Note: Descriptions are shown in the official language in which they were submitted.
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NUTRACEUTICAL SUPPLEMENT WITH LACTOBACILLUS RHAMNOSUS
FIELD
[0001] The present technology relates to compositions and uses thereof to aid
joint
support, aging, and sports medicine recovery and performance that include
Lactobacillus
rhamnosus.
BACKGROUND
[0002] This section provides background information related to the present
disclosure
which is not necessarily prior art.
[0003] Treatment of various musculoskeletal disorders and injuries is an
ongoing
problem. The body's joints, ligaments, muscles, nerves, tendons, and
structures that support
the limbs, neck, and back can be afflicted by degenerative diseases and
inflammatory
conditions that cause pain and impair normal activities. The body can also
suffer injuries
from strenuous activities, repetitive activities, or accidents that include
abrasions, contusions,
and fractures. Certain musculoskeletal issues arise from arthritis, aging, and
participation in
various physical or athletic activities.
[0004] Arthritis, for example, is a common disease resulting in inflammation
that
breaks down the lining of joints and cartilage. Osteoarthritis is the most
common form of
arthritis. It is the result of aging "wear and tear" and trauma to the joint,
such as sports
injuries or fractures. Major complaints of individuals with arthritis include
joint pain,
swelling, warmth, weakness, giving way of joint, instability, catching,
popping, stiffness,
poor sleep, muscle pain and fatigue. Autoimmune arthritides, such as
rheumatoid arthritis,
occurs when the body's immune system attacks itself. Osteoarthritis and
rheumatoid
arthritis are characterized by joint inflammation and cartilage degradation.
Mesenchymal
stem cells can rebuild cartilage, where the stem cells are resident in the
superficial zone of
articular cartilage. Treatment of arthritis has relied on relieving symptoms
by exercise,
braces, weight loss, medications, and surgery including total joint
replacement. Medications
such as non-steroidal anti-inflammatory drugs (NSAIDs) have risks that include
the stomach,
cardiovascular system and kidneys leading to ulcers, heart attacks and kidney
failure. There
is no disease modifying treatment for osteoarthritis. Osteoarthritis is
accelerated with
obesity/weight gain due to increased joint reactive forces.
[0005] Aging is a course of degeneration that is associated with the onset of
many
diseases. As people age, the prevalence of conditions associated with systemic
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inflammation, such as obesity, increases which is a common manifestation of
aging. The
body's ability to stimulate new bone marrow cells including mesenchymal stem
cells
decreases during aging, the result of which weakens the immune system and the
ability to
regenerate tissues. Conditions currently identified with an increased
prevalence with age
and increased inflammation include osteoarthritis, rheumatoid arthritis,
cardiovascular
disease, diabetes, obesity, Alzheimer's, chronic kidney disease, autoimmune
disease, cancer,
and other diseases. Symptoms of aging are associated with the underlying
oxidative stress,
increased inflammation, weakened immune system and the body's decreased
ability to form
new cells and tissues. Major symptoms of aging include fatigue, lethargy,
changes in
sleeping pattern, poor memory, poor vision, wrinkles, poor dentition, sexual
dysfunction,
decreased libido, type II diabetes, increased weight gain, fractures,
constipation, skin changes
including brown spots, loss of skin elasticity, menopause, hearing loss,
increased bone
fracture, arthritis, to name a few.
[0006] Injuries resulting from physical and athletic activities include muscle
strains,
joint sprains, ligament injuries, cartilage injuries, fractures and overuse
conditions. Joints
that are often attended to by an orthopedic surgeon for sports injury include
the shoulder,
knee, ankle, elbow, and wrist. Sports injuries can be accompanied by symptoms
including
pain, stiffness, swelling, feelings of instability, weakness, redness,
crepitance, bruising,
and/or mechanical symptoms such as locking or catching in a joint. Sports
medicine has
developed as a specialty for the prevention and treatment of sports-related
injuries that occur
at the ligament, muscle, tendon, and bone. Conventional treatment for sports
injuries
includes rest, ice, compression, elevation. Additional treatments include
physical therapy
and sports medicine rehabilitation, medication such as NSAIDs to reduce
inflammation that
exacerbates the injury and leads to surgery. There is no known medication or
current
treatment to accelerate the healing process after injury. Aging athletes are
at increased risk
of sports injury. An overweight, obese athlete is less physically fit and at
increased risk of
injury including arthritis due to increased joint reactive forces.
[0007] Mitigation of one or more symptoms or issues arising from joint
problems,
aging, and sports related injuries often includes the use of various anti-
inflammatory
treatments. Current anti-inflammatory treatments exhibit certain limitations.
For example,
the most commonly prescribed inflammatory/arthritis medications are the
various NSAID
drugs. NSAIDs include over the counter products, such as ibuprofen,
acetaminophen,
naproxen, and aspirin, and also include prescription formulations that of
naproxen and
celecoxib. Several of these medications are associated with one or more side
effects, some
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of which can be severe. Acetaminophen-associated overdoses, in particular,
account for
about 56,000 emergency room visits and about 26,000 hospitalizations yearly
and more than
450 deaths from liver failure. Acetaminophen is also the number one cause of
acute liver
failure and can result in kidney toxicity. NSAIDs can further induce nausea,
heartburn,
ingestion, abdominal pain, bleeding ulcer (GI complaints), where approximately
16,500
people per year die as a result of NSAID-associated gastrointestinal
complications and an
estimated 107,000 patients are hospitalized annually for NSAID-related GI
complications.
NSAIDs can further present complications relating to heart attack, stroke
(cardiovascular
events), congestive heart failure, atrial fibrillation, and kidney damage.
[0008] It would be advantageous to have an alternative to NSAIDs for the
treatment
of various health issues including musculo skeletal disorders and injuries.
SUM MARY
[0009] In concordance with the instant disclosure, the present technology
includes
compositions and methods that relate to treatment of various health issues
including
musculoskeletal disorders and injuries.
[0010] In one embodiment, a dietary supplement is provided that includes
Lactobacillus rhanmosus, ginger, and vitamin D, where the dietary supplement
can be
administered to aid a health issue related to one of joint support, aging, and
sports medicine.
[0011] In another embodiment, a dietary supplement is provided that includes
Lactobacillus rhamnosus, ginger, vitamin D, curcumin, and Boswellia extract,
where the
dietary supplement can be administered to provide joint support and mitigate
one or more
effects of arthritis.
[0012] In yet another embodiment, a dietary supplement is provided that
includes
Lactobacilhrs rhanmosus, ginger, vitamin D, Boswellia extract, and green tea
extract, where
the dietary supplement can be administered to mitigate one or more effects of
aging.
[0013] In yet another embodiment, a dietary supplement is provided that
includes
Lactobacillus rhanmosus, ginger, vitamin D, curcumin, and Epimedium extract,
where the
dietary supplement can be administered to mitigate one or more effects arising
from physical
or athletic activities.
[0014] Further areas of applicability will become apparent from the
description
provided herein. The description and specific examples in this summary are
intended for
purposes of illustration only and are not intended to limit the scope of the
present disclosure.
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DETAILED DESCRIPTION
[0015] The following description of technology is merely exemplary in nature
of the
subject matter, manufacture and use of one or more inventions, and is not
intended to limit
the scope, application, or uses of any specific invention claimed in this
application or in such
other applications as may be filed claiming priority to this application, or
patents issuing
therefrom. Regarding the methods disclosed, the order of the steps presented
is exemplary
in nature, and thus, the order of the steps can be different in various
embodiments. Except
where otherwise expressly indicated, all numerical quantities in this
description are to be
understood as modified by the word "about" in describing the broadest scope of
the
technology.
[0016] Although the open-ended term "comprising," as a synonym of non-
restrictive
terms such as including, containing, or having, is used herein to describe and
claim
embodiments of the present technology, embodiments may alternatively be
described using
more limiting terms such as "consisting of' or "consisting essentially of."
Thus, for any
given embodiment reciting materials, components, or process steps, the present
technology
also specifically includes embodiments consisting of, or consisting
essentially of, such
materials, components, or process steps excluding additional materials,
components or
processes (for consisting of) and excluding additional materials, components
or processes
affecting the significant properties of the embodiment (for consisting
essentially of), even
though such additional materials, components or processes are not explicitly
recited in this
application. For example, recitation of a composition or process reciting
elements A, B and
C specifically envisions embodiments consisting of, and consisting essentially
of, A, B and
C, excluding an element D that may be recited in the art, even though element
D is not
explicitly described as being excluded herein. Additionally, it should be
appreciated that all
natural supplements disclosed herein may be provided in the synthetic form and
used within
the scope of the present disclosure.
[0017] As referred to herein, all compositional percentages are by weight of
the total
composition, unless otherwise specified. Disclosures of ranges are, unless
specified
otherwise, inclusive of endpoints and include all distinct values and further
divided ranges
within the entire range. Thus, for example, a range of "from A to B" or "from
about A to
about B" is inclusive of A and of B. Disclosure of values and ranges of values
for specific
parameters (such as amounts, weight percentages, etc.) are not exclusive of
other values and
ranges of values useful herein. It is envisioned that two or more specific
exemplified values
for a given parameter may define endpoints for a range of values that may be
claimed for the
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parameter. For example, if Parameter X is exemplified herein to have value A
and also
exemplified to have value Z, it is envisioned that Parameter X may have a
range of values
from about A to about Z. Similarly, it is envisioned that disclosure of two or
more ranges of
values for a parameter (whether such ranges are nested, overlapping or
distinct) subsume all
possible combination of ranges for the value that might be claimed using
endpoints of the
disclosed ranges. For example, if Parameter X is exemplified herein to have
values in the
range of 1-10, or 2-9, or 3-8, it is also envisioned that Parameter X may have
other ranges of
values including 1-9, 1-8, 1-3, 1-2, 2-10, 2-8, 2-3, 3-10, 3-9, and so on.
[0018] The present technology is drawn to compositions that include the
probiotic
organism Lactobacillus rhanmosus and methods of administering such
compositions to aid
various health issues, including joint support, aging, and sports medicine
recovery and
performance. In addition to Lactobacillus rhaninosus, the compositions can
include a
member selected from the group consisting of curcumin, Boswellia extract,
ginger, vitamin
D, green tea extract, Epimedium extract, and combinations thereof. Curcumin
can be
provided by tumeric or extracted therefrom. Additionally, it should be
appreciated that
curcumin may be provided in a synthetic form and used within the scope of the
present
disclosure. Boswellia extract can be obtained from Boswellia serrata (i.e.,
Indian
Frankincense). Ginger can be obtained from the root of Zingiber officinale.
Vitamin D
can include cholecalciferol. Green tea extract can be obtained from Camellia
sinesnsis.
Epimedium extract can be obtained from Epimedium sagittatum (i.e., Horny Goat
Extract).
The compositions can be formulated for oral administration, including one or
more tablets or
capsules, liquid or slurry form, or as a powder or granulate. Other
ingredients can be
included, such as various excipients, including one or more antiadherents,
binders, coatings,
disintegrants, flavors, colors, lubricants, glidants, sorbents, preservatives,
and sweeteners.
Particular excipient examples include one or more of hypromellose, rice flour,
magnesium
stearate, cellulose, inulin, and silicon dioxide.
[0019] Various embodiments can provide compositions and uses thereof tailored
to
particular muscloskeletal disorders and/or injuries. In one embodiment, a
composition for
providing joint support and mitigating one or more effects of arthritis
comprises
Lactobacillus rhamnosus, vitamin D, curcumin, Boswellia extract, and ginger.
The
composition can be used to mitigate arthritic symptoms and pains, stimulate
the generation of
cartilage tissues from endogenous stem cells, and increase the body's
antioxidant activity.
In another embodiment, a composition for mitigating the effects of aging
comprises
Lactobacillus rhamnosus, Boswellia extract, ginger, green tea extract, and
vitamin D. The
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composition can be used to provide a protective effect on cells and tissues
against oxidative
stress, decrease inflammation, and regulate the immune system. In yet another
embodiment,
a composition for mitigating issues arising from physical and athletic
activities comprises
Lactobacillus rhaninosus, curcumin, ginger, vitamin D, and Epimedium extract.
The
composition can be used to provide protection against oxidative stress, anti-
inflammatory
action, and immuno-modulatory effects. The various components used in the
compositions
are approved by the U.S. Food and Drug Administration (FDA) for human use as
food and
diet supplements and can present limited or no side effects, such as gastric
irritation, stomach
ulcers, hypertension, cardiovascular diseases, and kidney dysfunction.
[0020] Lactobacillus rhanmoszts is approved by FDA for human use and can
provide
one or more activities attending to various health issues, including
musculoskeletal issues.
Lactobacillus rhanmoszts can reduce the inflammatory symptoms of rheumatoid
arthritis in
humans. Oral ingestion of Lactobacillus can improve the Health Assessment
Questionnaire
(HAQ) score and reduce the levels of plasma inflammatory cytokines.
Lactobacillus
rhatimosus can ameliorate arthritis in animals. Exopolysaccharide (BPS), the
major
component of Lactobaci/hts rhanmosus, has anti-arthritogenic properties. BPS
or BPS-
producing probiotics can suppress active arthritis. Lactobacillus rhanmostts
can suppress
inflammation in animals. Lactobacillus rhanmosus can reduce inflammatory
signaling in
immature intestines in animals. Thus, it can be expected to suppress
intestinal inflammatory
syndromes that present in newborns or children, such as necrotizing
enterocolitis (NEC),
idiopathic inflammatory bowel diseases (IBD), or infectious enteritis.
Lactobacillus
rhanmosus can reduce GI and respiratory infections. Lactobacilhts rhanmosus
can reduce
the risk for gastrointestinal and respiratory tract infections in pediatric
patients. Mice
treated with Lactobacillus rhanmosus showed attenuated weight gain on a high
fat diet (HD)
(not normal diet [ND]) compared to those with PBS (P). Mice receiving
Lactobacillus
rhanmosus showed faster glucose clearance (higher insulin sensitivity) on HD.
Lactobacillus rhanmosus can protect human colonic muscle. Lactobacillus
rhanmoszts can
attenuate lipopolysaccharide (LPS)-caused inflammation that produces higher IL-
6, lower IL-
(anti-inflammatory) and can restore the contractility of muscle tissue and
cells under
inflammation. Lactobacillus rhanmosus can increase insulin sensitivity.
Lactobacillus
rhaninosus can reduce oxidative stress in athletes during intense exercise.
Treatment with
Lactobacillus rhatnnosus can decrease the plasma levels of reactive oxygen
metabolites
(dROM) and biological antioxidant potential (BAP) in athletes who took intense
exercise
training. Lactobacillus rhanmostis can decrease the incidence of UV-induced
tumor
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formation. Hairless mice treated with Lactobacillus rhamnosus showed a delay
in tumor
expansion in both number and size. Lactobacillus rhamnosus can enhance both
natural and
acquired immunity. Treatment with Lactobacillus rhanmosus can increase the
plasma levels
of peripheral leucocytes and peritoneal macrophages (phagocytic activities)
and antibody,
which suggests that Lactobacillus rhamnosus increases both innate and acquired
immunity.
[0021] Accordingly, Lactobacillus rhamnosus provides several benefits,
including the
following. Lactobacillus rhamnosus is a unique probiotic strain. Lactobacillus
rhamnosus
can attenuate various types of arthritis.
Lactobacillus rhamnosus functions as antioxidant
which may make healthy cartilage cells. Lactobacillus rhamnosus can decrease
the
inflammatory cytokines that damage cartilage and joints. Lactobacillus
rhamnosus can
suppress the expression of inflammatory genes that cause pain and stiffness
accompanied
with arthritis. Lactobacillus rhamnosus can ameliorate rheumatoid arthritis.
Lactobacillus
rhamnosus can reduce oxidative stress and increases lifespan in the worm
model, C. elegan.
Lactobacillus rhamnosus can decrease inflammation. Lactobacillus rhamnosus can
improve
insulin-sensitivity and reduce fat accumulation. Lactobacillus rhamnosus can
provide
photoprotection against UV radiation. Lactobacillus rhamnosus shows anti-
oxidant benefits
in athletes during intense exercise training. Lactobacillus rhanmosus can
decrease the risk
of gastrointestinal and respiratory infections. Lactobacillus rhaninosus can
reduce adiposity
in animals on high fat diet. Lactobacillus rhanmosus can protect against UV-
induced
carcinogenesis. Lactobacillus rhamnosus is anti-inflammatory and immuno-
modulatory.
[0022] Compositions including Lactobacillus rhamnosus and uses thereof can
have
various physical amounts of Lactobacillus rhamnosus and various amounts of
colony
forming units (CFU) of Lactobacillus rhamnosus. Examples include physical
amounts
ranging from 1 mg to 1000 mg, 10 mg to 500 mg, 50 mg to 100 mg, and 67 mg.
Examples
further include CFU values ranging from 100 million to 1 trillion, 1 billion
to 100 billion, 5
billion to 50 billion, and 10 billion. Other amounts for the Lactobacillus
rhamnosus may also
be selected by one of ordinary skill in the art, as desired. These amounts can
be provided and
administered as a composition comprising a single dose or as multiple doses,
can be provided
as a single capsule or as multiple capsules, and can be admixed with other
components, for
example.
[0023] Curcumin is approved by FDA for human use and can provide one or more
activities attending to various health issues, including musculoskeletal
issues. As noted,
curcumin can be provided by tumeric or extracted therefrom. Additionally, it
should be
appreciated that curcumin may be provided in a synthetic form and used within
the scope of
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the present disclosure. Curcumin can have the same analgesic effect as
ibuprofen in
osteoarthritis patients. Curcumin can be as effective as ibuprofen for the
treatment of knee
osteoarthritis while it has fewer side effects than ibuprofen. Curcumin can
suppress the
activity of human synoviocytes that cause rheumatoid arthritis. Curcumin can
suppress the
production of inflammatory cytokines from synoviocytes that cause rheumatoid
arthritis in
patients. It can also suppress the expansion of human synoviocytes. Curcumin
can
suppress inflammation and arthritis in animals. Curcumin can suppress not only
the
production of inflammatory cytokines but also collagen-induced arthritis (CIA)
in animals.
Curcumin can enhance muscle regeneration after traumatic injury. Systemic
treatment with
curcumin can increase muscle mass (embryonic myosin heavy chain [EMEIC]) and
muscle
cells in two different injured muscle sites (Masseter & tibialis anterior
[TA]). Curcumin can
increase cardiac muscle repair and can ameliorate cardiac failure. Curcumin
can suppress
the production of malondialdehyde (MDA [A]), a lipid peroxide, and matrix
metalloproteases
(MMPs) that damage cardiac muscle, in an animal model of myocardial
infarction.
Curcumin can suppress oxidative stress that causes muscle damage. Curcumin can
suppress
the production of oxidants (hydrogen peroxide & NADPH-oxidase) and muscle
damage
markers (MCP-1 & CXCL14) in the muscle of animals that underwent extensive
exercise.
[0024] Accordingly, curcumin provides several benefits, including the
following.
Curcumin can be as safe and effective as an NSAlD in the treatment of knee
osteoarthritis.
Curcumin can be anti-inflammatory. Curcumin can reduce inflammatory cytokines
in
cartilage. Curcumin can show an anti-arthritic effect in animals. Curcumin can
promote
cartilage formation from mesenchymal stem cells. Curcumin can reduce
inflammation in
the synovial lining of human joints. Curcumin can suppress collagen-induced
arthritis in
animals. Curcumin can show a therapeutic effect for the treatment of
osteoarthritis.
Curcumin can promote muscle repair. Curcumin may prevent loss of muscle mass
and
stimulate muscle regeneration after traumatic injury. Curcumin can reduce
inflammation
and can enhance eccentric exercise-induced muscle damage. Curcumin can promote
cardiac
repair. Curcumin can decrease oxidative stress following downhill running-
induced muscle
damage. Curcumin can have a therapeutic effect for the treatment of
osteoarthritis.
[0025] Compositions including curcumin and uses thereof can employ various
amounts. Examples include amounts ranging from 1 mg to 1000 mg, 10 mg to 700
mg, 100
mg to 500 mg, and 350 mg. Other amounts for the curcumin may also be selected
by one of
ordinary skill in the art, as desired. These amounts can be provided and
administered as a
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composition comprising a single dose or as multiple doses, can be provided as
a single
capsule or as multiple capsules, and can be admixed with other components, for
example.
[0026] Boswellia extract is approved by FDA for human use and can provide one
or
more activities attending to various health issues, including musculoskeletal
issues. As
noted, Boswellia extract can include Boswellia serrata extract. Boswellia
extract can reduce
osteoarthritis symptoms. Osteoarthritis patients who received Boswellia plant
extract
reported decreased knee pain and swelling, increased knee flexion, and
increased walking
distance. Boswellia plant extract reduced osteoarthritis-related pain and
physical functions
(visual analog scale), Lequesne's functional index, and WOMAC pain and
stiffness.
Boswellia extract can reduce arthritis and inflammation in animals. Boswellia
extract can
reduce arthritic scores, paw edema, and the local tissue pro-inflammatory
cytokines tumor
necrosis factor alpha (TNF-a) and interleukin-1 beta (IL-1 p) in a Lewis rat
adjuvant arthritis
animal model. Boswellia extract can restore memory in animals. Orally ingested
Boswellia extract can reduce the escape latency and distance traveled but had
no influence on
swimming speed in the Morris water maze, suggesting that it enhances spatial
memory in
animals. Boswellia extract can have anti-tumor and anti-hyperlipidemic effects
in animals.
Orally ingested Boswellia plant extract can reduce TPA-induced skin
inflammation and
edema formation.
[0027] Accordingly, Boswellia extract provides several benefits, including the
following. Boswellia Extract significantly improves pain score and physical
function in
osteoarthritis patients. Boswellia extract can suppress the activation of the
inflammatory
immune system. Boswellia extract can block cartilage matrix breakdown and can
increase
type II collagen and aggrecan in human osteoarthritis chondrocytes. Boswellia
extract can
reduce cartilage-degrading enzymes and inflammation in osteoarthritis
patients. Boswellia
extract can be anti-inflammatory, anti-arthritic, and analgesic. Boswellia
extract can
decrease knee pain and can increase knee flexion and walking distance.
Boswellia extract
can decrease joint inflammation and spinal arthritis. Boswellia Extract can
improve
memory in an aging animal model. Boswellia extract can have anti-oxidant and
anti-
thrombotic effects. Boswellia extract can increase the elasticity of photo-
aged skin.
Boswellia extract can exert anti-tumor and anti-hyperlipidemic effects.
[0028] Compositions including Boswellia extract and uses thereof can have
various
physical amounts of Boswellia extract and various amounts of boswellic acid.
Examples
include physical amounts ranging from 1 mg to 1000 mg, 10 mg to 800 mg, 100 mg
to 600
mg, and 500 mg. Additionally, it should be appreciated that Boswellia extract
may be
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provided in a synthetic form and used within the scope of the present
disclosure. Other
amounts for the Boswellia extract may also be selected by one of ordinary
skill in the art, as
desired. Examples further include where the physical amount includes various
percentages of
boswellic acid, including 1% to 100%, 10% to 90%, 25% to 75%, and 65%. These
physical
amounts and percentages can be provided and administered as a composition
comprising a
single dose or as multiple doses, can be provided as a single capsule or as
multiple capsules,
and can be admixed with other components, for example.
[0029] Ginger is approved by FDA for human use and can provide one or more
activities attending to various health issues, including musculoskeletal
issues. As noted,
ginger can be obtained from the root of Zingiber officinale. Ginger can be as
effective as an
NSAID in reducing arthritic pain without gastropathy in patients. A ginger-
containing
supplement (Zinaxin) reduced arthritic pain (visual analogue scale [VAS]) to
the same extent
as diclofenac, an NSAID. Moreover, Zinaxin does not cause gastropathy that
accompanies
NSAID. Ginger can relieve osteoarthritis symptoms. Osteoarthritis patients
treated with
ginger showed a marked relief of osteoarthritis symptoms (based on Health
Assessment
Questionnaire) and progressing improvement without showing any sign of
negative effects.
Ginger can reduce inflammation and muscle soreness caused by exercise in
patients. In 98
patients treated with ginger for 6 weeks, there was a significant decrease in
muscle soreness
and inflammation.
[0030] Accordingly, ginger provides several benefits, including the following.
Ginger therapy can demonstrate a marked relief of osteoarthritis symptoms with
few side
effects. Ginger can suppress joint inflammation by reducing inflammatory
cytokines in
rheumatoid arthritis. Ginger can be as effective as the powerful steroid,
betamethasone, in
reducing osteoarthritis and rheumatoid arthritis. Ginger can reduce arthritis
pain as much as
diclofenac (NSAID) does in patients. Ginger can have antioxidant and
protective effects
against acetaminophen in animals. Ginger can have anti-oxidative, anti-
inflammatory, anti-
cancer, and anti-diabetic effects. Ginger can reduce muscle pain caused by
exercise.
Ginger can reduce muscle soreness caused by exercise. Long-term treatment with
ginger
can relieve osteoarthritis symptoms with few side effects.
[0031] Compositions including ginger and uses thereof can have various
physical
amounts of ginger where the physical amounts can include various concentration
ratios.
Examples include physical amounts ranging from 1 mg to 1000 mg, 10 mg to 800
mg, 100
mg to 600 mg, and 500 mg. Examples further include where the physical amount
relates to
a ginger concentrate range, including 1.5:1 to 20:1, 2:1 to 15:1, 5:1 to 10:1,
and 8:1.
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Equivalent concentrations of gingeroles (active ingredients of ginger) may
also be employed.
Additionally, it should be appreciated that ginger may be provided in a
synthetic form and
used within the scope of the present disclosure. Other amounts for the ginger
may also be
selected by one of ordinary skill in the art, as desired. These amounts can be
provided and
administered as a composition comprising a single dose or as multiple doses,
can be provided
as a single capsule or as multiple capsules, and can be admixed with other
components, for
example.
[0032] Vitamin D is approved by FDA for human use and can provide one or more
activities attending to various health issues, including musculoskeletal
issues. As noted,
vitamin D can include cholecalciferol. Vitamin D can decrease the effects of
osteoarthritis
in humans. Sunlight exposure and serum Vitamin D (25[OHP) levels are
positively
correlated with a decrease in knee cartilage loss. Thus, achieving vitamin D
sufficiency
may prevent and/or retard cartilage loss in knee osteoarthritis. Vitamin D
(1,25(OH)2D3)
can reduce the arthritic production of matrix metalloprotease-9 and
prostaglandin E2 (PGE2)
in human articular chondrocytes. Vitamin D can increase proper bone formation
and can
decrease unwanted aortic calcification on a high phosphate diet. In chronic
kidney disease,
patients lose their production of proper bone-forming factors and gain
unnecessary aortic
calcification. Paricalcitol containing Vitamin D can reverse both. Vitamin D
can increase
bone formation. Vitamin D (1,25(OH)2D3) can increase the expression of protein
factors
(type I collagen, osteopontin, sialoprotein, osteocalcin, alkaline
phosphatase, and BMP-2)
that enhance bone formation.
[0033] Accordingly, vitamin D provides several benefits, including the
following.
Vitamin D levels are positively associated with improvement in knee cartilage
volume.
Vitamin D can suppress inflammation in osteoarthritis and rheumatoid
arthritis. Vitamin D
can exert anti-inflammatory action on synovial lining cells. Vitamin D levels
can be
associated with bone mineral density and protection of cartilage in
osteoarthritic knee.
Vitamin D-deficient athletes can have a smaller heart size. Vitamin D-
deficiency can be
associated with lower body mass in professional football players. Vitamin D
directly affects
skeletal muscle structure and function. Vitamin D can prevent overuse-caused
injuries such
as stress fracture. Vitamin D can modulate the age-related decline in muscle
function and
benefits the aging athlete. Vitamin D can be used for DNA repair, thus
exerting an anti-
aging effect. Vitamin D can have protective effects on cardiovascular disease,
diabetes,
auto-immune disease and cancer. Vitamin D can be important for anti-aging of
the bone.
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Vitamin D can increase the expression of anti-aging genes. Vitamin D can
induce bone
formation in mesenchymal stem cells.
[0034] Compositions including vitamin D and uses thereof can have various
amounts
of vitamin D, including various ranges of international units (IU). Examples
include IU
values ranging from 1 IU to 10,000 IU, 10 IU to 7,500 IU, 100 IU to 5,000 IU,
500 IU to
2,000 II], and 1,000 IU. Additionally, it should be appreciated that vitamin D
may be
provided in a synthetic form and used within the scope of the present
disclosure. Other
amounts for the vitamin D may also be selected by one of ordinary skill in the
art, as desired.
These amounts can be provided and administered as a composition comprising a
single dose
or as multiple doses, can be provided as a single capsule or as multiple
capsules, and can be
admixed with other components, for example.
[0035] Green tea extract is approved by FDA for human use and can provide one
or
more activities attending to various health issues, including musculoskeletal
issues. As
noted, green tea extract can be obtained from Camellia sinesnsis and can
include various
weight percentages of polyphenols, catechins, and epigallocatechin gallate
(EGCG). Green
tea extract can suppress skin damage and aging caused by UV light. Cream
containing
green tea extract can reduce UV-induced photo-aging and subsequent
immunosuppression.
Green tea extract can increase learning and memory in old rats. Both young and
old rats
treated with green tea extract showed shorter time to find a safe place
(closed [dark] arm) and
to take a proper avoiding action to a pain-inducing condition. Green tea
extract can have
bone-forming and anti-fat effects. Green tea extract can increase the activity
of bone-
forming alkaline phosphatase (ALP) and suppresses the proliferation of fat
cells (adipocytes).
[0036] Accordingly, green tea extract provides several benefits, including the
following. Green tea extract can be effective in enhancing learning and
memory. Green
tea extract can have bone-forming and anti-obesity effects. Green tea extract
can have anti-
oxidant and anti-aging effects. Green tea extract can have a UV protective
effect. Green
tea extract can protect blood cells from aging-induced oxidative stress.
[0037] Compositions including green tea extract and uses thereof can have
various
physical amounts of green tea extract and various amounts of polyphenols,
catechins, and
epigallocatechin gallate. Examples include physical amounts ranging from 1 mg
to 1000
mg, 10 mg to 800 mg, 100 mg to 600 mg, and 500 mg. Examples further include
where the
physical amount includes polyphenols at 1% to 100 %, 10% to 100%, 50% to 100%,
and
98%. Examples further include where the physical amount includes catechins at
1% to 100
%, 10% to 100%, 50% to 100%, and 80%. Examples further include where the
physical
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amount includes epigallocatechin gallate at 1% to 100 %, 10% to 90%, 25% to
75%, and
50%. Additionally, it should be appreciated that green tea extract may be
provided in a
synthetic form and used within the scope of the present disclosure. Other
amounts for the
green tea extract may also be selected by one of ordinary skill in the art, as
desired. These
amounts can be provided and administered as a composition comprising a single
dose or as
multiple doses, can be provided as a single capsule or as multiple capsules,
and can be
admixed with other components, for example.
[0038] Epimedium extract is approved by FDA for human use and can provide one
or
more activities attending to various health issues, including musculoskeletal
issues. As
noted Epimedium extract can be obtained from Epinzediztin sagittatum (i.e.,
Horny Goat
Extract) and can have various weight percentages of icariin. Epimedium extract
can
increase the formation of bone cells. Treatment with epimedium extract
increased the
formation of bone cells from mesenchymal stem cells. Epimedium extract has a
vaso-
relaxant effect in animals. Icarrin, a major component of epimedium extract,
can reduce
blood pressure by relaxing coronary arterial vessel and increasing the
activity of the
antioxidant enzyme, eNOS, in the vessel. Epimedium extract can enhance
peripheral nerve
regeneration. Epimedium extract (Icarrin: major component) can promote
peripheral nerve
regeneration and improve the function of damaged nerves in a crush injury
animal model.
Epimedium extract can mimic one or more effects of testosterone. Icarrin (ICA
¨ a major
component of epimedium extract) can increase the levels of circulating serum
testosterone
and serum bone Gla-protein (BGP), a marker of bone growth.
[0039] Accordingly, Epimedium extract provides several benefits, including the
following. Epimedium extract can be a testosterone-mimetic. Epimedium extract
can
enhance the activities of the anti-oxidant enzymes, eNOS and NO. Epimedium
extract can
enhance the formation of bone cells from mesenchymal stem cells. Epimedium
extract can
improve bone formation and decrease bone absorption. Epimedium extract can
have
cardiovascular therapeutic effects. Epimedium extract can restore the function
of damaged
nerves and promote peripheral nerve regeneration.
[0040] Compositions including Epimedium extract and uses thereof can have
various
physical amounts of Epimedium extract with various weight percentages of
icariin.
Examples include physical amounts ranging from 1 mg to 1000 mg, 10 mg to 800
mg, 100
mg to 600 mg, and 500 mg. Examples further include where the physical amount
includes
various weight percentages of icariin, including 0.1% to 100%, 1% to 90%, 1%
to 50%, 5%
to 20%, and 10%.. Additionally, it should be appreciated that Epimedium
extract may be
13
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provided in a synthetic form and used within the scope of the present
disclosure. Other
amounts for the Epimedium extract may also be selected by one of ordinary
skill in the art, as
desired. These amounts can be provided and administered as a composition
comprising a
single dose or as multiple doses, can be provided as a single capsule or as
multiple capsules,
and can be admixed with other components, for example.
[0041] The compositions can be formulated in various ways, typically for oral
administration. Examples include forming the compositions into various tablets
or capsules,
providing the compositions in a liquid or slurry form, or providing the
compositions as
powders or granulates. The composition components can be entirely admixed
together into
a single portion, each provided as a separate portion, or various components
can be admixed
where the whole composition is provided by more than one portion but where a
total number
of portions is less than the number of components. Other dosage forms suitable
for oral
administration can be used.
[0042] Other ingredients can be included in the present compositions, such as
various
excipients, including one or more antiadherents (e.g., magnesium stearate),
binders (e.g.,
saccharides, gelatin, polymers), coatings (e.g., hydroxypropyl
methylcellulose, enterics such
as waxes, plastics, fibers etc.), disintegrants (e.g., polyvinylpyrrolidone,
carboxymethyl
cellulose, modified starches), flavors, colors, lubricants (e.g., talc,
silica, fats), glidants (e.g.,
fumed silica, talc, magnesium carbonate), sorbents, preservatives (e.g.,
antioxidants such as
vitamins A, E, and C), and sweeteners. Particular excipient examples include
one or more
of hypromellose, rice flour, magnesium stearate, cellulose, inulin, and
silicon dioxide.
[0043] The present compositions and methods of administering such compositions
can impact the way a body's stem cells respond to various health issues,
including
musculoskeletal issues. Stem cells include unique types of cells that have a
remarkable
potential to develop many different cell types. Stem cells can differentiate
into other cell
types, including fat, muscle, bone, cartilage, nerve, blood vessel, etc. They
are important in
early life during growth and repairing/replenishing tissues. Once a stem cell
develops into
specific cells, such as a fat cell, it typically can not form other tissues
such as bone, cartilage,
and muscle. The present compositions can impact stem cells and thereby reduce
the
inflammatory process and improve underlying symptoms. Keeping tissues healthy
also
leads to the possibility of disease modification and reducing
symptoms/progression of health
conditions.
[0044] Cells and tissues can be damaged with stress, lack of sleep, travel,
certain
medications, smoking, injury, aging, a poor diet, toxins, autoimmune diseases
and many other
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causes. Stem cells can participate in the healing and repair of these damaged
cells and
tissues. Stem cells offer a renewable source of cells and tissues to treat
multiple diseases, and
conditions including arthritis, aging, and sports medicine injuries. Damaged
cells and tissue
can lead to accelerated aging and injury, including sports injuries.
Currently, obesity is an
epidemic resulting in stem cells forming more fat leading to lack of bone,
cartilage, and blood
vessels resulting in diabetes, hypertension, osteoporosis, arthritis,
accelerated aging and sports medicine injuries.
[0045] The present compositions and uses thereof involve a paradigm shift in
the
thought of addressing the aging process. To slow the aging process, it is felt
that keeping
the cells that replenish the tissues (e.g., mesenchymal and hematopoietic stem
cells) healthy
is important. Aging declines the number of stem cells. Multiple scientific
studies
demonstrate the individual supplements in the present compositions can protect
such cells.
However, the combinations used in the present compositions revolutionize the
current
thought and approach to supporting joint health, anti-aging, and sports
medicine
performance/recovery. For example, without being bound by theory, it is
believed that the
present compositions and methods of using such compositions may direct a
person's own
stem cells to form muscle, bone, cartilage, nerve, blood vessel over fat. In
research studies,
individual supplements of the present compositions have demonstrated marked
improvement
of anti-oxidant function and the ability to down regulate inflammatory
chemicals and genes
responsible for contributing to the disease process. These effects have been
demonstrated
through extensive research, including clinical studies involving patients with
various
conditions. Individual components in the present compositions have
demonstrated the
ability to protect cells and promote chondrogenic differentiation of
mesenchymal stem cells
(MSCs).
EXAMPLES
[0046] An embodiment of a composition for providing joint support and
mitigating
one or more effects of arthritis comprises Lactobacillus rhanmosus, vitamin D,
curcumin,
Boswellia extract, and ginger. The composition is formulated as capsules, with
a serving
size of two capsules that contain ingredients or components as shown in the
following
TABLE 1.
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TABLE 1
Component
Amount per serving % Daily value
Vitamin D3 (as cholecalciferol) 1000 IU 250%
Ginger Root Extract 500 mg n/a
Boswellia Serrata Gum Extract 500 mg n/a
Curcumin as Tumeric Root Extract 350 mg n/a
Lactobacillus rhamnosus 10 billion CFU n/a
n/a = Daily Value not established.
Other Ingredients: Hypromellose (Capsule), Rice Flour, Magnesium Stearate.
[0047] Adipogenesis Testing with the Composition of TABLE 1:
[0048] Human bone marrow-derived Mesenchymal stem cells (MSC) differentiation
into adipocytes.
[0049] Frozen bone marrow mononuclear cells were purchased from Allcells
(Allcells, Emeryville, CA). After thawing, mononuclear cells were resuspended
in an a-
minimal essential medium a-MEM, Invitrogen, Carlsbad CA) supplemented with 10%
heat
inactivated fetal bovine serum (FBS, Invitrogen, Carlsbad, CA) and 1%
Antibiotics and
Antimycotic (Invitrogen, Carlsbad, CA). The cells were plated at a density of
1-5X106 cells
per 100 cm2 dish. The cultures were maintained at 37 C in a 5% CO2 incubator
and the
medium was changed after 48h and every 3-4 days thereafter. When the MSCs were
confluent, the cells were recovered by the addition of 0.25% trypsin/EDTA
(Invitrogen,
Carlsbad, CA). MSCs (Passage 2-3) were plated in either a 75-cm2 flask or a 24
well plate
and cultured in a-MEM with 20% FBS up to a density of 2.0X104 cells/cm2. The
medium
was replaced with adipogenic medium, and the cells were cultured for an
additional 14 days.
The adipogenic media consisted of complete culture medium supplemented with
DMEM-
high glucose, 10% (v/v) FBS, 10 g/m1 insulin, 0.5 mM dexamethasone (Sigma-
Aldrich, St.
Louis, MO), and 1% Antibiotics and Antimycotic (Invitrogen, Carlsbad, CA) in
the presence
and absence of the COX-1 inhibitor (2-Valeryloxybenzoic Acid, Cayman, Ann
Arbor MI)
and the COX-2 inhibitor (3-(4-methylsulphonylpheny1)-4-pheny1-5-
trifluoromethylisoxazol,
Cayman, Ann Arbor MI) with and without 20-HETE, 20-FIETE agonist (20-5,14-
HEDE) or
20-0H-PGE2. 20-HETE, 20-HETE agonist or 20-0H-PGE2 were added 3 times a week
at
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concentrations of 0.1 and 1 M. Inhibitors of COX-1 and COX-2 were added 3
times a week
at a dose of 10011M and 5p,M, respectively.
[0050] Oil Red 0 staining.
[0051] At day 14 of adipogenesis, 0.21% Oil Red 0 in 100% isopropanol (Sigma-
Aldrich, St. Louis, MO) was used. Briefly, adipocytes were fixed in 10%
formaldehyde,
washed in Oil-red 0 for 10 min, rinsed with 60% isopropanol (Sigma-Aldrich,
St. Louis,
MO), and the Oil red 0 eluted by adding 100% isopropanol for 10 min and OD
measured at
490 nm, for 0.5 sec reading. MSC-derived adipocytes were measured by Oil red 0
staining
(01)=490nm) after day 14. Each values of Oil red 0 staining were normalized by
cell
numbers (Values at OD=490nm).
[0052] In testing, it is observed that the formulation of TABLE 1 in the Oil
Red 0
staining test resulted in a statistically significant decrease in adipogenesis
relative to untreated
human MSC. Additionally, testing has shown that without the presence of
Lactobacillus
rhaninoszts, there was no statistically significant reduction in adipogenesis,
even with the
addition of the other ingredients of the formulation of TABLE 1. It is
believed that the
addition of Lactobacilha rhaninosus contributes to a synergistic effect in
combination with
the other ingredients, in a reduction of adipogenesis.
[0053] Anti-oxidative stress.
[0054] The percentage of live and dead cells was determined simultaneously by
measuring intracellular esterase activity and plasma membrane integrity using
the
LIVE/DEADO Viability/Cytotoxicity Assay Kit (Life Technologies). Briefly,
human MSC
were plated on 24 wells plate. On, next day cells were treated with
ingredients and 100 p,M
11202 for 12hr. The supernatant media from each well was collected into
microtubes and cells
were washed with 1X PBS, followed by collection of washes into respective
microtubes to
collect any floating or dead cells. The cells attached to the bottom of the
plate were incubated
in 1X PBS in a 37 C incubator. The microtubes were centrifuged at 2000 rpm
for 3 min to
get the cell pellet. After removing supernatant, cell pellet was resuspended
into 1X PBS
containing 2 M calcein AM and 4 f,tM ethidium / homodimer. Re-suspended cells
were
again poured into their respective wells and the plate was incubated at 37 C
for 15 min. The
cells were then imaged under 10X objective using fluorescence microscope
(Olympus IX71).
[0055] The anti-oxidative stress testing has shown that the other ingredients
of the
formulation of TABLE 1, when tested alone, did not result in a visually
significant
improvement in cell viability after the 12hr exposure to H202. However, the
formulation of
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TABLE 1 did result in a visually significant improvement in cell viability
after the 12hr
exposure to H202 These test results further support the belief that the
combination of the
ingredients has a synergistic effect in protecting cells against oxidative
stress and ultimate
cell death.
[0056] It is also believed that the formulation of TABLE 1 will have a
beneficial
effect on chondrogenic and osteogenic differentiation of human MSC.
Inflammation is
detrimental to chondrogenesis and chondrocyte formation from human MSC. The
formulation of TABLE 1 demonstrates anti-inflammatory effects, as inferred by
the
significant decrease in adipogenesis in the above-described experiments.
[0057] An embodiment of a composition for mitigating the effects of aging
comprises
Lactobacilhrs rhamnosus, Boswellia extract, ginger, green tea extract, and
vitamin D. The
composition is formulated as capsules, with a serving size of two capsules
that contain
ingredients or components as shown in the following TABLE 2.
TABLE 2
Component Amount
per serving % Daily value
Vitamin D (as cholecalciferol) 1000 IU 250%
Green Tea (leaf) Extract (Camellia sinensis) 500 mg n/a
(standardized to 98% polyphenols, 80% catechins,
and 50% EGCG)
Boswellia Serrata Extract 500 mg n/a
(standardized to 65% boswellic acid)
Ginger root (from 8:1 concentrate) (Zingiber 500 mg n/a
officinale)
Lactobacillus rhamnosus 10 billion CFU n/a
n/a = Daily Value not established.
Other Ingredients: Cellulose (Capsule), Inulin, Silicon Dioxide, Vegetable
Magnesium Stearate.
[0058] Adipogenesis Testing with the Composition of TABLE 2:
[0059] Human bone marrow-derived MSCs differentiation into adipocytes.
[0060] Frozen bone marrow mononuclear cells were purchased from Allcells
(Allcells, Emeryville, CA). After thawing, mononuclear cells were resuspended
in an a-
minimal essential medium a-MEM, Invitrogen, Carlsbad CA) supplemented with 10%
heat
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inactivated fetal bovine serum (FBS, Invitrogen, Carlsbad, CA) and 1%
Antibiotics and
Antimycotic (Invitrogen, Carlsbad, CA). The cells were plated at a density of
1-5X106 cells
per 100 cm2 dish. The cultures were maintained at 37 C in a 5% CO2 incubator
and the
medium was changed after 48h and every 3-4 days thereafter. When the MSCs were
confluent, the cells were recovered by the addition of 0.25% trypsin/EDTA
(Invitrogen,
Carlsbad, CA). MSCs (Passage 2-3) were plated in either a 75-cm2 flask or a 24
well plate
and cultured in a-MEM with 20% FBS up to a density of 2.0X104 cells/cm2. The
medium
was replaced with adipogenic medium, and the cells were cultured for an
additional 14 days.
The adipogenic media consisted of complete culture medium supplemented with
DMEM-
high glucose, 10% (v/v) FBS, 10 [ig/m1 insulin, 0.5 mM dexamethasone (Sigma-
Aldrich, St.
Louis, MO), and 1% Antibiotics and Antimycotic (Invitrogen, Carlsbad, CA) in
the presence
and absence of the COX-1 inhibitor (2-Valeryloxybenzoic Acid, Cayman, Ann
Arbor MI)
and the COX-2 inhibitor (3-(4-methylsulphonylpheny1)-4-pheny1-5-
trifluoromethylisoxazol,
Cayman, Ann Arbor MI) with and without 20-HETE, 20-HETE agonist (20-5,14-
FIEDE) or
20-0H-PGE2. 20-HETE, 20-HETE agonist or 20-0H-PGE2 were added 3 times a week
at
concentrations of 0.1 and l[iM. Inhibitors of COX-1 and COX-2 were added 3
times a week
at a dose of 100 M and 5 M, respectively.
[0061] Oil Red 0 staining.
[0062] At day 14 of adipogenesis, 0.21% Oil Red 0 in 100% isopropanol (Sigma-
Aldrich, St. Louis, MO) was used. Briefly, adipocytes were fixed in 10%
formaldehyde,
washed in Oil-red 0 for 10 min, rinsed with 60% isopropanol (Sigma-Aldrich,
St. Louis,
MO), and the Oil red 0 eluted by adding 100% isopropanol for 10 min and OD
measured at
490 nm, for 0.5 sec reading. MSC-derived adipocytes were measured by Oil red 0
staining
(0D=490nm) after day 14. Each values of Oil red 0 staining were normalized by
cell
numbers (Values at OD=490nm).
[0063] In testing, it is observed that the formulation of TABLE 2 in the Oil
Red 0
staining test resulted in a statistically significant decrease in adipogenesis
relative to untreated
human MSC. Additionally, testing has shown that without the presence of
Lactobacillus
rhamnosus, there was no statistically significant reduction in adipogenesis,
even with the
addition of the other ingredients of the formulation of TABLE 2. It is
believed that the
addition of Lactobacillus rhamnosus contributes to a synergistic effect in
combination with
the other ingredients, in a reduction of adipogenesis.
[0064] Anti-oxidative stress.
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[0065] The percentage of live and dead cells was determined simultaneously by
measuring intracellular esterase activity and plasma membrane integrity using
the
LIVE/DEAD Viability/Cytotoxicity Assay Kit (Life Technologies). Briefly,
human MSC
were plated on 24 wells plate. On, next day cells were treated with
ingredients and 100 uM
H202 for 12hr. The supernatant media from each well was collected into
microtubes and cells
were washed with 1X PBS, followed by collection of washes into respective
microtubes to
collect any floating or dead cells. The cells attached to the bottom of the
plate were incubated
in 1X PBS in a 37 C incubator. The microtubes were centrifuged at 2000 rpm
for 3 min to
get the cell pellet. After removing supernatant, cell pellet was resuspended
into 1X PBS
containing 2 uM calcein AM and 4 uM ethidium / homodimer. Re-suspended cells
were
again poured into their respective wells and the plate was incubated at 37 C
for 15 min. The
cells were then imaged under 10X objective using fluorescence microscope
(Olympus IX71).
[0066] The anti-oxidative stress testing has shown that the other ingredients
of the
formulation of TABLE 2, when tested alone, did not result in a visually
significant
improvement in cell viability after the 12hr exposure to H202. However, the
formulation of
TABLE 2 did result in a visually significant improvement in cell viability
after the 12hr
exposure to H202. These test results further support the belief that the
combination of the
ingredients has a synergistic effect in protecting cells against oxidative
stress and ultimate
cell death.
[0067] It is also believed that the formulation of TABLE 2 will have a
beneficial
effect on chondrogenic and osteogenic differentiation of human MSC.
Inflammation is
detrimental to chondrogenesis and chondrocyte formation from human MSC. The
formulation of TABLE 2 demonstrates anti-inflammatory effects, as inferred by
the
significant decrease in adipogenesis in the above-described experiments.
[0068] An embodiment of a composition for mitigating issues arising from
physical
and athletic activities comprises Lactobacillus rhanmosus, curcumin, ginger,
vitamin D, and
Epimedium extract. The composition is formulated as capsules, with a serving
size of two
capsules that contain ingredients or components as shown in the following
TABLE 3.
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TABLE 3
Component
Amount per serving % Daily value
Vitamin D (as cholecalciferol) 1000 IU 250%
Curcumin 350 mg n/a
Epidmedium sagittatum 500 mg n/a
(standardized to 10% icariin)
Ginger root (from 8:1 concentrate) (Zingiber 500 mg n/a
officinale)
Lactobacillus rhamnosus 10 billion CFU n/a
n/a = Daily Value not established.
Other Ingredients: Hypromellose (Capsule), Rice Flour, Magnesium Stearate.
[0069] Adipogenesis Testing with the Composition of TABLE 3:
[0070] Human bone marrow-derived MSCs differentiation into adipocytes.
[0071] Frozen bone marrow mononuclear cells were purchased from Allcells
(Allcells, Emeryville, CA). After thawing, mononuclear cells were resuspended
in an a-
minimal essential medium a-MEM, Invitrogen, Carlsbad CA) supplemented with 10%
heat
inactivated fetal bovine serum (FBS, Invitrogen, Carlsbad, CA) and 1%
Antibiotics and
Antimycotic (Invitrogen, Carlsbad, CA). The cells were plated at a density of
1-5X106 cells
per 100 cm2 dish. The cultures were maintained at 37 C in a 5% CO2 incubator
and the
medium was changed after 48h and every 3-4 days thereafter. When the MSCs were
confluent, the cells were recovered by the addition of 0.25% trypsin/EDTA
(Invitrogen,
Carlsbad, CA). MSCs (Passage 2-3) were plated in either a 75-cm2 flask or a 24
well plate
and cultured in a-MEM with 20% FBS up to a density of 2.0X104 cells/cm2. The
medium
was replaced with adipogenic medium, and the cells were cultured for an
additional 14 days.
The adipogenic media consisted of complete culture medium supplemented with
DMEM-
high glucose, 10% (v/v) PBS, 10 n/m1 insulin, 0.5 mM dexamethasone (Sigma-
Aldrich, St.
Louis, MO), and 1% Antibiotics and Antimycotic (Invitrogen, Carlsbad, CA) in
the presence
and absence of the COX-1 inhibitor (2-Valeryloxybenzoic Acid, Cayman, Ann
Arbor MI)
and the COX-2 inhibitor (3-(4-methylsulphonylpheny1)-4-phenyl-5-
trifluoromethylisoxazol,
Cayman, Ann Arbor MI) with and without 20-1IETE, 20-HETE agonist (20-5,14-
HEDE) or
20-0H-PGE2. 20-HETE, 20-HETE agonist or 20-0H-PGE2 were added 3 times a week
at
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concentrations of 0.1 and 104. Inhibitors of COX-1 and COX-2 were added 3
times a week
at a dose of 100p,M and 51AM, respectively.
[0072] Oil Red 0 staining.
[0073] At day 14 of adipogenesis, 0.21% Oil Red 0 in 100% isopropanol (Sigma-
Aldrich, St. Louis, MO) was used. Briefly, adipocytes were fixed in 10%
formaldehyde,
washed in Oil-red 0 for 10 min, rinsed with 60% isopropanol (Sigma-Aldrich,
St. Louis,
MO), and the Oil red 0 eluted by adding 100% isopropanol for 10 min and OD
measured at
490 nm, for 0.5 sec reading. MSC-derived adipocytes were measured by Oil red 0
staining
(0D=490nm) after day 14. Each values of Oil red 0 staining were normalized by
cell
numbers (Values at 0D=490nm).
[0074] In testing, it is observed that the formulation of TABLE 3 in the Oil
Red 0
staining test resulted in a statistically significant decrease in adipogenesis
relative to untreated
human MSC. Additionally, testing has shown that without the presence of
Lactobacillus
rhaninosus, there was no statistically significant reduction in adipogenesis,
even with the
addition of the other ingredients of the formulation of TABLE 3. It is
believed that the
addition of Lactobacillus rhaninosus contributes to a synergistic effect in
combination with
the other ingredients, in a reduction of adipogenesis.
[0075] Anti-oxidative stress.
[0076] The percentage of live and dead cells was determined simultaneously by
measuring intracellular esterase activity and plasma membrane integrity using
the
LIVE/DEADO Viability/Cytotoxicity Assay Kit (Life Technologies). Briefly,
human MSC
were plated on 24 wells plate. On, next day cells were treated with
ingredients and 100 p,M
H202 for 12hr. The supernatant media from each well was collected into
microtubes and cells
were washed with 1X PBS, followed by collection of washes into respective
microtubes to
collect any floating or dead cells. The cells attached to the bottom of the
plate were incubated
in 1X PBS in a 37 C incubator. The microtubes were centrifuged at 2000 rpm
for 3 min to
get the cell pellet. After removing supernatant, cell pellet was resuspended
into 1X PBS
containing 2 RM calcein AM and 4 pM ethidium / homodimer. Re-suspended cells
were
again poured into their respective wells and the plate was incubated at 37 C
for 15 min. The
cells were then imaged under 10X objective using fluorescence microscope
(Olympus IX71).
[0077] The anti-oxidative stress testing has shown that the other ingredients
of the
formulation of TABLE 3, when tested alone, did not result in a visually
significant
improvement in cell viability after the 12hr exposure to H202. However, the
formulation of
22
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TABLE 3 did result in a visually significant improvement in cell viability
after the 12hr
exposure to H202. These test results further support the belief that the
combination of the
ingredients has a synergistic effect in protecting cells against oxidative
stress and ultimate
cell death.
[0078] It is also believed that the formulation of TABLE 3 will have a
beneficial
effect on chondrogenic and osteogenic differentiation of human MSC.
Inflammation is
detrimental to chondrogenesis and chondrocyte formation from human MSC. The
formulation of TABLE 3 demonstrates anti-inflammatory effects, as inferred by
the
significant decrease in adipogenesis in the above-described experiments.
[0079] Example embodiments are provided so that this disclosure will be
thorough,
and will fully convey the scope to those who are skilled in the art. Numerous
specific
details are set forth such as examples of specific components, devices, and
methods, to
provide a thorough understanding of embodiments of the present disclosure. It
will be
apparent to those skilled in the art that specific details need not be
employed, that example
embodiments may be embodied in many different forms, and that neither should
be construed
to limit the scope of the disclosure. In some example embodiments, well-known
processes,
well-known device structures, and well-known technologies are not described in
detail.
Equivalent changes, modifications and variations of some embodiments,
materials,
compositions and methods can be made within the scope of the present
technology, with
substantially similar results.
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