U.S. patent application number 11/349832 was filed with the patent office on 2006-06-29 for composition for neutralizing house dust mite feces.
This patent application is currently assigned to Kimberly-Clark Worldwide, Inc.. Invention is credited to Duane G. Krzysik, David J. Tyrrell.
Application Number | 20060141074 11/349832 |
Document ID | / |
Family ID | 32297789 |
Filed Date | 2006-06-29 |
United States Patent
Application |
20060141074 |
Kind Code |
A1 |
Tyrrell; David J. ; et
al. |
June 29, 2006 |
Composition for neutralizing house dust mite feces
Abstract
Methods for neutralizing dust mite feces are disclosed. In one
embodiment, a botanical extract having a BAPNA-Trypsin Inhibition
(IC.sub.50.times.10.sup.-3 (%, v/v or w/v) of from about 0.01 to
about 500 and a sufficient solubility in water is introduced into
water to form an aqueous botanical extract solution. The aqueous
botanical extract solution is atomized into an area being treated
such that the botanical extract contacts and neutralizes the dust
mite feces located in the area. Particularly preferred botanical
extracts for use in the botanical extract aqueous solutions of the
present invention include Green Tea Extra and Grape Seed
Extract.
Inventors: |
Tyrrell; David J.;
(Appleton, WI) ; Krzysik; Duane G.; (Appleton,
WI) |
Correspondence
Address: |
SENNIGER POWERS (KCC)
ONE METROPOLITAN SQUARE
16TH FLOOR
ST LOUIS
MO
63102
US
|
Assignee: |
Kimberly-Clark Worldwide,
Inc.
|
Family ID: |
32297789 |
Appl. No.: |
11/349832 |
Filed: |
February 8, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10299868 |
Nov 19, 2002 |
7037535 |
|
|
11349832 |
Feb 8, 2006 |
|
|
|
Current U.S.
Class: |
424/729 ;
424/766; 424/769 |
Current CPC
Class: |
A01N 65/08 20130101;
A01N 65/00 20130101; A01N 65/00 20130101; A01N 61/00 20130101; A01N
65/08 20130101; A01N 25/02 20130101; A01N 65/08 20130101 |
Class at
Publication: |
424/729 ;
424/769; 424/766 |
International
Class: |
A01N 65/00 20060101
A01N065/00 |
Claims
1. An aqueous composition for neutralizing dust mite feces, the
composition comprising a water soluble botanical extract having a
BAPNA-Trypsin Inhibition (IC.sub.50.times.10.sub.-3 (%, v/v or
w/v)) of from about 0.01 to about 500.
2. The aqueous composition as set forth in claim 1 wherein the
BAPNA-Trypsin Inhibition is from about 0.01 to about 200.
3. The aqueous composition as set forth in claim 1 wherein the
BAPNA-Trypsin Inhibition is from about 0.01 to about 100.
4. The aqueous composition as set forth in claim 1 wherein the
BAPNA-Trypsin Inhibition is from about 0.01 to about 20.
5. The aqueous composition as set forth in claim 1 wherein the
BAPNA-Trypsin Inhibition is from about 0.01 to about 10.
6. The aqueous composition as set forth in claim 1 wherein the
botanical extract has a solubility in water of at least about 10%
(v/v or w/v).
7. The aqueous composition as set forth in claim 1 wherein the
botanical extract is selected from the group consisting of Apple
Green Tea, Arkin Special, Arnica Special, Avocado, Avocado GW,
Black Currant Green Tea, Cabbage Rose, Cat's Claw, Cemila Oleifera,
Centella, Cranberry Green Tea, Dandelion, Garcinia, Grape Seed,
Grapefruit Green Tea, Green Tea, Green Tea Concentrate, Green Tea
HS, Hexaplant Richter, Hibiscus Special, Hydrocotyle GR, Lavender,
Horse Chestnut, Milk Thistle, Orange Green Tea, Phytexcell Arnica,
Purple Coneflower, Sage GW, Sage Special, Sedaplant Richter, St.
John's Wort, Witchhazel GW, Yarrow, Grape Seed Extract, Green Tea
Extra, White Tea 50%, and combinations thereof.
8. A method of neutralizing dust mite feces, the method comprising:
introducing a protease inhibitory, water soluble agent into water
to form a protease inhibitory solution; and contacting the dust
mite feces with the protease inhibitory solution.
9. The method as set forth in claim 8 wherein the protease
inhibitory, water soluble agent is selected from the group
consisting of Apple Green Tea, Arkin Special, Arnica Special,
Avocado, Avocado GW, Black Currant Green Tea, Cabbage Rose, Cat's
Claw, Cemila Oleifera, Centella, Cranberry Green Tea, Dandelion,
Garcinia, Grape Seed, Grapefruit Green Tea, Green Tea, Green Tea
Concentrate, Green Tea HS, Hexaplant Richter, Hibiscus Special,
Hydrocotyle GR, Lavender, Horse Chestnut, Milk Thistle, Orange
Green Tea, Phytexcell Arnica, Purple Coneflower, Sage GW, Sage
Special, Sedaplant Richter, St. John's Wort, Witchhazel GW, Yarrow,
Grape Seed Extract, Green Tea Extra, White Tea 50% and combinations
thereof.
10. The method as set forth in claim 8 wherein the protease
inhibitory water soluble agent has a BAPNA-Trypsin Inhibition
(IC.sub.50.times.10.sup.-3 (%, v/v) of from about 0.01 to about
500.
11. A method of neutralizing dust mite feces, the method
comprising: solubilizing a botanical extact in a hydrophilic
solvent and introducing the solubilized botanical extract into
water to form an aqueous botanical extract solution, the botanical
extract having a BAPNA-Trypsin Inhibition
(IC.sub.50.times.10.sup.-3 (%, v/v or w/v)) of from about 0.01 to
about 500; and contacting the dust mite feces with the aqueous
botanical extract solution.
12. The method as set forth in claim 11 wherein the hydrophilic
solvent is selected from the group consisting of propylene glycol,
butylene glycol, ethylene glycol, hexylene glycol, pentylene
glycol, methyl propanediol, dipropylene glycol, glycerin, ethanol,
isopropyl alcohol, and mixtures thereof.
Description
REFERENCE TO RELATED APPLICATION
[0001] This divisional patent application claims priority from U.S.
patent application Ser. No. 10/299,868 filed on Nov. 19, 2002, the
entirety of which is hereby incorporated by reference.
BACKGROUND OF INVENTION
[0002] The present invention relates to a method of neutralizing
allergens produced by house dust mites. More particularly, the
present invention relates to a method and composition for
neutralizing house dust mite feces allergens by atomizing an
aqueous solution comprising a botanical extract having protease
inhibitory activity such that the atomized botanical extract
contacts the house dust mite feces and renders it substantially
non-allergenic.
[0003] It has been known for many years that common house dust is
an important cause of asthma, rhinitis and eczema in allergic
individuals. Common house dust mites (Dermatophagoides farinae and
Dermatophagoides pteronyssinus) are prevalent in house dust. Dust
mites are not insects, but are eight-legged arachnids, and are
related to ticks and spiders. Adult mites are approximately 250-300
micrometers in length, and are photophobic.
[0004] House dust mites are co-inhabitants of almost every house
and, because of their size and translucent body structure, cannot
be seen with the naked eye. Dust mites are found in almost all home
furnishing textiles and thrive in mattresses, pillows, cushions,
carpets, upholstery, and soft toys etc. An adult mite can live up
to about three months. The reactions caused by house dust mite
feces in sensitive people can range from itchy and watery eyes,
repeated sneezing and running nose, cough and bronchial asthma, to
eczema. The dust on which house dust mites thrive may be cotton,
wool lint, animal and human dander or hair, crumbs, pollens, and
molds.
[0005] House dust mites typically feed on human skin scales. In
addition to a food source, the other essential requirement for dust
mite growth is adequate humidity. Dust mites are 75% water by
weight and although they do not drink water, they must absorb water
vapor from the air to survive. Specialized glands above their pairs
of legs produce secretions high in sodium and potassium chloride,
which act to absorb water vapor from surrounding air. This can only
be accomplished if the surrounding humidity is sufficiently high.
Relative humidities of about 65-80% at temperatures ranging from
about 20 to 35.degree. C. are optimal for dust mite growth.
[0006] A major dust mite allergen is present in dust mite fecal
particles. Each dust mite produces about 20 fecal particles per
day, and more than 100,000 mites may be present in a gram of dust.
These fecal particles vary from about 10 to about 40 microns in
size, comparable to the size of pollen grains, and become airborne
during domestic activity such as making beds and vacuuming carpets.
Enzymes with serine protease and/or cysteine protease activities
from house dust mite feces have been shown to damage the airway
epithelium. This damage increases the propensity for house dust
mite allergens (including proteases) to penetrate the protective
epithelium lining, interact with immune cells, and can induce the
production of specific IgE molecules which can initiate an
inflammatory cascade culminating in an allergic response.
[0007] Group I allergens (Dermatophagoides farinae I, and
Dermatophagoides pternoyssinus I) are heat labile, 24,000 molecular
weight glycoproteins, and appear to be structural homologues and
have very similar N-terminal amino acid sequences. Group I
allergens are regarded as the most important and are excreted in
their highest concentrations by the mite's gastrointestinal tract
in the form of mite's fecal particles.
[0008] Group II allergens (Dermatophagoides farinae II, and
Dermatophagoides pternoyssinus II) are 15,000 molecular weight
proteins with almost identical N-terminal amino acid sequences that
are also secreted by the mite's gastrointestinal tract in the form
of fecal allergens, although not in as high a concentration as the
group I allergens. Most dust mite allergic individuals produce
antibodies to both the group I and group II allergens.
[0009] The control of dust mite population in the domestic
environment is one method of preventing house dust allergies. The
degree of cleanliness impacts the number of house dust mites and
the resulting allergen level. Common control measures include
vacuum cleaning, frequent washing, and treating the carpets and bed
spreads with insecticides, acaricides, and fungicides. Reducing
dust mite population by interfering with the food chain has also
been suggested.
[0010] Rao et al. in U.S. Pat. No. 6,060,075 disclose a composition
for controlling house dust mite population. The composition
comprises a plant derived acaricidal agent, which is disclosed as
neem seed kernel extract, and a plant derived disinfectant agent,
which is disclosed as an alcoholic extract of resins like stryax
benzoin. Rao et al. disclose that a bi-weekly spray of 200
microliters/100 milligrams of culture is required for eight weeks
to completely eliminate the house dust mites, and discloses that
re-establishment of house dust mites after treatment with
acaricides is a common problem due to the existence of nymph and
eggs. Rao et al., however, fail to disclose any compositions or
methods for neutralizing the feces allergen of dust mites, and
simply disclose a very narrow composition for killing house dust
mites, which does not necessarily neutralize the feces.
[0011] Miller in published U.S. Patent Application US2002/0022043
discloses a method for killing house dust mites in clothing and
other soft materials. Miller exposes woolen or other fabrics to the
vapors of certain plant oils, including wintergreen oil, lavandin
oil, Ylang-Ylang oil and others. The oils are aromatherapy-grade
oils produced by steam distillation. Miller generates vapors of the
oils numerous ways including: placing a few drops of the oil on a
paper towel, piece of cotton, or on a glass or plastic dish and
then placing the treated substrate in a closed environment with the
articles to be treated; spraying a mist of the oil onto the
substrate to be treated, or by heating the oil to cause
vaporization. Similar to Rao, Miller sets forth a method for
killing house dust mites, but does not disclose methods or
compositions for neutralizing house dust mite feces. Additionally,
the compositions set forth by Miller are oils which can be messy to
work with, can stain certain fabrics, and can be very difficult to
effectively vaporize.
[0012] McKechnie et al. in UK Patent Application GB 2,363,074
disclose a method for deactivating house dust mite allergens by
volatilizing an oil which comprises tea tree oil or another oil
comprising terpene compounds and contacting the volatilized oil
with the house dust mite feces. McKechnie et al. volatilize the oil
in numerous ways including: heating the oil to form vapors;
vaporizing the oil from a heated wick dipped into a reservoir of
the oil; and by utilizing an ultra-sonic jet nebuliser which
contains water with the oil floated on the surface of the water. As
with Miller discussed above, tea tree and other oils which are not
water soluble can be difficult to work with and difficult to
effectively vaporize.
[0013] Based on the foregoing, a simple, cost effective method of
neutralizing the feces allergens of house dust mites is needed. The
method preferably will involve simple, water soluble, compositions
which are easy to work with and easily atomizable, in contrast to
the oils disclosed in the prior art.
SUMMARY OF THE INVENTION
[0014] The present invention relates to neutralizing allergenic
dust mite feces by contacting the dust mite feces with botanical
extracts having protease inhibitory activity to reduce the
allergenicity of the dust mite feces. Substantially water soluble
botanical extracts, such as Green Tea Extra or Grape Seed Extract,
for example, are introduced into water to form an aqueous botanical
extract solution. The botanical extract, which may be in powder
form or in aqueous form, used to form the aqueous botanical extract
solution has a BAPNA-Trypsin inhibition (IC.sub.50.times.10.sup.-3
(%, v/v or w/v)) of from about 0.01 to about 500, and preferably
has a solubility in water of at least about 0.5% by weight if the
botanical extract is a solid or at least about 10% by weight if the
botanical extract is a liquid. Because the botanical extracts
suitable for use in the present invention have a high solubility in
water, they are easily and conveniently aerosolized such that large
areas can easily be treated. In one embodiment of the present
invention, the aqueous botanical extract solution is introduced
into an atomizer such that the aqueous botanical extract solution
can be atomized into a room and easily contact numerous areas where
dust mites are known to be present including, for example, bed
linens, pillows, and carpets. In a preferred embodiment, the median
drop size of the atomized aqueous botanical extract solution is
less than 1000 micrometers to ensure sufficient contact with the
dust mite feces.
[0015] Briefly, therefore, the present invention is directed to a
method of neutralizing dust mite feces. The method comprises
introducing a soluble botanical extract into water to form an
aqueous botanical solution and contacting the dust mite feces with
the aqueous botanical extract solution. The botanical extract has a
BAPNA-Trypsin Inhibition (IC.sub.50.times.10.sup.-3 (%, v/v)) of
from about 0.01 to about 500.
[0016] The invention is further directed to a method of
neutralizing dust mite feces. The method comprises introducing a
soluble botanical extract into water to form an aqueous botanical
solution and contacting the dust mite feces with the aqueous
botanical extract solution. The botanical extract has a
BAPNA-Trypsin Inhibition (IC.sub.50.times.10.sup.-3 (%, w/v)) of
from about 0.01 to about 500.
[0017] The invention is further directed to a method of
neutralizing dust mite feces. The method comprises introducing a
water soluble botanical extract into water to form an aqueous
botanical extract solution and contacting the dust mite feces with
the aqueous botanical extract solution. The botanical extract has a
BAPNA-Trypsin Inhibition (IC.sub.50.times.10.sup.-3 (%, v/v or
w/v)) of from about 0.01 to about 500 and a solubility in water of
at least about 0.5% by weight.
[0018] The invention is further directed to a method of
neutralizing dust mite feces. The method comprises introducing a
water soluble botanical extract into water to form an aqueous
botanical extract solution and contacting the dust mite feces with
the aqueous botanical extract solution. The botanical extract has a
BAPNA-Trypsin Inhibition (IC.sub.50.times.10.sup.-3 (%, v/v or
w/v)) of from about 0.01 to about 500 and a solubility in water of
at least about 1% (v/v or w/v).
[0019] The invention is further directed to an aqueous composition
for neutralizing dust mite feces. The composition comprises a water
soluble botanical extract having a BAPNA-Trypsin Inhibition
(IC.sub.50.times.10.sup.-3 (%, v/v or w/v)) of from about 0.01 to
about 500.
[0020] The invention is further directed to a method of
neutralizing dust mite feces. The method comprises introducing a
protease inhibitory, water soluble agent into water to form a
protease inhibitory solution and contacting the dust mite feces
with the protease inhibitory solution.
[0021] Other objects and features of this invention will be in part
apparent and in part pointed out hereinafter.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0022] In accordance with the present invention, it has been
discovered that certain botanical extracts, whether in powder form
or in liquid form, can be effectively utilized to reduce the
allergenicity of allergens including dust mite feces. Surprisingly,
substantially water soluble botanical extracts having a
BAPNA-Trypsin Inhibition (IC.sub.50.times.10.sup.-3 (%, v/v or
w/v)) of from about 0.01 to about 500 as described herein can be
introduced into water to form an aqueous botanical extract solution
which can be utilized in atomized form to treat large areas quickly
and easily to neutralize dust mite feces allergens. In preferred
embodiments, the botanical extracts used to form the botanical
extract solution have a solubility in water of at least about 0.5%
by weight, and more preferably at least about 1% by weight to
ensure sufficient solubility of the botanical extract into the
aqueous solution. Further, it is preferred that the median drop
size of the atomized aqueous botanical extract solution be less
than about 1000 micrometers to ensure effective coverage.
[0023] Dust mites and feces from dust mites are present throughout
many areas of homes as discussed above. Dust mite feces has been
repeatedly shown to contain high levels of serine proteases, which
are known to cause allergic responses in some humans when inhaled
into the nose, throat, and lungs. Through vigorous investigation
and experimentation, the inventors discovered that numerous
botanical extracts have strong protease inhibitory activity and
have high solubility in water. This combination of protease
inhibitory activity and solubility in water allows a concentrated
aqueous botanical extract solution to be prepared in accordance
with the present invention that can be contacted with the feces of
dust mites resulting in the neutralization of the feces such that
the dust mite feces become substantially non-allergenic. One
important aspect of the present invention is that the botanical
extracts act to neutralize the allergenic feces of the dust mite,
and do not simply kill some dust mites while leaving the feces to
continue to act as an allergen if inhaled. Because completely
killing every dust mite in an area, such as a bedroom, and keeping
the dust mites from returning is extremely difficult, if not
impossible, the present invention is substantially advantageous in
that the specific allergens themselves, the feces, are neutralized
through contact with the atomized botanical extract. As discussed
below, in some embodiments of the present invention, the botanical
extract solution described herein can be utilized in combination
with an additional agent capable of killing the dust mites. This
combination provides a highly effective anti-allergenic system.
[0024] Numerous water soluble botanical extracts which provide the
desired level of protease inhibitory activity can be used in the
aqueous solutions of the present invention to neutralize dust mite
feces. The botanical extracts useful in the practice of the present
invention may be in solid form or in aqueous form. Regardless of
whether the botanical extract is in solid form or aqueous form, as
used herein, the term "water soluble" means soluble in water at a
level of at least about 0.5% by weight, and preferably at least
about 1% by weight. Typically, aqueous botanical extracts will have
a much higher solubility in water. It will be recognized by one
skilled in the art that many of the liquid botanical extracts
described herein may be provided as mixtures of water, butylene
glycol, glycerin, and the extracted botanical, or similar
formulations. The extract formulation is soluble in water in part
due to the use of butylene glycol as the solvent. As such, the
botanical extracts themselves suitable for use in the present
invention may be water soluble or hydrophilic solvent soluble.
Common hydrophilic solvents include propylene glycol, butylene
glycol, ethylene glycol, hexylene glycol, pentylene glycol, methyl
propanediol, dipropylene glycol, and the like. Additionally,
solvents such as ethanol or isopropyl alcohol may be utilized as
solvents.
[0025] As used herein, the term "neutralize dust mite feces" means
that the dust mite feces is rendered less allergenistic; that is,
the dust mite feces, or components thereof, are a less potent
allergen and are less likely to result in an allergic reaction in a
human. Many botanical extracts are commercially available and
typically are in one of three forms: (1) powderous solid; (2)
aqueous solution comprising the botanical; or (3) oil. In solid
form, the botanical extract is typically about 100% pure botanical
extract, but may include a small percentage of other solids
resulting from the purification procedures used to collect the
botanical extract from the botanical source. When the botanical
extract is available only as an aqueous solution or oil, the exact
amount of botanical extract contained in the liquid may not be
precisely known. Regardless, liquid botanical extracts can be
tested and evaluated for use in the present invention as described
herein without knowing the precise amount of botanical present in
the solution.
[0026] In accordance with the present invention, the botanical
extracts suitable for use in the aqueous solutions are
substantially soluble in water. Preferably, the botanical extract
is soluble in water at a level of at least about 0.5% by weight,
and more preferably at least about 1% by weight. This solubility
ensures that the botanical extract will be sufficiently soluble for
introduction into the aqueous solutions as described herein such
that the solution can be easily atomized. As one skilled in the art
will recognize, higher solubilities are preferred.
[0027] Suitable botanical extracts for use with the methods and
compositions of the present invention include those botanical
extracts having serine protease inhibitory properties; that is,
botanical extracts that inhibit the activity of the enzyme serine
protease and reduce its ability to perform its intended cleaving
function on a given substrate. Specifically, botanical extracts
suitable for use include those botanical extracts having a
BAPNA-Trypsin Inhibition (IC.sub.50.times.10.sup.-3 (%, v/v or w/v)
of from about 0.01 to about 500, more preferably from about 0.01 to
about 200, still more preferably from about 0.01 to about 100, more
preferably from about 0.01 to about 20, and most preferably from
about 0.01 to about 10. Whether the percentage is reported in
volume/volume (v/v) or weight/volume (w/v) depends on whether the
botanical extract is a powder or a liquid. One skilled in the art
will recognize that the IC.sub.50 values set forth herein can
easily be converted to concentration percentages (representing the
concentration of a given botanical required to reduce the activity
of the target by 50%) by simply multiplying the value by 10.sup.-3;
for example, an IC.sub.50 value as reported herein of 500 would be
equal to a concentration of botanical extract of 0.5%; that is, a
concentration of 0.5% of the botanical would be required to reduce
the activity of the target enzyme by 50%.
[0028] The BAPNA-Trypsin Inhibition IC.sub.50 values described
herein are determined for botanical extracts according to the
BAPNA-Trypsin Inhibition Testing procedure set forth herein, and
well known to those skilled in the art.
BAPNA-Trypsin Inhibition Test For Determination of IC.sub.50 Values
of Botanical Extracts
[0029] Botanical extracts, whether in solid (powder) or liquid
form, are tested for their ability to inhibit a model serine
protease (porcine pancreatic trypsin) in solution as follows:
[0030] Step 1: Botanical extract testing solutions are prepared as
follows when the botanical extract is a liquid: a 10% (v/v)
starting solution is prepared by introducing 100 microliters of
liquid botanical extract into one milliliter of Phosphate Buffered
Saline (PBS), pH=7.4. If the liquid botanical extract is not
soluble at a concentration of 10% (v/v) in PBS, a starting solution
of 9% (v/v) is prepared and utilized as the starting solution
described herein. If the 9% (v/v) starting testing solution is not
soluble in PBS, starting testing solutions of 8% (v/v), 7% (v/v),
etc. are prepared until the desired solubility is obtained. Serial
dilutions of the starting solution for analysis are prepared using
PBS at a pH of 7.4 as the diluent. For example, 7 serial dilutions
of the 10% (v/v) starting solution can be made to prepare 8
different concentrations of botanical extract for testing: (1) 10%;
(2) 5%; (3) 2.5%; (4) 1.25%; (5) 0.63%; (6) 0.31%; (7) 0.16%; and
(8) 0.078%. Similar serial dilutions are made to prepare numerous
samples if the starting testing solution concentration is less than
10%.
[0031] Botanical extract testing solutions are prepared as follows
when the botanical extract is a solid: a 0.1% (w/v) starting
solution is prepared by introducing 1 milligram of solid botanical
extract into one milliliter of PBS, pH of 7.4. Serial dilutions of
the starting solution are prepared using PBS, pH of 7.4, as the
diluent. For example, 7 serial dilutions of the 0.1% (w/v) testing
solution can be made to prepare 8 different concentrations of
botanical extract for testing as described above.
[0032] Step 2: To the empty wells of a clear well plate, such as a
NUNC IMMUNO clear 96 well plate (VWR Scientific Products, Chicago,
Ill.), are added 150 microliters of 100 mM Tris buffer which has
been adjusted to a pH of about 8.0 utilizing HCL. The Tris buffer
is commonly known to those skilled in the art and can be prepared
using Tris powder or Tris liquid.
[0033] Step 3: To the wells of the well plate to be utilized for
testing the botanical extract (i.e., not the control wells to which
25 microliters of PBS is added in place of the botanical extract)
is added 25 microliters of the botanical extract solution prepared
under Step 1. For example, wells 1, 2, and 3 may have 25
microliters of the 10% (v/v) botanical extract solution added to
them to analyze the 10% botanical extract solution in triplicate.
Wells 4, 5, and 6 may then have 25 microliters of the 5% (v/v)
botanical extract solution added to them to analyze the 5%
botanical extract solution in triplicate, etc.
[0034] Step 4: A stock solution of the porcine pancreatic trypsin
is then prepared by adding the protease (15,200 units/milligram,
Sigma Chemical Company, St. Louis, Mo.) into 100 millimolar Tris
buffer, pH adjusted to 8.0 with HCl to yield a concentration of 4
micrograms of trypsin/milliliter. To each well is then added 25
microliters of the protease.
[0035] Step 5: The well plates are then incubated at room
temperature for 15 minutes.
[0036] Step 6: After incubation, 50 microliters of a 5 millimolar
solution of N-benzyl-arginine-p-nitroaniline (BAPNA, Sigma Chemical
Company, St. Louis, Mo.) is added to each of the wells. The BAPNA
substrate is prepared as a stock solution by preparing a 50
millimolar solution of BAPNA in dimethylsulfoxide and diluting the
solution to a 5 millimolar working solution with deionized water.
One skilled in the art will recognize that the final concentration
of botanical extract in the sample being tested is 1/10 of the
concentration of the botanical extract solution as prepared under
Step 1. For example, the 10% botanical extract solution prepared in
step 1 becomes a 1% concentration of botanical extract in the
well.
[0037] Step 7: After the BAPNA is added, the plate is inserted into
a SPECTRAmax PLUS Microplate Reader (Molecular Devices, Sunnyvale
Calif.), or similar instrument, and optical density measurements
(405 nanometers) are taken every 20 seconds for a period of 5
minutes to monitor the color change of the solution. When trypsin
cleaves the BAPNA substrate releasing the product p-nitroaniline, a
color change occurs. The amount of color change occurring at 405
nanometers per minute corresponds to the amount of product produced
per minute.
[0038] Reaction rates (optical density at 405 nanometers per
minute) are determined with each concentration of botanical extract
tested and the PBS control (no botanical extract). If the control
wells were prepared in duplicate or triplicate, for example, the
reaction rates from each of the wells were averaged. The data are
used to determine an IC.sub.50 value for each botanical extract
plotting trypsin activity (y-axis) versus botanical extract
concentration (percent w/v or v/v) (x-axis). IC.sub.50 is defined
as the concentration of the botanical extract that inhibits 50% of
the trypsin activity.
[0039] In accordance with the present invention, the following
botanical extracts have been found to have the desired protease
inhibitory properties and are suitable for use in an aqueous
solution for neutralizing dust mite feces as described herein:
Apple Green Tea, Arkin Special, Arnica Special, Avocado, Avocado
GW, Black Currant Green Tea, Cabbage Rose, Cat's Claw, Cemila
Oleifera, Centella, Cranberry Green Tea, Dandelion, Garcinia, Grape
Seed, Grapefruit Green Tea, Green Tea, Green Tea Concentrate, Green
Tea HS, Hexaplant Richter, Hibiscus Special, Hydrocotyle GR,
Lavender, Horse Chestnut, Milk Thistle, Orange Green Tea,
Phytexcell Arnica, Purple Coneflower, Sage GW, Sage Special,
Sedaplant Richter, St. John's Wort, Witchhazel GW, Yarrow, Green
Tea Extra, Grape Seed Extract and White Tea 50%. Preferred
botanical extracts include Green Tea Extra, Grape Seed Extract, and
White Tea 50%.
[0040] The soluble botanical extracts having sufficient protease
inhibitory activity described herein are introduced into water to
form an aqueous botanical extract solution which is subsequently
contacted with the dust mite feces to allow the botanical extract
to interact with and reduce the allergenicity of the dust mite
feces. The water source is not critical, and can be deionized
water, distilled water, tap water, and the like. Although not
required, it is preferred that the aqueous botanical extract
solutions as described herein be adequately preserved to
substantially prevent microbial contamination and thus improve the
overall quality of the solution. One skilled in the art will
recognize that there are a number of water soluble preservatives
commercially available which would be suitable for use with the
aqueous botanical extract solutions described herein.
[0041] The aqueous botanical extract solution can easily be
atomized into an area to be treated, such as a bedroom or other
room. As used herein, the term "atomized" means that the aqueous
botanical extract solution is reduced to a spray form from liquid
form, or volatilized. Atomization of the aqueous botanical extract
solutions of the present invention can be accomplished utilizing
various means including, for example, atomizers, aerosol sprayers,
mechanical sprayers, misters, humidifiers, foggers, fumigating
apparatuses, and the like. Both cold mist formulations and warm
mist formulations including the aqueous botanical extract solutions
are within the scope of the present invention. The aqueous
botanical solution can also contain a small amount of dyes to color
the product and/or a fragrance to impart a pleasing odor to the
solution. Both of these additives potentially enhance consumer
appeal of the product.
[0042] The precise method of volatilizing the aqueous botanical
extract solutions of the present invention to allow the botanical
extract to contact the dust mite feces is not critical so long as
the method employed can volatilize the aqueous solution
sufficiently. Because house dust mites are typically very small in
size, having a length of only about 250 microns to about 300
microns, it is preferable, although not critical, that the size of
the droplets produced by the atomizing means be of such a size that
they will sufficiently contact a majority of the dust mites and
feces present in an area. As such, it is preferred that at least
some of the atomized droplets of aqueous botanical extract solution
have a medium drop size of no more than 1000 micrometers, more
preferably no more than 500 micrometers, more preferably no more
than about 200 micrometers, and most preferably no more than about
100 micrometers or less. These median drop sizes will allow a
significant amount of the botanical extract to contact the dust
mite feces present. Alternatively, a pesticidal composition could
be introduced into the aqueous botanical extract solution and
atomized simultaneously.
[0043] In another embodiment of the present invention, the aqueous
botanical extract solutions described herein can be used in
combination with a pesticidal, acaricidal, or other suitable agent
which kills the dust mites upon application or treatment. This
combination of applications would serve to not only neutralize the
dust mite feces present, but also reduce the number of living dust
mites. For example, a pesticidal composition capable of killing
dust mites upon contact could first be sprayed or otherwise applied
to an area such as a bedroom, to kill dust mites. After the
pesticidal application, the aqueous botanical extracts of the
present invention could be atomized as described herein into the
area to neutralize the feces of the dust mites. Although the
aqueous botanical extract solutions described herein are highly
effective in neutralizing the dust mite feces when used alone, when
used in combination with an agent that kills dust mites, the
combination is also highly effective in controlling the amount of
dust mite allergens present.
[0044] The present invention is illustrated by the following
example which is merely for the purpose of illustration and is not
to be regarded as limiting the scope of the invention or manner in
which it may be practiced.
EXAMPLE 1
[0045] In this example, numerous botanical extracts, both in solid
(powder) form and in aqueous liquid form, were analyzed for serine
protease inhibitory activity using the BAPNA-Trypsin Inhibition
Test described herein. Although referred to throughout the Example
as "v/v," it should be realized that the concentration of the
botanical extract would be referred to a "w/v" if the botanical
extract was a solid. All of the botanical extracts tested were
either in solid or aqueous form, and all liquids were soluble at a
level of 10% by weight and all solids were essentially soluble at a
level of 1% by weight.
[0046] The botanical extracts, the name of the company where the
botanical extract was purchased, the location of the company, the
lot number (if available), the catalog number (if available) and
the IC.sub.50 results for the botanical extracts tested in this
Example are set forth in Table 1. Each liquid botanical extract
listed in Table 1 was prepared for analysis as follows: A 10% (v/v)
botanical extract solution in PBS (pH=7.4, Life Technologies,
Rockville, Md.) was prepared by introducing 100 microliters of
botanical extract into one milliliter of PBS. Serial dilutions of
the 10% botanical solution in PBS were then made to prepare the
following concentrations in addition to the 10% concentration; 5%,
2.5%, 1.25%, 0.63%, 0.31%, 0.16%, and 0.078%.
[0047] Each solid botanical extract listed in Table 1 was prepared
for analysis as follows: A 0.1% (w/v) botanical extract solution in
PBS (pH=7.4) was prepared by introducing 1 milligram of botanical
extract into one milliliter of PBS. Serial dilutions were prepared
from this solution for a total of eight testing solutions.
[0048] To the empty wells of a NUNC IMMUNO clear 96 well plate (VWR
Scientific Products, Chicago, Ill.) was added 150 microliters of a
100 millimolar Tris buffer (Sigma Chemical Company, St. Louis, Mo.)
with the pH adjusted to 8.0 utilizing HCl. Next, to allow for
testing in triplicate, each concentration of botanical solution was
added to three separate wells containing the Tris buffer. For
control purposes, three wells had 25 microliters of the PBS
solution introduced therein in place of any botanical extract
solution.
[0049] A stock solution of porcine pancreatic trypsin (15,200
units/milligram, Sigma Chemical Company, St. Louis, Mo.) was then
prepared by adding the porcine pancreatic trypsin into 100
millimolar Tris buffer, pH adjusted to 8.0 with HCL to yield a
concentration of 4 micrograms/milliliter. To each well of the plate
was then added 25 microliters of the 4 micrograms/milliliter
trypsin.
[0050] After the porcine pancreatic trypsin was added to the wells
of the plate, the well plates were incubated at room temperature
for 15 minutes.
[0051] After incubation, 50 microliters of a 5 millimolar solution
of N-benzyl-arginine-p-nitroaniline (BAPNA) was added to each of
the wells on the plate. The BAPNA substrate was prepared as a stock
solution by preparing a 50 millimolar solution of BAPNA in
dimethylsulfoxide and diluting the solution to a 5 millimolar
working solution with deionized water. This resulted in a total
concentration of botanical extracts being tested from a working
stock of 10% (v/v) botanical solution of (1) 1%; (2) 0.5%; (3)
0.25%; (4) 0.125%; (5) 0.063%; (6) 0.031%; (7) 0.016%; and (8)
0.0078%.
[0052] Immediately after the BAPNA was added, the plate was
inserted into a SPECTRAmax PLUS 384 Microplate Reader (Molecular
Devices, Sunnyvale, Calif.), and the optical density of each well
was measured at 405 nanometers every 20 seconds for 5 minutes.
[0053] After the optical density data was collected, an IC.sub.50
value was determined as set forth above for each botanical extract.
The results are set forth in Table 1. TABLE-US-00001 TABLE 1 Lot
Number/ IC.sub.50 (.times.10.sup.-3 Botanical Company Location
Catalog Number (% v/v or w/v)) Aloe Gel Tri-K Northvale, 970217/NA
1000 (no Industries N.J. effect) Apple Gattefosse Cedex, 23152 1000
(no Extract France effect) Apple Green Dragoco Totowa, N.J.
2/037050/L742477 11.5 Tea Arkin Dragoco Totowa, N.J.
2/032581/L647147 44.5 Special Arnica Dragoco Totowa, N.J.
2/034591/L641060 39 Special Avocado Dragoco Totowa, N.J.
2/034599/L645246 495 Avocado GW Dragoco Totowa, N.J.
2/031170/L603922 19 Black Dragoco Totowa, N.J. 2/036100/P331166
1000 (no Currant B effect) Black Dragoco Totowa, N.J.
2/037100/P331166 12.5 Currant Green Tea Cornflower Gattefosse
Cedex, 23593/5009 1000 (no France effect) Cabbage Gattefosse Cedex,
22223 14.5 Rose France Extract. Calendula Gattefosse Cedex,
24243/5015 1000 (no MCF 774 France effect) Hydro Cat's Claw
Bio-botanica Hauppauge, N.Y. 951341/9945A 16.5 Cemila Gattefosse
Cedex, 22423 4.5 Oleifera France Extract Centella Bio-botanica
Hauppauge, N.Y. 981177/9869A 176 Chamomile Bio-botanica Happauge,
980572/9831 1000 (no N.Y. effect) Chamomile Dragoco Totowa, N.J.
2/033021/694633 1000 (no Special effect) Chlorella Bio-botanica
Hauppauge, 951289/9835 1000 (no N.Y. effect) Concombre Gattefosse
Cedex, 19190 1000 (no GR 316 France effect) Cranberry B Dragoco
Totowa, N.J. 2/036600/P15193 1000 (no effect) Cranberry Dragoco
Totowa, N.J. 2/037600/4100723 6.5 Green Tea Dandelion Active
Lewisville, S72041A/316310-11 88.5 organics- Tx. Glenn Corp Dong
Quai Active Lewisville, S64418B/316320-11 1000 (no organics- Tx.
effect) Glenn Corp Drago-Oat- Dragoco Totowa, N.J. 2/060900/25493
1000 (no Active effect) Garcinia Bio-botanica Happauge, 951283/9861
400 N.Y. Ginseng GR Gattefosse Cedex, 23268/5030 1000 (no 471 Hydro
France effect) Glenn of Glenn Corp. St. Paul, 715 Oak Mn. Glenn of
Glenn Corp. St. Paul, 1000 (no Orange Mn. effect) Gotu Kola
Bio-botanica Happauge, 1000 (no PG 5:1 N.Y. effect) Grape
Gattefosse Cedex, 22151 1000 (no Extract France effect) Grape Seed
Active Lewisville, S76920B/318560-11 39 organics- Tx. Glenn Corp
Grape Seed Dragoco Totowa, N.J. 2/03199/P17400 0.048 Extract
Grapefruit Gattefosse Cedex, 23439 1000 (no France effect)
Grapefruit Dragoco Totowa, N.J. 2/037150/L4100211 12.5 Green Tea
Green Tea Bio-botanica Happauge, /9945 6 N.Y. Green Tea Active
Lewisville, 308463/300230-94 140 Conc. organics- Tx. Glenn Corp
Green Tea Dragoco Totowa, N.J. 2/031598/3066 0.15 Extra Green Tea
Alban Muller Northvale, 7114309/ 15.6 HS Intl N.J. Hexaplant
Chemisches Berlin, 732431/243 29.5 Richter Lab. Germany Hibiscus
Dragoco Totowa, N.J. 2/033115/L651028 91.5 Special Hydrocotyl
Gattefosse Cedex, 22842/5038 59 GR France Hydrolite-5 Dragoco
Totowa, N.J. 2/016020/27033 1000 (no effect) Kiwi Gattefosse Cedex,
23311 1000 (no France effect) White Gattefosse Cedex, 22571/5040
1000 (no Nettle France effect) Lavender Gattefosse Cedex, 21189 20
France Lemon Gattefosse Cedex, 24126 1000 (no Extract France
effect) Lily Gattefosse Cedex, 23410/5044 1000 (no France effect)
Horse Gattefosse Cedex, 22043/5046 36 Chestnut France Horse Indena-
Uppersaddle EG042 12.5 Chestnut International River, N.J. Sourcing
German Indena- Uppersaddle EG004 1000 (no Chamomile International
River, N.J. effect) Sourcing Matricaria Gattefosse Cedex, 21747
1000 (no Extract France effect) Sweet Gattefosse Cedex, 23316/5051
1000 (no Clover France effect) Milk Active Lewisville,
S76894A/344000-11 215 Thistle organics- Tx. Glenn Corp Nab Willow
Brooks S. 28392 1000 (no Bark Industries Plainfield, effect)
Extract N.J. Orange Dragoco Totowa, N.J. 2/037400/P327911 9.5 Green
Tea Phytexcell Croda Parsippany, 972/34656 245 Arnica N.J.
Phytexcell Croda Parsippany, 1004/34684 1000 (no Mulberry N.J.
effect) Phytoplenolin Bio-botanica Happauge, 980510/9870 760 N.Y.
Purple Bio-botanica Happauge, 951338/9852 21 Coneflower N.Y.
Raspberry Gattefosse Cedex, 23204 1000 (no France effect) Sage CL
Dragoco Totowa, N.J. 2/033294/L640225 1000 (no effect) Sage GW
Dragoco Totowa, N.J. 2/031770/L619604 20 Sage Dragoco Totowa, N.J.
2/033291/P312506 22 Special Sedaplant Chemisches Berlin, 732384/460
20 Richter Lab. Germany St. John's Dragoco Totowa, N.J.
2/032985/L658926 27 Wort W/S White Dragoco Totowa, N.J.
2/033141/L653324 1000 (no Mistle Toe effect) White Tea Dragoco
Totowa, N.J. 10521/C-14235 0.098 50% Witchhazel Dragoco Totowa,
N.J. 2/031340/L651033 99 GW Yarrow Bio-botanica Happauge,
951336/9958 80 N.Y.
[0054] As the data in Table 1 indicates, IC.sub.50 values
determined for the botanical extracts tested ranged from 0.048 to
1000 (no inhibition detected). This would indicate that the
botanical extract with an IC.sub.50 value of 0.048 (Grape Seed
Extract) is highly effective in neutralizing dust mite feces
whereas the botanical extract with a value of 1000 has little to no
effect.
[0055] In view of the above, it will be seen that the several
objects of the invention are achieved. As various changes could be
made in the above-described methods and compositions for
neutralizing dust mite feces without departing from the scope of
the invention, it is intended that all matter contained in the
above description be interpreted as illustrative and not in a
limiting sense.
* * * * *