U.S. patent application number 16/712302 was filed with the patent office on 2020-04-16 for aloe preparation for skin enhancement.
This patent application is currently assigned to Woodcliff Skincare Solutions, Inc.. The applicant listed for this patent is Woodcliff Skincare Solutions, Inc. University of Mississippi. Invention is credited to Denise DeBaun, Alvin Needleman, David Stanley Pasco, Alex Pashkowsky, Nirmal Derek Ceri Pugh.
Application Number | 20200113963 16/712302 |
Document ID | / |
Family ID | 40002847 |
Filed Date | 2020-04-16 |
View All Diagrams
United States Patent
Application |
20200113963 |
Kind Code |
A1 |
DeBaun; Denise ; et
al. |
April 16, 2020 |
Aloe Preparation For Skin Enhancement
Abstract
Methods for providing skin enhancement and pain relief to an
individual in need of treatment by administering to such an
individual an effective amount of an immunostimulatory Aloe vera
derived composition. Oral and topical methods and compositions are
provided. Aloe vera derived immunostimulatory compositions and
methods of producing such compositions are provided.
Inventors: |
DeBaun; Denise; (New York,
NY) ; Pasco; David Stanley; (Oxford, MS) ;
Pugh; Nirmal Derek Ceri; (Oxford, MS) ; Pashkowsky;
Alex; (New York, NY) ; Needleman; Alvin; (Las
Vegas, NV) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Woodcliff Skincare Solutions, Inc.
University of Mississippi |
Edison
University |
NJ
MS |
US
US |
|
|
Assignee: |
Woodcliff Skincare Solutions,
Inc.
Edison
NJ
University of Mississippi
University
MS
|
Family ID: |
40002847 |
Appl. No.: |
16/712302 |
Filed: |
December 12, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12599532 |
Nov 10, 2009 |
10543247 |
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PCT/US08/06089 |
May 9, 2008 |
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16712302 |
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60928924 |
May 11, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 36/886 20130101;
A61P 31/10 20180101; A61P 31/04 20180101; A61P 17/00 20180101; A61P
31/00 20180101; A61P 43/00 20180101; A61P 37/04 20180101; A61P
31/12 20180101; A61P 35/00 20180101; A61P 37/00 20180101; A61P
29/00 20180101; A61P 21/00 20180101; A61P 19/02 20180101; A61P
17/02 20180101; A61P 37/02 20180101 |
International
Class: |
A61K 36/886 20060101
A61K036/886 |
Claims
1. (canceled)
2. An immunostimulatory composition comprising: an effective amount
of an immunostimulatory Aloe vera-derived mixture and an Aloe
vera-derived material, wherein the immunostimulatory Aloe
vera-derived mixture comprises at least one Aloe vera-derived
lipo-polysaccharide, at least one Aloe vera-derived polysaccharide
and at least one Aloe vera-derived lipo-protein, wherein the
immunostimulatory Aloe vera-derived mixture exhibits an
immunostimulatory activity of at least 50% of a maximal NF-kappa
Beta directed luciferase expression by Escherichia coli bacterial
lipo-polysaccharide (ELPS) at a concentration of about 10 .mu.g/mL,
and the immunostimulatory activity is measured by NF-kappa Beta
directed luciferase expression in THP-1 monocytes at a
concentration of about 250 .mu.g/mL or less, wherein the Aloe
vera-derived material is selected from the group consisting of Aloe
vera gel concentrated at 1.times., Aloe vera gel concentrated at
10.times., Aloe vera gel concentrated at 200.times., Aloe vera
whole leaf spray dried powder concentrated at 100.times., Aloe vera
whole leaf decolorized liquid, acemannan, and Aloe vera
mucilaginous polysaccharide, wherein the amount of the Aloe
vera-derived material ranges from 10 wt % to about 90 wt % relative
to the total amount of the immunostimulatory Aloe vera-derived
mixture and the Aloe vera-derived material, and wherein the
composition is selected from the group consisting of a dispersion,
a solution, and a suspension.
3. The immunostimulatory composition of claim 2, wherein the Aloe
vera-derived polysaccharide comprises glucose in molar amount
ranging from about 25% to about 70%, rhamnose in a molar amount
ranging from about 0.5% to about 35%, galactose in a molar amount
ranging from about 5% to about 30%, mannose in a molar amount
ranging from about 3% to about 25%, and arabinose in a molar amount
ranging from about 0% and about 15%, based on the total molar
amount of the Aloe vera-derived polysaccharide, and wherein the
Aloe vera-derived polysaccharide has a molecular weight greater
than about 2,000,000 Daltons.
4. The immunostimulatory composition of claim 3, wherein the
immunostimulatory Aloe vera-derived mixture comprises between 0.01%
and 50% by weight of the Aloe vera-derived polysaccharide and
between 0.0001% and 10% by weight of the combination of the Aloe
vera-derived lipo-protein and Aloe vera-derived
lipo-polysaccharide.
5. The immunostimulatory composition of claim 4, wherein the
immunostimulatory Aloe vera-derived mixture comprises between 1%
and 20% by weight of the Aloe vera-derived polysaccharide.
6. The immunostimulatory composition of claim 4, wherein the
immunostimulatory Aloe vera-derived mixture comprises between 1%
and 15% by weight of the Aloe vera-derived polysaccharide.
7. The immunostimulatory composition of claim 4, wherein the
immunostimulatory Aloe vera-derived mixture comprises between
10-15% by weight of the Aloe vera-derived polysaccharide.
8. The immunostimulatory composition of claim 2, wherein the
immunostimulatory Aloe vera-derived mixture is obtained from Aloe
vera, wherein the Aloe vera includes at least one of Aloe vera
rind, Aloe vera mucilage, Aloe vera gel, or whole leaf Aloe
vera.
9. The composition of claim 2, further comprising: a pharmaceutical
carrier or excipient.
10. The immunostimulatory composition of claim 2, wherein the Aloe
vera-derived material is selected from the group consisting of an
immunostimulatory Aloe vera-derived material and a
non-immunostimulatory Aloe vera-derived material.
11. The immunostimulatory composition of claim 2, wherein the Aloe
vera-derived material is Aloe vera gel concentrated at 200.times.
and wherein the Aloe vera gel concentrated at 200.times. is present
in the composition in an amount of about 80% by weight relative to
the total amount of the Aloe vera-derived material and the
immunostimulatory Aloe vera-derived mixture.
12. A method of treating muscle or joint pain in a subject in need
thereof comprising administering to said subject an effective
amount of the immunostimulatory composition of claim 2.
13. The method of claim 12, wherein the subject is a human
being.
14. The method of claim 12, wherein the effective amount of the
immunostimulatory composition is between 0.2 milligrams and 1 gram
per day.
15. A method of improving the appearance, texture, firmness and/or
elasticity of skin and/or for providing accelerated growth of new
skin cells in a subject in need thereof comprising administering to
said subject an effective amount of the immunostimulatory
composition of claim 2.
16. The method of claim 15, wherein the subject is a human
being.
17. The method of claim 15, wherein the effective amount of the
immunostimulatory composition is between 0.0005 milligrams and 1
gram per day.
18. The immunostimulatory composition of claim 2, wherein the total
amount of the immunostimulatory Aloe vera-derived mixture and the
Aloe vera-derived material is present in the immmunostimulatory
composition in an amount between 0.05% and 5% by weight of the
total weight of the immunostimulatory composition.
19. The immunostimulatory composition of claim 18, wherein the
total amount of the immunostimulatory Aloe vera-derived mixture and
the Aloe vera-derived material is 0.5% by weight of the total
weight of the composition.
20. A method of treating muscle or joint pain in a subject in need
thereof comprising administering to said subject an effective
amount of the immunostimulatory composition of claim 11.
21. A method of improving the appearance, texture, firmness and/or
elasticity of skin and/or for providing accelerated growth of new
skin cells in a subject in need thereof comprising administering to
said subject an effective amount of the immunostimulatory
composition of claim 11.
22. The composition of claim 11, further comprising a
pharmaceutical carrier or excipient.
23. A method of treating muscle or joint pain in a subject in need
thereof comprising administering to said subject an effective
amount of the immunostimulatory composition of claim 22.
24. A method of improving the appearance, texture, firmness and/or
elasticity of skin and/or for providing accelerated growth of new
skin cells in a subject in need thereof comprising administering to
said subject an effective amount of the immunostimulatory
composition of claim 22.
25. An immunostimulatory composition, comprising: an effective
amount of an immunostimulatory Aloe vera-derived mixture, the
immunostimulatory Aloe vera-derived mixture comprising at least one
Aloe vera-derived lipo-polysaccharide, at least one Aloe
vera-derived polysaccharide, and at least one Aloe vera-derived
lipo-protein, wherein the immunostimulatory Aloe vera-derived
mixture exhibits an immunostimulatory activity of at least 50% of a
maximal NF-kappa Beta directed luciferase expression by Escherichia
coli bacterial lipo-polysaccharide (ELPS) at a concentration of
about 10 .mu.g/mL, and the immunostimulatory activity is measured
by NF-kappa Beta directed luciferase expression in THP-1 monocytes
at a concentration of about 250 .mu.g/mL or less, and wherein the
composition is selected from the group consisting of an dispersion,
a solution, and a suspension.
26. The immunostimulatory composition of claim 25, wherein the
immunostimulatory Aloe vera-derived mixture is insoluble in
water.
27. The immunostimulatory composition of claim 25, further
comprising: a complexing agent selected from the group consisting
of ethylenediaminetetraacetic acid (EDTA) or a salt thereof, such
as the disodium salt, citric acid, nitrilotriacetic acid, and the
salts thereof.
28. The immunostimulatory composition of claim 25, further
comprising: an alcohol selected from the group consisting of
ethanol, isopropanol, propylene glycol, and polyethylene
glycol.
29. The immunostimulatory composition of claim 2, wherein the
immunostimulatory Aloe vera-derived mixture exhibits an
immunostimulatory activity of at least 50% of a maximal NF-kappa
Beta directed luciferase expression by Escherichia coli bacterial
lipo-polysaccharide (ELPS) at a concentration of about 10 .mu.g/mL,
and the immunostimulatory activity is measured by NF-kappa Beta
directed luciferase expression in THP-1 monocytes at a
concentration of about 5 .mu.g/mL to about 250 .mu.g/mL.
30. The immunostimulatory composition of claim 2, wherein the
immunostimulatory Aloe vera-derived mixture is insoluble in
water.
31. The immunostimulatory composition of claim 2, further
comprising: a complexing agent selected from the group consisting
of ethylenediaminetetraacetic acid (EDTA) or a salt thereof, such
as the disodium salt, citric acid, nitrilotriacetic acid, and the
salts thereof.
32. The immunostimulatory composition of claim 2, further
comprising: an alcohol selected from the group consisting of
ethanol, isopropanol, propylene glycol, and polyethylene glycol.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date of
U.S. Provisional Patent Application No. 60/928,924, filed May 11,
2007, the disclosure of which is hereby incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] In one aspect, the present invention relates to the use of
bioactive compositions produced, isolated, extracted, based on or
derived from either an Aloe vera plant or a portion thereof, for
skin enhancement and for pain relief. The invention also relates to
immunostimulatory compositions, stabilized immunostimulatory
compositions, and methods of making and using such
compositions.
[0003] The genus Aloe (Liliaceae) is a shrubby tropical/subtropical
plant which has succulent and elongated leaves. Of the more than
360 Aloe species known, Aloe barbadensis Miller (Aloe vera Linne)
is the most widely used, both commercially and for its therapeutic
properties. Aloe vera plants contain two major juice materials:
first, a yellow exudate containing a high concentration of
anthraquinone compounds that has been used throughout the centuries
as a cathartic and for medicinal purges; and second, a clear
mucilaginous gel that has been used since ancient times to treat
burns and other wounds where it is thought to increase the rate of
healing and reduce the risk of infection. See Grindlay, D.
Reynolds, T., J. Ethnopharmcol. 1986, 16(2-3), 117-151; and Joshi,
S. P., J. Med. Aromat. Plant Sci. 1998, 20(3), 768-773.
[0004] For centuries, the Aloe plant has been considered to have,
and has been used, for its medicinal and therapeutic properties
without any clear understanding or scientific analysis of the bases
for such properties. Aristotle was aware that the healing
properties of Aloe would be invaluable to soldiers wounded in
battle and advised his student Alexander III ("the Great") to
conquer all lands that grew it, especially the island of Socotra
off the coast of eastern Africa. Pedanius Dioscorides, a physician
in the Roman army, mentioned medicinal Aloes in his encyclopedic
Greek herbal De Materia Medica (approximately 75 BC). Further, it
is known that the biological activities of fresh Aloe plant decay
quite rapidly.
[0005] Over that past few decades there has been a high level of
interest in developing an understanding of the active components of
Aloe that are responsible for its beneficial effects relating to
enhanced immune function. The research in this area has focused
largely on polysaccharides and other carbohydrate-containing
substances such as glycoproteins.
[0006] Active chemical substances and mixtures, including for
example, polysaccharides or other carbohydrate-containing
substances of Aloe leaves have been identified, isolated and
stabilized as described in U.S. Pat. No. 7,196,072, ("the '072
patent"), U.S. Pat. Nos. 5,902,796, 4,735,935, 4,851,224,
4,917,890, 4,957,907, 4,959,214, and 4,966,892.
[0007] One group of the active chemical substances has been
referred to as Aloe vera mucilaginous polysaccharides, which are
made up of a mixture of polysaccharides. The term "polysaccharides"
has been used loosely to include both oligomers and polymers of
carbohydrates or simple sugars and is used in a similar manner
herein. One group of such polysaccharides has been given the name
acemannan.
[0008] According to literature, acemannan is an ordered linear
polymer of substantially acetylated mannose monomers available from
Carrington Laboratories, Inc. (Irving, Tex.), and has been shown in
laboratory studies to increase up to 300%, in 48 hours, the
replication of fibroblasts in tissue culture, which are known to be
responsible for healing burns, ulcers and other wounds of the skin
and of the gastrointestinal lining.
[0009] Over a three year period, laboratory rats, the stomachs of
which react similarly to that of humans, were tested. Acemannan was
found to be equivalent to or superior to then current medications
used for the treatment of gastric ulcers. Most such products acted
to inhibit hydrochloric acid in the stomach. Acemannan worked on a
different principle and apparently did not alter the natural flow
of digestive acids. See U.S. Pat. No. 5,902,796, column 2, lines
20-25.
[0010] Acemannan has been shown to be an inducer of IL-I and
prostaglandin E2 (PGE2) production by human peripheral blood
adherent cells in culture. IL-I is an important macrophage product
reported in the literature to influence the activity and production
of lymphocytes, fibroblasts, B-lymphocytes and endothelial cells.
See Old, Scientific American, 258(5):59-60, 69-75 (1988).
[0011] IL-1 induces fibroblast proliferation which is fundamental
to wound healing. IL-1 also: (1) enhances bone marrow activity; it
may be therapeutic in individuals whose bone-marrow is depressed;
and (2) enhances the immune system in general.
[0012] According to literature, a series of experiments with mixed
lymphocyte cultures (MLC) has shown that acemannan increases the
alloantigenic response of these lymphocytes in a dose-related
fashion. Incubation of acemannan with monocytes permitted
monocyte-driven signals to enhance the T lymphocyte response to
leetin. Related studies on acemannan's effects on MLC have shown an
increase in phagocytosis and activity of natural killer cells. In
these in vitro test systems, acemannan was allegedly non-toxic and
an immunoenhancer.
[0013] Acemannan allegedly stimulates lymphocytes to secrete
lymphokines and also causes HIV-infected lymphocytes to produce
altered glycoproteins (GP-120) by a mechanism similar to that of
glucosidase I inhibitors. See Gruters et al., Nature
330:74-77(1987) and Pal et al., Intervirol. 30:27-(1989). Acemannan
is phagocytized and apparently pumped to the Golgi/glycoprotein
apparatus of the monocyte where it interferes directly with
glycoprotein synthesis.
[0014] Other uses of Aloe products have been described in, for
example, U.S. Pat. Nos. 5,106,616, 5,118,673, 5,308,838, 5,441,943,
and 5,443,830. The described studies have primarily focused on the
activities of bioactive chemical substances of Aloe vera as
antiviral agents, antitumor agents, immunostimulants,
immunomodulators, vaccine adjuvants, means of reducing
opportunistic infections, means of controlling inflammation, means
of stimulating the immune system and wound healing processes.
[0015] A number of other products based on
polysaccharide-containing fractions isolated from Aloe vera have
been published in the patent literature and elsewhere. For example,
a polyuronide with a molecular weight between 275,000 and 374,000
daltons is reported to be useful in treatment of surface wounds.
(See Farkas, A., U.S. Pat. No. 3,103,466 (1963).)
[0016] The 70 kD polysaccharide, Aloeferon, has also been reported
to have therapeutic potential. (See Madis, V. H.; Omar, M. M.;
Madis, V., U.S. Pat. No. 4,861,761 (1989).)
[0017] Other active components isolated from Aloe include a
polysaccharide between 420,000 and 520,000 daltons comprised of
equal amounts of glucose and mannose. (See Farkas, A., U.S. Pat.
No. 3,362,951 (1968).)
[0018] In addition, several groups have enzymatically prepared
altered polysaccharide compositions from the naturally occurring
carbohydrates in Aloe (See Strickland, F. M.; Pelley, R. P.;
Kripke, M. L., U.S. Pat. No. 5,824,659 (1998)).
[0019] Aloe vera polysaccharides have also been shown in controlled
studies to increase the rate of healing in animals. Aloe vera
polysaccharides have also been shown to be an effective treatment
for gastric ulcers in animal studies.
[0020] Anti-inflammatory activity of Aloe vera gel has been
reported by both oral testimonies and respected scientific
journals. Rubel [Cosmetics and Toiletries, 98:109-114 (1983)]
discussed the possible mechanism of the anti-inflammatory effect of
Aloe gel. Ukai et al., [Journal of Pharmacobio-Dynamics,
6(12):983-990 (1983)] noted anti-inflammatory activity of
polysaccharides extracted from the fruiting bodies of several
fungi. The polysaccharides allegedly demonstrated a significant
inhibitory effect on carrageenan-induced edema. One of the
polymers, O-acetylated-D-mannan (T-2-HN), in addition demonstrated
a more marked inhibitory effect than phenylbutazone on scald
hyperalgesia. Ukai et al., supra. The assertion in this article,
that the polysaccharide is allegedly free from protein and lipids
suggests that the anti-inflammatory effect is due to the acetylated
mannan only.
[0021] Other researchers have also reported anti-inflammatory
effects of complex polysaccharides [Saeki et al., Japanese Journal
of Pharmacology, 24(1):109-118 (1974)], glycoproteins [Arita et
al., Journal of Biochemistry, 76(4):861-869 (1974)] and sulfated
polysaccharides [Rocha et al., Biochemical Pharmacology,
18:1285-1295 (1969)].
[0022] It is therefore possible that mucilaginous gel, a component
of the Aloe vera plant, which is essentially a polysaccharide, and
components therein, are responsible in part for Aloe vera's
medicinal properties. The controversy over whether the
polysaccharide is a glucomannan, mannan, pectin, or of some other
composition, is allegedly resolved by a series of chemical
purification steps. Yagi et al., [Planta Medica, 31(1):17-20
(1977)], using a slightly modified extraction method; isolated
acetylated mannan (Aloe mannan) from Aloe arborescens Miller var.
natalensis. Ovodova [Khim, Prior, Soedin, 11(1):325-331 (1975)],
however, earlier isolated pectin as the main component of the same
Aloe species.
[0023] A number of other pharmacology studies have been conducted
on Aloe vera gel in recent times. Results have included more rapid
healing of radiation burns [Rowe, J. Am. Pharm. Assoc., 29:348-350
(1940)] and accelerated healing of wounds [Lushbaugh et al.,
Cancer, 6:690-698 (1953)]. Thermal burns treated with Aloe vera gel
heal much faster than untreated burns [Ashley et al., Plast.
Reconstr. Surg., 20:383-396 (1957), Rovatto, supra, Rodriguez-Bigas
et al., J. Plast. Reconstr. Surg., 81:386-389(1988)]. The gel is
useful in treating leg ulcers [El Zawahry et al., Int. J.
Dermatol., 12:68-73 (1973)] and in hastening post surgical healing
(Payne, Thesis submitted to Faculty of Baylor University, Waco,
Tex., MS Degree). Experimental evidence suggests that extracts of
Aloe vera have anti-infectious properties [Solar, Arch. Inst.
Pasteur Madagascar, 47:9-39 (1979)] and enhance phagocytosis
[Stepanova, Fiziol. Akt. Veshchestva, 9:94-97 (1977)].
[0024] During commercial processing and quality control of Aloe
products, microbiological analysis is recognized as an important
issue (Waller T A, et al. In Aloes: The genus Aloe; Reynolds T,
Ed.; CRC Press: NY, 2004; Chapter 8, pp 139-205). Microbiological
organisms can originate from both exogenous sources such as the
environment and from within the Aloe plant itself. The endogenous
bacterial flora are microorganisms that naturally exist within Aloe
that have evolved to grow well in that unique environment. The
primary interest in endogenous bacteria has been with respect to
controlling its potential overgrowth that can result with improper
post-harvest processing methods (e.g. lack of or incorrect
pasteurization). Furthermore, it has been highlighted that high
bacterial load in Aloe products is likely to result in loss of
biological activity. This has been reported by the observation that
high bacterial content (>100,000 organisms/gram of gel) is
correlated with loss of Aloe's ability to protect the skin immune
system from UVB-induced suppression (Waller T A, et al. In Aloes:
The genus Aloe; Reynolds T, Ed.; CRC Press: NY, 2004; Chapter 8, pp
139-205).
[0025] References in the art describe bacterial components in Aloe
with respect to endotoxin (also referred to as
lipo-polysaccharides) contamination as a confounding factor in
evaluating the biological activity of purported active substances
such acemannan polysaccharide. For example, this point was stressed
by Tizard (Tizard I R and Ramamoorthy L. In Aloes: The genus Aloe;
Reynolds T, Ed.; CRC Press: NY, 2004; Chapter 13, pp 311-332): "It
is difficult to produce Aloe carbohydrate solutions free of
contaminating endotoxin. Early studies on this material contained
small but significant quantities of bacterial endotoxin and it is
possible that some of the biological activities ascribed to
acemannan may have been endotoxin effects". Thus, bacterial
products in Aloe preparations are considered contaminants.
[0026] U.S. Pat. No. 5,902,796 describes various methods and
separation processes for obtaining different factors from Aloe
vera. One factor described is called a "microparticulate factor"
that can be obtained as a pellet by centrifugation at 20,000 g of
the supernatant obtained from a low speed centrifugation (180 g) of
an Aloe solution. Methods are also described for obtaining this
factor using filtration. This microparticulate factor is an
activator of macrophages. The size of this microparticulate factor
is estimated to be approximately 1 .mu.m.
[0027] The characterization of the microparticles was described in
a later paper (Ni Y, et al, International Immunopharmacology, 2004,
4: 1745-1755). The microparticles were reported to contain
galactose-rich polysaccharide(s) with the following composition:
40.2% galactose, 32.2% mannose, 20.6% glucose and other minor
sugars. This paper identifies the microparticles as degenerated
cellular organelles of mesophyll cells, as summarized: "Following
disruption of pulp by homogenization, three components were
isolated by sequential centrifugation. They were thin clear sheets,
microparticles and a viscous liquid gel, which correspond to cell
wall, degenerated cellular organelles and liquid content of
mesophyll cells based on morphological and chemical analysis".
Clearly these microparticles are not believed to be bacteria but
rather have a defined carbohydrate composition and are plant
structural components (i.e. degenerated cellular organelles of
mesophyll cells).
[0028] Gram positive micrococcus bacterial species are the most
prevalent Aloe-associated organisms. These bacteria have evolved to
grow well within the Aloe plant and do not grow very well in other
environments (Waller T A, et al. In Aloes: The genus Aloe; Reynolds
T, Ed.; CRC Press: NY, 2004; Chapter 8, pp 139-205).
[0029] Recent research indicates that consumption of foods
containing certain gram positive bacteria can have a beneficial
effect on the immune system. This has been most extensively studied
with respect to the ingestion of the probiotic lactic acid bacteria
Lactobacillus and Bifidobacterium strains found in yoghurt and
similar foods (See, Nutr Rev., 2006, 64: 1-14). Many of these
immune enhancing effects do not require viable bacterial cells
since they can be mimicked by consumption of heat killed
organisms.
[0030] Problems often associated with aging include a loss of skin
elasticity and muscle or joint pain. These symptoms may or may not
be related and while significant steps are being made in the
understanding of the aging process, much is still unknown.
Treatments for skin elasticity are often cosmetic in nature only.
By covering the skin with a cosmetic, a more youthful appearance
can be obtained in some instances. Some products actually glue the
loose skin making it appear more taught. There are also surgical
methods which can involve removing excess skin and/or tightening
the skin in procedures commonly referred to as "face lifts". If,
however, skin elasticity could be improved by the use of
preparations which are ingested or applied topically, and which
actually effect the elasticity of the skin directly, surgery could
be postponed or avoided completely and fewer cosmetic products
might be needed.
[0031] Similarly, there are many possible causes of joint pain
including arthritis and many strategies to treat them. These
include topical products, pharmaceutical preparations and even
joint replacement surgery. Still, as to either joint pain, or skin
elasticity, improvements in cure methods are necessary. If a
product whose active ingredient is all-natural could be used to
provide primary relief, or could be combined with other strategies,
the results would be highly desirable.
SUMMARY OF THE INVENTION
[0032] The present invention relates to, in one aspect, Aloe vera
derived materials exhibiting at least a certain amount of
immunostimulatory activity ("IAvDs"). The degree of
immunostimulatory activity required to qualify as an IAvD as
measured by NF-kappa Beta directed luciferase expression in THP-1
monocytes at a concentration of about 250 .mu.g/mL or less is at
least about 50% of the maximal NF-kappa Beta directed luciferase
expression achieved by bacterial lipo-polysaccharide at a
concentration of about 10 .mu.g/mL. The standard for this
measurement is crude bacterial lipo-polysaccharide "ELPS" (E. coli,
serotype 026:B6) obtained from Sigma Chemical Co.).
[0033] Another aspect of the invention is compositions comprising a
mixture of lipo-proteins, and lipo-polysaccharides, and optionally,
also at least one polysaccharide, all of which can be isolated from
Aloe vera, although the mixture can be produced synthetically. The
mixtures are referred to herein as "PLL". IAvD and PLL mixtures may
contain two or more materials, in any combination. These mixtures
may be blended with pharmaceutical carriers or excipients.
[0034] In a preferred embodiment, an immunostimulatory composition
comprises a mixture of at least any two of (1) aloe-derived
polysaccharides, (2) aloe-derived lipo-proteins, and (3)
aloe-derived lipo-polysaccharides, the mixture exhibiting an
immunostimulatory activity of at least about 50% of a maximal
NF-kappa Beta directed luciferase expression achieved by a
bacterial lipo-polysaccharide at a concentration of about 10
.mu.g/mL, wherein the immunostimulatory activity is measured by
NF-kappa Beta directed luciferase expression in THP-1 monocytes at
a concentration of about 250 .mu.g/mL or less.
[0035] The mixture may comprise aloe-derived polysaccharides,
aloe-derived lipo-proteins, and aloe-derived
lipo-polysaccharides
[0036] In a preferred embodiment, the PLL is a particular mixture,
referred to herein as "AloeEx", comprising 1) the Aloe vera derived
polysaccharide disclosed in the '072 patent, the disclosure of
which is hereby incorporated by reference, 2) Aloe-derived
lipo-proteins "ALP", and 3) Aloe-derived lipo-polysaccharides
"ALPS". Preferably, this mixture includes sufficient
immunostimulatory activity to qualify as an IAvD as described
herein.
[0037] Preferably, the PLL comprises between about 0.01% and about
50% by weight of the Aloeride polysaccharide and between about
0.0001% and about 10% ALPs and ALPSs (the total combined amount of
ALPs and ALPSs) by weight. In other embodiments, the Aloeride
polysaccharide comprises between about 1% and about 15% by weight
and between about 0.0001% and about 10% ALPs and ALPSs by weight.
In another embodiment, the Aloeride polysaccharide is about 10-15%
by weight and the ALPs and ALPSs are between about 0.0001% to about
10% by weight of the IAvD.
[0038] It is believed that these ALPs and ALPSs are actually from
microorganisms associated with Aloe vera. It is also believed that
ALPs and ALPSs possess independent immunostimulatory properties.
These may be used in addition to, or instead of immunostimulatory
Aloe-derive polysaccharides, including for example, those of the
'072 patent, to provide various benefits in the treatment or
prevention of, among other things, any condition previously
described in the art in conjunction with prior Aloe derivatives,
such as treatment of burns and wounds, treatment of ulcers of the
skin and of the gastrointestinal tract, enhancing bone marrow
activity in individuals whose bone marrow is depressed, enhancing
the immune system in general, as an antiviral agent, antitumor
agent, immunostimulant, immunomodulator, vaccine adjuvant, means
for reducing infections, means of controlling inflammation, means
of stimulating the wound healing process, means for increasing the
rate of healing, inhibiting scald hyperalgesia, rapid healing of
radiation burns and thermal burns, treating leg ulcers, hastening
post surgical healing, anti-infectious applications, enhancing
phagocytosis, treatment of immunodeficiency disorders, cancer,
infectious diseases or other diseases and conditions, amelioration
of symptoms, decreasing muscle and joint pain, improving metabolic
processes, improving the appearance, texture, firmness and/or
elasticity of skin, and as a dietary supplement or nutritional
aid.
[0039] In another aspect of the invention, immunostimulatory
composition comprises aloe-derived lipo-proteins. The aloe-derived
lipo-proteins exhibit an immunostimulatory activity of at least
about 50% of a maximal NF-kappa Beta directed luciferase expression
achieved by a bacterial lipo-polysaccharide at a concentration of
about 10 .mu.g/mL, wherein the immunostimulatory activity is
measured by NF-kappa Beta directed luciferase expression in THP-1
monocytes at a concentration of about 250 .mu.g/mL or less.
[0040] In another aspect of the invention, the composition is a
pharmaceutical composition comprising one or more IAvD. In one
embodiment, the composition is a pharmaceutical composition
comprising one or more PLL in combination with pharmaceutical
carriers or excipients. In a preferred embodiment, the PLL is
AloeEx. These pharmaceutical compositions may be used for any of
the purposes described immediately above in connection with
PLLs.
[0041] Certain IAvDs that are useful in accordance with the present
invention may be difficult to work with and formulate. There may be
problems dispersing or dissolving particular IAvDs and producing
homogenous mixtures thereof. This can be particularly problematic
when formulating dosage forms. Nonhomogenous mixtures risk an
unacceptable level of dose variation from dosage form to dosage
form and therefore dose to dose. Finding suitable processing aides,
solvents or excipients that are compatible with IAvDs and do not
negatively affect the immunostimulatory properties or stability of
the IAvDs and even more preferably do not unnecessarily dilute the
activity thereby requiring larger quantities of more expensive
materials, would therefore be highly desirable.
[0042] In accordance with another aspect of the invention, various
"second" materials can be used in connection with a PLL or IAvD. It
has been discovered that combinations with "second" materials can
allow for the production of a homogenous dispersion, emulsion,
suspension or solution and the like of a PLL or IAvD. These
"second" materials may or may not be Aloe-derived materials and
indeed may or may not be IAvDs (for example, they may not be
sufficiently immunostimulatory to qualify as an IAvD as defined
herein).
[0043] In one preferred embodiment, however, these second materials
are derived from Aloe vera. In particular, it has been found that
certain highly active first IAvDs can be dispersed, suspended,
emulsified and/or solubilized by using a second Aloe-derived
material, which, in some preferred embodiments are second IAvDs,
e.g., Aloe-derived materials that meet the activity requirements
set forth herein for IAvDs.
[0044] In other preferred embodiments the second materials are
Aloe-derived materials which, if they have any immunostimulating
activity at all, exhibit activity at a level below that of an IAvD.
These second, non-IAvD Aloe-derived materials generally have a
significant polysaccharide content wherein about 50% or more of the
polysaccharides are of a molecular weight of about 1,500,000
daltons or less. For example, MagnAloe AG at 250 .mu.g/ml exhibits
less than 25% activation.
[0045] Moreover, these second materials should also improve or
enhance the processability of the first IAvD. The amount of this
second Aloe-derived material used (whether a second IAvD or a
second non-IAvD) should be an amount which is sufficient to allow
for the production of, as desired, a homogenous dispersion,
solution, suspension, or emulsion of a first IAvD. Generally at
least about 10% of this second Aloe-derived material is required,
by weight percent, relative to the weight percent of the first IAvD
and the second Aloe-derived material.
[0046] In a preferred embodiment in accordance with the present
invention, the first material is a PLL and the second material is
an Aloe-derived material which is not an IAvD, has a polysaccharide
content as discussed above (generally at least 50% less than
1,500,000 daltons) and permits the formation of a stable,
homogeneous dispersion, solution, suspension, or emulsion. In a
particularly preferred embodiment, the PLL is AloeEx and the second
material is MagnAloe AG. Methods of making these formulations are
also contemplated.
[0047] In still another aspect of the invention, there is provided
a method of treating muscle or joint pain, for improving the
appearance, texture, firmness and/or elasticity of skin and/or for
providing accelerated growth of new skin cells comprising
administering, to an individual in need thereof, a therapeutic dose
of an IAvD or PLL composition. The composition may be administered
orally, topically or transdermally, once or several times a day and
may be administered alone, with a pharmaceutically acceptable
carrier or excipient, and/or with other active or inactive
ingredients.
[0048] In one embodiment of this aspect, the IAvD is a PLL.
[0049] In a particular embodiment, the PLL is AloeEx.
[0050] In another embodiment, the IAvD composition comprises an
Aloe derived polysaccharide or mixture of polysaccharides, such as,
without limitation, an Aloe-derived polysaccharide such as that
described in the '072 patent.
[0051] Another aspect of the invention is a method for preparing an
IAvD comprising providing an Aloe vera source material; extracting
a liquid Aloe solution from the Aloe vera source material;
filtering the liquid Aloe solution; adding a flocculent, thereby
creating aggregates containing microorganisms or microorganism
components/fragments; and collecting the aggregates.
[0052] In another embodiment, the collected aggregates can be
processed to remove anthraquinones, if present.
[0053] In an embodiment, the Aloe vera source material is selected
from the group consisting of Aloe vera gel, whole leaf Aloe vera,
Aloe vera rind, Aloe vera mucilage and Aloe vera pups. Aloe vera
pups are small Aloe vera plants usually considered a waste
product.
[0054] In a preferred embodiment, the Aloe vera source material
comprises Aloe vera rind and Aloe vera mucilage. Other Aloe based
materials, such as spent Aloe materials typically considered as
waste materials from other processes may be used.
[0055] In another aspect of the invention, method for preparing a
chemically standardized Aloe vera derived material comprises:
providing an Aloe vera derived material; determining the content of
2,3-dihydroxypropyl cysteine in the Aloe vera derived material; and
comparing the amount of 2,3-dihydroxypropyl cysteine in the Aloe
vera derived material to the amount of 2,3-dihydroxypropyl cysteine
in a standard preparation, thereby determining a standardized value
for the Aloe vera derived material.
[0056] In one embodiment, the method further comprises purifying
the Aloe vera derived material prior to determining the content of
2,3-dihydroxypropyl cysteine in the Aloe vera derived material.
[0057] In yet another aspect of the invention a method for
preparing a biologically standardized Aloe vera derived material
comprises: providing an Aloe vera derived material; determining the
level of activation of immune cells of the Aloe vera derived
material; and comparing the level of activity of the Aloe vera
derived material to a standard immunostimulatory value, thereby
determining a standardized activity value of the Aloe vera derived
material.
[0058] In one embodiment, the method further comprises purifying
the Aloe vera derived material prior to determining the level of
activation of immune cells of the Aloe vera derived material.
[0059] In another aspect of the invention a method of enhancing
immune function comprises administering, to an individual in need
thereof, a therapeutic dose of at least one IAvD.
BRIEF DESCRIPTION OF THE DRAWINGS
[0060] FIG. 1 depicts the glcosyl residue composition of AloeEx
Batch 56 (ALOE 056) by mole % of total carbohydrate.
[0061] FIG. 2 depicts a statistically significant decrease in mean
skin rebound time in seconds (an indicator of an increase in skin
elasticity or firmness).
[0062] FIG. 3 identifies skin elasticity study demographics by
gender and age.
[0063] FIG. 4 depicts study panelist demographics.
[0064] FIG. 5 identifies measurement randomization parameters.
[0065] FIG. 6 identifies treatment/group randomization.
[0066] FIGS. 7A-7D depict the statistical analysis of Cutometer
measurements for both the test material group and the placebo
group.
[0067] FIGS. 8A-8B depict questionnaire results for test subjects
assigned the active treatment.
[0068] FIGS. 9A-9B depict questionnaire results for test subjects
assigned the placebo treatment.`
[0069] FIGS. 10A-10B depict that TLR2 is involved in NF-kappa B
activation by AloeEx.
[0070] FIG. 11--depicts the response of THP-1 cells to AloeEx
(batch 056) and AloeEx crude fractions.
[0071] FIG. 12--depicts proteinase K digestion and SDS
polyacrylamide gel analysis of 4% SDS extract of AloeEx phenol
layer fraction.
[0072] FIG. 13--depicts lipoprotein lipase digestion of 4% SDS
extract of AloeEx phenol layer fraction.
[0073] FIG. 14--depicts the response of THP-1 cells to different
Aloe vera leaf parts.
DETAILED DESCRIPTION
[0074] While the specification concludes with the claims
particularly pointing and distinctly claiming the invention, it is
believed that the present invention will be better understood from
the following description.
[0075] All percentages and ratios used herein are by weight of the
total composition and all measurements made are at 25.degree. C.
and normal pressure unless otherwise designated.
[0076] All temperatures are in Degrees Celsius unless specified
otherwise.
[0077] The present invention can comprise (open ended) or consist
essentially of the components of the present invention as well as
other ingredients or elements described herein. As used herein,
"comprising" means the elements recited, or their equivalent in
structure or function, plus any other element or elements which are
not recited. The terms "having" and "including" are also to be
construed as open ended unless the context suggests otherwise. As
used herein, "consisting essentially of" means that the invention
may include ingredients in addition to those recited in the claim,
but only if the additional ingredients do not materially alter the
basic and novel characteristics of the claimed invention.
Preferably, such additives will not be present at all or only in
trace amounts. However, it may be possible to include up to about
10% by weight of materials that could materially alter the basic
and novel characteristics of the invention as long as the utility
of the compositions (as opposed to the degree of utility) is
maintained.
[0078] All ranges recited herein include the endpoints, including
those that recite a range "between" two values. Terms such as
"about," "generally," "substantially," and the like are to be
construed as modifying a term or value such that it is not an
absolute, but does not read on the prior art. Such terms will be
defined by the circumstances and the terms that they modify as
those terms are understood by those of skill in the art. This
includes, at very least, the degree of expected experimental error,
technique error and instrument error for a given technique used to
measure a value.
[0079] Note that while the specification and claims may refer to a
tablet or other dosage form of the invention as, for example,
containing particles having a certain particle size or
distribution, or a certain type of, for example, a filler, it may
be difficult to tell from the final dosage form that the recitation
is satisfied. However, such a recitation may be satisfied if the
materials used prior to final blending and formulation, for
example, meet that recitation.
[0080] In another example, while it might be difficult to know the
weight gain that the coating contributed to a coated particle or
its particle size distribution as it actually exists in a finished
dosage form, if it is determined that the coated particles used to
make the dosage form, prior to, in the case of a tablet, for
example, a final blending and compression step, did exhibit the
desired coating level and/or particle size, that is sufficient.
[0081] Indeed here, it might be difficult to establish the content
of a particular component in a particular formulation or the
activity of a particular component in the final formulation. If
this can be established based on the starting or intermediate
materials, it is sufficient. Indeed, as to any property which
cannot be ascertained from the finished product directly, it is
sufficient if that property resides in the formulation just prior
to final production. It will be appreciated that immunostimulatory
activity sufficient to define an IAvD as described herein may be
established any time before or after formulation.
[0082] As used herein, treatment and/or improvement, when used in
connection with joint pain or skin elasticity, or indeed other
conditions described herein, refers to any lessening, whether
permanent or temporary, lasting or transient, of joint or muscle
pain or any increase in skin elasticity that can be attributed to
or associated with administration of the PLL and/or IAvD
composition. Treatment and improvement also include use of products
as described herein to prevent a condition or its recurrence. Note
also that improvement and treatment are used interchangeably,
unless the text or context dictate otherwise.
[0083] In particular, to qualify as an IAvD herein, a material must
substantially contribute to the immunostimulatory properties of the
composition. As used herein, the phrase "substantially contributes"
to the immunostimulatory properties of the composition refers to an
immunostimulatory effect as described herein.
[0084] As used herein, the terms immunostimulatory,
immunostimulatory effect or immunostimulatory properties and/or
activity relate to the ability of IAvD's in accordance with the
invention to cause macrophage activation, and in particular,
immunostimulatory activity as indicated by increased expression of
NF-kappa Beta directed luciferase expression in THP-1 monocytes as
described previously (equal to about 50% that of ELPS at 10
.mu.g/mL at a concentration of 250 .mu.g/mL or less). This may be
measured based on any individual Aloe-derived component or
collection or mixture thereof. Thus, activity can, for example,
reflect that of an Aloe-derived polysaccharide or its combination
with ALP and/or ALPS. A method for measuring this type of
macrophage activation, herein incorporated by reference, is
disclosed in the '072 patent, column 5, line 12 to column 6, line
22.
[0085] Generally, macrophage activation can be measured using a
luciferase reporter gene assay in THP-1 human monocytic cells. This
assay measures immunostimulatory activity as indicated by increased
expression of a NF-kappa Beta-driven luciferase reporter. THP-1
cells are cultured in RPMI 1640. medium supplemented with fetal
bovine serum (10% v/v) and amikacin (60 mg/L) at 37.degree. C.,
under 5% CO.sub.2 and 95% air. Actively growing cells are
transiently transfected using DEAE-dextran (10
.mu.g/l.times.10.sup.6 cells) and the pBIIXLUC reporter plasmid (1
.mu.g/l.times.10.sup.6 cells) containing two binding sites for
NF-kappa Beta. Transfection solution containing THP-1 cells are
incubated for 7 minutes in 37.degree. C. water bath. The
transfected cells are then resuspended in 10% FBS, RPMI 1640 medium
and plated out in 96-well plates at a cell density of
2.times.10.sup.5 cells per well. After 24-hours test samples are
added to transfected cells. Four hours after addition of samples,
cells are harvested and luciferase activity measured. Cells are
harvested using Packard filter plates and lysed using 30 .mu.L of
luciferase mix (1:1, LucLite.TM. luciferase:1.times.PBS, 1 mM Ca
and Mg). Luciferase light emission is measured using a Packard
microplate scintillation counter in single photon mode. Activation
is reported as a percentage relative to maximal activation of
NF-kappa Beta by 10 .mu.g/mL ELPS, used as a positive control.
Other assay methods are contemplated.
[0086] As used herein, the term non-immunostimulatory Aloe
vera-derived material and other words indicating this concept are
defined as an Aloe vera-derived material which does not meet the
immunostimulatory requirements of an IAvD as defined herein.
[0087] The term Aloe-derived lipo-protein or "ALP" refer to
bacterial lipo-proteins from bacteria endogenous to Aloe vera. This
does not mean that the bacteria must come from Aloe vera as the
bacteria could be separately cultured or cultivated. Gram positive
micrococcus bacterial species are the most prevalent
Aloe-associated organisms. These bacteria have evolved to grow well
within the Aloe plant and do not grow very well in other
environments (Waller T A, et al. In Aloes: The genus Aloe; Reynolds
T, Ed.; CRC Press: NY, 2004; Chapter 8, pp 139-205). Gram positive
bacteria contain lipoproteins, but no lipo-polysaccharides. As
disclosed herein, lipoproteins are believed to be a significant
contributor to in vitro macrophage activation from gram positive
bacteria. Since gram positive bacteria are the most prevalent type
of bacteria in Aloe, lipoproteins represent an important active
derived from Aloe.
[0088] Recent research indicates that consumption of foods
containing certain gram positive bacteria can have a beneficial
effect on the immune system. This has been most extensively studied
with respect to the ingestion of the probiotic lactic acid bacteria
Lactobacillus and Bifidobacterium strains found in yoghurt and
similar foods (reviewed in Ezendam and van Loveren, Nutr Rev.,
2006, 64: 1-14). Many of these immune enhancing effects do not
require viable bacterial cells since they can be mimicked by
consumption of heat killed organisms.
[0089] As used herein, the terms Aloe vera derived
lipo-polysaccharide and ALPS refer to a large molecule comprising a
lipid and a polysaccharide (carbohydrate) joined by a covalent
bond. ALPSs are a major component of the outer membrane of
Gram-negative bacteria found in or associated with Aloe vera,
contributing greatly to the structural integrity of the bacteria,
and protecting the membrane from certain kinds of chemical attack.
ALPS are endotoxins, and induce a strong response from normal
animal immune systems.
[0090] LPS generally act as the prototypical endotoxin, because it
binds the CD14/TLR4/MD2 receptor complex, which promotes the
secretion of pro-inflammatory cytokines in many cell types, but
especially in macrophages. An "LPS challenge" in immunology is the
exposing of the subject to an LPS which may act as a toxin. LPS
also increases the negative charge of the cell membrane and helps
stabilize the overall membrane structure. LPS are additionally an
exogenous pyrogen (external fever-inducing composition). ALPs are
different from ALPSs, also a desirable component of the PLLs of the
invention, in that their immunostimulatory action is believed to be
mediated through toll-like receptor 2, whereas ALPS are believed to
be mediated through toll-like receptor 4.
[0091] Some IAvD compositions have no ALPs or ALPSs present that
substantially contribute to the immunostimulatory properties of the
composition. Other IAvD compositions however, include no
polysaccharides, other than ALPSs that substantially contribute to
the immunostimulatory properties of the composition. Still other
IAvD compositions have only ALPs which substantially contribute to
the immunostimulatory properties of the composition.
[0092] Preferably, there are two or more of polysaccharides, ALPS
and/or ALPSs, any or all of which substantially contribute
individually or synergistically to the immunostimulatory activity
of the IAvD composition.
[0093] Some IAvD compositions include ALPS and/or ALPSs having
immunostimulatory activity which are derived from microorganisms
such as certain bacteria, yeasts, fungi, molds and the like found
in or otherwise associated with Aloe vera. These ALPS and ALPSs may
also be recovered from microorganisms which have been cultured, so
long as that genus and species are found on or associated with Aloe
vera. These ALPs and ALPSs, which may be found on or within the
Aloe vera plant, which may be concentrated or synthesized, may be
used in addition to, or instead of polysaccharides to provide
various benefits in the treatment of, among other things, joint and
muscle pain, and enhancing the appearance, texture, firmness,
and/or elasticity of skin. PLL's containing Aloe-derived
polysaccharides, like that described in the '072 patent, as well as
both ALPs and APLS's are contemplated for these uses.
[0094] As used herein, the term Aloe-derived material refers to any
material contained in or present on Aloe vera including but not
limited to Aloe-derived polysaccharides, lipo-proteins and
lipo-polysaccharides. Such materials can be isolated from the Aloe
plant and/or synthesized.
[0095] As used herein, the term synthetic or synthesized refers to
materials which are manmade and/or manufactured and which have the
same or similar compositions of those materials found in nature. In
the case of ALPs and ALPSs, it also refers to separately
cultured.
[0096] As used herein, the term "AloeEx" refers to an
immunostimulatory PLL mixture, extracted from Aloe vera or produced
synthetically, comprising the polysaccharide of the '072 patent as
well as ALP and ALPS. A PLL such as AloeEX can be isolated from the
rind and/or the mucilage of Aloe vera leaves (i.e. the region
between the inner gel and the outer rind) and may be obtained from
Aloe waste materials (i.e. spent rinds used in other processes and
considered a waste product by current industry standards).
[0097] AloeEx often has an immunostimulatory activity, as measured
by the techniques described herein, of 5 .mu.g/mL. This means that
AloeEx at a concentration of about 5 micrograms/milliliter
increases NF-kappa B directed luciferase expression in THP-1
monocytes comparable to levels 50% of those realized by the use of
ELPS at a concentration of 10 .mu.g/mL, or more. Of course, batch
to batch variations can occur within this level of activity.
[0098] As used herein, the term Aloeride (as distinguished from the
trademark ALOERIDE.RTM. discussed below) refers to an
immunostimulatory polysaccharide composition, isolated from Aloe
vera or produced synthetically, wherein the polysaccharides have an
apparent molecular weight greater than about two million daltons
and comprise glucose, galactose, mannose and arabinose as reported
in the '072 patent. In particular, the Aloeride polysaccharide
composition may comprise between about 25% and about 70% glucose,
between about 0.5% and 35% rhamnose, between about 5% and about 30%
galactose, between about 3% and about 25% mannose and between about
0% and about 15% arabinose (in mole %). In the '072 patent the
Aloeride polysaccharide is also called NP18298. For the purpose of
the current invention, Aloeride and NP18298 refer to the same
composition.
[0099] As used herein, the term MagnAloe AG refers to a powdered
mixture of Aloe-derived polysaccharides having a molecular weight
distribution as described at
http://www.firstAloevera.com/magnAloe_information.htm. Any mixture
comprising high molecular weight Aloe-derived polysaccharide
materials is contemplated for the purpose of an IAvD or non-IAvD as
described herein.
[0100] Preliminary findings leading to the '072 patent suggested
that detectable levels of immunostimulation in crude Aloe juice and
gel could not be accounted for by previously known Aloe components.
Commercial Aloe preparations (200 .mu.g/mL) only resulted in
negligible NF-kappa Beta activation in the macrophage assay.
Aloeride "NP18298", which accounted for this activity, was found to
comprise a polysaccharide-containing fraction where the
polysaccharides present had an apparent molecular weight above 2
million daltons. Its major glycosyl components reported therein
included glucose, galactose, mannose and arabinose. Based on the
degree and methods of purification used and reported in the '072
patent, it is not believed that Aloeride included substantial
levels of ALPs and/or ALPSs.
[0101] In investigating alternate sources and methods of scaling-up
production of Aloeride-containing materials, it was subsequently
discovered that other fractions, fractions which could include the
fraction described in the '072 patent, along with other materials
including ALPs and ALPSs, possessed desirable immunostimulatory
activity.
[0102] The methods described in the '072 patent, using the starting
materials described, provided a relatively pure
polysaccharide-containing fraction, devoid of sufficient ALPs and
ALPSs to provide significant additional immunostimulatory
activity.
[0103] Without wishing to be bound by any particular theory of
operation, it is believed that ALPs and ALPSs possess separate
immunostimulatory properties and may be used in addition to, or
instead of polysaccharides to provide various benefits in the
treatment of, among other things, joint and muscle pain, and skin
elasticity enhancement.
[0104] The results of testing indicate that AloeEx which comprises
the Aloeride polysaccharide, ALPs and ALPSs, are collectively
responsible for the macrophage activation properties
(immunostimulatory properties) of AloeEx.
[0105] IAvD's may include PLL's, AloeEx, mixtures of ALPs and
ALPSs, polysaccharides and the Aloeride polysaccharide (which is
substantially free from ALPs and ALPSs). IAvD's may also include
other materials, including commercial polysaccharide preparations
having immunostimulatory derived compositions and which have been
shown to cause activation of NF-kappa Beta directed luciferase
expression in THP-1 cells to the extent that they meet the criteria
for IAvD's set forth herein.
[0106] As stabilized formulations and compositions of IAvD's are
desirable, such formulations may include second Aloe-derived
non-IAvD materials in combination with IAvD materials. Second
Aloe-derived non-IAvD materials include materials with lower
immunostimulatory activity and at least 50% of the polysaccharides
generally having a molecular weight of 1,500,000 daltons or
less.
[0107] If the IAvD is mixed with a second Aloe-derived material,
whether a second IAvD or non-IAvD, of course, the percentage of
individual carbohydrates, and indeed the overall molecular weight
can be changed. Moreover, the overall activity of the mixture may
change. For example, a mixture of AloeEx and MagnAloe AG 20%:80%
mixture will generally have a lower overall molecular weight for
the polysaccharides, will have a lower overall activity than AloeEx
and will have mannose as the most prevalent carbohydrate.
[0108] Non-limiting examples of these second, non-IAvD Aloe-derived
materials include those selected from the group consisting of
MagnAloe AG, Aloe vera gel 1.times. (either natural, ground filet,
micronized filet or decolorized), Aloe vera gel 10.times. (either
natural or decolorized), Aloe vera gel 200.times. (either
dehydrated powder or spray dried powder), Aloe vera whole leaf
spray dried powder 100.times., Aloe vera whole leaf decolorized
liquid (either 1.times., 2.times., 4.times., or 10.times.)
acemannan, Aloe vera mucilaginous polysaccharide (AVMP) and
Manapol.
[0109] The amount of the second Aloe-derived material present in
the formulation generally is at least about 10% to about 90% by
weight relative to the amount of the total of the second
Aloe-derived material and the first IAvD. The greater the ability
of the second Aloe-derived material to solubilize, suspend,
emulsify and/or disperse the first IAvD, as well as the greater the
activity of the second Aloe-derived material, the less that may be
needed. In one preferred embodiment, the second Aloe-derived
material is MagnAloe AG and the amount of MagnAloe AG is greater
than the amount of AloeEx, and in a particularly preferred
embodiment, they are provided in a 4:1 weight ratio MagnAloe AG to
AloeEx. In the resulting homogenous mixture formed by the two,
MagnAloe AG would form about 80% thereof. As described and defined
herein, MagnAloe AG is not believed to qualify as an IAvD as
defined herein.
[0110] Other IAvD's and non-IAvD's include commercial
polysaccharide preparations such as Aloe vera mucilaginous
polysaccharide (AVMP) and Manapol, available from Carrington
Laboratories Inc. (Irving, Tex.) having immunostimulatory derived
compositions and which have been shown to cause activation of
NF-kappa Beta directed luciferase expression in THP-1 cells at 200
.mu.g/mL. In particular, AVMP and Manapol are believed to be
non-IAvD's having reported activation in the range of 2-4% relative
to maximal activation of NF-kappa Beta by 10 .mu.g/mL ELPS.
[0111] Any of the abovementioned compositions may be utilized in
the present invention either alone or in combination.
[0112] Skin Treatment
[0113] One aspect of the present invention is a method of improving
the appearance, texture, firmness, and/or elasticity of skin or
treating decreased skin elasticity in a subject in need thereof. A
subject in need thereof is any mammal, particularly, a human, in
need of enhanced or improved skin elasticity. This is accomplished
by administering to a subject in need thereof an effective amount
of an IAvD composition for a period of time sufficient to obtain
improvement in the subjects' skin elasticity. Neither improvement
nor treatment in accordance with the present invention in the
context of skin elasticity requires more than some discernable,
measurable improvement therein on a subjective basis. Unless stated
otherwise, this can be an improvement for any subject on a
subjective basis based on, for example, visual and tactile
observation.
[0114] However, often improvements are measured based on a
statistically significant improvement. As determined by ANOVA,
tests as measured by any one of the tests described herein when the
methods are utilized with a defined group of subjects to be tested
in a clinical study. For example, the Cutometer parameter Ur/Ue
describes skin elasticity as it relates to stretching and
contracting. Seventy percent of Group 1 subjects showed an
improvement in Cutometer parameter Ur/Ue. Relative to baseline,
this significant increase in parameter Ur/Ue is equivalent to a 29%
improvement at Week 12 for subjects who ingested the invention
supplement.
[0115] The amount of improvement necessary to achieve statistical
significance will be dictated by a number of factors including,
without limitation, the number of subjects, the length of the test,
the techniques used for measuring improvement in elasticity, the
condition of the subject, the type of dosage form, the daily dose
used and the like. Moreover, it is not a requirement of the present
invention that statistical significance be achieved using each and
every testing methodology, so long as it is obtained in at least
one test of elasticity.
[0116] The IAvD compositions of the invention can be administered
through any normal route. However, oral and topical routes,
including transdermal routes, are preferred. Of course, the route
of administration can dictate much of the design of the dosage
forms used to accomplish same.
[0117] The amount of IAvD composition found in a single dose will
be affected by whether or not it is topically applied or ingested
and for what purpose it is used. Generally, however, and
irrespective of dosage form selected, the amount of IAvD
composition used in accordance with the present invention used for
treating skin elasticity ranges from between about 0.0025 mg to
about 1 g per day. This means the amount of IAvDs, not the total
amount of Aloe-derived material. It may not be possible to
distinguish the individual components of the IAvD formulations. It
is sufficient for these purposes that the IAvDs possess these
properties prior to being mixed with other materials.
[0118] For example, an IAvD and an Aloe-derived non-IAvD may be
present or an IAvD may be present.
[0119] IAvD formulations for improved skin elasticity may be given
as a single dose or as divided doses throughout the day and
generally the number of doses given will range from between 1 to
about 6 per day, more preferably 1 to 4 per day, most preferably 1
to 2 per day. This is based on an IAvD exhibiting activity
characterized by increased NF-kappa B directed luciferase
expression in THP-1 cells to levels about 50%, as described herein,
of those achieved by ELPS having a concentration of about 10
.mu.g/mL when tested at a concentration of about 5 .mu.g/mL (that
generally found in AloeEx). Note that there is variation in this
level of monocyte/macrophage activation batch to batch, so more or
less PLL may be required to provide an equivalent level of
activity. Moreover, other IAvDs may be more or less
immunostimulatory when compared to PLL's. An amount of each of
these IAvDs that would give comparable activity to PLL's may be
needed.
[0120] Accordingly, the amount of the IAvD composition found in
each dosage form in accordance with the present invention can range
from between about 0.0005 milligram to about 1 gram depending upon
the daily dose, the number of doses as well as the route of
administration.
[0121] Most preferably, in terms of improved skin elasticity and/or
accelerated skin cell renewal by oral administration, the amount of
IAvD composition ranges from between about 0.2 milligram (mg) to
about 5 gram (g), more preferably from between about 5 mg to about
1 g, per day most preferably about 10 mg to about 500 mg. And when
provided topically the amount ranges from between about 0.0005 mg
to about 2.5 mg, more preferably from between about 0.0025 and
about 0.25 mg, and even more preferably between about 0.0025 mg to
about 0.025 mg. These amounts are based on the use of AloeEx at an
activity of about 5 micrograms/milliliter (.mu.g/mL). Amounts of
other IAvD's which yield similar results are contemplated.
[0122] In a preferred embodiment in accordance with the present
invention, the daily dosing of IAvD compositions continue for at
least 7 days, more preferably at least 2 weeks, more preferably at
least 2 months. Most preferably, the materials of the present
invention are administered continuously throughout a subject's life
to improve and maintain skin elasticity. However, the amount
administered for treatment may, eventually, be reduced to
accomplish maintenance of a certain desired level of skin
elasticity or accelerated skin cell renewal. Thus, after some
period of treatment, once skin elasticity has improved, a lower
dose may be administered for maintenance thereof.
[0123] When applied topically, the dosage may be spread directly
onto an afflicted area. It may in some cases be rubbed into the
skin. In other instances, it is not manipulated but is allowed to
be absorbed or to remain on the application area as applied. The
IAvD may also be applied on occlusive bandage or some other type of
wrap placed over it.
[0124] Alternatively, the IAvD may be formulated into a patch.
These topical applications may be designed to deliver the IAvD to
the surface of the skin or to some layer or layers of the skin
itself. Alternatively, it may be formulated to deliver the IAvD
transdermally for deeper local or systemic distribution. More about
the types of materials that can be used to formulate oral and
topical dosage forms are discussed herein.
[0125] When administered orally, IAvD compositions may be provided
in a tablet, capsule, powder, sachet, lozenge, gum candy or any
other ingestible form. The IAvD may also be provided as a liquid or
as a solid which is first dissolved in or mixed with a liquid
before ingestion. Dosage forms may be swallowable or may be solids
in the mouth and disintegrate in the mouth prior to ingestion. The
IAvD may even be sprinkled on food and ingested therewith.
[0126] Note that "accelerated cell regeneration" is an end point in
its own right and methods of enhancing accelerated cell
regeneration and products which accomplish this using IAvD
compositions are also contemplated. Accelerated cell regeneration
is the regeneration of skin cells under dead layers of skin which
are desirably exfoliated. This is accomplished using the same
techniques and products as described in connection with skin
elasticity.
[0127] Joint/Muscle Pain
[0128] Another aspect of the present invention is the improvement
or the treatment of joint and/or muscle pain in subjects in need
thereof by the use of IAvD compositions in accordance with the
present invention. Generally, the routes of administration and
dosage forms previously discussed in accordance with skin
elasticity apply equally well to these methods. Topical application
can affect a particular muscle or muscle group and/or a particular
joint. Systemic administration (orally or transdermally) may reduce
pain at that same location and/or may provide broader relief over
many muscle groups and/or joints.
[0129] Also as noted previously in connection with skin elasticity,
treatment in accordance with this aspect of the present invention
means a reduction in joint pain and/or muscle pain which can be
measured subjectively on a subject-by-subject basis. However, more
preferably, a successful product in accordance with the present
invention will provide adequate treatment in a statistically
significant way when given to a group of subjects treated over an
extended period of time. Desirably, at least 75% of subjects will
manifest a lower frequency of muscle and/or joint pain, a reduction
in pain intensity or a reduction in pain duration after twelve
weeks of treatment. This can be pain generally or in a specific
muscle or joint.
[0130] Improvement in a subject's condition in the context of joint
pain and/or muscle pain is often determined by a subjective rating
system scored by each individual rating his or her pain at a
specific site or generally before, during and/or after
treatment.
[0131] Also as previously noted, the amount of IAvD composition
administered in accordance with this aspect of the present
invention will vary depending upon the route of administration, the
area to be treated, the degree and type of pain and the like.
However, the amount generally will be in the same ranges as
previously described in connection with the methods of improving
skin elasticity. It is also possible in both this method and
methods of improving skin elasticity to administer the IAvD
composition both orally and topically at the same time or within
the same day, or on some alternative or complementary schedule
thereby providing both general and more specifically targeted
relief or improvement.
[0132] Any IAvD composition may be used in accordance with the
present invention. For example, any PLL, any AloeEx or any Aloeride
polysaccharide formulation exhibiting immunostimulatory properties
as described herein may be used. The present invention includes
pharmaceutical compositions and methods of using an IAvD,
optionally in combination with acceptable pharmaceutical carriers
or excipients.
[0133] Compositions
[0134] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose.
More specifically, a therapeutically effective amount means an
amount effective to prevent development of or to alleviate the
existing symptoms of the subject being treated. Determination of
the effective amounts is well within the capability of those
skilled in the art, especially in light of the detailed disclosure
provided herein.
[0135] The amount of the composition administered will be dependent
upon the condition being treated, the subject being treated, on the
subject's weight, the severity of the affliction, the manner of
administration and the judgment of the individual's physician or of
the individual.
[0136] As discussed above, suitable routes of administration may,
for example, include oral, transmucosal, topical or
transdermal.
[0137] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
powderizing, levigating, emulsifying, encapsulating, entrapping or
lyophilizing processes.
[0138] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries which facilitate processing of the
compositions and compounds into preparations which can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0139] For oral administration, the compositions can be
administered alone or formulated readily by combining the active
compositions with pharmaceutically acceptable carriers well known
in the art. Such carriers enable the compositions of the invention
to be formulated as tablets, pills, dragees, capsules, liquids,
gels, syrups, slurries, suspensions and the like, for oral
ingestion by a patient to be treated.
[0140] Pharmaceutical preparations for oral and/or topical use can
be obtained using a solid excipient, optionally grinding or milling
the individual components of a preparation and/or a resulting
mixture, and processing the components and/or mixture of granules,
after adding suitable auxiliaries, if desired, to obtain for
example, creams, lotions, serums, powders, effervescent
formulations, tablets or dragee cores. Granulation is, however, but
one of numerous known techniques which may be used.
[0141] Suitable excipients are, in particular, fillers such as
sugars, including lactose, sucrose, mannitol, or sorbitol;
cellulose preparations such as, for example, maize starch, wheat
starch, rice starch, potato starch, gelatin, gum tragacanth, methyl
cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
[0142] If desired, disintegrating agents may be added, such as PVP,
sodium starch glycolate, cross-linked polyvinyl pyrrolidone, agar,
or alginic acide or a salt thereof such as sodium alginate.
[0143] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active composition doses.
[0144] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compositions
may be dissolved or suspended in suitable liquids, such as fatty
oils, liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for such administration.
[0145] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in a conventional
manner.
[0146] For topical administration, emulsions, microemulsions, or
other topical creams, lotions, milks, washes, ointments, serums or
gels may also be prepared. Direct emulsification of AloeEx may be
possible, for example preparation of an AloeEx oil-in-water
emulsion wherein the hydrophobic core or an oil phase is largely
composed of AloeEx. Other surfactants or formulary excipients may
be added to physically stabilize the emulsion.
[0147] Pharmaceutical preparations suitable for topical
administration are especially creams, ointments and gels and also
pastes, foams, tinctures and solutions that contain from
approximately 0.05 to approximately 5% IAvD composition by weight.
Preferably the preparations are about 0.5% IAvD composition by
weight. These compositions include the total amount of Aloe-derived
materials including, as appropriate, at least one IAvD and any
other Aloe based materials. In the formulations discussed in the
clinical studies, for example the 0.5% IAvD composition was made up
of a 1:4 mixture of AloeEx and MagnAloe AG.
[0148] Creams or lotions may be oil-in-water emulsions that
typically contain more than 50% water. As oily base there are used
especially fatty alcohols, especially those containing from 12 to
18 carbon atoms, for example lauryl, cetyl or stearyl alcohol,
fatty acids, especially those containing from 10 to 18 carbon
atoms, for example palmitic or stearic acid, liquid to solid waxes,
for example isopropyl myristate, wool wax or beeswax, and/or
hydrocarbons, especially liquid, semi-solid or solid substances or
mixtures thereof, for example petroleum jelly (petrolatum) or
paraffin oil. Suitable emulsifiers are surface-active substances
having predominantly hydrophilic properties, such as corresponding
non-ionic emulsifiers, for example fatty acid esters of
polyalcohols or ethylene oxide adducts thereof, especially
corresponding fatty acid esters with (poly)ethylene glycol,
(poly)propylene glycol or sorbitol, the fatty acid moiety
containing especially from 10 to 18 carbon atoms, especially
partial glycerol fatty acid esters or partial fatty acid esters of
polyhydroxyethylene sorbitan, such as polyglycerol fatty acid
esters or polyoxyethylene sorbitan fatty acid esters (Tweens), and
also polyoxyethylene fatty alcohol ethers or fatty acid esters, the
fatty alcohol moiety containing especially from 12 to 18 carbon
atoms and the fatty acid moiety especially from 10 to 18 carbon
atoms, especially those having approximately from 2 to 23 ethylene
glycol or ethylene oxide units, such as
polyhydroxyethylenecetylstearyl ether (for example Cetomacrogol),
polyhydroxyethylene-(4)-lauryl ether and
polyhydroxyethyleneglycerol fatty acid ester (for example Tagat S),
or corresponding ionic emulsifiers, such as alkali metal salts of
fatty alcohol sulfates, especially having from 12 to 18 carbon
atoms in the fatty alcohol moiety, for example sodium lauryl
sulfate, sodium cetyl sulfate or sodium stearyl sulfate, which are
usually used in the presence of fatty alcohols, for example cetyl
alcohol or stearyl alcohol.
[0149] Additives to the aqueous phase are agents that prevent the
creams from drying out, for example humectants, such as
polyalcohols, such as glycerol, sorbitol, propylene glycol and/or
polyethylene glycols, and also preservatives, perfumes, etc.
[0150] Ointments or lotions may be water-in-oil emulsions that
typically contain up to 70%, but preferably from approximately 20%
to approximately 50%, water or aqueous phase. Suitable as fatty
phase are especially hydrocarbons, for example petroleum jelly,
paraffin oil and/or hard paraffins, which, in order to improve the
water-binding capacity, preferably contain suitable hydroxy
compounds, such as fatty alcohols or esters thereof, for example
cetyl alcohol or wool wax alcohols, or wool wax.
[0151] Emulsifiers are corresponding lipophilic substances, such as
sorbitan fatty acid esters (Spans), for example sorbitan oleate
and/or sorbitan isostearate. Additives to the aqueous phase are,
inter alia, humectants, such as polyalcohols, for example glycerol,
propylene glycol, sorbitol and/or polyethylene glycol, and also
preservatives, perfumes, etc.
[0152] Microemulsions are isotropic systems based on the following
four components: water, an emulsifier, for example of the type
indicated above, such as a surfactant, for example emulgin, a
lipid, such as a non-polar oil, for example paraffin oil, and an
alcohol containing a lipophilic group, for example
2-octyldodecanol. If desired, other additives may be added to the
microemulsions.
[0153] Fatty ointments are water-free and contain as base
especially hydrocarbons, for example paraffin, petroleum jelly
and/or liquid paraffins, also natural or partially synthetic fat,
such as fatty acid esters of glycerol, for example coconut fatty
acid triglyceride, or preferably hardened oils, for example
hydrogenated groundnut oil or castor oil, also fatty acid partial
esters of glycerol, for example glycerol mono- and di-stearate, and
also, for example, the fatty alcohols increasing the
water-absorption capacity, emulsifiers and/or additives mentioned
in connection with the ointments.
[0154] With gels, a distinction is made between aqueous gels,
water-free gels and gels having a low water content, which gels
consist of swellable, gel-forming materials. There are used
especially transparent hydrogels based on inorganic or organic
macromolecules. High molecular weight inorganic components having
gel-forming properties are predominantly water-containing
silicates, such as aluminium silicates, for example bentonite,
magnesium aluminium silicates, for example Veegum, or colloidal
silicic acid, for example Aerosil. As high molecular weight organic
substances there are used, for example, natural, semi-synthetic or
synthetic macromolecules. Natural and semi-synthetic polymers are
derived, for example, from polysaccharides containing a great
variety of carbohydrate components, such as celluloses, starches,
tragacanth, gum arabic and agar-agar, and gelatin, alginic acid and
salts thereof, for example sodium alginate, and derivatives
thereof, such as lower alkylcelluloses, for example methyl- or
ethyl-cellulose, carboxy- or hydroxy-lower alkylcelluloses, for
example carboxymethyl- or hydroxyethyl-cellulose. The components of
synthetic gel-forming macromolecules are, for example, suitably
substituted unsaturated aliphatic compounds such as vinyl alcohol,
vinylpyrrolidine, acrylic or methacrylic acid. Examples of such
polymers are polyvinyl alcohol derivatives, such as polyviol,
polyvinylpyrrolidines, such as collidone, polyacrylates and
polymethacrylates, especially having a molecular weight of from
approximately 80,000 to approximately 1 million, or salts thereof,
such as Rohagit S, Eudispert or Carbopol. Customary additives, such
as preservatives, stabilizers, or perfumes, may be added to the
gels.
[0155] Foams are administered, for example, from pressurized
containers and are liquid oil-in-water emulsions in aerosol form;
unsubstituted or halogenated hydrocarbons, such as alkanes, for
example propane or butane, or chlorofluoro-lower alkanes, for
example dichlorodifluoromethane and dichlorotetrafluoroethane, are
used as propellant. As oil phase there are used, inter alia,
hydrocarbons, for example paraffin oil, fatty alcohols, for example
cetyl alcohol, fatty acid esters, for example isopropyl myristate,
and/or other waxes. As emulsifiers there are used, inter alia,
mixtures of emulsifiers having predominantly hydrophilic
properties, such as polyoxyethylene sorbitan fatty acid esters, and
emulsifiers having predominantly lipophilic properties, such as
sorbitan fatty acid esters. The customary additives, such as
preservatives, etc., are also added.
[0156] Tinctures and solutions generally have an ethanolic base, to
which water may be added and to which there are added, inter alia,
polyalcohols, for example glycerol, glycols and/or polyethylene
glycol, as humectants for reducing evaporation, and fat-restoring
substances, such as fatty acid esters with low molecular weight
polyethylene glycols, that is to say lipophilic substances that are
soluble in the aqueous mixture, as a replacement for the fatty
substances removed from the skin by the ethanol, and, if necessary,
other adjuncts and additives. Suitable tinctures or solutions may
also be applied in spray form by means of suitable devices.
[0157] The manufacture of the topically administrable
pharmaceutical preparations is effected in a manner known per se,
for example by dissolving or suspending the active ingredient in
the base or, if necessary, in a portion thereof. When the active
ingredient is administered in the form of a solution, it is
generally dissolved in one of the two phases before emulsification;
when the active ingredient is administered in the form of a
suspension, it is mixed with a portion of the base after
emulsification and then added to the remainder of the
formulation.
[0158] An IAvD composition or combination of IAvD compositions may
by applied as a topical medication for local effect, such as
enhanced skin elasticity and/or accelerated skin cell growth or
regeneration. This is a treatment which is applied to the skin and
acts directly at the site of application. It must be placed at the
site of pathology, and it directly absorbs through the skin and
exerts its effect. It is believed that there is no systemic
absorption needed for the effect to occur.
[0159] An IAvD composition or combination of IAvD compositions may
be applied as a topical medication for systemic effect. This is a
treatment which is applied to the skin and acts at a site distant
from the site of application to reduce muscle or joint pain,
enhance skin elasticity or both. The medication may be applied
anywhere on the body and achieve its effect. The formulation must
absorb through the skin, enter the bloodstream, reach effective
levels in the blood, and then travel to its site of action to exert
its effect.
[0160] It will be known to persons of ordinary skill in the art
that skin penetration enhancers may be used to facilitate systemic
delivery of the IAvD composition or compositions or even deeper
muscle or joint penetration. In this regard, penetration enhancers
have been used to increase the permeability of the dermal surface
to drugs, and are often proton accepting solvents such as dimethyl
sulfoxide (DMSO) and dimethylacetamide. Other penetration enhancers
that have been studied and reported as effective include
2-pyrrolidine, N,N-diethyl-m-toluamide (Deet),
1-dodecal-azacycloheptane-2-one N,N-dimethylformamide,
N-methyl-2-pyrrolidine, calcium thioglycolate, hexanol, fatty acids
and esters, pyrrolidone derivatives, derivatives of 1,3-dioxanes
and 1,3-dioxolanes, 1-N-dodecyl-2-pyrrolidone-5-carboxylic acid,
2-pentyl-2-oxo-pyrrolidineacetic acid,
2-dodecyl-2-oxo-1-pyrrolidineacetic acid,
1-azacycloheptan-2-one-2-dodecylacetic acid, and aminoalcohol
derivatives, including derivatives of 1,3-dioxanes, among many
others.
[0161] An IAvD composition or combination of IAvD compositions may
be included in dosage forms used to treat any responsive condition,
such as, joint pain, muscle pain, loss of skin elasticity or skin
wrinkles.
[0162] The pharmaceutical compositions also may comprise suitable
solid or gel phase carriers or excipients. Examples of such
carriers or excipients include but are not limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as polyethylene
glycols.
[0163] Stable compositions for treatment, prevention, or
amelioration of any of the conditions, diseases, afflictions,
wounds and the like, including, without limitation one or more
symptoms of joint or muscle pain and for enhancing skin elasticity
are provided. The compositions provided herein are stable
formulations of an immunostimulatory Aloe vera extract or a
derivative thereof, in a pharmacologically suitable fluid that are
stable during long term storage. The compositions are may be
suitable for direct administration to a subject in need thereof or
may be in a concentrated form. Pharmacologically suitable fluids
include, but are not limited to, polar fluids, including protic
fluids. In certain embodiments herein, the compositions are aqueous
solutions. In other embodiments, the compositions are non aqueous
or emulsions.
[0164] The IAvD compositions may be formulated with a
pharmacologically suitable fluid or solvent. Pharmacologically
suitable fluids include, but are not limited to, polar solvents,
including, but not limited to, compounds that contain hydroxyl
groups or other polar groups. Such solvents include, but are not
limited to, water or alcohols, such as ethanol, isopropanol, and
glycols including propylene glycol, polyethylene glycol,
polypropyleneglycol, glycol ether, glycerol and polyoxyethylene
alcohols.
[0165] Polar solvents also include protic solvents, including, but
not limited to, water, aqueous saline solutions with one or more
pharmaceutically acceptable salt(s), alcohols, glycols or a mixture
thereof. For a saline solution as the solvent or as a component
thereof, particularly suitable salts are those which display no or
only negligible pharmacological activity after administration.
[0166] The compositions provided herein also may include excipients
and additives. The particular excipient or additive for use in the
compositions for long term storage provided herein may be
determined empirically using methods well known to those of skill
in the art. Excipients and additives are any pharmacologically
suitable and therapeutically useful substance which is not an
active substance. Excipients and additives generally have no
pharmacological activity, or at least no undesirable
pharmacological activity. The excipients and additives include, but
are not limited to, surfactants, stabilizers, complexing agents,
antioxidants, or preservatives which prolong the duration of use of
the finished pharmaceutical formulation, flavorings, vitamins, or
other additives known in the art.
[0167] Complexing agents include, but are not limited to,
ethylenediaminetetraacetic acid (EDTA) or a salt thereof, such as
the disodium salt, citric acid, nitrilotriacetic acid and the salts
thereof. In one embodiment, the complexing agent is EDTA.
Preservatives include, but are not limited to, those that protect
the solution from contamination with pathogenic particles,
including benzalkonium chloride or benzoic acid, or benzoates such
as sodium benzoate. Antioxidants include, but are not limited to,
vitamins, provitamins, ascorbic acid, vitamin E or salts or esters
thereof.
[0168] The compositions provided herein also may include a
cosolvent, which increases the solubility of additives or the
active ingredient(s). The particular cosolvent for use in the
compositions for long term storage provided herein may be
determined empirically using methods well known to those of skill
in the art. Cosolvents for use herein include, but are not limited
to, hydroxylated solvents or other polar solvents, such as alcohols
such as isopropyl alcohol, glycols such as propylene glycol,
polyethylene glycol, polypropylene glycol, glycol ether, glycerol,
and polyoxyethylene alcohols.
[0169] In other of embodiments, the compositions further contain a
buffer, including, but not limited to, citric acid/phosphate,
acetate, barbital, borate, Britton-Robinson, cacodylate, citrate,
collidine, formate, maleate, McIlvaine, phFosphate, Prideaux-Ward,
succinate, citrate-phosphate-borate (Teorell-Stanhagen), veronal
acetate, MES (2-(N-morpholino)ethanesulfonic acid), BIS-TRIS
(bis(2-hydroxyethyl)imino-tris-(hydroxymethyl)methane), ADA
(N-(2-acetamido)-2-iminodiacetic acid), ACES
(N-(carbamoylmethyl)-2-aminoethanesulfonic acid), PIPES
(piperazine-N,N'-bis(2-ethanesulfonic acid)), MOPSO
(3-(N-morpholino)-2-hydroxypropanesulfonic acid), BISTRIS PROPANE
(1,3-bis(tris(hydroxymethyl)methylamino)propane), BES
(N,N-bis(2-MOPS hydroxyethyl)-2-aminoethanesulfonic acid),
(3-(N-morpholino)propanesulfonic acid), TES
(N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), HEPES
(N-(2-hydroxyethyl)piperazine-N'-(2-eth-anesulfonic acid), DIPSO
(3-(N,N-bis(2-hydroxyethyl)amino)-2-hydroxypropanesulfonic acid),
MOBS (4-(N-morpholino)-butanesulfonic acid), TAPSO
(3-(N-tris(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic
acid), TRIZMA.RTM. (tris(hydroxymethylaminomethane), HEPPSO
(N-(2-hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid),
POPSO (piperazine-N,N'-bis(2-hydroxypropanesulfonic acid)), TEA
(triethanolamine), EPPS (N-(2-hydroxyethyl)piperazine-N'-acid),
(3-propanesulfon-ic acid), TRICINE
(N-tris(hydroxy-methyl)methylglycine), GLY-GLY (glycylglycine),
BICINE (N,N-bis(2-hydroxyethyl)glycine), HEPBS
(N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)), TAPS
(N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), AMPD
(2-amino-2-methyl-1,3-propanediol), and/or any other buffers known
to those of skill in the art.
[0170] The particular buffer and buffer concentration of a given
composition provided herein may be determined empirically using
standard stability assays well known to those of skill in the
art.
[0171] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration. Compositions comprising a composition of the
invention formulated in a compatible pharmaceutical carrier may
also be prepared, placed in an appropriate container, and labeled
for treatment of an indicated condition. Suitable conditions
indicated on the label may include treatment of a condition.
[0172] The formulation may also contain optional ingredients
including, for example, herbs, vitamins, minerals, enhancers,
colorants, sweeteners, flavorants, inert ingredients, and the like.
Such optional ingredients may be either naturally occurring or
concentrated forms. Selection of one or several of these
ingredients is a matter of formulation, design, consumer preference
and end-user.
[0173] In accordance with another aspect of the present invention,
various materials have been found which can be used to allow for
the production of a homogenous dispersion, emulsion, suspension,
solution and the like of an IAvD. In particular, it has been found
that certain highly active, but generally insoluble IAvDs can be
dispersed, suspended, emulsified and/or solubilized by using a
second Aloe-derived material. That second Aloe-derived material may
be a second IAvD or a material which has insufficient activity to
qualify as an IAvD. The amount of the second Aloe-derived material
should be an amount which is sufficient to allow for, as desired,
the production of a homogenous dispersion, solution, homogeneous
suspension, or homogeneous emulsion of the first IAvD. Generally at
least about 10% of the second Aloe-derived material is required, by
weight percent, relative to the weight percent of the total weight
of the first IAvD and the second Aloe-derived material.
[0174] In a particularly preferred embodiment in accordance with
the present invention, the first IAvD is AloeEx. Any Aloe-derived
material that can improve the processability of the first IAvD may
be used. The second Aloe-derived material generally is composed of
polysaccharides in which at least 50% of the polysaccharides have
molecular weights of 1,500,000 daltons or less. This second
Aloe-derived material, can include, without limitation, an Aloe
based material selected from the group consisting of MagnAloe AG,
Aloe vera gel 1.times. (either natural, ground filet, micronized
filet or decolorized), Aloe vera gel 10.times. (either natural or
decolorized), Aloe vera gel 200.times. (either dehydrated powder or
spray dried powder), Aloe vera whole leaf spray dried powder
100.times., Aloe vera whole leaf decolorized liquid (either
1.times., 2.times., 4.times., or 10.times.), acemannan, Aloe vera
mucilaginous polysaccharide (AVMP) and Manapol. MagnAloe AG is a
powder available from First Aloe of Costa Rica, S.A. Information
can be obtained at www.firstAloevera.com/magnAloe_information.htm.
MagnAloe AG has polysaccharide levels of over 800 kiladaltons (KDa)
as indicated below. This is on average 40% higher than spray dried
200-1 Aloe vera powder sold on the market. MagnAloe AG is reported
to contain 42.65 mg/g of polysaccharides having a molecular weight
(MW) of 50-200 KDa, 118.5 mg/g having a MW of 200-800 KDa, 366.2
mg/g having a MW 800-1400 KDa, 155.8 mg/g having a MW 1400-2000 KDa
of a total polysaccharide of 683.1 mg/g. The water solubility of
the MagnAloe generally is about 15%.
[0175] Aloe vera gel 1.times. (either natural, ground filet,
micronized filet or decolorized), Aloe vera gel 10.times. (either
natural or decolorized), Aloe vera gel 200.times. (either
dehydrated powder or spray dried powder), Aloe vera whole leaf
spray dried powder 100.times., Aloe vera whole leaf decolorized
liquid (either 1.times., 2.times., 4.times., or 10.times.) are
available for Improve USA, Inc., 215 Dalton Drive, Suite D, DeSoto,
Tex. 75115. The water solubility of Aloe vera gel 200.times.
generally is about 90-100%.
[0176] Acemannan, Aloe vera mucilaginous polysaccharide (AVMP) and
Manapol are available from Carrington Laboratories, Irving, Tex. In
one particularly preferred embodiment, the single most prevalent
carbohydrate in the IAvD is glucose, and the single most prevalent
carbohydrate in the second Aloe-derived material is mannose.
[0177] In one embodiment, the amount of the first IAvD ranges from
about 10% to about 90% by weight based on the total amount of the
first IAvD and second Aloe-derived material and the amount of the
second material ranges from about 90% to about 10% based on the
amount of the first IAvD and second Aloe-derived material. In one
preferred embodiment, the amount of the second material is greater
than the amount of the first IAvD, and in a particularly preferred
embodiment, they are provided in a 4:1 weight ratio second material
to first IAvD. In the resulting homogenous mixture formed by the
two, the second material would form about 80% thereof. In still
another preferred aspect, the first IAvD is AloeEx and the second
material is an Aloe-derived material that includes polysaccharides
in which at least 50% of the polysaccharide have molecular weights
of about 1,500,000 daltons or less, such as MagnAloe AG and the
amount of the AloeEx ranges from about 10% to about 90% by weight
based on the amount of the AloeEx and MagnAloe and the amount of
the MagnAloe AG ranges from about 90% to about 10% based on the
amount of the AloeEx and MagnAloe AG. In one preferred embodiment,
the amount of the MagnAloe AG is greater than the amount of the
AloeEx, and in a particularly preferred embodiment, they are
provided in a 4:1 weight ratio MagnAloe AG to AloeEx. In the
resulting homogenous mixture formed by the two, the MagnAloe AG
would form about 80% thereof.
[0178] In one embodiment, AloeEx and a second material, preferably
a second Aloe-derived material may be formulated by mixing raw
AloeEx with water which has been heated to approximately 80.degree.
C. The mixture is agitated for at least 10 minutes to homogenize
the material. The mixture is then filtered, preferably with a
filter having a pore size no larger than 15 microns. The resulting
filtrate may be utilized in formulating a variety of IAvD
compositions as described herein.
[0179] Homogenization of AloeEx may or may not require heat
depending on the equipment utilized. For example homogenizers may
be used as part of the process to render a uniform particulate
suspension. A commonly known "color" homogenizer may be used to
reduce AloeEx particle size to formulate an aqueous AloeEx
suspension and/or emulsion.
[0180] A characterization of AloeEx is shown in FIG. 1. The
material forms a brown to green flaked powder.
[0181] Referring to FIG. 1, a chart depicts one lot of AloeEx
material (batch 56) with associated sugar content based on the mole
% of total carbohydrates.
[0182] FIG. 1 shows the results of glycosyl composition analysis
performed by The University of Georgia, Complex Carbohydrate
Research Center by combined gas chromatography/mass spectrometry
(GC/MS) of the per-O-trimethylsilyl (TMS) derivatives of the
monosaccharide methyl glycosides produced from the sample by acidic
methanolysis.
[0183] Methyl glycosides were first prepared from a dry sample
provided by the inventors by methanolysis in 1 M HCl in methanol at
80.degree. C. (18-22 hours), followed by re-N-acetylation with
pyridine and acetic anhydride in methanol (for detection of amino
sugars). The samples were then per-O-trimethylsilylated by
treatment with Tri-Sil (Pierce) at 80.degree. C. (0.5 hours).
[These procedures were carried out as previously described in
Merkle and Poppe (1994) Methods Enzymol. 230:1-15; York, et al.
(1985) Methods Enzymol. 118:3-40.] A significant level of
precipitate was noted immediately upon addition of Tri Sil. GC/MS
analysis of the TMS methyl glycosides was performed on an HP 5890
GC interfaced to a 5970 MSD, using an All Tech EC-1 fused silica
capillary column (30 m.times.0.25 mm ID).
[0184] The monosaccharides were identified by their retention times
in comparison to standards, and the carbohydrate character of these
were authenticated by their mass spectra. For interpreting the mass
spectral data, fragment ion 73 is the characteristic base fragment
for all TMS methyl glycosides, 204 and 217 are characteristic of
neutral sugars, and 173 is characteristic of amino sugars. Fragment
217 is also characteristic of uronic acids and fragment ion 298 is
characteristic of the sialic acids.
[0185] FIG. 1 identifies the glcosyl residue composition by mole %.
Values are expressed as mole percent of total carbohydrate. The
total percent carbohydrate was calculated to be 8.6%. The
constituent values measured were glucose 61.1%, arabinose 9.5%,
galactose 9.2%, galacturonic acid 7.9%, mannose 6.6%, xylose 1.6%,
ribose 1.5%, rhamnose 1.4% and fucose 1.1%.
[0186] Clinical Study--Skin Elasticity--Topical Formulation
[0187] One clinical study, as outlined below demonstrates the
effect of one IAvD composition, sold under the trademark
ALOERIDE.RTM.. ALOERIDE.RTM. may be administered topically to
improve skin elasticity and may also be used as an ingredient in a
nutritional supplement.
[0188] The effect of an ALOERIDE.RTM. gel 0.5% Product #10849LL,
Lot #11939 on skin elasticity or firmness, was determined in a
clinical study by Consumer Product Testing, Inc. of Fairfield, N.J.
The test formulation (ALOERIDE.RTM. gel 0.5%) comprised a 4:1
mixture by weight of MagnAloe AG and AloeEx and was mixed with
water, alcohol (10%) and a small amount of Carbopol (0.5%) then
dispersed using a homogenizer. The dispersion of AloeEx, water,
alcohol and a small amount of carbopol was used to make up the test
gel suspension.
[0189] The test product was supplied in small containers sufficient
to last for 8 weeks when applied to defined facial areas (alcohol
performs as a preservative).
[0190] The clinical trial was a double test of AloeEx function:
Carbopol tends to be drying, a negative test performance factor
estimated at (-) 15%. The clinical trial proved the ability of
AloeEx/MagnAloe AG to: 1) Reduce fine lines and wrinkles in a
statistical majority of the test subjects at 4 weeks and at 8
weeks; 2) The ability of AloeEx to overcome any negative dryness
associated with carbopol.
[0191] AloeEx used in this clinical trial was
non-dispersible/insoluble and difficult to homogenize. When mixed
1:4 with MagnAloe AG, however, a homogenous gel/emulsion was
realized.
[0192] Thirty-one qualified male and female subjects, ranging in
age from 40 to 65 years, were selected for study. The test material
indicated a significant reduction in skin rebound time at both the
4 week and 8 week evaluation intervals. Study participants
experience up to a 72% improvement in skin elasticity or firmness
at the conclusion of the study (FIG. 2) and 100% of the study
participants experienced general improvement in skin elasticity.
Study demographics based on age and gender are shown in FIG. 3.
[0193] Subjects reported to the laboratory with cleansed faces of
the first day of testing for a base measurement. Prior to
conducting any readings or scoring, subject equilibrated in an
environmentally controlled room for 15 minutes at ambient
conditions; temperature at 21.degree. C.+/-2.degree. C.; relative
humidity at 40%+/-2%.
[0194] Skin elasticity (firmness) was measured using the pinch test
as disclosed by Shanahan. R. W., et al, Parameters for Assessing
the Efficacy of Skin Care Products. Drug & Cosmetic Industry,
140, No. 1, 42-48, 1987. Skin elasticity or firmness scores were
obtained by an expert tester by grasping the skin of the lateral,
suborbital area of the eyes between thumb and forefinger. The
resultant folds were released and timing of all visible movement of
the skin, from release until no further motion, was observed and
measured to the nearest one hundredth of a second. Scores were
recorded on a Data Recording form for statistical analysis of the
measurements.
[0195] Improvements in skin elasticity are considered substantiated
if the is a statistically significant (P less than or equal to 0.05
using the dependent t-Test) decrease in the mean score between the
baseline measurements and the subsequent 4 week and 8 weeks post
treatment scoring intervals.
[0196] FIG. 2 shows a statistically significant decrease in mean
skin rebound time in seconds (an indicator of an increase in skin
elasticity or firmness). The mean of all baseline measurements was
2.09 seconds and 1.96 seconds for left and right measurements
respectively. A significant decrease was observed at the week 4
test interval yielding 1.60 seconds and 1.53 seconds respectively.
Further decreases in mean skin rebound time to 1.19 seconds and
1.18 seconds, for left and right, were measured at the week 8 test
interval. As previously discussed, the results were statistically
significant as p was calculated to be less than or equal to zero
for all measurement intervals (FIG. 2).
[0197] Another clinical study, as outlined below demonstrates the
effect of another IAvD composition administered orally to improve
muscle and/or joint pain and skin elasticity.
[0198] Clinical Study--Skin Elasticity--Joint/Muscle Pain--Oral
Formulation
[0199] A study was conducted by Clinical Research Laboratories,
Inc. to evaluate the efficacy of an oral supplement of the instant
invention designed to improve skin firmness and elasticity and to
diminish muscle and joint pain during a 12-week period. (A
Double-Blind Placebo Controlled Pilot Study to Assess the Safety
and Efficacy of an Oral Supplement Designed to Diminish Aging,
study number CRL34706 herein incorporated by reference in its
entirety). Placebo tablets used in this clinical study contained
microcrystalline cellulose, croscarmellose sodium, stearic acid,
silicon dioxide, and magnesium stearate. The active tablets
contained the same inactive ingredients as the placebo tablets with
50 mg of a 4:1 mixture of MagnAloe AG and AloeEx. These tablets
were given twice daily, orally, at the time of arising and once at
bedtime.
[0200] This study was initiated on Aug. 30, 2006 and was completed
on Dec. 15, 2006. Initially, safety screening was performed as
follows on each subject.
[0201] Safety Evaluations Blood Analysis
[0202] Blood was drawn from each subject at designated study
visits, and the following blood tests were performed:
[0203] Complete Blood Count (CBC) with differential and platelet
count.
[0204] Chemistry Panel+HDL AST (GOT), ALT (GPT), gamma Gt, LDH
(LD), Total Protein (TP), Uric Acid, (UA, total Billirubin, Urea
Nitrogen (UN), total cholesterol (HDL, LDL, TG), Na, K, Ca, Cl.
[0205] Urine Analysis
[0206] A urine specimen was obtained from each subject at
designated study visits, and the following urine tests were
performed:
[0207] Glucose, Protein, Uribilinogen, Blood, pH
[0208] Sitting Vital Signs
[0209] An assessment of sitting vital signs (i.e., pulse, blood
pressure and respirations) was recorded at the screening visit.
[0210] Pregnancy Test
[0211] All panelists (with the exception of post-menopausal women)
were given a urine pregnancy test at the screening visit.
[0212] Dermal Evaluations
[0213] In order to determine the effects of treatment on the skin,
a CRL Technician performed dermal evaluations on each subject's
face to examine for the presence of erythema, edema and dryness.
The extent and/or severity of evidence of dermal irritation was
recorded utilizing the following scoring scale:
[0214] Scoring Scale for Erythema, Edema and Dryness
[0215] 0=None 1=Mild 2=Moderate 3=Severe
[0216] Efficacy Evaluations
[0217] Cutometer
[0218] The suction is generated by a variable vacuum pump, and the
depth of penetration of the skin into the probe is measured
optically with an accuracy of 0.01 mm. The probe is attached to a
computer, which controls the vacuum application and plots skin
deformation as a function of time. From the resulting curve, a
number of variables can be extrapolated including immediate,
delayed and final distention and immediate retraction (Elsner
1990).
[0219] These variables estimate the elastic, viscoelastic and
purely viscous behavior of the skin (Barel et al. 1995).
[0220] The following parameters were measured:
[0221] The final distention, Uf, measured at 10 seconds. The
immediate distention, Ue, measured at 0.1 seconds. The delayed
distention, Uv. Immediate retraction, Ur
[0222] The deformation parameters are extrinsic parameters
dependent on skin thickness.
[0223] In order to circumvent the measurement of skin thickness,
the following ratios were used to evaluate the elastic nature of
the skin (Elsner 1990, Cua et al. 1990, Krushe and Wonet 1995 and
Dobrev 1995):
[0224] Ur/Ue is the biological elasticity of the skin. It measures
the ability of the skin to regain its initial configuration after
deformation. A value of 1 indicates 100% elasticity.
[0225] Uv/Ue is the ratio between delayed and immediate
deformation, i.e., it is the viscoelastic to elastic ratio. An
increase in the value of this ratio indicates that there has been
an increase in the viscoelastic portion of the deformation and/or
relative decrease of the elastic part.
[0226] Ur/Uf is a measure of the net elasticity of the skin.
[0227] This study consisted of a pre-screening visit, during which
time subjects were examined by a Clinical Research Technician to
determine eligibility. Eligible subjects exhibited mild to moderate
fine lines and wrinkles on the crow's feet area of the face and
experienced chronic muscle and/or joint pain. At this time,
subjects were informed not to eat or drink anything after 9:00 p.m.
on the evening prior to the scheduled screening visit, in
preparation for the blood and urine analysis.
[0228] Prior to study initiation, subjects arrived at the CRL
laboratory for the scheduled screening visit. Blood was drawn from
each perspective subject and the forearm utilized for blood
collection was recorded on a case report form. A urine specimen was
obtained from each panelist for urine analysis, an assessment of
sitting vital signs (i.e., pulse, blood pressure and respirations)
was performed and recorded, and all panelists (with the exception
of pre-menopausal subjects) were given a urine pregnancy test.
Subjects discontinued the use of all oral supplements for the
remainder of the conditioning period and for the duration of the
study, with the exception of the oral supplements provided to each
subject during the study.
[0229] To standardize the skin condition of the study population at
baseline and minimize variability attributable to use of different
skin care regimens, a one week conditioning period began the study.
Subjects were required to undergo a one week conditioning period,
during which time a glycerin soap was used for all facial washing
prior to study initiation. At the screening visit, each subject was
provided with detailed study instructions, a bar of glycerin soap
to use for all facial washing, Cetaphil.RTM. Daily Facial
Moisturizer SPF 15 to use once per day, and a Daily Diary to record
soap usage and verify compliance. Subjects began cleansing the face
with the soap twice daily (morning and night), beginning seven days
prior to the baseline visit. At this screening visit, every subject
completed a screening questionnaire.
[0230] Qualified subjects had normal blood and urine test results
and each panelist's vital signs were within the normal range, as a
result of the previously mentioned screening visit safety
evaluations. Additionally, eligible subjects who agreed to
follow-up visits and study requirements and met all of the
Inclusion/Exclusion Criteria, were to be impaneled for the
study.
[0231] Upon returning to the CRL testing facility for the baseline
visit, subjects acclimated to ambient laboratory conditions for a
period of 30 minutes prior to evaluations. Following the
acclimation period, a dermal evaluation of the face of each subject
was performed by a trained CRL Technician, for the presence of
erythema, edema and dryness. For each subject, either the right or
the left side of the face was selected for measurement, as
determined by a computer-generated randomization code (FIG. 4).
Measurement designation remained consistent throughout the study.
Cutometer readings were captured from the cheekbone region below
the eye on the same side of the face. All measurements were
appropriately recorded. Each subject completed a baseline consumer
perception questionnaire in order to evaluate the type and
frequency of muscle and joint pain.
[0232] A specified test material was randomly assigned to each
subject, such that Group 1 panelists received the active tablets,
and Group 2 received the placebo tablets. Subject group
designations were determined in accordance with a
computer-generated randomization code. Treatment designation/group
assignment remained consistent throughout the study (FIGS.
5-6).
[0233] Each subject was provided with a designated test material,
detailed study instructions and a new Daily Diary in which to
record daily times of supplement use, daily application of the
Cetaphil.RTM. Daily Facial Moisturizer SPF 15, and any suggestive
comments pertaining to supplement usage.
[0234] The printed study instructions detailed study procedures for
each visit and the appointment dates and times of the return
visit(s) to Clinical Research Laboratories, Inc. Subjects were
instructed to continue the use of the provided glycerin soap, twice
daily, as well as the use of the Cetaphil.RTM. Daily Facial
Moisturizer SPF 15, once per day.
[0235] Each subject was contacted, via telephone, by a CRL
Technician for a Week 4 compliance check. After 8 weeks of
ingesting the designated oral supplement, subjects reported to the
CRL testing facility and each panelist acclimated to ambient
temperature and humidity for a period of 30 minutes. Following
acclimation, a dermal evaluation of the face of every subject was
performed by a trained CRL Technician. Cutometer readings were
taken. These procedures and evaluations were performed and
recorded, as previously explained for the baseline study
interval.
[0236] Subjects reported to Clinical Research Laboratories, Inc.
following 12 weeks of supplement use and each subject acclimated to
ambient temperature and humidity for a period of 30 minutes. Blood
was drawn from each subject and the forearm used for blood
collection was recorded on a case report form. A final urine
specimen was obtained from each panelist. A dermal evaluation of
the face was performed by a trained CRL Technician, for the
presence of erythema, edema and dryness. Cutometer readings were
captured from the cheekbone region below the eye on the same side
of the face. All measurements were appropriately recorded.
[0237] At Week 12, all subjects completed a final questionnaire and
unused portions of test materials and Daily Diaries were
returned.
[0238] Statistical Methods
[0239] The primary efficacy parameters yielding significant results
for this study are Cutometer measurements and consumer perception
questionnaires.
[0240] One-way ANOVA tests (repeat-measurement) were applied to
each group to determine if there were differences between baseline
and Week 8, and baseline and Week 12 for all the parameters. If
overall F-test was significant, Tukey's multiple comparison test
was further applied to determine where the significant differences
exist. The ANOVA test uses the F-test to determine whether there
exists a significant difference among means of treatments or time
intervals. When the F test rejects the null hypothesis, it is
usually necessary to undertake a thorough analysis of the nature of
the factor-level effects. Tukey test is a multiple comparison
procedure. This is the method which examines or compares more than
one pair of means or proportions at the same time but controls the
overall significance level to be the same as that which is
specified for a single pair comparison.
[0241] The comparisons between Group 1 (subjects assigned to the
invention test material supplement) and Group 2 (subjects assigned
to the placebo test material supplement) were based on percent
changes from baseline at each time interval (Week 8 and Week 12).
Two-sample t-tests were applied to determine if significant
differences exist between Group 1 and Group 2. (FIGS. 8A-8D)
[0242] Two-tailed p-values of 0.05 or less was the criteria for
significance, corresponding to a confidence level of 95%. All
statistical computations were performed using Microsoft Excel or
SAS.RTM. Statistical Analysis Software.
[0243] Results
[0244] Twenty subjects completed the study. One subject (#7)
discontinued study participation for reasons unrelated to the test
material.
[0245] Adverse Event
[0246] On Nov. 20, 2006, subject #6 (assigned to the placebo
product) reported that on Nov. 17, 2006, following approximately 9
weeks of ingesting the placebo tablets, she noticed a red rash and
hive-like bumps in the cheek area on both sides of her face and on
her upper chest. The subject also reported that she experienced
itching of these areas. On Nov. 20, 2006, upon examination by a CRL
Certified Dermatologist, the subject was diagnosed with poison ivy.
Subject #6 continued study participation. The Investigator
determined that this adverse event of moderate severity was
absolutely not related to the use of the test material. Follow-up
telephone calls were made by a CRL Technician, and the information
from each conversation was documented as follows: On Nov. 22, 2006,
the subject reported that the hives had disappeared and the facial
redness was diminishing.
[0247] On Nov. 27, 2006, subject #6 indicated that the rash was
drying and nearly all of the redness was gone. On Dec. 8, 2006, the
subject reported that the poison was gone and the adverse facial
skin condition had completely resolved.
[0248] Instrumental Measurements, Dermal Evaluations And
Questionnaire Responses
[0249] For both groups of subjects, FIGS. 7A-D summarize the
Cutometer measurements for each panelist at each designated study
interval. Table VI lists the statistical variables determined for
comparisons of the differences between Week 8 and Week 12
measurements to baseline. For each subject, Table VII lists the
baseline, Week 8 and Week 12 dermal evaluation scores for the
presence of erythema, edema and dryness. Table VIII lists the
questionnaire data for the screening questionnaire [combining both
groups of subjects] based on percentages. Tables IX and X list the
questionnaire data for the baseline and Week 12 (final) consumer
perception questionnaires for Group 1 (subjects assigned to the
active tablets) and for Group 2 (subjects assigned to the placebo
tablets) based on percentages. Open-ended questions are also
included in these questionnaire tables.
[0250] Conclusion
[0251] For each subject, study procedures included blood and urine
analyses, sitting vital signs, a pregnancy test (with the exception
of pre-menopausal panelists), Cutometer measurements and consumer
perception questionnaires.
[0252] Blood Analysis
[0253] For each subject, a blood analysis was performed at the
baseline and Week 12 study intervals, as indicated above. No
clinically significant findings were determined at Week 12 relative
to that which was revealed at baseline.
[0254] Urine Analysis
[0255] A urine specimen was obtained from each subject at baseline
and at the Week 12 study interval, as specified above. No
clinically significant findings were determined at Week 12 relative
to that which was revealed at baseline.
[0256] Efficacy Evaluations
[0257] As assessed by the Cutometer parameter Ur/Ue, statistically
significant differences existed between baseline and Week 12 for
Group 1 subjects (assigned to the raw ingredient test material
supplement, 10853LLA). The Cutometer parameter Ur/Ue describes skin
elasticity as it relates to stretching and contracting. Seventy
percent of Group 1 subjects showed an improvement in Cutometer
parameter Ur/Ue. Relative to baseline, this significant increase in
parameter Ur/Ue is equivalent to a 29% improvement at Week 12,
suggesting that skin elasticity and distensibility improved for
Group 1 subjects who ingested 10853LLA, the invention supplement.
FIGS. 7A-8D summarize the statistical analysis of study results.
FIG. 7D show a statistically significant improvement of Ur/Ue from
baseline to week 12 (p=0.0109)
[0258] Questionnaire responses which were analyzed via Z-score
analysis consisted of question numbers 10 through 15 in the Week 12
(Final) questionnaire. Favorable consumer perception questionnaire
responses of the study population at Week 12 (final visit), which
were applicable to Z-score analysis, as listed below, were measured
with statistical significance (Z-scores greater than or equal to
the absolute value of 1.96) in questionnaires completed by each
subject.
[0259] A significant proportion of the Group I study population
assigned to 10853LLA, the active tablets, perceived improvements
following 12 weeks of supplement use and subjects responded
favorably with regard to the following treatment effects:
[0260] Approximately 85% of subjects agreed that after using the
product for 12 weeks, they experienced muscle and/or joint pain
less frequently.
[0261] Approximately 85% of subjects agreed that after using the
product for 12 weeks, the overall pain has been less intense.
[0262] Approximately 90% of subjects agreed that after using the
product for 12 weeks, the pain did not last as long.
[0263] Approximately 85% of subjects agreed that the product had a
positive effect on their joint and/or muscle pain. The detailed
statistical results are shown in FIGS. 8A-B.
[0264] For the Group 2 study population assigned to 10853LLB, the
placebo tablets, no favorable responses were reported following 12
weeks of supplement use. The results are shown in FIGS. 9A-B.
STUDY REFERENCES
[0265] Basel, Courage and Clarys, Handbook of Non-Invasive Methods
and the Skin, Serup and Jemec, eds. 1995. [0266] Blichman and
Serup, Acta Derm Venereol, 1988. [0267] Cua, Maibach and Wilhelm,
Dermatologica Research, 1990. [0268] Dobrev, Sixth Congress of
Dermatology and Venerology, Pleve, Bulgaria, May 11-13, 1995.
[0269] Elsner, Eighth International Symposium "Bioengineering and
the skin," Stresa/ltalia, June 1990. [0270] Frankowski, Chen and
Huth, Photonics West Annual Symposium, Electronics Imaging 2000,
San Jose, January 23-28, 2000. [0271] Fiorentini, Becheroni, Iorio.
Int. J. Cosmet. Sci. 26-29. 1988. [0272] Friedman, Skover, Payonk
and Geronemus, Seminars in Cutaneous Medicine and Surger, 200, 21
(4):266-273. [0273] Krushe and Worre, Archives of Dermatologica
Research. 287-293, 1995. [0274] Morganti, Randazzo and Cardillo.
Appl. Cosmet. 10-12, 1986. [0275] Randeau, Kurdian, Sirvent, Closs
and Girard, Cosmetics and Toiletries Manufacture Worldwide 2002.
[0276] Simon, Arzliche Kosmetologie, 256-259, 1984.
[0277] All references mentioned above are expressly are expressly
incorporated by reference into the instant application.
[0278] Method for Monocyte Activation Assay
[0279] Monocyte activation assay was used to evaluate
immunostimulatory activity. The THP-1 human monocyte cell line was
transfected with a luciferase reporter gene construct containing
two copies of the NF-kappa B motif for HIV/IgK as described
previously. This monocyte activation assay is an example of an in
vitro test system that can be used for bioactivity based
standardization of Aloe vera extracts and product material.
[0280] Method for Toll-Like Receptor 2 (TLR2) Expression Vector
Experiment
[0281] HEK 293 cells were cultured in DMEM/F12 medium supplemented
with fetal bovine serum (10%) and 1% penicillin-streptomycin at
37.degree. C., under 5% CO.sub.2. Actively growing cells were
transiently transfected with the appropriate plasmid(s) using
electroporation (at 150 V and one 70-ms pulse). Following
electroporation, cells were plated at a density of 5.times.10.sup.4
cells in 200 .mu.l/well of culture medium. After 48 hours, agents
to be tested were added to transfected cells. Six hours after
addition of samples, cells were harvested and luciferase activity
measured. The NF-kappa B plasmid construct (pBIIXLUC) contained two
copies of NF-kappa B motif from HIV/IgK. Plasmids co-expressing
human CD14 and human TLR2 (pDUO-hCD14/TLR2) were purchased from
InvivoGen. Zymosan was purchased from Sigma, Staphylococcus aureus
peptidoglycan from Fluka and ultra pure Salmonella minnesota LPS
from List Biological Laboratories, Inc.
[0282] AloeEx Activates NF-Kappa B In Vitro Through a
TLR2-Dependent Process.
[0283] FIGS. 10A-10B depict that AloeEx (batch 2-5-05) acts through
TLR2. HEK 293 cells (cell line with minimal TLR expression) were
transiently transfected with a luciferase reporter gene construct
containing two binding sites for NF-kappa B, alone (FIG. 10A), or
in combination with the pDUO-hCD14/TLR2 expression plasmid (FIG.
10B). The pDUO-hCD14/TLR2 expression plasmid expresses proteins
supporting TLR2-(CD14 and TLR2) dependent NF-kappa B activation.
Forty-eight hours after transfection, cells were treated with
either AloeEx TLR2 agonists peptidoglycan (PG) or zymosan (Z), or
TLR4 agonist ultrapure S. minnesota lipo-polysaccharide (LPS) at
the indicated concentrations (.mu.g/ml). Luciferase activity was
determined 6 hours after addition of agents.
[0284] Neither the TLR2 specific (peptidoglycan or zymosan) or TLR4
specific (ultrapure LPS) ligands nor AloeEx activated NF-kappa
B-dependent luciferase expression in HEK 293 cells transfected with
the reporter plasmid alone (FIG. 10A). AloeEx activated NF-kappa B
in cells supporting TLR2-dependent (expressing CD12 and TLR2)
activation (FIG. 10B). Peptidoglycan and zymosan also activated
NF-kappa B in cells expressing TLR2 and CD14 as compared with
ultrapure LPS which did not.
[0285] Presence of Alps and Alp in AloeEx
[0286] The inventors have demonstrated that AloeEx contains
bacterial LPS and bacterial lipo-proteins that are most likely
derived from microorganisms that are associated with Aloe vera. The
presence of these bacterial components in AloeEx are believed to
contribute to the therapeutic properties of products derived from
AloeEx.
[0287] AloeEx was extracted with 90% aqueous phenol: water (1:1) at
70.degree. C. Upon cooling the extract and subsequent
centrifugation two layers were formed (water and phenol) and a
pellet of insoluble material. The water layer contains ALPS. Due to
the non-polar lipid moiety on lipo-proteins, the phenol layer
contain the ALPs. Using this procedure, AloeEx (batch 056) was
separated into a water layer fraction, a phenol layer fraction and
insoluble material. The water layer fraction was freeze-dried (8.9%
yield). Material in the phenol layer was precipitated by addition
of 2 volumes of ether:acetone (1:5) and 4 volumes of ethyl acetate.
The phenol layer precipitate was washed extensively with ethyl
acetate and isopropanol and then dried at 60.degree. C. (45%
yield). The original AloeEx material, water layer fraction, phenol
layer fraction and insoluble material were all tested in the
monocyte assay (FIG. 11). Twenty-fours hours following transfection
with the NF-kappa B luciferase reporter plasmid, cells were treated
with the indicated sample for 4 hours (concentrations in .mu.g/ml).
Luciferase activity was determined and is reported as a percent of
maximal light output from LPS-treated cells. Both the water layer
and phenol layers exhibited substantial monocyte activation
believed to be due to bacterial ALPS and ALP, respectively. The
insoluble material was also active as indicated by its ability to
enhance NF-kappa B activation by 35% when tested at 5 .mu.g/ml.
[0288] Characterization of Alps in AloeEx Phenol Layer Fraction
[0289] The phenol layer fraction material was extracted three times
with 4% sodium dodecyl sulfate (SDS) (26 mg/ml) at 100.degree. C.
for 30 minutes. SDS insoluble material was removed by
centrifugation and when tested in the monocyte assay contained only
about 1-2% of the total activity present in the original phenol
layer fraction.
[0290] The SDS extract of the phenol layer fraction was incubated
with 0.1 mg/ml (3.9 units/ml) proteinase K from Tritirachium album
(Sigma) in 50 mM TRIS (pH 7.5), 5 mM -mercaptoethanol, and 5 mM
CaCl.sub.2 for 2 hours at 50.degree. C. The digest was then heated
at 98.degree. C. for 10 minutes. A control sample, without
proteinase K, was run under identical conditions. For SDS
polyacrylamide gel analysis, extract (proteinase K treated and
untreated) was mixed with 1 volume of Tris-Tricine sample buffer
(Bio-Rad) and loaded in nonadjacent lanes of a 16.5% Tris-Tricine
precast gel (Ready Gel, Bio-Rad). The amount of sample loaded
represented the SDS extractable material present in 130 .mu.g of
AloeEx phenol layer fraction. Wide molecular weight range protein
marker (Sigma) was run in the gel. Individual gel lanes were cut
into 12 equal sections (0.5 cm/section) and extracted with 150
.mu.l of 1.5% octylglucoside, 2.5 mM TRIS (pH 7.5), 5 mM CaCl.sub.2
at 95.degree. C. for 5 minutes. Supernatants of samples were
collected and evaluated for activity in the monocyte activation
assay.
[0291] FIG. 12 indicates that protein does not appear to be
directly responsible for the activation of the monocytes by AloeEx
phenol layer fraction, but rather that protein is part of the
molecule that is responsible for this activity. This is indicated
by the reduction in the apparent size of the active compounds
following proteinase K treatment as determined by fractionation on
an SDS-polyacrylamide gel. Following proteinase K treatment there
was a substantial reduction in activity for the region between 66
and 6.5 kDa, while there was an increase in activity for the region
between 6.5 kDa and the gel front. This result is similar to those
obtained with bacterial lipoproteins in that proteinase K digestion
does not reduce the activity of the lipoproteins (i.e. the protein
component of the lipo-protein is not necessary for monocyte
activation) but proteinase K treatment does reduce the size of the
lipo-proteins when fractionated on an SDS-polyacrylamide gel. The
black bars labeled "C" and "PK" in FIG. 12 represent the activity
of the untreated and proteinase K treated AloeEx phenol layer
fraction, respectively, before fractionation on the gel.
[0292] To determine sensitivity of the 4% SDS extract of the AloeEx
phenol layer fraction to lipo-protein lipase, the SDS was removed
using SDS-out reagent (Pierce) in the presence of 0.88%
octylglucoside. The sample was then adjusted to a final
concentration of 10 .mu.M AEBSF protease inhibitor cocktail
solution (Sigma) and 0.2% BSA (Sigma, No. A-9418). The sample was
incubated at 37.degree. C. for 16 hours with 39,600 units/ml (1
mg/ml) of lipo-protein lipase from Pseudomonas species (Sigma No.
L9656). Control samples (without lipo-protein lipase) were run
under identical conditions. Activity of lipo-protein lipase treated
and untreated samples were evaluated using the monocyte activation
assay.
[0293] The results as depicted in FIG. 13 show that the activity
present in the 4% SDS extract of AloeEx phenol layer fraction is
completely abrogated by treatment with lipo-protein lipase. This
result together with the results presented in. FIG. 12 confirms
that the activity in the AloeEx phenol layer fraction is due to
ALPs. This procedure demonstrated that AloeEx contains ALPs that
are believed to be derived from microorganisms that are associated
with Aloe vera. The presence of this bacterial component, combined
with other components in AloeEx, are believed to significantly
contribute to the therapeutic properties of this product.
[0294] Characterization of Alps in AloeEx
[0295] The following summarizes the amount of ALPS present in
AloeEx (batch 056) and the two fractions derived from this material
(water layer fraction and phenol layer fraction).
[0296] AloeEx (batch 056) 625-6250 EU/mg
[0297] AloeEx Water Layer Fraction 62,500-625,000 EU/mg
[0298] AloeEx Phenol Layer Fraction <4.8 EU/mg
[0299] The amount of ALPS was estimated using Pyrosate LAL assay
kit (Associates of Cape Cod Inc) according to manufacture's
instruction. As expected, the water layer fraction contained
essentially all of the ALPS present in AloeEx, as compared to the
phenol layer fraction. This data demonstrated that AloeEx contains
ALPS that is believed to be derived from microorganisms that are
associated with Aloe vera. The presence of this bacterial component
combined with other components in AloeEx are believed to contribute
to the therapeutic properties of this product.
[0300] The Rind and Mucilage Contain the Majority of the Monocyte
Activating Components in Aloe Vera
[0301] The Aloe vera leaf can be divided into three basic
structural parts: the gel, the outer rind and the mucilage (the
layer between the gel and the outer rind). An experiment was
conducted to determine which part of the Aloe leaf contained the
highest level of monocyte activating components. From one Aloe vera
leaf, the gel was carefully cut-out by passing a knife parallel to
the inner rind surface. After removal of the gel, the leaf was
scraped over the edge of a vial to collect the mucilage part and to
separate it from the outer rind. All three components and a section
of the whole leaf Aloe were freeze-dried and ground to a fine
powder. Each sample was then extensively extracted with methanol to
remove non-polar substances, such as anthraquinones, that have an
inhibitory effect in the monocyte activation assay. Samples with
non-polar substances removed were prepared as a fine suspension in
water. Twenty-fours hours following transfection with the NF-kappa
B luciferase reporter plasmid, THP-1 cells were treated with the
prepared samples for 4 hours. Luciferase activity was determined
and is reported as a percent of maximal light output from
ELPS-treated cells. FIG. 14 depicts the response of the different
Aloe vera leaf parts and that of the whole leaf material. This data
suggests that the mucilage layer and outer rind contain the
majority of the monocyte activating components in the Aloe leaf. In
this experiment, the percent that each leaf part comprises of the
total dry weight of the Aloe leaf was: gel (8.7%), mucilage layer
(6.2%) and rind (85.1%).
[0302] Since the outer rind and mucilage layer contain the majority
of the monocyte activating components, and these parts also
comprise the majority of the dry solids in the Aloe leaf, the
inventors conclude that the outer rind and mucilage layer are of
commercial interest. Typically, the outer rind is discarded as a
waste product after the gel has been removed or after the whole
Aloe leaf has been processed to create Aloe juice. Another waste
product is Aloe pups (small Aloe plants that grow off of the larger
plants). Since the Aloe pups contain mostly rind with very little
gel and are considered of little commercial value. The rind and
Aloe pups waste products can be processed to produce for example,
dietary supplements that enhance immune function. The dried rind
could be ground to a powder and processed to remove anthraquinones.
This material could be further processed by extraction to produce
crude extracts and purified fractions. The objective of the crude
extracts and purified fractions would be to concentrate the amount
of AloeEx and/or microorganisms and microorganism
fragments/components associated with Aloe vera.
[0303] Monocyte Activating Components in AloeEx are Extractable and
Soluble in Detergent
[0304] For development of various products (e.g. for cosmetics) it
may be desirable to solublize the immune enhancing components in
AloeEx material. The immune enhancing components would include
Aloeride polysaccharide, ALPS and ALPSs derived from microorganisms
that are associated with Aloe vera. As previously described, a
significant portion of the immune enhancing components of AloeEx
material can be fractionated into a water layer fraction and phenol
layer fraction. The immune enhancing activity present in the phenol
layer fraction (ALPS) can be completely extracted/solubilized with
4% SDS at 100.degree. C. The immune enhancing activity present in
the water layer (ALPS and Aloeride polysaccharides) is water
soluble and also SDS soluble.
[0305] Extraction of AloeEx with SDS represents a potential method
to solublize a significant portion of the immune enhancing
components present in this material. The data suggest that other
known detergents (including those that are more compatible with
product formulation) could also be potentially useful in
extraction/solublization of the immune enhancing components in
AloeEx material. This method of detergent extraction could also be
used to solubilize the monocyte activating components (i.e.
Aloeride polysaccharide and microorganism components associated
with Aloe vera) from raw materials such as whole leaf, rind, Aloe
pups, etc.
[0306] Method for Producing an Extract by Isolation of
Microorganisms and Microorganism Components/Fragments Associated
with Aloe Vera
[0307] As demonstrated, bacterial products from microorganisms
associated with Aloe vera are responsible for a significant portion
of the immune enhancing properties of AloeEx. It is shown that
AloeEx accounts for a majority of the in vitro monocyte/macrophage
activation potential of Aloe vera. Therefore, it is believed that
bacterial products from microorganisms are responsible for a
signifcant portion of the immune enhancing properties of Aloe
vera.
[0308] Extracts that concentrate the amount of microorganisms
and/or microorganism components/fragments associated with Aloe vera
can be prepared from crude Aloe material and/or Aloe vera raw
material, including waste material (e.g. leftover rinds, Aloe pups,
etc.). Crude extracts can be processed to create purified fractions
by extracting the bacterial lipo-proteins and/or other bacterial
components using a solvent. These fractions would contain enhanced
levels of bacterial components associated with Aloe vera. Solvents
which may be used to prepare a purified fraction include detergents
such as SDS, water, water miscible solvents such as alcohol, and
combinations of these solvents or other solvents well known in the
art.
[0309] The following procedure could be used to prepare an extract
that concentrates the microorganisms and/or microorganism
components/fragments associated with Aloe vera. Aloe vera raw
material (gel, whole leaf, rind, pups, etc.) can be liquified using
methods known in the art. The liquid can be subjected to coarse
filtration to remove fibrous material. A food-grade flocculant
(clarifier or similar substance) can be added to the liquified Aloe
to create aggregates of particles (bacterial, other microorganisms,
microorganism fragments/components). These aggregrated particles
could then be separated from the remaining liquid using methods
known in the art. Such methods would include centrifugation,
filtration, settling followed by decanting, filtering aggregates
out of the liquid using a net, etc. The final collected material
can be also processed to remove anthraquinones, if present, using a
technique such as washing/extracting with alcohol. If desired, the
resulting material can be dried.
[0310] Method for Producing a Standardized Aloe Vera Product
[0311] Biological and chemical standardization represent two
methods that can be used to standardize Aloe vera products that
contain the immune enhancing agents described in the present
invention. Both methods can be used for standardizing either
extract material or the raw material. The purpose of
standardization is to ensure that each batch of product material
contains the same level of active component(s).
[0312] In one method a bioactivity standardized Aloe vera product
containing an effective amount of immunostimulatory activity is
prepared. In this method, Aloe vera product material is tested in
vitro for activation of immune cells and the bioactivity is then
compared to a standard preparation immunostimulatory value to
determine a standardized activity value of the product. An example
of an in vitro test system is the monocyte activation assay
previously described. Bioactivity based standardization of product
material is important when the chemical content of the active
components do not correlate with biological activity due to unknown
structure-activity relationships and/or complex interactions
between multiple actives. Under such circumstances, the amount of
active substances is not sufficient to reflect the potency of the
product material and standardization through the use of a
biological assay is desirable. Bioactivity standardization has been
used by the pharmaceutical industry for biologics such as insulin
and cytokines.
[0313] In another method a chemically standardized Aloe vera
product containing an effective amount of immunostimulatory
lipo-proteins originating from microorganisms associated with Aloe
vera is prepared. A chemical marker which may be used for
standardization is 2,3-dihydroxypropyl cysteine. This modified
cysteine amino acid is thought to be unique to lipo-proteins that
are immunostimulatory from prokaryotic organisms. In this method,
Aloe vera product material is tested for the amount of
2,3-dihydroxypropyl cysteine and then compared to the amount of
2,3-dihydroxypropyl cysteine in a standard preparation to determine
a standardized value of the product.
[0314] In certain embodiments a food grade flocculent, clarifier or
coagulant may be used to form microorganism aggregates. Examples
include, but are not limited to, for example, alum, aluminium
chlorohydrate, aluminum sulfate, calcium oxide, iron(III) chloride,
iron(II) sulfate, polyacrylamide, sodium aluminate, sodium silicate
and combinations thereof. Natural products which can also be used
as flocculants include Chitosan, Moringa oleifera seeds, Papain,
species of Strychnos (seeds) Isinglass and combinations thereof.
Other such materials are well known to those skilled in the
art.
[0315] Although the invention herein has been described with
reference to particular embodiments, it is to be understood that
these embodiments are merely illustrative of the principles and
applications of the present invention. It is therefore to be
understood that numerous modifications may be made to the
illustrative embodiments and that other arrangements may be devised
without departing from the spirit and scope of the present
invention as defined by the appended claims.
* * * * *
References