U.S. patent application number 11/991696 was filed with the patent office on 2009-12-31 for composition of whey growth factor extract for reducing muscle inflammation.
This patent application is currently assigned to Murray Goulburn Co-Opeartive Co Limited. Invention is credited to David Cameron-Smith, Michelle Rowney.
Application Number | 20090324733 11/991696 |
Document ID | / |
Family ID | 37835315 |
Filed Date | 2009-12-31 |
United States Patent
Application |
20090324733 |
Kind Code |
A1 |
Rowney; Michelle ; et
al. |
December 31, 2009 |
Composition of Whey Growth Factor Extract for Reducing Muscle
Inflammation
Abstract
The invention relates to the production of compositions
containing milk products for use as a nutritional supplement. More
specifically, it relates to compositions containing whey growth
factor extract, as well as their use in methods for reducing muscle
inflammation in individuals undertaking resistance exercise
training. According to one aspect of the invention, there is
provided the use of a composition comprising whey growth factor
extract, isolated from a milk product by cation exchange
chromatography, to reduce exercise-induced muscle inflammation.
Inventors: |
Rowney; Michelle; (Port
Campbell, AU) ; Cameron-Smith; David; (Ashburton,
AU) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP;FLOOR 30, SUITE 3000
ONE POST OFFICE SQUARE
BOSTON
MA
02109
US
|
Assignee: |
Murray Goulburn Co-Opeartive Co
Limited
Brunswick
AU
|
Family ID: |
37835315 |
Appl. No.: |
11/991696 |
Filed: |
September 8, 2006 |
PCT Filed: |
September 8, 2006 |
PCT NO: |
PCT/AU2006/001323 |
371 Date: |
September 2, 2009 |
Current U.S.
Class: |
424/535 ;
514/1.1 |
Current CPC
Class: |
A23L 33/19 20160801;
A61K 38/1709 20130101; A61K 35/20 20130101; A23J 1/205 20130101;
A23V 2002/00 20130101; A61P 21/00 20180101; A23L 33/40 20160801;
A23C 21/06 20130101; A61P 29/00 20180101; A23V 2300/30 20130101;
A23V 2002/00 20130101; A23V 2200/316 20130101; A23V 2250/54252
20130101 |
Class at
Publication: |
424/535 ;
514/12 |
International
Class: |
A61K 35/20 20060101
A61K035/20; A61K 38/16 20060101 A61K038/16 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 9, 2005 |
AU |
2005904980 |
Claims
1. A muscle anti-inflammatory composition comprising whey growth
factor extract, isolated from a milk product by cation exchange
chromatography.
2. A muscle anti-inflammatory composition comprising whey growth
factor extract, isolated from a milk product by a process
comprising the steps; a) applying the milk product to a SP
Sepharose cation exchange column, b) washing the column with a
buffer of low ionic strength, c) eluting the WGFE fraction with a
buffer containing in the range 0.4-0.5M NaCl, or equivalent ionic
strength, at pH 6.5.
3. A muscle anti-inflammatory composition comprising whey growth
factor extract, isolated from skim milk by a process comprising the
steps; a) applying the skim milk to a SP Sepharose cation exchange
column, b) washing the column with a buffer of less than 0.008M
NaCl, c) eluting the WGFE fraction with a buffer containing 0.4M
NaCl, at pH 6.5.
4. The muscle anti-inflammatory composition as claimed in any one
of claims 1 to 3, further comprising an additional protein
source.
5. The composition of claim 4, wherein the additional protein
source is whey protein.
6. The composition of claim 5, wherein the whey protein is whey
protein isolate (WPI).
7. The composition of claim 6, wherein the whey protein isolate
comprises: TABLE-US-00005 Moisture 5.0% Fat 0.5% pH (5% solution)
6.3 Ash 3.7% Lactose 0.5% Protein (TN .times. 6.38) 90.0% Sodium
0.7% Phosphorous 0.3% Calcium 0.15%
8. The composition of any one of claims 1 to 3, wherein the whey
growth factor extract is isolated from a source selected from the
group consisting of cheese whey, rennet casein whey, acid casein
whey and skim milk.
9. The composition of claim 8, wherein the whey growth factor
extract is isolated from skim milk.
10. A composition according to any one of claims 1 to 3, when used
as a muscle anti-inflammatory agent.
11. A method of reducing muscle inflammation in a subject
undertaking resistance exercise training comprising administering
to the subject a composition comprising an effective amount of the
muscle strength-enhancing composition of any one of claims 1 to
3.
12. The method of claim 11 wherein the composition is administered
in a daily dose of at least 5 mg/kg body weight to 12.5 mg/kg body
weight whey growth factor extract.
13. The method of claim 11, wherein the composition comprises an
additional protein source.
14. A method of reducing muscle inflammation in a subject
undertaking resistance exercise training comprising administering
to the subject a composition comprising an effective amount of whey
growth factor extract, isolated from a milk product by cation
exchange chromatography, and an additional protein source taken
separately.
15. The method of claim 13 or 14, wherein the composition is
administered in a daily dose of at least 5 mg/kg body weight to
12.5 mg/kg body weight whey growth factor extract, and at least 225
mg/kg body weight of additional protein source.
16. The method of claim 15 wherein the additional protein source is
whey protein.
17. The method of claim 14, wherein the administration to the
subject is once per two or three days up to at least once per
day.
18. The method of claim 14, wherein the administration to the
subject is before, and/or immediately after, resistance exercise
training.
19. The method of claim 14, wherein the administration is
immediately after resistance exercise training.
20. The method of claim 18, wherein the administration is between
20 minutes and two hours after exercise.
21. A method of treating resistance exercise induced muscle
inflammation in a subject, the method comprising administering to
the subject whey growth factor extract, isolated from a milk
product by cation exchange chromatography.
22. A method of treating resistance exercise induced muscle
inflammation in a subject, the method comprising administering to
the subject whey growth factor extract, isolated from a milk
product by a process according to claim 2 or 3.
23. A method of treating resistance exercise induced muscle
inflammation in a subject, the method comprising administering to
the subject the composition of any one of claims 1 to 3.
24. A food, drink, tablet or capsule comprising the composition of
any one of claims 1 to 3.
25. The food of claim 24, being in the form of a nutritional bar or
snack food.
26. A method of manufacturing a food, drink, tablet or capsule
reduce muscle inflammation in a subject undertaking resistance
exercise training the method comprising manufacturing the
composition of any one of claims 1 to 3.
27-29. (canceled)
Description
FIELD OF THE INVENTION
[0001] The invention relates to the production of compositions
containing milk products for use as a nutritional supplement. More
specifically, it relates to compositions containing whey growth
factor extract, as well as their use in methods for reducing muscle
inflammation in individuals undertaking resistance exercise
training.
BACKGROUND OF THE INVENTION
[0002] The present invention is to be understood in light of what
has previously been done in the field. However, the following
discussion is not an acknowledgement or admission that any of the
material referred to was published, used or part of the common
general knowledge in Australia as at the priority date of the
application.
[0003] The use of nutritional supplements by humans, and even
animals, to improve general health or to improve, for example,
athletic performance, is known. Nutritional supplements are not
intended to provide all the nutrients necessary for a complete
diet, but instead are generally intended to complement the dietary
intake such that it becomes more nutritionally complete. It is
recognised that vitamins, minerals and other substances found in
such supplements play important physiological roles and that a
deficiency of certain vitamins, minerals and/or other components of
supplements has been linked to development of certain diseases, a
decrease in general health or lower performance in athletes.
[0004] Conversely, nutritional supplements are known to enhance a
variety of physiological states, under various conditions. There
are many targets for nutritional supplements, for example sick
patients, convalescing patients, elderly persons and persons
undergoing strenuous exercise regimes who wish to improve their
performance and/or recovery from such exercise.
[0005] The nutritional requirements of bodybuilders and persons
engaged in strenuous physical exercise are quite particular,
whether to decrease body fat and increase lean muscle mass, and or
to improve recovery from the strenuous exercise. Recovery includes
the ability to overcome exercise-induced inflammation.
[0006] Inflammation is a localised response to injury or
destruction of tissues. It is characterised in the acute form by
pain, heat, redness, swelling, and loss of function. Accordingly,
reduced muscle inflammation includes, but is not limited to a
reduction in any one or more of the aforementioned symptoms in
muscle. The inflammatory response antagonises muscle protein
synthesis and contributes to much of the physical discomfort
experienced by persons 1 to 2 days after engaging in strenuous
exercise, including resistance exercise training (Macintyre D L, et
al., Sports Med. 1995; 20(1):24-40; MacIntyre D L, et al., Eur J
Appl Physiol. 2001; 84(3):180-6). If the inflammation is severe,
anti-inflammatory drugs such as corticosteroids may be prescribed.
However, the use of such drugs can have deleterious side-effects
and, in many cases, is prohibited for athletes in competition.
[0007] Weight-bearing or resistance training exercise elicits a
wide-range of responses in the activated muscle. There are anabolic
signalling pathways activated by the stretch and strain of muscles
and by growth factors which may be synthesised locally or circulate
in the blood stream.
[0008] Modulation of the expression of various genes is an
essential process in regulating the sequence of cellular events
needed to activate stem cells that are located within the muscle
bed (satellite cells) (Anderson & Wozniak, Can J Physiol
Pharmacol. 2004; 82(5):300-10) to rapidly proliferate, before
maturing and ultimately fusing with existing muscle fibres or
joining together to create new muscle fibres. Regulators of the
activation of satellite cells include Syndecan-3 (a transmembrane
heparin sulphate proteoglycan essential for satellite cell
proliferation), and Pax-7 (a protein that is essential for
satellite cell activation and necessary for muscle tissue repair)
(Seale et al., Dev Biol. 2004; 275(2):287-300; Cornelison et al.,
Dev Biol. 2001; 239(1):79-94). Other genes involved in the
exercise-induced muscle repair and inflammation process include the
small molecular weight chemokines (CCL2 and CCL4) and the early
response genes, jun-B and c-fos.
[0009] If a natural food product, such as a product derived from
milk, could be obtained having beneficial effects on exercise
recovery it would be a readily available and safe therapeutic
supplement.
[0010] It is known in the prior art to produce a milk product from
cows maintained in a specific hyperimmune state, created by
administering periodic booster immunisations with sufficiently high
doses of antigens, which can be used to treat various diseases
including arthritis, dermatitis and neoplastic diseases (EP 0
064103 A1, Beck LR). Normal cow's milk however does not contain the
specific `anti-inflammatory factor` present in milk produced from
the aforementioned hyper-immunised animals, although it is believed
to contain low levels of other anti-inflammatory factors. A person
of skill in the art would appreciate that production of the
anti-inflammatory milk products according to EP 0 064103 A1 is very
expensive and time consuming.
[0011] It is also known in the prior art to produce
anti-inflammatory milk products comprising colostrum which is rich
in immunoglobulins. Colostrum is the pre-milk produced immediately
after birth before the breast secretions stabilize into milk. Prime
colostrum from cows is obtained within the first six hours after
calving and contains four times the protein found in milk from the
same cow obtained 48 hours later. The immunoglobulin fraction of
colostrum is loaded with antibodies, lactoferrin and immune
enhancers. Lactoferrin is thought to enhance the anti-inflammatory
effect of colostrum, and is the subject of U.S. Pat. No. 6,475,511
(Gohlke MB). Immunoglobulins are thought to act locally on immune
function in the gut.
[0012] Protein supplementation has been used widely by persons
undertaking resistance exercise training to promote muscle protein
synthesis in order to repair muscle tissue and facilitate muscle
growth. The nutritional supplementation may be provided in the form
of a drink or food and includes protein powders to be mixed with
liquid for use, nutritional bars and snack foods, tablets, capsules
and other preparations. Suitable protein sources commercially
available include hydrolysed milk proteins, caseinates, soy protein
isolates and milk protein concentrates prepared from
ultra-filtrated skim milk. Nutritional supplements which are based
on other protein sources, such as whey protein, are also available
and can be provided in the form of fruit juices, but are thought to
be inadequate because they do not also provide a lipid source (WO
02/15720). In addition, it has been considered that some
milk-derived proteins are not readily absorbed by the gut, or do
not survive the harsh environment of the digestive system to have a
therapeutic effect.
[0013] Whey growth factor extract (WGFE) is one such milk product
which was thought to be susceptible to at least partial loss of
biological activity once ingested due to fragmentation of molecules
such as growth factors. Moreover, by virtue of the process for its
isolation, WGFE contains only low levels of immunoglobulins which,
as described above, are thought to be important anti-inflammatory
factors.
[0014] Nevertheless, the capacity of whey protein supplementation
to provide a benefit to persons engaged in resistance exercise
training has been reported to provide a benefit. Whey protein
isolate (WPI) and milk protein isolate (MPI), each having a
different composition to whey growth factor extract, have been
reported to be effective with bodybuilders in rapidly gaining lean
muscle mass while reducing body fat. WPI is high in branched-chain
amino acids and considered to be fast-acting, whereas MPI is mainly
casein which is more slowly metabolised and is effective in
promoting muscle growth. The use of WPI, or whey protein
concentrate, in combination with the amino acids glutamine,
leucine, isoleucine and valine to improve muscular fatigue in a rat
model is the subject of WO 2004/049830 A1 (Tsuchita H et al.). The
authors measured Tyrosine release from soleus muscle as the
indicator of fatigue.
[0015] The present inventors have found that a composition
comprising whey growth factor extract altered the expression of
genes involved in exercise-induced muscle inflammation. In a
separate study conducted by the inventors, trained athletes
administered whey growth factor extract reported a reduction in
post-exercise soreness. Importantly, in terms of the invention,
whey growth factor extract may be isolated from normal bulk milk
products without a requirement to maintain cows in a hyperimmune
state or to harvest colostrum which is in limited supply relative
to other milk products.
SUMMARY OF THE INVENTION
[0016] The invention relates to a WGFE composition and method of
using the composition which enables persons undergoing resistance
exercise training to reduce their level of muscle inflammation over
that obtained with compositions of the prior art.
[0017] It is thus an object of the present invention to provide a
composition and method of using same which is improved/more
efficacious than compositions of the prior art for reducing muscle
inflammation.
[0018] According to one aspect of the invention, there is provided
a skeletal muscle inflammation-reducing composition comprising whey
growth factor extract, isolated from a milk product by cation
exchange chromatography.
[0019] In a further aspect of the invention, there is provided a
muscle anti-inflammatory composition comprising whey growth factor
extract, isolated from a milk product by a process comprising the
steps;
[0020] a) applying the milk product to a SP Sepharose cation
exchange
[0021] column,
[0022] b) washing the column with a buffer of low ionic
strength,
[0023] c) eluting the WGFE fraction with a buffer containing in the
range 0.4-0.5M NaCl, or equivalent ionic strength, at pH 6.5.
[0024] According to another aspect of the invention, there is
provided a muscle anti-inflammatory composition comprising whey
growth factor extract, isolated from a milk product by a process
comprising the steps;
[0025] a) applying the milk product to a SP Sepharose cation
exchange
[0026] column,
[0027] b) washing the column with a buffer of 0.008M NaCl or
less,
[0028] c) eluting the WGFE fraction with a buffer containing 0.4M
NaCl, or equivalent ionic strength, at pH 6.5.
[0029] In a further aspect of the invention, there is provided a
composition according to the above wherein the whey growth factor
extract is isolated from a milk product selected from whole milk,
cheese whey, rennet casein whey, acid casein whey, or concentrates
thereof, or skim milk.
[0030] In a further aspect of the invention, there is provided a
composition according to the above when used as a muscle
anti-inflammatory agent.
[0031] In a further aspect of the invention there is provided a
method of reducing skeletal muscle inflammation in subjects
undertaking resistance exercise training comprising administering
to the subjects an efficacious amount of a composition comprising
whey growth factor extract.
[0032] In a further aspect of the invention there is provided a
method of reducing skeletal muscle inflammation in subjects
undertaking resistance exercise training comprising administering
to the subjects an efficacious amount of a composition comprising
whey growth factor extract, isolated from a milk product by a
process comprising the steps;
[0033] a) applying the milk product to a SP Sepharose cation
exchange column,
[0034] b) washing the column with a buffer of low ionic
strength,
[0035] c) eluting the WGFE fraction with a buffer containing in the
range 0.4-0.5M NaCl, or equivalent ionic strength, at pH 6.5.
[0036] In a further aspect of the invention there is provided a
method of reducing skeletal muscle inflammation in subjects
undertaking resistance exercise training comprising administering
to the subjects an efficacious amount of a composition comprising
whey growth factor extract, isolated from a milk product by a
process comprising the steps;
[0037] a) applying the milk product to a SP Sepharose cation
exchange column,
[0038] b) washing the column with a buffer of 0.008M NaCl or
less,
[0039] c) eluting the WGFE fraction with a buffer containing 0.4M
NaCl, or equivalent ionic strength, at pH 6.5.
[0040] In another aspect of the invention there is provided a
method of reducing skeletal muscle inflammation in subjects
undertaking resistance exercise training wherein the amount of whey
growth factor extract administered, per daily dose, is at least 5
mg/kg body weight to 12.5 mg/kg body weight. Preferably the daily
dose of whey growth factor extract is at least 25 mg/kg body
weight.
[0041] In a further aspect of the invention there is provided a use
of whey growth factor extract, isolated from a milk product by
cation exchange chromatography, for the production of a medicament
for the treatment of a subject in need of reduced skeletal muscle
inflammation.
[0042] In a further aspect of the invention there is provided a use
of whey growth factor extract, isolated from a milk product by a
process described above, for the production of a medicament for the
treatment of a subject in need of reduced skeletal muscle
inflammation.
[0043] According to another aspect of the invention, there is
provided a skeletal muscle inflammation-reducing composition
comprising whey growth factor extract, isolated from a milk product
by cation exchange chromatography, and an additional protein
source.
[0044] According to a further aspect of the invention the
additional protein source is whey protein, preferably whey protein
isolate (WPI), more preferably whey protein isolate wherein the
whey protein isolate comprises:
TABLE-US-00001 Moisture 5.0% Fat 0.5% pH (5% solution) 6.3 Ash 3.7%
Lactose 0.5% Protein (TN .times. 6.38) 90.0% Sodium 0.7%
Phosphorous 0.3% Calcium 0.15%
[0045] In a further aspect of the invention there is provided a
method of reducing skeletal muscle inflammation in a subject
undertaking resistance exercise training comprising administering
to the subject an efficacious amount of a composition comprising
whey growth factor extract and an additional protein source, such
as whey, preferably WPI.
[0046] In a further aspect of the invention there is provided a
method of reducing muscle inflammation in a subject undertaking
resistance exercise training comprising administering to the
subject a composition comprising an effective amount of whey growth
factor extract, isolated from a milk product by cation exchange
chromatography, and an additional protein source taken
separately.
[0047] Preferably the amount of additional protein source
administered, per daily dose, is at least 225 mg/kg body weight
(dry weight) and preferably at least 435 mg/kg body weight (dry
weight).
[0048] In a further aspect of the invention the administration of
the composition of the invention is once per two or three days up
to at least once per day, preferably before and/or immediately
after resistance exercise training, more preferably immediately
after resistance exercise training, most preferably between 20
minutes and two hours after exercise.
[0049] In a further aspect of the invention there is provided a use
of whey growth factor extract, isolated from a milk product by
cation exchange chromatography, and an additional protein source
for the production of a medicament for the treatment of a subject
in need of reduced skeletal muscle inflammation.
[0050] In a further aspect of the invention, there is provided a
food or drink comprising the composition of the invention for use
in a method to reduce skeletal muscle inflammation in a subject
undertaking resistance exercise training.
[0051] In yet a further aspect of the invention there is provided a
use of the composition of the invention for the manufacture of a
food or drink to reduce skeletal muscle inflammation in a subject
undertaking resistance exercise training.
DESCRIPTION OF THE DRAWINGS
[0052] FIG. 1: Fold change in Myo D, myogenin, and myostatin gene
(mRNA) expression in the vastus lateralis muscle of adult subjects,
at rest and following 3 hours of resistance exercise training
performed at the start of the trial [pre-training] and following 12
weeks of treatment [post-training] with 20 g WPI (Group A), 1 g
WGFE+20 g WPI (Group B1), or 2 g WGFE+20 g WPI (Group B2). Results
are presented as mean.+-.SEM.
[0053] FIG. 2: Fold change in Pax 7 and Syndecan 3 gene (mRNA)
expression in the vastus lateralis muscle of adult subjects, at
rest and following 3 hours of resistance exercise training
performed at the start of the trial [pre-training] and following 12
weeks of treatment [post-training] with 20 g WPI (Group A),1 g
WGFE+20 g WPI (Group B1), or 2 g WGFE+20 g WPI (Group B2). Results
are presented as mean.+-.SEM.
[0054] FIG. 3: Percentage of Pax 7 positive nuclei in tissue
sections from the vastus lateralis muscle of adult subjects,
following 3 hours of resistance exercise training performed at the
start of the trial [pre-training] and following 12 weeks of
treatment [post-training] with 20 g WPI (Group A), 1 g WGFE+20 g
WPI (Group B1), or 2 g WGFE+20 g WPI (Group B2) as analysed by
immunohistochemical staining.
[0055] FIG. 4: Fold change in IGF-1 Binding Protein 4 and 5 gene
(mRNA) expression in the vastus lateralis muscle of adult subjects,
at rest and following 3 hours of resistance exercise training
performed at the start of the trial [pre-training] and following 12
weeks of treatment [post-training] with 20 g WPI (Group A), 1 g
WGFE+20 g WPI (Group B1), or 2 g WGFE+20 g WPI (Group B2). Results
are presented as mean.+-.SEM.
[0056] FIG. 5: Fold change in Jun B and c-fos gene (mRNA)
expression in the vastus lateralis muscle of adult subjects, at
rest and following 3 hours of resistance exercise training
performed at the start of the trial [pre-training] and following 12
weeks of treatment [post-training] with 20 g WPI (Group A), 1 g
WGFE+20 g WPI (Group B1), or 2 g WGFE+20 g WPI (Group B2). Results
are presented as mean.+-.SEM.
[0057] FIG. 6: Fold change in CCL2, 4 and 7 gene (mRNA) expression
in the vastus lateralis muscle of adult subjects, at rest and
following 3 hours of resistance exercise training performed at the
start of the trial [pre-training] and following 12 weeks of
treatment [post-training] with 20 g WPI (Group A), 1 g WGFE+20 g
WPI (Group B1), or 2 g WGFE+20 g WPI (Group B2). Results are
presented as mean.+-.SEM.
[0058] FIG. 7: TNF-alpha production (ng/ml) by RAW cells exposed to
lipopolysaccharide (LPS) and cultured in the presence of colostrum
(sample 2BC), WPI (sample 4BC) at 100 mg/ml, WGFE (sample 6BC) at
100 mg/ml, various milk fractions, or LPS alone (control). WGFE and
LPS were also added to the culture simultaneously (sample
WGFE+LPS). The values plotted represent the arithmetic mean.+-.SEM
of the average response obtained in 3 independent experiments.
[0059] FIG. 8: Comparison of TNF-alpha production (ng/ml) by RAW
cells exposed to lipopolysaccharide (LPS) and cultured in the
presence of 100 mg/ml WPI or 100 mg/ml WGFE.
DETAILED DESCRIPTION OF THE INVENTION
[0060] The invention relates to a composition containing WGFE and
uses thereof to enable a subject undergoing resistance exercise
training to reduce their level of muscle inflammation over that
obtained with compositions of the prior art.
[0061] According to one aspect of the invention, there is provided
a skeletal muscle inflammation-reducing composition comprising whey
growth factor extract, isolated from a milk product by cation
exchange chromatography.
[0062] The whey growth factor extract for use in the invention may
be isolated from milk, skim milk, milk derivatives, whey,
colostrum, and colostrum derivatives by, for example, the method
described in Australian Patent No. 645589 (PCT/AU91/00303) which is
incorporated herein by reference. This method essentially relies on
strong cation exchange chromatography to selectively extract basic
proteins from the starting material to constitute whey growth
factor extract.
Process for Producing WGFE Fraction
[0063] A preferred method of producing WGFE for use in the
invention is to use a column packed with SP (sulphopropyl)
Sepharose To the column a flow of a dairy product, preferably
skimmed milk, is applied until the volume of milk applied is up to
1000 times the volume of the resin packed into the column. The milk
remaining in the column is removed with a buffer of low ionic
strength (<0.008M NaCl or equivalent) for 10 min. The WGFE
fraction is eluted from the column with a buffer containing sodium
ions equivalent to 0.4-0.5M NaCl (though other cations would be
suitable), most preferably 0.4M NaCl.
[0064] The mobile phase may have a pH within a broad range, such as
4.5-9.0, preferably 5.5-7.5, most preferably about 6.5. At the
upper and lower limits both protein stability and the ability of
proteins to bind to the cation exchange resin become influenced. A
pH in the range 5.5-7.5 provides the highest WGFE yields.
[0065] The type of cation exchange resin suitable for adsorption of
the WGFE components may include resins such as Sepharose cation
exchange resin beads. For example, SP Sepharose Big Beads and CM
Sepharose beads (products of GE Healthcare) which contain
sulfopropyl functional groups and carboxymethyl groups,
respectively, are suitable. The size of the cation exchange resin
beads is preferably in the range from 45-300 .mu.m. Both SP
Sepharose beads in the range 45-165 .mu.m and in the range 100-300
.mu.m are suitable for WGFE purification according to the
invention.
[0066] One of the further treatments to which the WGFE fraction can
be subjected is desalting by, for example, dialysis or
ultrafiltration.
[0067] Accordingly, in a further aspect of the invention, there is
provided a composition according to the above wherein the whey
growth factor extract is isolated from whey or skim milk. The whey
used as starting material may be cheese whey, rennet casein whey,
acid casein whey, or concentrates thereof. The amounts of whey
growth factor extract and protein source to use according to the
invention are to be sufficient for reduced muscle inflammation or
to have a therapeutic effect.
[0068] In another aspect of the invention there is provided a
dosage regime wherein the amount of whey growth factor extract
administered, per daily dose, is at least 5 mg/kg body weight to
12.5 mg/kg body weight. Preferably the daily dose of whey growth
factor extract is at least 25 mg/kg body weight.
[0069] In a further aspect of the invention there is provided a
method of reducing skeletal muscle inflammation in subjects
undertaking resistance exercise training comprising administering
to the subjects an efficacious amount of a composition comprising
whey growth factor extract.
[0070] When the composition includes an additional protein source,
which may be any protein source suitable for consumption such as
WPI, post-exercise inflammation is reduced compared to subjects
administered a protein source alone. The protein source may be
obtained from whole milk, preferably whey protein and more
preferably whey protein isolate (WPI). One such whey protein
isolate is available commercially under the trade name NatraPro.TM.
by Murray Goulburn Co-Op Company Ltd. A typical composition of
NatraPro.TM. WPI includes:
TABLE-US-00002 Moisture 5.0% Fat 0.5% pH (5% solution) 6.3 Ash 3.7%
Lactose 0.5% Protein (TN .times. 6.38) 90.0% Sodium 0.7%
Phosphorous 0.3% Calcium 0.15%
[0071] Preferably the composition according to the invention also
contains a protein source and, in a dosage regime, the amount of
additional protein source per daily dose administered is at least
225 mg/kg body weight (dry weight) and preferably at least 435
mg/kg body weight (dry weight). The additional protein source could
be formulated with the WGFE composition or formulated to be
administered separately to the WGFE composition.
[0072] In a further aspect of the invention there is provided a use
of whey growth factor extract for the production of a medicament
for the treatment of a subject in need of reduced skeletal muscle
inflammation. For example, athletes may require resistance exercise
training to build up their muscle strength to enhance their
performance, or to overcome a debilitating injury which has caused
muscle wasting. The muscle inflammation may be caused by, for
example, a `corked` thigh, or a torn muscle. A medicament
comprising the composition of the invention may further assist in
the subject recovering more rapidly to return to their training
regime and/or to compete. Muscle inflammation may also occur in
those with a propensity for bruising, such as the elderly.
[0073] In a further aspect of the invention there is provided a
method of reducing skeletal muscle inflammation in subjects
undertaking resistance exercise training comprising administering
to the subjects an efficacious amount of a composition comprising
whey growth factor extract and an additional protein source.
[0074] It will be apparent to those skilled in the art that the
administration of the composition of the invention may be on the
days of training or on the days of training and/or other days,
providing the regimen of administration results in reduced skeletal
muscle inflammation. Preferably administration is on the days of
exercise and more preferably administration occurs either just
before and/or immediately after the exercise. More preferably,
administration occurs between 20 minutes and 2 hours after
exercise. Accordingly, in a preferred aspect of the invention,
there is provided a method as described above wherein the
administration is immediately after the exercise. Subjects
administered 1 g WGFE plus 20 g WPI, or 2 g WGFE plus 20 g WPI
immediately after resistance exercise training showed increased
modulation of the expression of genes involved in inflammation
compared to subjects administered WPI alone (see Examples and
Results). In addition, subjects administered WGFE reported reduced
post-exercise soreness compared to those administered WPI,
supporting a role for WGFE in reducing post-exercise muscle
inflammation.
[0075] Moreover, the composition of the invention may be used to
reduce muscle inflammation in non-human mammals such as horses,
greyhounds and others in which reduced muscle inflammation is
desirable.
[0076] In a further aspect of the invention, there is provided a
food or drink comprising the composition of the invention for use
in a method to reduce skeletal muscle inflammation in subjects
undertaking resistance exercise training.
[0077] In yet a further aspect of the invention there is provided a
use of the composition of the invention for the manufacture of a
food or drink to reduce skeletal muscle inflammation in subjects
undertaking resistance exercise training.
[0078] WPI typically contains about 90% w/v protein; therefore 20 g
of WPI contains about 18 g of protein as a nutritional source.
[0079] Whey growth factor extract typically contains about 85% w/v
protein; therefore 2 g of whey growth factor extract contains about
1.7 g of protein as a nutritional source.
[0080] It will be appreciated that the present invention described
herein is not to be limited to specific examples of features
disclosed.
EXAMPLES
Example 1
Clinical Trial
[0081] A clinical trial was conducted in which 20 young males
participated in a three month randomised double-blinded resistance
training program. Whey growth factor extract was prepared according
to the method broadly described in Australian Patent No. 645589
(PCT/AU91/00303), more specifically described supra. Each whey
protein formulation contained an artificial sweetener
(Nutrasweet.TM., Nutrasweet Company, USA). Whey protein
formulations were consumed immediately after each exercise session,
with each subject completing three supervised exercise sessions per
week.
[0082] Subjects were randomly assigned to one of three supplement
groups:
[0083] Group A: 20 g NatraPro (WPI) per dose; n=7
[0084] Group B1: 20 g NatraPro (WPI) plus 1 g WGFE per dose;
n=6
[0085] Group B2: 20 g NatraPro (WPI) plus 2 g WGFE per dose;
n=7
[0086] A typical composition of NatraPro.TM. WPI includes:
TABLE-US-00003 Moisture 5.0% Fat 0.5% pH (5% solution) 6.3 Ash 3.7%
Lactose 0.5% Protein (TN .times. 6.38) 90.0% Sodium 0.7%
Phosphorous 0.3% Calcium 0.15%
Muscle Analysis
[0087] Muscle samples were collected from the vastus lateralis
muscle of the right leg using the percutaneous needle biopsy
technique. Excised muscle tissue was visually inspected, dissected
free of any fat or connective tissue and blotted to remove excess
blood and immediately frozen in liquid nitrogen for subsequent
analysis. A portion of the muscle tissue was mounted in an aqueous
mounting medium and frozen in isopentane cooled in liquid nitrogen
for subsequent immunohistochemical analysis.
Immunohistochemistry
[0088] Serial sections (10 .mu.m) of each sample were mounted on
microscope slides for analysis of myosin heavy chain fibre type. An
immunohistochemical technique based on the fast and slow isoforms
of myosin was used to examine fibre type distribution and muscle
cross-sectional area based on the protocol of Behan. Cellular
localisation of proteins was performed using standard
immunohistochemical techniques and antibodies raised against the
proteins of interest.
RNA Extraction & Gene Expression Analysis
[0089] RNA was extracted from skeletal muscle samples using the
ToTALLY RNA kit and reagents (Ambion Inc.) according to the
manufacturer's instructions. Total RNA concentrations and quality
were determined using the Agilent 2100 Bioanalyzer (Agilent
Technologies, Inc.). Subsequently, RNA was reverse transcribed into
cDNA using the AMV reverse transcriptase kit protocols and reagents
(Promega). Analysis of gene expression was performed on the Applied
Biosystems 7500 Real-Time PCR System using gene specific primers
designed using Primer Express 2.0 software.
Protein Expression Analysis
[0090] Standard western blotting techniques and an antibody
directed towards a common section of myosin heavy chain protein
isoforms (Zymed) was employed to examine total Myosin Heavy Chain
protein content.
Results
[0091] Results are presented as mean.+-.SEM and significance
calculated by two-way ANOVA using Bonforoni post hoc tests. No
significant differences in age, weight, height, or BMI values were
observed both pre and post training.
TABLE-US-00004 TABLE 1 Subject Characteristics NatraPro NatraPro
NatraPro Subject WPI (A) WPI (B 1) WPI (B 2) Characteristics (n =
7) (n = 6) (n = 7) Age 20.4 .+-. 0.6 19.5 .+-. 0.6 19.0 .+-. 0.4
Height 182.7 .+-. 3.3 180 .+-. 5.2 182.3 .+-. 4.1 Weight Pre
Training 79.9 .+-. 4.0 80.3 .+-. 4.6 79.3 .+-. 6.0 Post Training
80.1 .+-. 3.2 80.0 .+-. 4.7 80.9 .+-. 5.9 BMI Pre Training 24.5
.+-. 0.8 24.3 .+-. 1.0 23.9 .+-. 1.1 Post Training 24.07 .+-. 1.1
24.2 .+-. 1.0 24.4 .+-. 1.0
Gene Expression Analysis
[0092] There was no observed, consistent effect of training or
treatments on the expression of MyoD and myogenin whilst training
significantly increased the expression of muscle LIM protein (MLP)
(FIG. 1). The expression of Pax 7 and Syndecan 3, two markers of
satellite cell recruitment and proliferation, was increased 2-fold
and 7-fold, respectively, following 12 weeks of training and is
supported by protein expression data (FIGS. 2 and 3). The
expression of IGFBP4 and IGFBP5 (FIG. 4) were markedly increased in
group B1 at 12 weeks in response to acute exercise. The normal
post-exercise up-regulation of the early response genes jun-B and
c-fos which are linked to muscle inflammation (Chen Y W et al., J.
App. Physiol. 2003; 95(6):2485-94) was attenuated in both B1 and B2
supplement groups suggesting a reduced stress response in these
groups (FIG. 5). At 12 weeks both the resting and post-exercise
expression levels of CCL4 and CCL7 were increased following a
single bout of resistance exercise in groups B1 and B2
(significantly) compared with control group A (FIG. 6).
Example 2
Effect of WGFE on LPS--Induced TNF-Alpha Expression by Raw
Cells
[0093] An in vitro study was conducted to test the effect of
various milk product fractions on LPS-induced pro-inflammatory
TNF-alpha expression by RAW cells. RAW cells were grown in the
presence of LPS and then colostrum (sample 2BC), WPI (sample 4BC)
at 100 mg/ml, WGFE (sample 6BC) at 100 mg/ml, various milk
fractions, was added. In one sample no milk product was added
following LPS stimulation (`+ve` control). WGFE and LPS were also
added to the culture simultaneously (sample WGFE+LPS). The values
plotted represent the arithmetic mean.+-.SEM of the average
response obtained in 3 independent experiments (FIG. 7). A
comparison was done between the effect of WGFE and WPI on
inhibition of LPS-induced TNF-alpha expression (FIG. 8).
[0094] Whey growth factor extract was prepared as for Example
1.
[0095] The data supports that WGFE reduces the inflammation
response in muscle following resistance exercise training. In vitro
data suggests that WGFE reduces inflammation by inhibiting
TNF-alpha expression. The data also supports that WGFE in
combination with an additional protein source such as WPI decreases
the post-exercise inflammatory response to a greater extent than
observed when WPI, a protein source known to be used by subjects
undertaking resistance exercise training, is administered
alone.
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