Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
INSECT REPELLENT COMPOSITIONS COMPRISING
DI HYDRONEPETALACTONE
Field of the Invention
The present invention relates to the field of insect repellency, and
the use of dihydronepetalactone stereoisomers generally as repellent
materials.
Background of the Invention
Repellent substances generally cause insects to be driven away
from, or to reject, otherwise insect-acceptable food sources or habitats. At
least 85% of insect repellent sales in the United States are for insect
repellents containing N,N-diethyl-m-toluamide (DEET) as their primary
active ingredient. Further, Consumer Reports tests indicated that products
with the highest concentration of DEET lasted the longest against
mosquitoes. Although an effective repellent, DEET possesses an
unpleasant odor and imparts a greasy feeling to the skin. Furthermore,
although it has recently been re-registered for use in the U.S. by the EPA,
concerns have been raised as to its safety, particularly when applied to
children (Briassoulis, G.; Narlioglou, M.; Hatzis, T. (2001) Human &
Experimental Toxicology 20(1), 8-14). Some studies have suggested that
high concentrations of DEET may give rise to allergic or toxic reactions in
some individuals. Other disadvantages associated with DEET include:
1) it is a synthetic chemical, that is, it is. not derived from natural
sources;
2) it exhibits a limited spectrum of activity - it is not, for example, as
effective as might be desired against black-legged or deer ticks (Schreck,
C.E., Fish, D. & McGovern, T.P. (1995) Journal of the American Mosquito
Control Association 11(1), 136-140); 3) DEET dissolves or mars many
plastics and painted surfaces; and 4) DEET may plasticize some inert
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ingredients typically used in topical formulations which leads to lower user
acceptability. ,
As a result of the above limitations, DEET-free products with
repellent activity are finding favor with consumers. In particular, demand
for compositions containing natural products is increasing. New candidate
repellents should possess a desirable balance of properties, and will
preferably reach or exceed the positive properties of DEET, and/or not
suffer from its negative properties (Hollon, T. (2003) The Scientist June 16
2003, 25-26). Potential substitutes for DEET should desirably then exhibit
a combination of excellent repellency, high residual activity and low toxicity
to humans (or pets) and the environment. Moreover, there is increasing
demand for repellent compounds that can be obtained from, or
synthesized from, natural plant materials and that are pleasant to use.
Any candidate to replace DEET should exhibit repellency to a wide variety
of insects considered noxious by humans, including, but not limited to,
biting insects, wood-boring insects, noxious insects, household pests, and
the like.
Many plant species produce essential oils (aromatic oils) which are
used as natural sources of insect repellent and fragrant chemicals [Hay,
R.K.M., Svoboda, K.P., Botany, in `Volatile Oil Crops: their biology,
chemistry and production'. Hay, R.K.M., Waterman, P.G. (eds.). Longman
Group UK Limited (1993)]. Citronella oil,, known for its general repellence
towards insects, is obtained from the graminaceous plants Cymbopogon
winterianus and C. nardus. Examples of plants used as sources of
fragrant chemicals include Melissa off/cinalis (Melissa), Peri/la frutescens
(Perilla), Posostemon cablin (Patchouli) and various Lavandula spp.
(Lavender). All of these examples of plants yielding oils of value are
members of the Labiatae (Lamiaceae) family. Plants of the genus Nepeta
(catmints) are also members of this family, and produce an essential oil
that is a minor item of commerce. This oil is very rich in a class of
monoterpenoid compounds known as iridoids [Inouye, H. Iridoids. Methods
in Plant Biochemistry 7:99-143 (1991)], more specifically the
methylcyclopentanoid nepetalactones [Clark, L.J. et al. The Plant Journal,
11:1387-1393 (1997)] and derivatives.
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Iridoid monoterpenoids have long been known to be effective
repellents to a variety of insect species (Eisner, T. (1964) Science
146:1318-1320; Eisner, T. (1965) Science 148:966-968; Peterson, C. and
Coats, J. (2001) Pesticide .Outlook 12:154-158; Peterson, C. et al. (2001)
Abstracts of Papers American Chemical Society 222 (1-2): AGRO73).
Studies of the repellency of catnip oil (predominantly nepetalactone)
showed that it was repellent towards a number of insect species on short-
term exposure, but not to a number of other species (Eisner, T. (1964)
Science 146:1318-1320).
U.S. Patent 4,663,346 discloses insect repellants with compositions
containing bicyclic iridoid lactones (e.g., iridomyrmecin). Further, U.S.
Patent 4,869,896 discloses use of these bicyclic iridoid lactone
compositions in potentiated insect repellent mixtures with DEET. U.S.
Patent 6,524,605 discloses insect repellents comprising nepetalactones
derived from the catmint plant N. cataria, and the differential efficacy of
nepetalactone stereoisomers as insect repellents.
Compositions containing dihydronepetalactones (DHN), a class of
iridoid monoterpenoids derived from nepetalactones (shown in Figure 1),
are known to provide insecticidal effects. For example, a study of the
composition of the secretion from anal glands of the ant Iridomyrmex
nitidus showed that isodihydronepetalactone'was present in appreciable
amounts, together with isoiridomyrmecin (Cavill, G.W.K., and D.V. Clark.
(1967) J. Insect Physiol. 13:131-135). Isoiridomyrmecin was known at the
time to possess good `knockdown' insecticidal activity.
Cavill et al. (1982) (Tetrahedron 38:1931-1938), discloses the
presence of dihydronepetalactones in the insect repellent secretion of an
ant but the compound iridodial is said to be the principal repellent
constituent.
Jefson, M., et al. (1983) (J. Chemical Ecology 9:159-180) disclose
dihydronepetalactone to exhibit effective repellency in the vapor phase to
ants over a period of 25 seconds. Longer times were not investigated.
After 25 seconds of exposure to vapors from the pure
dihydronepetalactone, approximately 50-60% of Monomorium destructor
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ants ceased to feed. No indication was given in regard to the duration of
the repellent effect.
Summary of the Invention
One embodiment of this invention is an insect repellent
composition of matter that is or includes a dihydronepetalactone, or a
mixture of dihydronepetalactone stereoisomers, represented by the
general formula:
0
0
Another embodiment of this invention is a composition of matter
that repels insects when applied to a human, animal or inanimate host that
includes a dihydronepetalactone, or a mixture of dihydronepetalactone
stereoisomers, represented by the general formula set forth above.
A further embodiment of this invention is a composition of
matter that repels one or more insects selected from the group consisting
of bees, black flies, chiggers, fleas, green head flies, mosquitoes, stable
flies, ticks, wasps, wood-boring insects, houseflies, cockroaches, lice,
roaches, wood lice, flour and bean beetles, dust mites, moths, silverfish,
and weevils, that includes a dihydronepetalactone, or a mixture of
dihydronepetalactone stereoisomers, represented by the general formula
set forth above.
Yet another embodiment of this invention is a composition of
matter that has a mean complete protection time that is statistically
indistinguishable from that of N,N-diethyl-m-toluamide that includes a
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dihydronepetalactone, or a mixture of dihydronepetalactone
stereoisomers, represented by the general formula set forth above.
Yet another embodiment of this invention is an insect repellent
composition of matter that includes, in an amount of about 0.001 % to
about 80% by weight, a dihydronepetalactone, or a mixture of
dihydronepetalactone stereoisomers, represented by the general formula
set forth above.
Yet another embodiment of this invention is a process for
fabricating an insect repellent composition or an insect repellent article of
manufacture by providing as the composition or article, or incorporating
into the composition or article, a dihydronepetalactone, or a mixture of
dihydronepetalactone stereoisomers, represented by the general formula
set forth above.
Yet another embodiment of this invention is a method of
imparting, augmenting or enhancing the insect repellent effect of an article,
by incorporating into the article a dihydronepetalactone, or a mixture of
dihydronepetalactone stereoisomers, represented by the general formula
set forth above.
Yet another embodiment of this invention is a method of
repelling insects from a human, animal or inanimate host by exposing the
insects to a dihydronepetalactone, or a mixture of dihydronepetalactone
stereoisomers, represented by the general formula set forth above. The
insects repelled may be, for example, one or more of mosquitoes, stable
flies and ticks.
Yet another embodiment of this invention is the use of a
dihydronepetalactone, or a mixture of dihydronepetalactone
stereoisomers, represented by the general formula above to repel insects
from a human, animal or inanimate host. The insects repelled may be, for
example, one or more of mosquitoes, stable flies and ticks.
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Yet another embodiment of this invention is a process for the
production of a dihydronepetalactone of formula (XVI) by hydrogenating a
nepetalactone of formula (XV) according to the following scheme:
0 0
0
6 0
(XV) (XVI)
in the presence of palladium supported on a catalyst support that is not
SrCO3.
Applicants have found that dihydronepetalactones perform well as a
new class of effective insect repellent compounds without the
disadvantageous properties characteristic of prior-art compositions. When
used as an insect repellent, DHN prevents damage to plants and animals,
including humans, or to articles of manufacture, by making insect food
sources or living conditions unattractive or offensive.
Brief Description of the Drawings
Figure 1 shows the chemical structures of the naturally-occurring.
iridoid (methylcyclopentanoid) nepetalactones.
Figure 2 shows the total ion chromatograms from combined gas
chromatography/mass spectrometry (GC-MS) analysis of a distilled
nepetalactone-enriched fraction from commercially-available catmint oil
(A), together with that of the material produced from this fraction by
hydrogenation (B).
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Figure 3 shows the mass spectra of the major constituents of the
nepetalactone-enriched fraction (A) and the hydrogenated material (B)
identified by GC-MS analysis in Fig. 2.
Figure 4 shows the 13C NMR analysis performed on a distilled
nepetalactone-enriched fraction of commercially-available catmint oil.
Figure 5 shows the 13C NMR spectrum obtained from analysis of
the dihydronepetalactones produced by hydrogenation of a distilled
nepetalactone-enriched fraction of commercially-available catmint oil
Figure 6 shows the distribution of probing density with time, during
tests of various repellents against female Aedes aegypti mosquitoes in an
in vitro repellency test.
Figure 7 shows the 13C NMR analysis of trans,cis-nepetalactone.
Figure 8 shows the 13C NMR analysis of dihydronepetalactones
derived from hydrogenation of trans,cis-nepetalactone.
Figure 9 shows the distribution of probing density with time, during
tests of dihydronepetalactones derived from hydrogenation of trans,cis-
nepetalactone against female Aedes aegypti mosquitoes in an in vitro
repellency test.
Figure 10 shows the distribution of landing density with time, during
tests of various repellents against stable flies (Stomoxys calcitrans) in an
in vitro repellency test.
Figure 11 shows the distribution of probing density with time, during
tests of various repellents against female anopheles mosquitoes
(Anopheles albimanus) in an in vitro repellency test.
Description of a Preferred Embodiment of the Invention
A nepetalactone is a compound having the general structure:
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0
7a 1
7 20
6
4
4
4a
Four chiral centers are present within the methylcyclopentanoid backbone
of nepetalactone at carbons 4, 4a, 7 and 7a as shown above; (7S)-
nepetalactones are produced by several plants and insects.
5 Dihydronepetalactones are known as minor constituents of the
essential oils of several labiate plants of the genus Nepeta (Regnier, F.E.,
et al. (1967) Phytochemistry 6:1281-1289; DePooter, H.L., et al. (1988)
Flavour and Fragrance Journal 3:155-159; Handjieva, N.V. and S.S.
Popov (1996) J. Essential Oil Res. 8:639-643). Dihydronepetalactones
are defined by Formula 1:
O
9 2 C
8 3
7 6 4
5
Formula 1
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wherein 1, 5, 6 and 9 indicate the four chiral centers of the molecule and
the structure shown is intended to encompass all stereoisomers of
dihydronepetalactone. The structures of dihydronepetalactone
stereoisomers that may be derived from (7S)-nepetalactones are shown
below.
0 0
H H
O O
FI H
(1 S,5S,9S,6R)-5,9-dimethyl-3- (1 S,9S,5R,6R)-5,9-dimethyl-3-
oxabicyclo[4.3.01-nonan-2-one oxabicyclo[4.3.0]-nonan-2-one
O O
H LH
0 0
H H
(1 S,5S,9S,6S)-5,9-dimethyl3- (1 S,9S,6S,5R)-5,9-dimethyl-3
oxabicyclo[4.3.0]-nonan-2-one oxabicyclo[4.3.0]-nonan-2-one
O O
H ~ _H
0 = 0
FI H
(9S,5S,1R,6R)-5,9-dimethyl-3- (9S,1R,5R,6R)-5,9-dimethyl-3-
oxabicyclo[4.3.0]-nonan-2-one oxabicyclo[4.3.0]-nonan-2-one.
0 0
O 0
H H
(9S,6S,1R,5S)-5;9-dimethyl-3- (9S,6S,1R,5R)-5,9-dimethyl-3
oxabicyclo14.3.0]-nonan-2-one oxabicyclo[4.3.0]-nonan-2-one
A "dihydronepetalactone" (DHN) will be understood to encompass
any and all dihydronepetalactone stereoisomers and mixtures thereof,
unless a particular isomer or mixture is specified. When
dihydronepetalactone is prepared from a naturally occurring source of
nepetalactone some variation in molar concentration of stereoisomers is
expected. Preparation from a naturally occurring source is, however, a
preferred method of preparation.
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Regnier et al, op.cit., discloses the preparation of DHN from
nepetalactone by the catalyzed hydrogenation of nepetalactone isolated
from the essential oils of plants of the genus Nepeta (catmints). One
preferred and convenient method for synthesis of dihydronepetalactone is
thus by hydrogenation of nepetalactone obtained in relatively pure form
from the essential oils isolated by various means from plants of the genus
Nepeta (catmints). Catalysts such as platinum oxide and palladium
supported on strontium carbonate give dihydronepalactone in 24-90%
yields (Regnier et al. op.cit.).
Methods for isolation of essential oils are well known in the art, and
examples of methodology for oil extraction include (but are not limited to)
steam distillation, organic solvent extraction, microwave-assisted organic
solvent extraction, supercritical fluid extraction, mechanical extraction and
enfleurage (initial cold extraction into fats followed by organic solvent
extraction).
The essential oils isolated from different Nepeta species are well
known to possess different proportions of each naturally-occurring
stereoisomer of nepetalactone (Regnier et al. op. cit.; DePooter, et al.
op.cit.; Handjieva et al op.cit.). Thus DHN prepared from oil derived from
any Nepeta species will necessarily be a mixture of stereoisomers
thereof, the constitution of that mixture depending upon the particular
species of Nepeta from which it is derived.
As discussed herein above, four chiral centers are present within
the methylcyclopentanoid backbone of the nepetalactone at carbons 4, 4a,
7 and 7a as shown:
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0
7 7a 2 Co
6
4a
A total of eight pairs of dihydronepetalactone enantiomers are
possible after hydrogenation of nepetalactone. Of these, the naturally
occurring stereoisomers described thus far are (9S)-
5 dihydronepetalactones. Preferred repellent materials in accordance with
the present invention include a mixture of any or all of the possible
stereoisomers of dihydronepetalactone. More preferred repellent
materials include a mixture of (9S)-dihydronepetalactones. Most preferred
are (9S)-dihydronepetalactone stereoisomers derived from (7S)-
nepetalactones. This includes the compounds commonly known as
cis, trans-nepetalactone, cis, cis-nepetalactone, trans,cis-nepetalactone,
and trans, trans-nepetalactone; as illustrated' in Figure 1. The predominant
stereoisomers produced by N. cataria (cis,trans and trans,cis-) are
preferred.
Upon completion of the hydrogenation reaction, the resulting
mixture of isomer products may be separated- by a conventional method
(e.g., preparative liquid chromatography) to yield each highly purified pair
of dihydronepetalactone diastereomers. This permits the use of various
different diastereomers as are found to be most effective against particular
insects. It is preferable to isolate a specific nepetalactone isomer from a
plant to convert to its corresponding pair of diastereomers by
hydrogenation.
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In addition to variation in nepetalactone stereoisomer content
between different Nepeta species, intra-species variation is also known to
exist. Plants of a given species may produce oils with different
compositions depending on the conditions of their growth or growth stage
at harvest. In fact variation in oil composition independent of growth
conditions or growth stage at harvest has been found in Nepeta racemosa,
(Clark, L.J., et al. op.cit.). Plants of a single species exhibiting different
oil
compositions are termed chemotypes. In Nepeta racemosa, chemotypes
exhibiting marked differences in the proportion of different nepetalactone
stereoisomers exist. Thus, the preferred process for producing specific
dihydronepetalactone enantiomers is hydrogenation of an oil from a
Nepeta chemotype known to contain specific nepetalactone
stereoisomers.
An insect as repelled by the composition of this invention includes
any member of a large group of invertebrate animals characterized, in the
adult state (non-adult insect states include larva and pupa), by division of
the body into head, thorax, and abdomen, three pairs .of legs, and, often
(but not always) two pairs of membranous wings. This definition therefore
includes but is not limited to a variety of biting insects (e.g., ants, bees,
black flies, chiggers, fleas, green head flies, mosquitoes, stable flies,
ticks,
wasps), wood-boring insects (e.g., termites), noxious insects (e.g..,
houseflies, cockroaches, lice, roaches, wood lice), and household pests
(e.g., flour and bean beetles, dust mites, moths, silverfish, weevils). In one
embodiment, for example, the DHN compositions of the present invention
are effective insect repellents against a wide spectra of common insect
pests, such as those mentinoed above and also including biting insects,
wood-boring insects, noxious insects, and household pests, most
particularly mosquitoes, stable flies, and ticks such as deer ticks.
In a further embodiment the DHN compositions of this invention are
effective to repel any one or more of the members of the group consisting
of bees, black flies, chiggers, fleas, green head flies, mosquitoes, stable
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flies, ticks, wasps, wood-boring insects, houseflies, cockroaches, lice,
roaches, wood lice, flour and bean beetles, dust mites, moths, silverfish,
and weevils. The insects repelled may also, however, be one or more
those that are selected from a subgroup of the foregoing formed by
omitting any one or more members from the whole group as set forth in
the list in the first sentence of this paragraph. As a result, the repelled
insect(s) may in such instance not only be those selected from any
subgroup of any size that may be formed from the whole group as set forth
in the list above, but may exclude the members that have been omitted
from the whole group to form the subgroup. The subgroup formed by
omitting various members from the whole group in the list above may,
moreover, be an individual member of the whole group such that the
repelled insect excludes all other members of the whole group.
A host is any plant or animal affected by insects. Typically, hosts
are considered to be insect-acceptable food sources or insect-acceptable
habitats. Hosts can be animals (including without limitation pets and/or
other domesticated animals), humans, plants or a so-called "insect
susceptible article", encompassing any inanimate article which is affected
by insects. This may include buildings, furniture, and the like.
In a further embodiment of the present invention, DHN is
incorporated into a host such as an insect susceptible article to produce an
insect repellent article for the purpose either of deterring insects from
landing on the article, or from occupying the air surrounding the article.
Contemplated in this embodiment are those instances in which an article
may already exhibit some degree of insect repellency prior to treatment
with a DHN composition of the invention. In such instances it is
contemplated that the insect repellency of the article will be enhanced by
the application of the DHN composition of the invention.
An insect repellent is any compound or composition which deters
insects from a host. It will be appreciated that such usage makes no
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distinction among compounds that have highly ephemeral effects as
compared to those that exhibit long term beneficial effects, and/or those
that require very high surface concentrations before there is an observable
effect on insect behavior.
The term "insect repellent" thus indicates a compound or
composition conferring on a host protection from insects when compared
to no treatment at all. "Protection" desirably results in a statistically
significant reduction in numbers of insects, and may, for example, be
usefully determined by measuring mean complete protection time ("CPT")
in tests in which insect behavior toward treated animals, including humans,
and treated inanimate surfaces is observed. Mean CPT refers to the
mean length of time over repetitions of tests in which the time before the
first landing, probing or biting (in the case of a biting insect) or crawling
(in
the case of a crawling insect such as a tick or chigger) on a treated
surface is observed [see e.g. US EPA Office of Prevention, Pesticides and
Toxic Substances product performance .test guidelines OPPTS 810.3700;
and Fradin, M.S., Day, J.F. (2002) New England Journal of Medicine 347,
13-18]. In one exemplary embodiment of this invention, the insect
repellent composition hereof has a mean CPT that is statistically
indistinguishable from that of DEET. In the test in which this condition of
the respective mean CPT performances of a DHN composition and DEET
are shown to be statistically indistinguisable, the test conditions (including
amounts of active ingredients) utilized must of course be identical, or, if
not
identical, must differ only in ways that do not prevent utilization of the
results for the purposes of documenting the existence of the condition
described.
As noted above, DHN compared favorably in performance with
DEET. Moreover, DHN is advantageously prepared from naturally
occurring nepetalactone derived from plants whereas DEET, and many
other insect repellents, are not prepared from natural sources - an
important consumer consideration when choosing an effective repellent.
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Preparation from natural sources also offers the potential for low
production costs.
It is a particularly surprising aspect of the present invention that
DHN provides a considerable improvement over the odor of DEET while
exhibiting effective insect repellency. The DHN compounds and
compositions of this invention possess a pleasant fragrance. The
fragrance notes of the DHN materials make them useful in imparting,
altering, augmenting or enhancing the overall olfactory component of an
insect repellent composition or article, for example, by utilizing or
moderating the olfactory reaction contributed by one or more other
ingredients in the composition. Specifically, the DHN compositions of the
invention may be utilized to either mask or modify the odor contributed by
other ingredients in the formulation of the final repellent composition or
article, and/or to enhance consumer appeal of a product by imparting a
characteristic perfume or aroma.
It will be appreciated that the effectiveness of DHN or any insect
repellent depends upon the surface concentration of the active ingredient
on the host surface to which it is applied. Many compounds known in the
art to exhibit insect repellency do so, however, only in relatively
concentrated form. See, for example, McGovern et al in U.S. 4,416, 881,
which discloses the use of repellent concentrations of 6.25-25%. In other
situations representative of the art, it is often found that concentrations of
DEET much below 1 % require repeated application to achieve an effective
surface concentration, yet concentrations above 30% result in excessive
surface concentration, which is both wasteful and conducive to the
production of undesirable side effects. A further advantage of this
invention is consequently that DHN not only provides effective insect
repellency at concentrations similar to those employed for DEET, DHN
may be employed at concentrations up to and including- neat DHN (i.e. the
composition hereof may, if desired, contain 100% by weight DHN). The
property of effective repellency in DHN provides many options for
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economical utilization of the DHN active ingredient over a wide range of
levels of concentration.
In one embodiment of the present invention, DHN is incorporated in
effective amounts into a composition- suitable for application to a host plant
or animal, preferably to human skin. Suitable compositions include DHN
and a vehicle, preferably alcohol such as iso-propyl alcohol, a lotion such
as numerous skin creams such as are known in the art, or a silicaceous
clay. Preferably the DHN is present in the insect repellent composition of
the invention at a concentration of about 0.1 % to 30% by weight,
preferably about 0.5% to 20% by weight, and most preferably about 1 % to
15% by weight.
For an insect repellent to be effective the evaporation rate of the
active ingredient from the host's skin or the treated article must be
sufficiently high to provide a vapor density which has the desired effect on
the target insects. However, a balance must be struck between
evaporation rate and the desired duration of the insect repellent effect
too high an evaporation rate will deplete the insect repellent on the surface
causing a loss in efficacy. Numerous extrinsic factors affect the
evaporation rate, such as the ambient temperature, the temperature of the
treated surface, and the presence or absence of air movement. The
composition of this invention has a skin surface evaporation rate of at least
a minimum effective evaporation rate, and preferably has a skin surface
evaporation rate of at least a minimum effective evaporation rate for at
least five hours.
In most cases, penetration into and through the skin is an
undesirable mode of loss of compound from the skin surface. For
example, insect repellents are known to be absorbed into human skin,
making potential toxicity a concern on the one hand, and clearly removing
the absorbed amount of repellent from insect repellent activity. Similar
considerations must be made for insect repellent articles.
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While DHN provides effective insect repellency under typical
conditions of use, it may under some circumstances be desirable to
reduce the rate of evaporation thereof. A variety of strategies may be
employed to reduce the evaporation rate of DHN if so desired. For
example, one method is to combine the DHN with a polymer or other inert
ingredient, forcing the DHN to migrate through the mixture to the surface
thereof before it can evaporate. However, if the result is dilution of the
concentration of DHN that can be applied to the host's skin surface or that
is present on the surface of an insect repellent article, thus reducing the
overall potency of the formulation, this must be factored into the
evaporation strategy selected. Alternatively, the active ingredient is micro-
encapsulated to control rates of loss from the host's skin surface or insect
repellent article. In still another alternative, a precursor molecule may be
prepared, which slowly disintegrates on the skin surface or insect repellent
article to release the active ingredient.
For example, release of the active ingredient may be, for example,
by sub-micron encapsulation, in which the active ingredient is
encapsulated (surrounded) within a skin nourishing protein just the way air
is captured within a balloon. The protein may be used at, for example, a
20% concentration. An application of repellent contains many of these
protein capsules that are suspended in either a water-based lotion, or
water for spray application. After contact with skin the protein capsules
begin to breakdown releasing the encapsulated dihydronepetalactone.
The process continues as each microscopic capsule is depleted then
replaced in succession by a new capsule that contacts the skin and
releases its active ingredient. The process may take up to 24 hours for
one application. Because a protein's adherence to the skin is so effective,
these formulas are very resistant to perspiration (sweat-off), and water.
When applied they are dry and comfortable with no greasiness. This
system results in very effective protection, but it is only effective when
used on skin because clothing, does not have the capability to release the
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proteins. An alternative system uses a polymer to encase the repellent,
which slows down early evaporation leaving more dihydronepetalactone
available for later evaporation. This system can often increase a
repellent's length of effectiveness by 25% to 50% over comparable non-
entrapped products, but often feels greasy because of the presence of the
polymer. In another alternative, a synergist is used to keep stimulating
the evaporation of the dihydronepetalactone in the composition.
In the present invention, a variety of carriers or diluents for the
above-disclosed dihydronepetalactones can be used. The carrier allows
the formulation to be adjusted to an effective concentration of repellant,
molecules. When formulating a topical insect repellent suitable for human
or animal skin, preferably, the repellant molecules are mixed in a
dermatologically acceptable carrier. The carrier may further provide water
repellency, prevent skin irritation, and/or soothe and condition skin.
15' Factors to consider when selecting a carrier(s) for any formulation of
insect
repellent include commercial availability, cost, repellency, evaporation
rate, odor, and stability. Some carriers can themselves have repellent
properties. The carrier, moreover, should preferably also be one that will
not be harmful to the environment.
Suitable for the present invention are one or more commercially
available organic and inorganic liquid, solid, or semi- solid carriers or
carrier formulations known in the art for formulating insect repellent
products. For example the carrier may include silicone, petrolatum, or
lanolin.
Examples of organic liquid carriers include liquid aliphatic
hydrocarbons (e.g., pentane, hexane, heptane, nonane, decane and their
analogs) and liquid aromatic hydrocarbons. Examples of other liquid
hydrocarbons include oils produced by the distillation of coal and the
distillation of various types and grades of petrochemical stocks, including
kerosene oils that are obtained by fractional distillation of petroleum.
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Other petroleum oils include those generally referred to as agricultural
spray oils (e.g., the so-called. light and medium spray oils, consisting of
middle fractions in the distillation of petroleum and which are only slightly
volatile). Such oils are usually highly refined and may contain only minute
amounts of unsaturated compounds. Such oils, moreover, are generally
paraffin oils and accordingly can be emulsified with water and an
emulsifier, diluted to lower concentrations, and used as sprays. Tall oils,
obtained from sulfate digestion of wood pulp, like the paraffin oils, can
similarly be used. Other organic liquid carriers can include liquid terpene
hydrocarbons and terpene alcohols such as alpha-pinene, dipentene,
terpineol, and the like.
Other carriers include aliphatic and aromatic alcohols, esters,
aldehydes, ketones, mineral oil, higher alcohols, finely divided organic and
inorganic solid materials. In addition to the above-mentioned liquid
hydrocarbons, the carrier can contain conventional emulsifying agents
which can be used for causing. the dihydronepetalactone compounds to be
dispersed in, and diluted with, water for end-use application.
Aliphatic monohydric alcohols include methyl, ethyl, normal-propyl,
isopropyl, normal-butyl, sec-butyl, and tert-butyl alcohols. Suitable
alcohols include glycols (such as ethylene and propylene glycol) and
pinacols. Suitable polyhydroxy alcohols include glycerol, arabitol,
erythritol, sorbitol, and the like. Finally, suitable cyclic alcohols include
cyclopentyl and cyclohexyl alcohols.
Additionally, conventional or so-called "stabilizers" (e.g., tert-butyl
sulfinyl dimethyl dithiocarbonate) can be used in conjunction with, or as a
component of, the carrier or carriers comprising the compositions of the
present invention.
Solid carriers that can be used in the compositions of the present
invention include finely divided organic and inorganic solid materials.
Suitable finely divided solid inorganic carriers include siliceous minerals
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such as synthetic and natural clay, bentonite, attapulgite, fuller's earth,
diatomaceous earth, kaolin, mica, talc, finely divided quartz, and the like,
as well as synthetically prepared siliceous materials, such as silica
aerogels and precipitated and fume silicas. Examples of finely divided
solid organic materials include cellulose, sawdust, synthetic organic
polymers, and the like. Examples of semi-solid or colloidal carriers include
waxy solids, gels (such as petroleum jelly), lanolin, and the like, and
mixtures of well-known liquid and solid substances which can provide
semi-solid carrier products, for providing effective repellency within the
scope of the instant invention.
Insect repellent compositions of the present invention containing the
dihydronepetalactones may also contain adjuvants known in the art of
personal care product formulations, such as thickeners, buffering agents,
chelating agents, preservatives, fragrances, antioxidants, gelling agents,
stabilizers, surfactants, emolients, coloring agents, aloe vera, waxes, other.
penetration enhancers and mixtures thereof, and therapeutically or
cosmetically active agents.
Therapeutically or cosmetically active ingredients useful in the
compositions of the invention include fungicides, sunscreening agents,
20, sunblocking agents, vitamins, tanning agents, plant extracts, anti-
inflammatory agents, anti-oxidants, radical scavenging agents, retinoids,
alpha-hydroxy acids, emollients, antiseptics, antibiotics, antibacterial
agents or antihistamines, and may be present in an amount effective for
achieving the therapeutic or cosmetic result desired.
The composition of this invention may also be blended with a
non-dihydronepetalactone insect repellent, such as those included in the
consisting of: benzil, benzyl benzoate, 2,3,4,5-bis(butyl-2-ene)
tetrahydrofurfural, butoxypolypropylene glycol, N-butylacetanilide, normal-
butyl-6,6-dimethyl-5,6-dihydro-1,4-pyrone-2-carboxylate, dibutyl adipate,
dibutyl phthalate, di-normal-butyl succinate, N,N-diethyl-meta-toluamide,
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dimethyl carbate, dimethyl phthalate, 2-ethyl-2-butyl-1,3-propanediol, 2-
ethyl- 1,3-hexanediol, di-normal-propyl isocinchomeronate, 2-
phenylcyclohexanol, p-methane-3,8-diol, and normal-propyl N, N-
d iethylsuccinamate.
The DHN composition of the invention may include any number of
the above recited adjuvants in order to meet the requirements of any
particular application. The specific proportions of each ingredient will
similarly be dictated by the requirements of the application. However, the
compositions of the invention should preferably comprise at least about
0.001 % by weight DHN, or about 0.001 % to about 80% by weight DHN, or
about 0.01 % to about 30% by weight of DHN, or about 0-.1 % to about 30%
by weight of DHN, preferably about 0.5% to about 20% by weight, most
preferably about 1 % to about 15% by weight. In general, the composition
of the repellent should contain sufficient amounts of active insect repellant
material to be efficacious in repelling the insect from the host over a
prolonged period of time (preferably, for a period of at least several hours).
Dihydronepetalactones may be utilized in the present invention in
the form of individual diastereomers or a mixture of various diastereomers,
or combined with other insect repellents. DHN maybe employed at any
concentration level suitable for the particular need, including neat.
However, it is contemplated that the amount of DHN in an insect repellent
composition or repellent article in accordance with the present invention
will generally not exceed about 80% by weight.
The compositions of the invention may be formulated and packaged
so as to deliver the product in a variety of forms including as a solution,
suspension, cream, ointment, gel, film or spray, depending on the
preferred method of use. The carrier may be an aerosol composition
adapted to disperse the dihydronepetalactone into the atmosphere by
means of a compressed gas.
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Desirable properties of a topical insect repellent article include low
toxicity, resistance to loss by water immersion or sweating, low or no odor
or at least a pleasant odor, ease of application, and rapid formation of a
dry tack-free surface film on the host's skin. In order to obtain these
properties, the formulation for a topical insect repellent article should
permit insect-infested animals (e.g., dogs with fleas, poultry with lice, cows
with ticks, and humans) to be treated with an insect repellent composition
of the present invention by contacting the skin, fur or feathers of such an
animal with an effective amount of the repellent article for repelling the
insect from the animal host. Thus, dispersing the article into the air or
dispersing the composition as a liquid mist or fine dust will permit the
repellent composition to fall on the desired host surfaces. Likewise,
directly spreading of liquid/semi-solid/solid repellent article on the host is
an effective method of contacting the surface of the host with an effective
amount of the repellent composition.
Particularly because of the pleasant aroma associated with DHN, a
further embodiment of the present invention is the incorporation of DHN
into products which are not primarily associated with insect repellency in
order to provide an effective degree of repellency thereto. Included among
such products (but not thereto limited) are colognes, lotions, sprays,
creams, gels, ointments, bath and shower gels, foam products (e.g.,
shaving foams), makeup, deodorants, shampoo, hair lacquers/hair rinses,
and personal soap compositions (e.g., hand soaps and bath/shower
soaps).
Further contemplated in the present invention are those
embodiments wherein DHN provides effective insect repellency in a
variety of articles that are susceptible to attack by insects by incorporation
therein. In a typical embodiment the articles are outdoors, but need not
be. Among the articles contemplated are included, but not limited to, air
3a fresheners, candles, various scented articles, fibers, sheets, textile
goods,
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paper, paint, ink, clay, wood, furniture (e.g., for patios and decks),
carpets,
sanitary goods, plastics, polymers, and the like.
In one embodiment, the dihydronepetalactone is combined with a
polymer to provide moldability, reduction of evaporation rate, and
controlled release. Such a polymer may be biodegradeable Suitable
polymers include but are not limited to high density polyethylene, low
density polyethylene, biodegradable thermoplastic polyurethanes,
biodegradable ethylene polymers, and poly(epsilon caprolactone)
homopolymers and compositions containing the same, as disclosed for
example in U.S. 4,496,467, U.S. 4,469,613 and U.S. 4,548,764. Preferred
biodegradeable polymers include DuPont Biomax biodegradeable
polyester and poly-L-lactide.
This invention also involves a process for manufacturing DHN in
which a palladium catalyst is used. The term "catalyst" as used herein
refers to a substance that affects the rate of a chemical reaction (but not
the reaction equilibrium) and emerges from the process chemically
,unchanged.
The process for the production of a dihydronepetalactone of
formula (XVI) involves hydrogenating a nepetalactone of formula (XV)
according to the following scheme:
0 0
0 0
(XV) (XVI)
in the presence of palladium supported on a catalyst support that is not
SrCO3.
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The term "promoter" as used herein is a compound that is added to
enhance the physical or chemical function of a catalyst. A chemical
promoter generally augments the activity of a catalyst and may be
incorporated into the catalyst during any step in the chemical processing of
the catalyst constituent. The chemical promoter generally enhances the
physical or chemical function of the catalyst agent, but can also be added
to retard undesirable side reactions. A "metal promoter" refers to a
metallic compound that is added to enhance the physical or chemical
function of a catalyst.
Hydrogenation of nepetalactone is effected in the presence of a
suitable active metal hydrogenation catalyst. Acceptable solvents,
catalysts, apparatus, and procedures for hydrogenation in general can be
found in Augustine, Heterogeneous Catalysis for the Synthetic Chemist,
Marcel Decker, New York, N.Y. (1996). Many hydrogenation catalysts are
effective, including (without limitation) those containing as the principal
component iridium, palladium, rhodium, nickel, ruthenium, platinum,
rhenium, compounds thereof, combinations thereof, and the supported
versions thereof.
The metal catalyst used in the process of this invention may be
used as a supported or as an unsupported catalyst. A supported catalyst
is one in which the active catalyst agent is deposited on a support material
by spraying, soaking or physical mixing, followed by drying, calcination,
and if necessary, activation through methods such as reduction or
oxidation. Materials frequently used as support are porous solids with high
total surface areas (external and internal) which can provide high
concentrations of active sites per unit weight of catalyst. The catalyst
support may enhance the function of the catalyst agent; and supported
catalysts are generally preferred because the active metal catalyst is used
more efficiently. A catalyst which is not supported on a catalyst support
material is an unsupported catalyst.
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The catalyst support can be any solid, inert substance including, but
not limited to, oxides such as silica, alumina, titania, calcium carbonate,
barium sulfate, and carbons. The catalyst support can be in the form of
powder, granules, pellets, or the like. A preferred support material of the
present invention is selected from the group consisting of carbon, alumina,
silica, silica-alumina, titania, titania-alumina, titania-silica, barium,
calcium,
compounds thereof and combinations thereof. Suitable supports include
carbon, SiO2, CaCO3, BaSO4 and AI203. Moreover, supported catalytic
metals may have the same supporting material or different supporting
materials.
In one embodiment of the instant invention, a more preferred
support is carbon. Further preferred supports are those, particularly
carbon, that have a surface area greater than 100-200 m2/g. Further
preferred supports are those, particularly carbon, that have a surface area
of at least 300 m2/g.
Commercially available carbons which may be used in this
invention include those sold under the following trademarks: Bameby &
SutcliffeTM, DarcoTM, NucharTM, Columbia JXNTM, Columbia LCKT"",
Calgon PCBTM, Calgon BPLT"", WestvacoTM, NoritTM and Barnaby Cheny
NBTM. The carbon can also be commercially available carbon such as
Calsicat C, Sibunit C, or Calgon C (commercially available under the
registered trademark Centaur ).
Preferred combinations of catalytic metal and support system
include palladium on carbon such as in ESCAT#142 catalyst (Englehard).
While the weight percent of catalyst on the support is not critical, it
will be appreciated that the higher the weight percent of metal, the faster
the reaction. A preferred content range of the metal in a supported
catalyst is from about 0.1 wt% to about 20 wt% of the whole of the
supported catalyst (catalyst weight plus the support weight). A more
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preferred catalytic metal content range is from about 1 wt% to about 10
wt% by weight of the whole of the supported catalyst. A further preferred
catalytic metal content range is from about 3 wt% to about 7 wt% by
weight of the whole of the supported catalyst.
Optionally, a metal promoter may be used with the catalytic metal in
the method of the present invention. Suitable metal promoters include:
1) those elements from groups 1 and 2 of the periodic table; 2) tin,
copper, gold, silver, and combinations thereof; and 3) combinations of
group 8 metals of the periodic table in lesser amounts.
Temperature, solvent, catalyst, pressure and mixing rate are all
parameters that affect the hydrogenation. The relationships among these
parameters may be adjusted to effect the desired conversion, reaction
rate, and selectivity in the reaction of the process.
Within the context of the present invention the preferred
temperature is from about 25 C to 250 C, more. preferably from about
50 C to about 150 C, and'most preferred from about 50 C to 100 C. The
hydrogen pressure is preferably about 0.1 to about 20 MPa, more
preferably about 0.3 to 10 MPa, and most preferably about 0.3 to 4 MPa.
The reaction may be performed neat or in the presence of a solvent.
Useful solvents include those known in the art of hydrogenation such as
hydrocarbons, ethers, and alcohols. Alcohols are most preferred,
particularly lower alkanols such as methanol, ethanol, propanol, butanol,
and pentanol. Where the reaction is carried out according to the preferred
embodiments, selectivites in the range of at least 70% are attainable
where selectivites of at least 85% are typical. Selectivity is the weight
percent of the converted material that is dihydronepetalactone where the
converted material is the portion of the starting material that participates
in
the hydrogenation reaction.
The process of the present invention may be carried out in batch,
sequential batch (i.e. a series of batch reactors) or in continuous mode in
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any of the equipment customarily employed for continuous processes
(see, for example,,H.S. Fogler, Elementary Chemical Reaction
Engineering, Prentice-Hall, Inc., NJ, USA). The condensate water formed
as the product of the reaction is removed by separation methods
customarily employed for such separations.
Upon completion of the hydrogenation reaction, the resulting
mixture of dihydronepetalactone isomer products may be separated by a
conventional method, such as for example, by distillation, by
crystallization, or by preparative liquid chromatography to yield each highly
purified pair of dihydronepetalactone enantiomers. Chiral chromatography
may be employed to separate enantiomers.
The present invention is further described in but not limited by the
following specific embodiments.
Examples
In the following examples, the notation "w/v" refers to the weight in
grams of the active ingredient per 100 mL of solution.
Other abbreviations employed are as follows: "h" means hour(s),
"min" means minute(s), "sec" means second(s), "d" means day(s), "mL"
means milliliters, "L" means liters, "m/z" means mass (m). to charge (z)
ratio, "ppm" means parts per million, "mol %" means percentage
expressed on a molar basis, "Hz" means Hertz (1/sec), and "psig" means
pounds per square inch guage.
EXAMPLE 1
Preparation of nepetalactones by fractional steam distillation of oil of
Nepeta cataria
A sample of commercially-available catnip oil, prepared by steam
distillation of herbaceous material from the catmint Nepeta cataria, was
obtained (Berje, Bloomfield, NJ, USA). Combined gas chromatography -
mass spectrometry (GC-MS) of the oil as received indicated that the
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principal constituents were nepetalactone stereoisomers (Fig. 1).
However, as purchased, the oil is a highly contaminated natural product,
and it is desirable to refine the extract to a purified nepetalactone. We
fractionally distilled to remove contaminants with higher and lower boiling
points than the nepetalactones.
Thus the nepetalactone fraction was prepared by fractional
distillation of the as-received oil (21 pot; 12in. x 1 in. packed column with
0.24" SS packing; variable reflux head; ca. 2mm Hg, with fractions
collected between 80 C and 99 C). Figure 2A presents the GC-MS total
ion chromatogram of the nepetalactone-enriched fraction prepared by
fractional distillation of the commercial sample of Nepeta cataria essential
oil. The conditions employed were: column HP5-MS, 25m x 0.2mm; oven
120 C, 2 min, 15 C/min, 210 C, 5 min.; He @ I ml/min. Peaks with m/z
166 are nepetalactones; the unlabelled peaks correspond to minor
sesquiterpenoid contaminants.
In Figure 3A, the mass spectrum of the major peak (6.03 min,
nepetalactone) in Figure 2A is shown. 1 H and 13C NMR analysis of the oil
and the purified material was also carried out, and the 13C data is
presented (Figure 4). The 13C chemical shifts for the four possible
stereoisomers reported in the literature were compared to the spectra
taken for the sample. Three stereoisomers were detected and the
amounts were quantified based on the carbonyl region at around 170 ppm.
The chemical shifts, for both the original oil and the enriched material, are
provided in Table 1. Each carbon atom of nepetalactone is identified, as
shown in Figure 4.
Table 1
13C Chemical Shifts and Mol% Values of
Nepetalactone Stereoisomers Present in Commercial Sample of
Essential Oil of Catmint (Nepeta cataria) and in
Fraction Purified by Steam Distillation
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ESSENTIAL OIL PURIFIED FRACTION
cis, trans- trans, cis- cis, cis- cis, trans- trans, cis- Cis, cis-
ATOM 6 (ppm) 6 (ppm) 8 (ppm) 6 (ppm) S (ppm) 8 (ppm)
a 170.9 170.1 170.8 170.1
b 133.7 135.9 134.2 133.7 135.9 134.2
c 115.3 120.4 115.3 120.4
d 40.8 37.3 39.6 40.8 37.4 39.5
e 49.4 49.1 46.4 49.5 49.1 46.3
f 39.7 32.1 38.4 39.8 32.1 38.4
g 33.0 30.0 32.7 33.1 30.0 32.7
h 30.9 26.1 30.4 31.0 26.1 30.5
j 20.2 17.5 17.1 20.3 17.6 17.2
i 15.4 14.2 14.7 15.5 14.2 14.8
Mol% 80.20% 17.70% 2.10% 84.50% 14.30% 1.20%
This analysis indicated that in the oil, nepetalactones were present
in the following proportions: 80.2 mol% cis,trans-nepetalactone, 17.7 mol%
trans,cis-nepetalactone and 2.1 mol% cis,cis-nepetalactone. The data
indicated the proportions of nepetalactones in the purified material were
84.5 mol% cis,trans-nepetalactone, 14.3 mol% trans, cis-n e petal acton e
and 1.2,mol% cis,cis-nepetalactone. GC-MS analysis of this purified
fraction indicated that it consisted predominantly of these nepetalactones
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(m/z 166), accompanied by trace amounts of the sesquiterpenoids
caryophyllene and humulene (data not shown).
EXAMPLE 2
Preparation of dihydronepetalactones
107 g of the distilled nepetalactone fraction of the catmint oil
prepared as described in Example 1 was dissolved in ethanol (200 ml) and
placed in a Fisher-Porter bottle with 12.7g 2% Pd/SrCO3 (Aldrich 41,461-
1) as catalyst. The tube was evacuated and backfilled with H2 twice, then
charged with H2 at 30 psig. After 48 h stirring at room temperature, the
tube was vented and the contents filtered over CeliteTM to remove catalyst.
The solvent was removed under vacuum, yielding a clear oil.
GC-MS analysis (column HP5-MS, 25m x 0.2mm; Oven 120 C,
2 min, 15 C/min, 210 C, 5 min.; He @ 1 ml/min) was conducted on this
material. The total ion chromatogram is presented in Figure 2B. This
analysis indicated that the principal component (65.43% area; Rt 7.08 min)
represented a dihydronepetalactone isomer, with m/z 168; the mass
spectrum of this component is presented in Figure 3B. This spectrum
contains an ion with m/z 113, diagnostic for dihydronepetalactones
(Jefson, M., et al. op.cit.). Five additional peaks, representing the
remaining dihydronepetalactone diastereomers which might be derived
from the three nepetalactones present in the starting material were also
represented in the chromatogram. These occurred at Rt 5.41 min, 6.8%
area, m/z 168; Rt 5.93 min, area 1.2%, m/z 168; Rt 6.52min, 4.88% area,
mass 168; Rt 6.76min, 13.8% area, m/z 168 and Rt 7.13 min, 1.25% area,
m/z 168. No residual nepetalactones were detected by GC-MS.
1 H, 13C and a series of 2D NMR analyses were also performed.
The carbonyl region of the 13C NMR spectrum (Figure 5) showed at least
five spin systems, one of them in larger amounts than the other four (ca.
75%). Very little residual nepetalactone was detected.
Based on the analysis of coupling constants and the intensities of
the different NOE cross peaks observed, the stereochemistry of the
principal component of the material was determined to be the
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0
f e a 0
d b
c
dihydronepetalactone of Formula 2 (9S,5S,I R,6R)-5,9-dimethyl-3-
oxabicyclo[4.3.0]nonan-2-one).
Formula 2
The distance between the methyl group (i) and proton (d) is longer
than the distance between the methyl group (j) and the proton (e), an
observation consistent with the cis-trans stereochemical configuration.
The stereoisomer isodihydronepetalactone (9S,5R,1 R,6R)-5,9-
dimethyl-3-oxabicyclo[4.3.0]nonan-2-one; (Formula 3) was similarly
identified by 13C chemical shifts and is present in 3.6%.
0
f e a 0
9
h d b
C
Formula 3
Thus the GC-MS and NMR data indicate that hydrogenation of the
mixture of nepetalactone stereoisomers yielded the corresponding
dihydronepetalactone diastereomers, as expected. The pair of
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diastereomers (Formula 2 and Formula 3) derived from cis,trans-
nepetalactone (84.5 Mol% of the starting material) were the predominant
dihydronepetalactones, at 78.6% of the mixture following hydrogenation.
EXAMPLE 3
Repellency testing of a dihydronepetalactone mixture
The DHN prepared in accordance with Example 2 (designated
"mDHN") was evaluated for its repellent effects against female Aedes
aegypti mosquitoes. I
Approximately 250 female Aedes aegypti mosquitoes were
introduced into a chamber containing 5 wells, each covered by a
Baudruche (animal intestine) membrane. Wells were filled with bovine
blood, containing sodium citrate (to prevent clotting) and ATP (72 mg ATP
disodium salt per 26 ml of blood), and heated to 37 C. A volume of 25 l
of isopropyl alcohol, containing one of the test specimens shown in
Table 2, was applied to each membrane.
Table 2
Experimental Design Applied for Repellency Testing
Purpose Compound Concentration
Untreated Control Isopropyl alcohol 100%
Positive Control Isopropyl alcohol with DEET 1.0% (w/v)
Experimental Samples Isopropyl alcohol with 1.0% (w/v)
Dihydronepetalactones
2.5% (w/v)
5.0% (w/v)
After 5 min, 4 day-old female mosquitoes were added to the
chamber. The number of mosquitoes probing the membranes for each
treatment was recorded at 2 min intervals over 20 min. Each datum
represents the mean of three replicate experiments.
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Table 3 presents the amount of time taken before the female A.
aegypti mosquitoes first probed each treated membrane The numbers in
parantheses are the standard error of the mean (SEM) for the three
replicates.
Table 3
Effect of Dihydronepetalactone Concentraton on
Mean Time to "First Probe"
Repellent Concentration Mean Time (min) (SEM)
Isopropyl alcohol (untreated 4.66 (0.66)
control)
I% DEET (positive control) 12.0 (0.0)
1% mDHN 8.0 (1.15)
2.5% mDHN 9.33 (3.33)
5% mDHN 19.33 (0.66)
Mosquitoes began probing the untreated control well within 4.6 min.
Dihydronepetalactones at 5% concentration was found to discourage
mosquito "first probing" for approximately 19 min, compared to 12 min for
DEET (at 1 % w/v).. Lower concentrations of dihydronepetalactones (1 %
and 2.5% w/v) were found to inhibit first probing for an average of 8 and
9.3 min, respectively.
The distribution of landing/probing density by female A. aegypti on
membranes treated with dihydronepetalactones was analyzed over time,
and is shown graphically in Figure 6. The total number of probes
permitted on each membrane during the course of the experiments were
determined, and the results are summarized in Table 4. DHN at 5%
concentration was found to almost eliminate mosquito probes for
20 minutes; only a single probe was recorded over the entire 20 min test
time, while DEET (1 % w/v) permitted an average of 4.55 mosquitoes to
land. Again, lower concentrations of DHN (1 % and 2.5% w/v) were found
to exhibit repellency (as compared to the untreated control), but at lower
levels than the positive control (DEET at I% w/v).
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Table 4
Number of Probes Permitted
According to Repellency Concentration
Repellent Concentration Mean Number of probes
(SEM)
Isopropyl alcohol (untreated 58.66 (4.48)
control)
1 % DEET (positive control) 4.55 (0.29)
1% mDHN 14.0 (6.8).
2.5% mDHN 6.33 (1.2)
5% mDHN 0.33 (0.33)
Again, -the data shows that at all concentrations tested
dihydronepetalactones were repellent, although significantly increased
repellency with respect tot % DEET was observed only at 5% (w/v).
EXAMPLE 4
Preparation of dihydronepetalactones from trans,cis-nepetalactone
A number of plants were grown from seed of the catmint Nepeta
racemosa (Chiltern Seeds, Cumbria, UK). Leaf pairs plucked from
individual plants were immersed in ethyl acetate and after 2h the solvent
was removed and the extracts analyzed by gas chromatography. Plants
producing preponderantly trans,cis-nepetalactone in their oils were thus
identified (Clark, L.J., et al. op.cit.), and grown to maturity. Leaf material
from these plants was harvested, freeze-dried, extracted into ethyl acetate,
and the extracts concentrated. Nepetalactone was purified from the
concentrated extract by silica gel chromatography in hexane/ethyl acetate
(9:1) followed by preparative thin-layer chromatography on silica using the
same solvent mixture. After removal of the solvent and re-dissolving in
hexane, the trans,cis-nepetalactone was crystallized on dry ice. GC-MS
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and NMR (1H and 13C) analysis confirmed the identity of the crystalline
material as trans,cis-nepetalactone. The 13C chemical shifts (Fig. 7),
compared to the chemical shifts of Table 1, are shown in Table 5.
Table 5 13C chemical shifts of the nepetalactone sample prepared in
Example 4, compared to the chemical shifts of trans,cis-nepetalactone
(from Table 1)
Atom trans, cis- Sample
nepetalactone
S (ppm) S (ppm)
a 170.1 170.3
b 135.9 136.0
c 120.4 120.5
d 37.3 37.5
e 49.1 49.3
f 32.1 32.2
g 30.0 30.1
h 26.1 26.3
j 17.5 17.7
i 14.2 14.4
Hydrogenation of the trans,cis-nepetalactone thus prepared was
carried out in ethanol using ESCAT#142 catalyst (Englehart) at 50 C for
4h. GC-MS and NMR (1H and 13C) confirmed that the trans,cis-
nepetalactone had been quantitatively converted to the corresponding
dihydronepetalactone stereoisomers, with one in significant excess. NMR
analysis of the major diastereomer: 1 H NMR (500 MHz, CDCI3): d 0.97 (d,
3H, J = 6.28 Hz), 0.98 (d, 3H, J = 6.94 Hz) d 1.24 (m, 2H), 1.74 (m, 1 H),
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1.77 (m, 2H), 1.99 (m, 2H), 2.12 (dd, 1 H, J = 6.86 and 13.2 Hz), 2.51 (m,
1 H), 3.78 (tr, 1 H, J = 11.1 Hz), 4.33 (dd, 1 H, J = 5.73 and 11.32 Hz); 13C
(500 MHz, CDCI3): d 15.43, 18.09, 27.95, 30.81, 31.58, 35.70, 42.5 1,
51.40, 76.18, 172.03. The '3C NMR spectrum (Fig. 8) indicated that this
major diastereomer constituted ca. 93.7% of the preparation. Based on the
observed couplings for the methylene to the lactone oxygen, the
stereogenic methine carbon bearing methyl group, the methyl group itself
and the bridgehead methine, it is concluded that the diastereomer is most
likely the (1 S,9S,5R,6R)-5,9-dimethyl-3-oxabicyclo[4.3.0]nonan-2-one) of
Formula 4.
0
Formula 4
The magnitude of the observed couplings are consistent with
dihedral angles between the protons on vicinal carbon atoms in the above
structure according to the Karplus equation (ref. Spectrophotometric
Identification of Organic Compounds, 4th. edition, Robert M. Silverstein, G.
Clayton Bassler and Terence C. Morill, 1981, page 208-210).
EXAMPLE 5
Repellency Testing of Dihydronepetalactones Prepared by Hydrogenation
of trans,cis-Nepetalactone
The dihydronepetalactone prepared in Example 4, Formula 4, was
tested for repellency against Aedes aegypti mosquitoes essentially as
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described in Example 3. The experimental design is summarized in Table
.6, and all data presented is from five replicate experiments.
Table 6
Experimental Design Applied for Repellency Testing
Purpose Compound Concentration
Untreated Control Isopropyl alcohol 100%
Positive Control Isopropyl alcohol with DEET 1.0% (w/v)
Experimental Samples Isopropyl alcohol with DHN 1.0% (w/v)
0.5% (w/v)
0.2% (w/v)
Table 7 presents the effect of DHN concentration with respect to the
amount of time taken before the female A. aegypti mosquitoes first probed
each membrane.
Table 7
Effect of Dihydronepetalactone Concentraton on
Mean Time to "First probe"
Repellent Concentration Mean Time (min)
(SEM)
Isopropyl alcohol (untreated control)- 8.0 (1.67)
1 % DEET (positive control) 14.8 (3.2)
1% DHN 16.0(2.09)
0.5% DHN 9.6 (2.48)
0.2% DHN 8.4 (1.16)
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Dihydronepetalactone at 1% concentration was found to discourage
mosquito "first probing" for approximately 16 min. DEET at the same
concentration, exhibited a mean time to first probe of 14.8 min. Lower
concentrations of dihydronepetalactone (0.5% and 0.2% w/v) were found
to inhibit first probing for an average of 9.6 and 8.4 min, respectively.
The distribution of probing density by female A. aegypti on
membranes treated with dihydronepetalactones was analyzed over time,
as shown graphically in Figure 9. The total number of probes permitted on
each membrane during the course of the experiments were determined,
and the results are summarized in Table 8. DHN at 1.0% concentration
was found to completely eliminate mosquito probing for 10 minutes, while
DEET (I% w/v) permitted mosquitoes to initiate probing by 6 min. Again,
.lower concentrations of dihydronepetalactone (0.5% and 0.2% w/v) were
found to exhibit repellency (as compared. to the untreated control), but at
lower levels than the positive control (DEET at 1 % (w/v)).
Table 8
Number of Probes Permitted
According to Repellent and Concentration
During 20 minute Observation Period
Repellent Concentration Mean Number of Probes
(SEM)
Isopropyl alcohol (untreated 41.4 (18.46)
control)
1 % DEET (positive control) 4.8 (3.2)
1%DHN 4.0(2.16)
0.5% DHN 16.2 (5.49)
0.2% DHN 23.2 (5.97)
Percentage repellency was calculated for each repellent treatment
at each observation time using the following equation:
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%Repellency = 100 - [(T/C) x 100]
where:
T = the mean number of mosquitoes probing a treated well for that
replicate at time tx
C = the mean number of mosquitoes probing the IPA control well at time tx
The resulting percentages were then arcsine transformed and an ANOVA
was conducted using the calculated repellency from all five replicates.
Multiple comparisons of means were conducted using the Student-
Newman-Keuls test. The mean arcsines from ANOVA were then
converted back into percentages. The results are shown in Table 9.
Table 9
Mean percentage repellencies as calculated from the ANOVA
Repellent Treatment Mean (%)
1 % DEET (positive control) 92.4
1% DHN 96.1
0.5% DHN 66.7
0.2% mDHN 62.5
1 % DHN ranked first in repellency, and was statistically
indistinguishable from 1 % DEET.
EXAMPLE 6
Repellency Testing of Dihydronepetalactones Against Stable Flies
(Stomoxys calcitrans)
DHN derived from hydrogenation of trans,cis-nepetalactone
(consisting principally of 1 S,9S,5R,6R-5,9-dimethyl-3-
oxabicyclo[4.3.0]nonan-2-one; Formula 4), designated "Experimental
Sample #1 ", and the mixture of dihydronepetalactones prepared according
to Example 2 (designated Experimental Sample #2; mDHN) , were tested
for repellency against Stomoxys calcitrans, essentially as described in
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Example 3. The DHN used here differed from that prepared in Example 4
in that it was derived from hydrogenation (using a Pd/SrCO3 catalyst) of
trans,cis-nepetalactone crystallized from commercial oil (Berje, NJ). In
these experiments, an additional positive control compound- was included,
namely p-menthane-3,8-diol (PMD), obtained from Takasago International
Corp. (USA), Rockleigh, NJ. The experimental design is summarized in
Table 10, and all data presented is an average of five replicate
experiments.
Table 10
Experimental Design Applied for Repellency Testing against Stable
Flies
Purpose Compound Concentration
Untreated Control Isopropyl alcohol 100%
Positive control #1 Isopropyl alcohol with PMD 1.0% (w/v)
Positive Control #2- Isopropyl alcohol with DEET 1.0% (w/v)
Experimental Isopropyl alcohol with 1.0% (w/v)-
Sample #1 Dihydronepetalactone (DHN)
Experimental Isopropyl alcohol with 1.0% (w/v)
Sample #2 Dihydronepetalactone diastereomer
mix (mDHN)
In these tests, an accurate time to "first landing" could not be
determined, since some landings occurred before the first exposure period
of 2 min in three or more of the five replicates for each test variable.
The distribution of landing density by stable flies on membranes
treated with dihydronepetalactones was analyzed over time, as shown
graphically in Figure 10. The total number of landings permitted on each
membrane during the course of the experiments were determined, and the
results are summarized in Table 11. Landings commenced on exposure of
the insects to the test wells, and appeared to peak after ca. 5 min,
gradually decreasing thereafter over time. Overall, the number of landings
on membranes treated with dihydronepetalactones at I% concentration
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were significantly fewer than observed on untreated (IPA) membranes,
and equivalent to those observed with DEET (1 % w/v). p-Menthane-3,8-
diol (PMD) was less effective in repelling landings than either the
dihydronepetalactones or DEET throughout the course of the experiment,
and although some initial repellency could be observed, this compound
became ineffective after 6 min. Again, this data indicates that 1 %
dihydronepetalactones exhibited equivalent repellent activity to 1 % DEET.
Table 11
Number of Landings Permitted
During 20 minute Test
Repellent Treatment Mean Number of Landings (SEM)
Isopropyl alcohol (untreated- control) 44.0 (8.59)
1 % PMD (positive control #1) 33.6 (9.21)
1 % DEET (positive control #2) 17.8 (4.96)
1% DHN 21.2 (3.-35)-
1% mDHN .18.8 (8.59)
Percentage repellency and statistical analyses were carried out as
described in Example 5, and the results presented in Table 12.
Table 12
Mean percentage repellencies as calculated from the ANOVA
Repellent Treatment Mean (%)
1 % PMD (positive control #1) 4.7
1 % DEET (positive control #2) 55.5
1% DHN 43.2
1% mDHN 49.8
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mDHN, DEET and DHN performed statistically equally well, providing 43.2
to 55.5% repellency, and were statistically better than PMD, which gave
only 4.7% repellency when compared to IPA.
EXAMPLE 7
Repellency Testing of Dihydronepetalactones Against Anopheles
Mosquitoes (Anopheles albimanus)
DHN derived from hydrogenation of trans,cis-nepetalactone
(consisting principally of 1 S,9S,5R,6R-5,9-dimethyl-3-
oxabicyclo[4.3.0]nonan-2-one; Formula 4) designated "Experimental
Sample #1", and the mixture of dihydronepetalactones prepared according
to Example 2 (designated as "Experimental Sample #2"; mDHN) were
tested for repellency against one hundred unfed adult female A.
albimanus, essentially as described in Example 3. The DHN used here
differed from that prepared in Example 4 in that it was derived from
hydrogenation (using a Pd/SrCO3 catalyst) of trans,cis-nepetalactone
crystallized from commercial oil (Berje, Bloomfield, NJ). PMD was again
included as a further control. The experimental design is summarized in
Table 13, and all data presented is the average of five replicate
experiments.
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Table 13 Experimental Design Applied for Repellency Testing
against Anopheles Mosquitoes
Purpose Compound Concentration
Untreated Control Isopropyl alcohol 100%
Positive control #1 Isopropyl alcohol with PMD 1.4% (w/v)
Positive Control #2 Isopropyl alcohol with DEET 1.0% (w/v)
Experimental Isopropyl alcohol with 1.0% (w/v)
Sample #1 Dihydronepetalactone (DHN)
Experimental Isopropyl alcohol with 1.0% (w/v)
Sample #2 Dihydronepetalactone diastereomer
mix (mDHN)
In these tests, an accurate time to "first probing" could not be
determined, since some probes occurred before the first exposure period
of 2 min in two or more of the five replicates for each test variable. The
distribution of probing density by anopheles mosquitoes on membranes
treated with dihydronepetalactones was analyzed over time, as shown
graphically in Figure 11. Probing commenced on exposure of the insects
to the test wells, and gradually increased thereafter over time. Overall, the
number of probes on membranes treated with dihydronepetalactones at
1 % concentration were significantly fewer than observed on untreated
(IPA) membranes throughout the experiment.
The total number of probes permitted on each membrane during the
course of the experiments were determined, and the results are
summarized in Table 14. The data indicates that 1 %
dihydronepetalactones exhibited higher repellent activity compared to
equivalent concentrations of either DEET or PMD against A. albimanus.
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Table 14
Number of Landings Permitted
During 20 minute Test
Repellent Treatment Mean Number of Probes (SEM)
Isopropyl alcohol (untreated control) 66.2 (15.53)
I% PMD (positive control #1) 47.2 (8.57)
1 % DEET (positive control #2) 50.4 (13.01)
1% DHN 38.0(9.71)
1% mDHN 34.8 (6.26)
Percentage repellency and statistical analyses were carried out as
described in Example 5, and the results presented in Table 15.
Table 15
Mean percentage repellencies as calculated from the ANOVA
Repellent Treatment Mean (%)
1% PMD (positive control #1) 11.5
1 % DEET (positive control #2) 13.3
1%DHN 32.9
1%mDHN 46.1
mDHN was statistically superior to DEET and provided 46.1% repellency.
DHN, while statistically equal to mDHN, was also statistically equal to
DEET and provided 32.9% repellency. DEET and PMD, which provided
13.3% and 11.5% repellency respectively, were statistically equal in
efficacy.
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Example 8
Repellency of dihydronepetalactones towards the deer tick, Ixodes
scapularis
DHN derived from hydrogenation of trans,cis-nepetalactone (consisting
principally of 1 S,9S, 5R, 6 R-5,9-d i m ethyl-3-oxa bi cyclo[4.3. 0] non an-2-
one;
Formula 4) prepared as in Example 7, and the mixture of
dihydronepetalactones prepared according to Example 2 were tested for
repellency against I. Scapularis, with DEET included in the test as a
positive control.
A volume of 25 I of each compound (30% (w/v) in isopropanol) was
applied within 4cm diameter circles drawn on the left forearms of six male
human volunteers. Each volunteer had two repellents applied individually
within two circles on this forearm; a single 4cm diameter circle drawn on
the other arm was left untreated. to act as a control for tick attractiveness.
Laboratory-reared unfed nymphs of the deer tick Ixodes scapularis were
brought within 1 mm of the untreated circles on cotton buds (Q-tip ). If
normal questing behavior was observed, and/or the insect crawled onto
the untreated area, it was deemed qualified and then presented to a
treated area. A qualified tick which quested at or crawled onto the treated
area within 60s was recorded as having not been repelled. A qualified tick
which did not quest or ceased questing within 60s an/or retreated from the
treated area was recorded as repelled. Additionally, a qualified tick that
crawled onto the treated area but fell off within an additional 60s was
recorded as repelled.
Each volunteer had 5 qualified ticks offered to each treated circle at
approximately hourly intervals. Exposures continued until 3 out of any
group of 5 offered ticks were deemed 'attracted'. The first non-repelled tick
was defined as the first attracted tick which was followed by a second
attracted tick within the same or following exposure period. The time of the
first confirmed attracted tick was deemed to be the time at which complete
repellency `broke down' for that volunteer.
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Table 16
Mean complete protection times for DHN, mDHN and DEET at 30% (w/v),
topically applied to human volunteers, towards the deer tick Ixodes
scapula
Repellent Treatment Mean (SEM)
30% DEET (positive control) 124.0 (69.95)
30% DHN 109.0 (58.64)
30% mDHN 85.25 (28.76)
The data (Table 16) indicates that DEET offered a mean complete
protection time from the deer tick Ixodes scapularis of 124min, whilst DHN õ
was, similarly effective for 109min, and mDHN (mixed diastereomers of
DHN) for 85min. Thus it is clear that both DHN and mDHN are repellent
towards the deer tick. An ANOVA of the protection times was conducted,
which showed that DHN, mDHN and DEET,were statistically
indistinguishable in the longevity of their repellency to these ticks.
Example 9
Repellency of dihydronepetalactones applied to human subjects towards
the mosquito Anopheles albimanus
The DHN derived from hydrogenation of trans,cis-nepetalactone
(consisting principally of 1 S,9S,5R,6R-5,9-dimethyl-3-
oxabicyclo[4.3.0]nonan-2-one; Formula 4), prepared as in Example 7, and
the mixture of dihydronepetalactones prepared according to Example 2,
were tested for,repellency, against A. albimanus, with DEET included in the
test as a positive control, using adult human volunteers. Test cages (2 x 2
x 2 feet). with two sleeved entry ports on each of two opposite sides were
used, with a hand rest in the center. The sides and top were screened and
the base was equipped with a mirror to facilitate observations. Two
hundred adult female mosquitoes, which had never received a blood meal
and which had been deprived of their normal diet of 10% sucrose 24h prior
to use, were released into the test cage. Each volunteer was pre-qualified
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as attractive through having 10 insects land on their untreated forearms
within 30s of insertion into the cage.
A volume of I.Oml of each compound (either 5% or 10% (w/v) in
isopropanol) was applied to 250cm2 areas on the forearms of six male
human volunteers, the remainder of the limbs having been made
inaccessible to insects. Each volunteer had different repellents applied
individually onto each forearm. After allowing the applied repellents to dry
for 30min, the forearms were placed into the test cage for 5min periods at
30min intervals, and the number of mosquitoes probing or biting during
each exposure period recorded. Breakdown of repellency was recorded for
each repellent on each volunteer. Breakdown was defined as the time at
which the first confirmed bite occurred; the first confirmed bite was defined
as a bite which was followed by a second bite either within the same or the
next exposure period. The data is presented in Table 17 as mean
complete protection time. The data indicates that both DHN and mDHN
conferred complete protection from bites for significant periods of time
(eg., at 10% (w/v) for 3.5 and 5 hours, respectively), and comparable to
that afforded by DEET at the same concentration.
The data was analyzed using ANOVA, and this showed that the 5%
and 10%mDHN solutions were statistically indistinguishable in efficacy
from 5% and 10% DEET, respectively. The 5% and 10% solutions of DHN,
although statistically equal to the corresponding solutions of mDHN,
provided lesser protection times.
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Table 17
Mean complete protection times of dihydronepetalactones at 5% and 10%
(w/v topically applied to human volunteers, towards female Anopheles
albimanus mosquitoes
Repellent Treatment Mean (h) (SEM)
5% DEET (positive control) 4.0 (0.5)
5% DHN 1.8 (0.12)
5% mDHN 3.0 (0.54)
10% DEET (positive control) 6.2 (0.63)
10% DHN 3.5 (0.29)
10% mDHN 5.0 (0.61) -
48
