U.S. patent application number 11/277489 was filed with the patent office on 2007-09-27 for process for the isolation and stabilization of low-molecular weight aminoglycans from waste egg shells.
Invention is credited to Bomi Patel Framroze.
Application Number | 20070225484 11/277489 |
Document ID | / |
Family ID | 38534375 |
Filed Date | 2007-09-27 |
United States Patent
Application |
20070225484 |
Kind Code |
A1 |
Framroze; Bomi Patel |
September 27, 2007 |
Process for the Isolation and Stabilization of Low-Molecular Weight
Aminoglycans from Waste Egg Shells
Abstract
Processes for the isolation of low-molecular weight aminoglycan
compound of formula I from a hitherto unexploited natural source of
waste egg shells which is made up of alternating glucuronic acid
and N-acetyl glucosamine units comprising the steps of: ##STR1##
(a) pre-preparation of the waste egg-shells for extraction of the
embryonic low molecular weight aminoglycan compound of formula I
using a polar organic solvent in water; (b) extraction of the low
molecular weight aminoglycan compound of formula I as its soluble
salt using an aqueous polar salt solution; (c) isolation of a
purified low molecular weight aminoglycan compound of formula I by
gel formation out of the aqueous salt mixture by using a polar
organic solvent followed by filtration or centrifuging; (d)
stabilization of the isolated aminoglycan extract by sequential
introduction of organic oils into a semi-dried gel to form ordered
sheets of aminoglycan compound of formula I with no visible
cross-linking of the carbohydrate polymer.
Inventors: |
Framroze; Bomi Patel;
(Mumbai, IN) |
Correspondence
Address: |
Bomi P. Framroze;3 Shree Sadan
4A Carmichael Road
Mumbai
400026
IN
|
Family ID: |
38534375 |
Appl. No.: |
11/277489 |
Filed: |
March 25, 2006 |
Current U.S.
Class: |
536/18.7 |
Current CPC
Class: |
C08B 37/0003 20130101;
C07D 309/10 20130101; C08B 37/0063 20130101; C07H 17/04
20130101 |
Class at
Publication: |
536/018.7 |
International
Class: |
C08B 37/00 20060101
C08B037/00 |
Claims
1. A process for the isolation of low-molecular weight aminoglycan
compounds of formula I from waste egg shells, ##STR3## wherein M
may be one or more instances of Na, Ca, K, Mg and n is an integer
between 20 and 40; said Process comprising the steps of: (a)
pre-preparation of the waste egg-shells for extraction of the
embryonic low molecular weight aminoglycan compound of formula I
using a polar organic solvent in water, wherein the pretreated egg
shells are mixed thoroughly with the polar organic solvent in water
at temperatures between 25.degree. C. and 40.degree. C. for 1 hour
to 4 hours, followed by decantation of the supernatant and the
eggshells carried forward for extraction; (b) extraction of the low
molecular weight aminoglycan compound of formula I as its water
soluble salt using an aqueous polar salt solution, wherein the
eggshells from step (a) are vigorously shaken with the aqueous
polar salt solution at 25.degree. C. to 40.degree. C. for 6 to 24
hours, followed by decantation, filtration or centrifugation to
collect the aqueous layer containing the dissolved aminoglycan
compound of formula I; (c) isolation of a purified low molecular
weight aminoglycan compound of formula I by gel formation out of
the aqueous salt mixture by using a polar organic solvent, wherein
the solution from step (b) is subjected to sequential step wise
addition of the polar organic solvent in an amount between 75% and
150% volume/volume of the polar organic solvent between 10.degree.
C. and 20.degree. C. in 1 hour to 2 hours and the gel formed is
allowed to stand for 4 hours to 12 hours to complete precipitation,
followed by decantation, filtration or centrifugation to isolate
semi dried aminoglycan compound of formula I containing between 4%
to 8% of moisture; (d) stabilization of the isolated aminoglycan
compound of formula I from step (c) by sequential introduction of
organic oils into the semi-dried gel to form the aminoglycan
compound of formula I.
2. The process as claimed in claim 1, wherein the polar organic
solvent used in step (a) is selected from the group consisting of
an alcohol, acetone, methylethylketone or 1,4-dioxane.
3. The process as claimed in claim 1, wherein the said aqueous
polar salt solution used in step (b) is an organic acid salt
selected from the group consisting of sodium, potassium, calcium or
magnesium salt of citrate, glutamate, acetate, pyrrolidone
carbonate, tartrate, glycinate, sulfate sulfite, nitrate, carbonate
or oxalate.
4. The process as claimed in claim 1, wherein the polar organic
solvent used in step (c) is a lower alcohol selected from methanol,
ethanol, propanol or butanol, or an organic ether selected from
diethylether, tetrahydrofuran, methylal or ethylal.
5. The process as claimed in claim 1, wherein the organic oils used
in step (d) are the oils obtained from plant sources.
6. The process as claimed in claim 5, wherein the said organic oil
is selected from jojoba, almond, sage, rosemary, lavender,
sandalwood or aloe oil.
Description
FIELD
[0001] Embodiments of the invention relate to a method for simply
and efficiently isolating and stabilizing ultra low molecular
weight aminoglycans from waste egg shells.
BACKGROUND
[0002] Embodiments of the invention relate to processes for
isolating, stabilizing and formulating low molecular weight
aminoglycans from waste egg shells. The aminolycan extract is
useful for the preparation of cosmetic creams with skin
moisturizing and anti-wrinkle properties.
[0003] Nakano et al. (Poult Sci. (1991), Vol. 70(12), pp. 2524-8)
have shown that the chemical composition of glycosaminoglycan
fractions from the comb and wattle of single comb white Leghorn
roosters consist of very large molecular weight glycosaminoglycans
that have applications in cartilage replacement therapy.
[0004] Balazs et al (U.S. Pat. No. 4,141,973) has described a
process to isolated pure hyaluronic acid from animal tissue bearing
molecular weights in the range of 1 MD to 6 MD useful as a
replacement for synovial fluids and vitreous humor.
[0005] Heaney et al. (Biochim Biophys Acta. (1976), Vol. 18;451(1),
pp. 133-42) have shown that the organic part of the chicken's egg
shell consists of collagen, proteins and polysaccharides which are
probably present as glycoproteins and glycosaminoglycans. They
further identified the organic components by chromatography to
yield glycosaminoglycans with a minimum molecular weight of 30,000
Daltons. Sedimentation velocity analysis in a density gradient,
showed that the polysaccharides contained equimolar amounts of
glucosamine (36.3% s/w) and glucuronic acid 35.6% w/w.
Identification of the degradation products showed the
glycosaminoglycan to be mainly hyaluronic acid.
[0006] Stahl et al (U.S. Pat. No. 6,537,795) have described a
process to produce and isolate aminoglycans from cultivated strains
of streptococci fermentation. These aminoglycans are characterized
by extreme high molecular weights above 6 MD and are useful for
cartilage replacement therapy.
[0007] Related processes for isolations and purifications of
glycosaminoglycans from other natural sources and animal tissues
may also be found in U.S. Pat. No. 5,824,658, U.S. Pat. No.
6,660,853, U.S. Pat. No. 6,451,326. The references discussed within
these patents are hereby incorporated herein by reference.
SUMMARY
[0008] Embodiments of the invention provide novel processes for the
isolation of low-molecular weight aminoglycan compounds of formula
I from a hitherto unknown natural source of waste egg shells made
up of alternating glucuronic acid and N-acetyl glucosamine units
##STR2##
[0009] comprising:
[0010] (a) pre-preparation of the waste egg-shells for extraction
of the embryonic low molecular weight aminoglycan compound of
formula I using a polar organic solvent dissolved in water
[0011] (b) extraction of the low molecular weight aminoglycan
compound of formula I as its soluble salt using an aqueous polar
salt solution
[0012] (c) isolation of a purified low molecular weight aminoglycan
compound of formula I by gel formation out of the aqueous salt
mixture by using a polar organic solvent followed by filtration or
centrifuging
[0013] (d) stabilization of the isolated aminoglycan extract by
sequential introduction of organic oils into a semi-dried gel to
form ordered sheets of aminoglycan compound of formula I with no
visible cross-linking of the carbohydrate polymer
[0014] Embodiments of the invention relate more particularly to
step (b), wherein the aqueous polar salt solution can be the
sodium, potassium, calcium or magnesium salts of citrate,
glutamate, acetate, pyrrolidonecarbonate, tartrate, glycinate,
sulfate, sulfite, nitrate, carbonate, oxalate to yield a solution
containing aminoglycan compound of formula I wherein M may be one
or more instances of Na, Ca, K, Mg and n is an integer between 20
and 40, which is suitable for selective gelation and isolation.
[0015] The processes described herein are novel methods to
selectively and simply yield a low-molecular weight aminoglycan
compound of formula I from waste egg shells. More specifically the
processes of the invention, compared to the procedures for
isolating aminoglycans disclosed in the prior art, are
differentiated by; [0016] a) identification of an novel hitherto
unused source, egg shell waste, which is otherwise difficult to
dispose of and causes significant negative impact to the
environment, [0017] b) contains very low concentrations of
detrimental proteins and nucleotides, [0018] c) requires no
expensive and inefficient separations of organic and inorganic
materials from the egg shell waste, [0019] d) are simpler
extractions involving mild reagents and solvents materials and
[0020] e) requires no acetylation or other derivatization for
example using acetic anhydride and sulfuric acid as described in
U.S. Pat. No. 5,679,657 to achieve the desired viscosity and
threading properties needed for cosmetic applications.
[0021] The aminoglycan compound of formula I are of an unusually
low molecular weight and are yet stabilized without derivatization
to show excellent dermal penetration to reduce surface wrinkles in
skin and exhibit an excellent softening and moisturizing effect as
well.
DETAILED DESCRIPTION
[0022] Egg shell waste produced from the egg processing industry is
usually washed with solvents and treated to eliminate unpleasant
smells before being used as landfill. The calcium carbonate of the
shells is only usable upon extensive separation and cleaning
procedures which makes the process commercially uneconomical. There
is no specific need to pulverize the egg shells within a narrowly
bound range either since the processes of the present invention are
not dependent on the separation of the inner membrane from the egg
shell as in the complex process and equipment described by MacNeil
(U.S. Pat. No. 6,176,376) to arrive at pure calcium carbonate.
[0023] We have identified processes to selectively isolate valuable
organic compounds, specifically aminoglycan compound of formula I
from crushed egg shells without the expensive separation of the
organic and inorganic components.
[0024] Crushed egg shells may be treated with warm water or warm 5%
ethanol solution and filtered to remove adhered organic wastes from
the surface of the shells. The ratio of organic mass to calcium
carbonate may be between 1% to 15% w/w. Greater ratios of organic
mass would indicate unwashed egg mass present in the crushed egg
shells which can lead to the presence of detrimental protein and
nucleotide products in the aminoglycan extract. It is noted that
unlike other sources of aminoglycans such as animal tissue and
fermentation broths as known in the prior art, the use of egg shell
waste as shown herein is unique in the absence of significant
antigenic protein and nucleotide components in the extracted media
leading to easier methods of extracting purified aminoglycan
compound of formula I. The egg shells may be additionally
pre-treated with ultraviolet light to destroy microbes which may be
present even after liquid cleaning.
[0025] The next step comprises treatment of the above egg shell
mass to a highly selective extraction of the carbohydrate component
in the form of its water-soluble salt. The processes involve
suspending the egg shell mass in 1:2 to 1:10 volume of solution
containing 5% to 40% by weight citrate, glutamate, acetate,
pyrrolidonecarbonate, tartrate, glycinate, sulfate, sulfite,
nitrate, carbonate and oxalate salts of sodium, potassium, calcium
or magnesium or a combination of the above salt solutions as
needed. More specifically the mono-valent salts of organic acids
are preferred. The suspension is held for 1 to 24 hours, more
preferably for 6 to 12 hours, with periodic vigorous shaking at
temperatures ranging between 10 C and 35 C. The suspension is
subsequently filtered or centrifuged to remove the aqueous solution
containing the appropriate salt of the aminoglycan compounds of
formula I. The egg shell mass thus separated shows a much looser
binding of the membranes to the egg shell and hence may be more
easily treated using processes known in the art to separate the
pure calcium carbonate containing egg shell from the organic
residue.
[0026] The next step comprises the gel precipitation of the
aminoglycan in its appropriate salt form from the aqueous solution.
The processes involve reducing the polarity of the aqueous solution
and hence the solubility of the aminoglycan by the sequential
addition of any aqueous miscible organic solvent such as alcohols,
acetone, dimethylformamide, N-methylpyrrolidinone or 1,4-dioxane.
The organic solvent is added in lots with mild stirring and cooling
to maintain the temperature of the reaction between 20 C to 25 C to
yield a white gel formation suspended in the aqueous layer. The
solution is allowed to stand for 2 to 24 hours until gelation is
complete and subsequently filtered or centrifuged to yield a
semi-dry extract of aminoglycan compound of formula I. It is
important to not allow the extract to be completely dried since a
certain amount of the aqueous phase is required during the
stabilization process carried out next.
[0027] The final step comprises the stabilization of the
low-molecular weight aminoglycan compound of formula I by ordering
the molecules in a lipophilic environment to prevent cross-linking
which is characteristic of non-acetylated and low-molecular weight
aminoglycans as described in the prior art. The process involves a
sequential addition of two oils whose total weight ratio to the
aminoglycan extract is between 1:0.5 and 1:3 of aminoglycan to oils
and wherein the individual oil ratios between the two oils are 3:1
to 8:1. The first oils should be more hydrophobic in nature and may
be oils found typically in plant nuts. Specifically almond and
jojoba oils are more preferred as the first oil. The second oil
component should be more hydrophilic in nature and may be oils
typical isolated from herbs and spices from the vegetative parts of
plants. Specifically sage, rosemary and lavender oils are more
preferred as the second oils.
[0028] The molecular weight of aminoglycan compound of formula I
thus isolated is difficult to measure directly and hence I have
relied on the measure of intrinsic viscosity to determine the
molecular weight. The intrinsic viscosities of various solutions
containing aminoglycan compound of formula I were found to lie
between 4 cm3/gm and 7 cm3/gm and when plotted against standard
solutions of hyaluronic acid salts (Mol. Wt. approximately 1.2 MD)
led to the assignment of a unique natural ultra low molecular
weight for aminoglycan compound of formula I between the range of
15,000 Daltons and 28,000 Daltons. An ultra low molecular weight
aminoglycan compound of formula I from a natural source has not
been previously described in the prior art (for example as
summarized by Balazs et al. in U.S. Pat. No. 4,582,865).
Experimental Results
EXAMPLE 1
[0029] 500 grams of pre-treated egg shell waste with approximately
10% organic content was added to an open mouth glass container with
a screw top. To this was added 750 ml of a 5% aqueous solution of
sodium citrate and the container sealed and placed on a shaker for
24 hours at moderate speeds. After 24 hours the entire mixture was
transferred into a filter funnel and the solid egg shell waste was
separated from the aqueous suspension. The solids were washed
1.times. with 250 ml of 5% aqueous solution of sodium citrate and
the combined aqueous layers were washed once with 250 ml of
methylene chloride to remove potential proteinaceous matter and the
aqueous layer then transferred to a 2L beaker. The beaker was
placed in a cold water bath and a slow addition of absolute
methanol was initiated with slow stirring. After about 200 ml of
methanol addition was completed, a cloudy white precipitate began
to form and the stirring was stopped. An equal additional quantity
of methanol was added slowly and the beaker allowed to stand for 12
hours to make sure gelation was complete. The entire mass was
transferred to a filter funnel and filtered to give a cream colored
gel of aminoglycan compound of formula I. The precipitate was dried
until a moisture content of 5-7% was measured. The final weight of
the gel of aminoglycan compound of formula I was 42 grams.
EXAMPLE 2
[0030] The gel material containing aminoglycan compound of formula
I from Example 1 was mixed with 4 grams of jojoba oil at 15-20 C
and stirred vigorously for 20 minutes. The resultant gel was warmed
to 25 C and allowed to gently stir for 1 hour. To this mass was
added 1 gram of sage oil and the resultant gel was further stirred
gently for 10 minutes. The gel is then allowed to slowly cool to 10
C over 4 hours whereby the aminoglycan compound of formula I is
ordered into a secondary structure that is stable in the absence of
circulating air at room temperature for at least 3 months.
EXAMPLE 3
[0031] The above example 1 was repeated with a 10% aqueous solution
of potassium tartrate to yield 46 grams of the gel of aminoglycan
compounds of formula I.
EXAMPLE 4
[0032] The above example 1 was repeated with a 20% solution of
sodium acetate to yield 43 grams of the gel of aminoglycan compound
of formula I.
EXAMPLE 5
[0033] The above example 4 was repeated with a final gelation using
ethanol instead of methanol to yield 47 grams of the gel of
aminoglycan compound of formula I.
EXAMPLE 6
[0034] The above example 5 was repeated with a final gelation using
acetone instead of ethanol to yield 41 grams of the gel of
aminoglycan compound of formula I.
EXAMPLE 7
[0035] The above example 1 was repeated with a 10% solution of
sodium carbonate to yield 24 grams of the gel of aminoglycan
compound of formula I.
EXAMPLE 8
[0036] The above example 1 was repeated with a 25% solution of
calcium carbonate to yield 14 grams of the gel of aminoglycan
compound of formula I.
EXAMPLE 9
[0037] 10 grams of the above stabilized gel made as per the
procedure shown in Example 2 is added to 50 ml of distilled water
containing 3 ml of glycerin and stirred to a uniform suspension. To
this suspension is added a melt consisting of 10 grams of
emulsifying wax, 10 grams of paraffin wax, 4 grams of white beeswax
and 13 grams of a mix of cosmetically useful plant oils such as
almond, lavender, sandalwood and walnut and the mixture stirred
vigorously to give a uniform cream with excellent physical
characteristics and anti-wrinkle properties.
[0038] With respect to the above isolated and stabilized gels of
aminoglycan compound of formula I the following analytical and
usefulness tests were conducted.
Absence of Chondroitin Sulfate
[0039] It is known in the prior art that all commercial sources of
aminoglycans are usually closely associated with other tissue
components such as Chondroitin sulfate (Arkins and Sheehan,
Structure of Hyaluronic Acid, Nature New Biol 235, 253, 1972 and
Bettelheim and Philpott, Electron Microscopic Studies of Hyaluronic
Acid--Protein Gels, Biochim Biophys Acta 34, 124, 1959). The gel
extract isolated as per the methods described above contains less
than 2% Chondroitin sulfate probably due to the low association
possible with the extra small size of the aminoglycan compound of
formula 1 isolated herein.
Absence of Proteins
[0040] Since proteins are potentially antigenic, it is essential
for cosmetic formulations to isolate any aminoglycan gel
essentially free of proteins. The gel extract from Example 1 was
subjected to the highly sensitive colorimetric test for detecting
the presence of proteins described by Lowry et al. (J. Biol. Chem.,
193, 265-275, 1951) No positive result was obtained indicating the
presence of proteins to be less than 0.1% by weight.
[0041] The absence of any appreciable protein concentration is a
distinct difference from other glycolaminoglycan compounds isolated
from other natural sources such as Rooster Comb and fermentation
broths. It has been reported (Kludas, U.S. Pat. No. 5,055,298) that
these aminoglycans are usually covalently linked with proteins to
form proteoglycans. Clinically relevant removal of all of these
proteins, which are not components of human skin, has proved to be
difficult and not easily accomplished. The presence of these
proteins in various other aminoglycan extracts has been identified
as a cause of significant inflammatory responses on skin surfaces,
making their use in cosmetic formulations challenging.
Absence of Nucleotides
[0042] Ultraviolet spectroscopy has been used to show the absence
of potentially antigenic DNA and RNA nucleotides in the aminoglycan
compound of formula I extracted herein. A 1% solution of the
aminoglycan extract from Example 1 in 10% sodium chloride solution
was prepared. This solution was subjected to ultraviolet
spectroscopy at 257 nanometers to measure the level of nucleotides
in the solution. The absence of any absorption at this wavelength
was taken as a measure of the absence of nucleotides in the
aminoglycan extract from Example 1.
Viscosity
[0043] A small sample of the gel was freeze-dried to give a white
solid with a thread like structure which slowly dissolved in water.
A solution of 1 gram of the powder was made up in 1000 ml of a
phosphate buffer at pH 7. Viscosity was determined with an Ostwald
viscosimeter at a temperature of 25 C. The relative viscosity of
the solution was measured as 0.76 to 0.80. When compared to
aminoglycans of known higher molecular weight this viscosity
measurement leads to molecular weights for aminoglycan compound of
formula I between 15 kD and 28 kD.
Glucosamine Presence
[0044] The presence of glucosamine in the aminoglycan compound of
formula I was determined by the method of Elson and Morgan (Biochem
J, Vol. 27, (1933), p. 1894,) on material that had been hydrolyzed
for 6 hours with 5N hydrochloric acid at 100 C and evaporated to
dryness. The glucosamine content of the aminoglycan compound of
formula I was between 38% and 41% which matches the expected
calculated value.
Thread Forming Ability
[0045] It is well documented in the prior art that the higher the
thread forming ability the more moisturizing is the effect of the
aminoglycan. Many derivatives of high and medium molecular weight
aminoglycans such as acetylation and co-polymerization (U.S. Pat.
No. 5,679,657) have been used to increase the intrinsic threading
value of aminoglycans isolated from animal and bacterial sources.
It is unexpectedly observed that the ultra low molecular weight
aminoglycan compound of formula I isolated herein shows a
remarkably high thread forming ability and may account for part of
the high anti-wrinkle effects observed. In a humidity chamber at a
temperature of 25 C and relative humidity of 50%, 1 cm of a glass
rod was immersed in a 1% aqueous solution of aminoglycan extract
from Example 1 and the thread length obtained upon lowering the
beaker at a velocity of 10 cm/min was observed. The thread length
of the aminoglycan of this invention was observed to be between 2.8
cm and 3.5 cm which is considerably longer than the 0.8 cm to 1.3
cm observed for commercially available sodium hyaluronate and even
better than the lengths observed for derivatized aminoglycans.
Anti-Wrinkle Properties
[0046] The anti-wrinkle properties of the cream produced as per the
method described in Example 9 was tested using a 3D imaging system
to measure depths of surface wrinkles. The method described by S.
Jaspers et al, ("Microtopometry Measurement of Human Skin in vivo
by a new Digital Optical Projection System", Preprints 5th Congress
of the International Society for Skin Imaging, Wien 1997) was used
to show a 25% to 38% reduction in wrinkle depth after 4 weeks of
daily use.
* * * * *