there remains a need for a natural, safe substance which acts to
repel
biting insects when topically applied to the skin. The active fractions) of
such a substance
is also needed.
SUMMARY OF T1~~'E INVENTION
The present invention provides a method for repelling biting insects
comprising the
step of topically applying emu oil, active fractions of emu oil or
compositions comprising
emu oil and citronella to the skin of a subject.
BR_TFF DESCRIPTION OF THE FIGL1RFS
Figure 1 shows the number of mosquitoes present on filter paper treated with
water,
sucrose or sucrose and diluted emu oil (sample 776) at 2.5 minute intervals.
The solid
triangles show water-treated filter paper controls. The open squares show
sucrose-treated
filter paper. The solid squares show sucrose-treated paper overlaid with
undiluted sample
776. The open triangles show sucrose-treated paper overlaid with a 50%
dilution of
sample 776. The open circles show sucrose-treated paper overlaid with sample
776 diluted
to 25%.
Figure 2 shows the number of mosquitoes present on filter paper treated with
water,
sucrose, or sucrose and fractions of sample 776 at 5 minute intervals. The
solid squares
show water-treated paper. The open squares show sucrose-treated paper
overlaid. The
solid triangles show sucrose-treated paper overlaid with a sample 776. The
open triangles
show sucrose-treated paper overlaid with fraction F 1. The solid circles show
sucrose-
treated paper overlaid with fraction F2.
Figure 3 shows the'H NMR spectrum of the F1 fraction of emu oil.
Figure 4 shows the 1H NMR spectrum of the F2 fraction of emu oil.
Figure 5 shows the average number of mosquitoes biting test limbs per ten
minute
intervals. .
Throughout this application, various publications, patents and published
patent
applications are referred to by an identifying citation.
2

CA 02248038 2003-10-28
The present invention provides a method of repelling biting insects using emu
oil, a
natural and safe substance. In a preferred embodiment, pure emu oil is applied
to the skin.
In another preferred embodiment, diluted emu oil is topically applied. In yet
another
preferred embodiment, an active fraction of emu oil is applied to the skin. In
a further
preferred embodiment, a composition comprising emu oil and citronella is
applied to the
skin.
The following examples are presented as a further guide to the practitioner of
ordinary skill in the art, and are not to be construed as limiting the
invention in any way.
Z'he effect of emu oil on freatency of mosaui~ lands a_n_d bites
To determine if emu oil was an effective mosquito repellent, pure emu oil
(Zoogen,
Inc., Davis, Ca) was applied to one hand of a volunteer. The other hand was
left untreated.
Each hand was placed in a nylon mesh cage containing mosquitoes (Aedes
aegypti) and the
number of mosquitoes which landed and/or bit in 30 seconds was recorded. The
experiment was performed in duplicate. Results of the experiments were
averaged and are
summarized in Table 1.
Table 1
emu-oil treated handuntreated hand
(lands/bites) (lands/bites)
Test 1 0/0 11/11
Test 2 1 /0 26/26
These results demonstrate that topically applied emu oil is an effective
mosquito
repellent. It greatly reduces the number of mosquitoes which land, and
completely
eliminates biting.
3

CA 02248038 1998-09-02
WO 97133594 PCT/LTS97/04101
ldxample 2 ,
The effectiveness of emu oil as a mosguito repellent over time
To determine how long topically applied emu oil maintains eff cacy as a
mosquito '
repellent, the treated hand was exposed to a cage of mosquitoes at 15, 30 and
60 minutes
after application. The number of lands and bites were compared at each time
point with
the untreated hand. Results from duplicate experiments were averaged and are
presented
in Table 2.
Table 2
Time after applicationNumber of lands Number of lands on
on emu- untreated hand
oil treated hand
4 18
30 2 12
60 10 12
These results show that emu oil remains an effective mosquito repellent for at
least
30 minutes.
l~xample 3
The effectiveness of diluted emu oiI
To determine the effectiveness of diluted emu oil, the emu oil was diluted
with
ethyl acetate to a fixed percentage, applied to one hand and inserted into a
mosquito cage.
The number of lands were recorded. The experiments were performed in duplicate
at each
dilution level. Results are shown in Table 3.
r
4

CA 02248038 1998-09-02
WO 97/33594 PCT/LTS97/04101
Table 3
Percent emu Number of landsNumber of landsAverage Number
oil Exp't 1 Exp't 2 of lands
0 10 9 9.5
0.50 10 10 10
1.0 5 5 5
5.0 6 4 5
10.0 4 5 4.5
25.0 2 I 1.5
50.0 1 1 I
75.0 1 1 1
100.0 0 0 0
These results demonstrate that dilute amounts of emu oil effectively repel
mosquitoes. At
a dilution as low as I %, emu oil reduces by one-half the number of mosquitoes
which land.
At 25% emu oil, the number of mosquito lands drops to one-tenth of lands on an
untreated
hand. Thus, emu oil is an effective insect repellent at a concentration of 1 %
or higher.
Exam In a 4
Fractionation of emu oil and the effectiveness of the fractions
An 850 mg sample (776) of emu oil was steam-distilled and fractionated using
silica flash chromatography (Baker silica gel, 40 p.m), essentially as
described in Still et al.
(1978) J. Organic Chem. 43:2923. Two major components of the sample were
eluted from
the column with 100% hexane and 25% ethyl acetate/hexane. Fractions were
analyzed by
thin layer chromatography (TLC) on silica plates developed with 50%
hexane/ethyl
acetate. Components on the TLC plates were observed by exposing the plates to
UV light
(indicating UV chromophores) and spraying plates with vanillin/sulfuric acid
(indicating
the presence of higher alcohols, sterols, phenols or essential oils). 430 mg
of a clear oil,
termed F2, was found to be UV active and reactive to vanillin/sulfuric acid.
The second
component, 380 mg of a pale yellow oil termed F1, was not UV active and did
not stain
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CA 02248038 1998-09-02
WO 97/33594 PCT/US97/04101
with vanillin. F1 and F2 fractions were analyzed by 1H NMR (300 MHz, CDCl3) as
shown
in Figures 3 and 4.
A mosquito repellent bioassay was performed by treating wedges of filter paper
,
with sucrose overlaid with either an aliquot of the crude sample 776, F I or
F2. Filter paper
treated with water or sucrose served as controls. Samples of F 1 and F2 were
tested at full- '
strength or diluted with corn oil to 50% or 25% of full-strength. At regular
time intervals,
the number of mosquitoes which landed and fed on the wedges of filter paper
was
recorded. Results are shown in Figures 1 and 2.
As shown in Figure 1, even diluted to 25% of full-strength, the crude sample
{776)
greatly reduces the number of mosquitoes landing on the sucrose paper. In
addition,
Figure 2 shows that both the F 1 and F2 fractions of sample 776 at full-
strength and diluted
to 50% or 25% of full-strength were effective in repelling mosquitoes when
compared to
the sucrose-treated control.
The effect of emu oil as a tick repellent
To determine if emu oiI was an effective tick repellent, a test subject's
hands were
treated with emu oil while the fingers of the hand were left untreated. As a
positive
control, Ultrathon (3M, Minneapolis, MN) was applied to the hand and the
forgers were
left untreated. An untreated hand was used as a negative control. Unfed
nymphal Western
Black-legged ticks were placed on the fingers of the hands and observed as
they climbed
toward the treated or untreated skin of the hand. Ticks crossing onto the
treated skin were
scored as "crossing." Those not crossing were scored as "repelled." Ticks were
removed
after a single score was recorded. Repellency is calculated as the proportion
of all trials in
which a tick is repelled. For example, 8 repels in 10 trials provides a
repellency of 80%.
In this study, each subject tested a tick at 15 minute intervals for 2 hours
and 15 minutes.
The results are shown below:
Negative control - untreated skin - 0% repellency
Positive control Ultrathon (3M) - 70% repellency
Emu oil - 40% repellency
There wa.s no indication that the repellency declined over the two hour test
period.
6

CA 02248038 1998-09-02
WO 97/33594 PCT/US97/04101
$xample 6
The effect of a composition of emu oil and citronella on freauencv of mosauito
bites
To determine if citronella would enhance the mosquito repelling effect of
emu oil, the repellent activity of emu oil alone and a composition comprising
emu oil and
citronella (30 drops of 100% oil of citronella/25 mL of pure emu oil) were
compared to a
positive control, Ultrathion~ (3M, Minneapolis, MN, approximately 31% DEET).
Oil of
citronella, Cymbopagon nardus, was obtained from Aura Cacia, Weaverville, CA.
I O Testing was conducted in the Florida Keys, namely Big Pine Key and Little
Pine
Key. During testing, the air temperature was 24-26°C, with clear skies
and slight wind.
Aedes taeniorhynchus accounted for >99% of mosquitoes that approached or
attacked
subjects.
In the first study, three subjects applied pure emu oil, one subject applied
Ultrathon, and two subjects served as negative controls. In the second study,
oil of
citronella was added to the emu oil. Three subjects applied the composition
comprising
emu oil and citronella, two subjects applied Ultrathon, and two subjects
served as negative
controls. Six of the participants were employees of the Monroe County,
Florida, Mosquito
Control Service. Treatments were evenly divided among leg and arm surfaces.
The
surface areas of treated surfaces were calculated for each subject in advance
of the
application. The test materials were applied at dosage rate of approximately
3 mLJ650 square centimeters of surface area.
Test subjects counted and recorded bites in a series of IO minute periods.
Counts
were recorded on data sheets. In the first study, the testing period was two
hours, with
12 consecutive 10 minute recording periods. In the second study, the test was
interrupted
during two brief windy periods by the need to move to more productive locales
(i.e., places
- with higher biting rates) within the test sites. As a result, the total
testing period including
the interruption was two hours and twenty-five minutes.
Ambient biting rates were measured throughout the study by the subjects with
untreated control Limbs. The average ambient biting rates were sufficient for
testing in
both studies and ranged between 17 and 70 bites per 10 minute interval (mean =
38.5; see
Figure 5).
7

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Both emu oiI and the positive control, Ultrathon, repelled mosquitoes over the
test
periods. A composition comprising emu oil and oil of citronella was
particularly effective
as a repellent (see Figure S). The repellent properties of emu oil alone
reduced the number
of bites to an average of 5.7 bites per ten minutes. The composition
comprising emu oiI
S and citronella approximated the positive control, Ultrathon, with 0.8 bites
per ten minutes
for the emu oil/citronella composition compared to 0.2 bites per ten minutes
for Ultrathion.
Over the testing period, emu oil, a composition comprising emu oil and
citronella and
Ultrathon substantially reduced biting rates from Aedes tae~ciorhynchus. A
composition
comprising emu oil and citronella was much more effective than emu oil alone
and offers
_protection comparable to that offered by mosquito repellents registered by
the U.S.
Environmental Protection Agency. No pattern of diminishing repellency over
time was
observed for emu oil and citronella.
Having now fully described this invention, it will be appreciated by those
skilled in
1 S the art that the same can be performed within a wide range of equivalent
parameters,
concentrations and conditions without departing from the spirit and scope of
the invention
and without undue experimentation. While this invention has been described in
connection
with specific embodiments thereof, it will be understood that it is capable of
further
modifications. This application is intended to cover any variations, uses, or
adaptations of
the inventions following, in general, the principles of the invention and
including such
departures from the present disclosure as come within known or customary
practice within
the art to which the invention pertains and as may be applied to the essential
features
hereinbefore set forth as follows the scope of the appended claim.
8

Dessin représentatif