CA1223252A - Elastin hydrolyzate - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A soluble elastin partial hydrolyzate is comprised of po-lypeptides having at least 3.5 desmosine and isodesmosine residues/
1,000 amino acid residues. The hydrolyzate is useful in cosmetic preparations.

Description

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ELASTIC HYDROLYZATE

BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
This invention relates to the preparation of elastic hydra-lusts.
DESCRIPTION OF THE PRIOR ART
Elastic is the major component of elastic fibers found prim manly on connective tissue in conjunction with collagen and polyp saccharides. Major concentrations of elastic are found in blood vessels. Another source of elastic is in the ligaments, and more particularly the ligamentum Nash, prominent in the necks of grazing animals and on their hides. The ligamentum Nash is a preferred source of elastic because of the high concentration of elastic therein.
Elastic is known to have a highly distinctive amino acid composition. Although similar to collagen in that one-third of the amino acid residues are Gleason, elastic is rich in praline, and in contrast to collagen, elastic contains very little hydroxyproline, no hydroxylysine, and is very low in polar amino acids. Elastic is very rich in nonpolar aliphatic residues such as isoleucine~ Lawson, valise and ala nine. Elastic, as present in mature animals, is highly cross-linked, therefore making it very difficult to syllables. This dense cross-link;ng is attributable to the desmosine and isodesmosine residues which are highly functional and cross-link both intro- and interfibrillarly. It is believed that the desmosine and ;sodesmosine cross-linking gives the elastic fibers their elasticity. The demo-sine residue can be represented by the structural formula:

I

T
--N--C--C
I

Shea HEN NO
SUE
Ho SUE_ SHEA CHIHUAHUAS OH
Old Jo KIWI

N

Of ~12 (Shea CHIC N--O H H

And the isodesmosine residue can be represented by the structural formula:

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H
- -- - No N -H
Cull -(C1~2)2 (SCHICK--- I

H

I .. (SHEA --C-34 No H

I

This highly cross-linked structure is extremely difficult to syllables and purify, especially in the case of mature animals such as horses, cows and the like, which are aged and thus contain elastic which has an extremely high cross-link density.
Prior art methods of solubilizing elast;n primarily include the use of elasticize to hydrolyze the peptize linkages to provide an -I, acceptable product I` Solubilized elastic has found utility in the cosmetic no aye . However, its manufacture has been limited to small quantities and it is not necessarily of acceptable purities because of the enzymatic residue required for the hydrolysis Further, partially hydrolyzed elastics have been produced;
however, the processing conditions for their production reduce the amount of desmosine and isodesmosine amino acid residues which are recovered in the elastic hydrolyzate. Therefore, many of the elastic hydrolyzates lose the primary elastin-characterizing amino acids, i.e.
desmosine and isodesmosine. One particular method of producing elastic by non enzymatic means is disclosed in "ELASTIC" by Berg et at., Cosmetics & Toiletries, Vol. 94, October, 1979.

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Characteristically, elastic in its natural state in mature animals, and particularly the ligamentum Nash, is present at a level of at least 3.5 combined desmosine and isodesmosine residues/1,000 Amelia acid residues. Thus, in order to produce a hydrolyzed elastic which retains the basic characteristics of elastic, it is necessary to recover substantially all of the desmosine and isodesmosine residues which are present in the starting material.
In accordance with the present invention, the method of pro during a soluble elastic partial hydrolyzate in pure Form is provided wherein the desmosine and isodesmosine residues are substantially recovered.
BRIEF DESCRIPTION OF THE INVENTION
A method of preparing soluble partially hydrolyzed elastic is provided. The method involves treating insoluble elastic with a per oxide and subsequently partially hydrolyzing the treated elastic and recovering substantially pure soluble partially hydrolyzed elastic.
A soluble elastic partial hydrolyzate is comprised of polyp peptizes having at least 3.5 desmosine and isodesmosine residues/1,000 amino acid residues.
DETAILED DESCRIPTION OF THE_ INVENTION
The elastin-containing material may be obtained from a variety of sources well known to those skilled in the art. Preferably, the source of elastic is natural insoluble elastic from bovine hide or the bovine ligamentum Nash. Although the hides themselves may be used as a source of elastic, it is preferred that the bovine ligament tug Nash be used since it has about 80 percent by weight elastic therein.
When the ligament Nash are used as a source of elastic, just as are the hides, the collagen constituents must be removed therefrom. In order to remove the collagen constituents therefrom, the collagen is solubilized by the use of an acid solution containing citric acid, tartaric acid, weak hydrochloric acid or the like. The acid should be sufficiently weak so that it does not hydrolyze the elastic, but must be sufficiently strong so as to syllables the collagen.

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Preferably, the acid should be sufficient to provide a pi to the aqueous solution of 3.5 to 4.5. In addition to the acid hydrolysis of the collagen, organic peroxides may be used in the same solution in order to initiate the destabilization of the desmosine and is odes-cosine cross-linkages. The treatment of the ligaments with the aqueous acid solution is for about 12 to 18 hours at room tempera-lure with agitation. Subsequent to the acid treatment, the liquid is drained and the ligaments washed with cold water. The ligaments are then autoclave at 2.2 to 3 atmospheres pressure for about 2 to 8 hours. After autoclavation, the ligaments are then treated with a base such as sodium hydroxide or potassium hydroxide in order to hydrolyze the elastic cross-linkages, along with sodium sulfate at a level of 1 to 1.5 molar in water to prevent overselling of the elastic fibers. Sufficient base should be provided to establish a pi of 10 to 12. The ligaments are immersed in the solution for about 12 to 18 hours, removed from the solution, and again washed. The ligaments are then neutralized with dilute acid, preferably a mineral acid such as hydrochloric acid or sulfuric acid. After neutralize-lion, the ligaments are washed with cold water. The ligaments are then placed in an appropriate vessel and shredded in the presence of an acidic solution at a pi of 2.2 to 3.2, and after shredding, the shredded ligaments are boiled in water for 3 to 5 hours After boiling, no ligament strands should be visible and there is an apparent home-generous solution. Subsequent to boiling, the solution is allowed to cool and it is filtered. The pi ox the solution is adjusted with dilute sodium hydroxide to a pi of about 6. To the solution having the adjusted pi is added 0.1 to 0.2 percent of an organic or inorganic peroxide or a combination thereof, along with a filtering aid, such as diatomaceous earth or the like and/or charcoal. The solution is then boiled for 1 to 5 hours and filtered. Additional peroxide treat-mints may be provided depending on the extent of cross-linking of the elastic starting material. The pi of the elastic solution is adjusted to about 5 to 6 and the solution is filtered. Stabilizers may be added to prevent degradation of the elastic partial hydrolyzate upon storage.

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The peroxides useful in destabilizing the desmosine and isodesmosine bonds are the organic and inorganic peroxides, and most preferably, the inorganic peroxides. Exemplary of inorganic per-oxides are hydrogen peroxide and ammonium per sulfate. Ammonium per-sulfate is represented by the structural formula:

O O

N OWE O O S O N H
11 -.
.--Organic peroxides may be used and are selected on the bass of decomposition half-life in the presence or absence of a suitable accelerator. Typical organic peroxides are tertiary-but~l hydropower-oxide, bouncily peroxide, laurel peroxide, dicumene hydroperoxide and : 10 the like. Combinations of various peroxides may be used in treating the insoluble elastic. Preferably, the peroxide is present at a level of about 0.025 to 0.5 percent by weight based on the weight of the insoluble elastic, and more preferably, about 0.1 to 0.25 percent by weight based on the weight of the insoluble elastic. It has been I found that hydrogen peroxide and ammonium per sulfate mixed together in approximately equal quantities are useful in treating the insoluble elastic. It is believed that the peroxide destabilizes the desmosine and isodesmosine cross-linkages, which is required to partially hydra-lyre and syllables the elastic. Preferably, the insoluble elastic is treated with the peroxide in the presence of water at above room temperature and for the final hydrolysis under refluxing conditions.
When ammonium per sulfate and hydrogen peroxide are used in about equal quantities as the peroxide constituents, the elastic is treated in the presence thereof under above ambient temperature conditions and below reflex at 12 to 18 hours and under reflex for 3 to 5 hours, and more preferably, 3 to 4 hours.

LO 3 2 So Although the peroxide destabilizes the desmosine and is-desmosine cross-linkages, it is necessary to further treat with acid to perform the complete hydrolysis. Most preferably, the acidic solution is composed of water and a mineral acid such as hydrochloric ac;cl, sulfuric acid or the like. However, weaker acids such as acetic acid may also be used but are not preferred. In order to hydrolyze the elastic it it necessary to heat the elastic in the presence of the acid, preferably at reflex and at atmospheric pros-sure. In the case of a solution of 3 to 10 percent hydrochloric acid, and preferably 5 to 7 percent hydrochloric acid, refluxing treatment is between 2 to 8 hours, and more preferably, 3 to 5 hours.
The substantially pure partially hydrolyzed elastic in act cordons with the above procedure has an average molecular weight of 8,000 to 15,000, with a substantial number of hydrolyzate molecules having a molecular weight between 500 and 1,000. Thus, the water soluble partial hydrolyzate has a molecular weight range of 500 to 15,000. It has been found upon analysis that the procedure according to the invention recovers substantially all of the desmosine and isodesmosine amino acid residues from the elastic, thus maintaining elastin-like characteristics to the hydrolyzate. Further, it has been found that the soluble elastic partial hydrolyzate in accordance with the invention has at least 3.5 desmosine and isodesmosine residues/
1,000 amino acid residues. In the case where some polypeptides are extracted during the hydrolysis procedure, it has been found that the desmosine and isodesmosine remain so that higher levels than 3.5 residues/1,000 residues and up to 6 residues/1,000 residues are pro-sent in the elastic partial hydrolyzate, evidencing the fact that all the desmosine and isodesmosine residues present in the starting material are recovered in the hydrolyzate.
The dissolved partially hydrolyzed elastic may be further purified by treatment with activated carbon or the like. Further, if the solution is hazy it may be further treated with hydrogen peroxide or a similar peroxide to make the solution transparent. Further ~L~2~3~'3~

stabilizers and antioxidant may be added to impart prolonged shelf life to the elastic solution. Typical stabilizers are sorbic acid, sodium bonniest and the like. The hydrolyzate is soluble in water and is parboil as a solution of up to 40 percent by weight at am-blent temperature.
If a non aqueous solution of the partially hydrolyzed elastic is desired, the water may be removed from the elastic solution by evaporation or the like, and it may be redissolved in propylene guy-got, dipropylene glycol or the like.
The invention is further illustrated by reverence to the lot-lowing examples.

In an appropriately sized vessel were charged 100 pounds of fresh beef neck ligaments (ligamentum Nash). water at 85C was added until the ligaments were fully immersed. One thousand grams of citric cold, 50 grams of ammonium per sulfate, and 500 milliliters of 30 percent hydrogen peroxide were charged to the vessel with agitation. The agitation was continued for 18 hours without the addition of heat thereto. The ammonium per sulfate and hydrogen per-oxide began the attack on the desmosine and isodesmoslne cross-linkages while the citric acid solubilized any collagen or other pretenses impurities in the ligaments. After agitation for 18 hours, the liquid and fat were drained from the vessel and the lima-mints were washed with cold water for approximately 1 hour, or until all the loose fat was removed. The ligaments were placed in an auto-crave at 40 pi (2.8 atmospheres) for about 6 hours. After auto-elevation, any excess liquid and fat were drained from the autoclave and the ligaments were washed in cold water for about 1 hour. For each 7.5 pounds of ligaments the following solution was added thereto:
10 liters of water, 2.2 pounds of sodium hydroxide and 3 pounds of sodium sulfate. The pi of the solution was 10 to 12. The ligalnents were immersed in this solution and allowed to stand for 18 hours. The solution was drained from the ligaments and the ligaments were washed with cold water for approximately 1 hour. The ligaments were charged to an appropriately sized stainless steel vessel and water was added thereto until fully immersed. Two liters of 37 percent Hal were added Lucy to the Bessel, agitated until homogeneous, and the ligaments were allowed to stand in the solution at ambient temperature overnight.
The pi ox the solution was about 7. After standing for 18 hours, the liquid was drained and the ligaments were washed with cold water for about 1 hour.
The washed ligaments were placed in an appropriately sized vessel and water was added thereto until fully immersed. Approxi-mutely 2.3 liters of 37 percent Hal were added to the water. The contents of the vessel were agitated with a high-speed rotatable mixer for 2 to 3 hours and the ligaments were fully shredded. The dispersion of shredded ligaments and water was placed in an appropriate vessel and heated at reflex for about 4 hours. After 4 hours, no ligament strands were visible. The solution was cooled to room them-portray and filtered. The filtered solution was placed in 5 gallon pails and adjusted to a pi of 6 with S normal aqueous sodium hydroxide.
To each 5 gallons of solution were added 100 milliliters of 30 percent hydrogen peroxide and 60 grams of ammonium per sulfate, along with 50 grams of diatomaceous earth and 75 grams of animal charcoal as a clarifier. The material was then placed in the appropriate vessel and heated For 2 to 3 hours under reflex and allowed to cool. After cooling, the material was filtered through fine filter paper and to each 40 pounds of material were added 80 milliliters of 30 percent hydrogen peroxide and 40 grams of ammonium per sulfate. The material was allowed to stand overnight. After standing overnight, the ye was adjusted to 5-5.5 with aqueous ammonium hydroxide and the solution was adjusted to 10 percent solids with water. The material was again filtered through fine filter paper, and to each 40 pounds of material were added 40 grams of sorbic acid, 20 grams of sodium bonniest and 40 grams of Germ all 115 antioxidant. The elastic partial hydrolyzate 30 produced in accordance with the Example had the -following analysis:
Total nitrogen 1.5 percent Elastic partial hydrolyzate 8.43 percent Dry matter 12 percent Ash 3 percent pi 5 to 5.5 Z

Amino acid analysis of the material showed that there were 3.9 combined desmosine and isodesmosine residues/l,OOO residues of amino acid residues.
The average molecular weight of the elastic was about 10,000, hazing a molecular weight distribution between 500 and 20,000.
' EXAMPLE 2 One hundred kilograms of collagen containing material, in-eluding raw hide, untanned tannery wastes, limed splits and trimming scraps were charged to a suitable vessel containing 300 liters of water having therein 10 kilograms of calcium hydroxide, Kilograms of sodium hydroxide and 5 kilograms of sodium chloride. The fibrous protein counterweighing material was allowed to stand in the aqueous soul-lion at ambient temperature for 5 days. After 5 days, the vessel was drained and the fibrous protein containing material free of hair and fats was neutralized to a pi of 7 by treating with an aqueous soul-lion of 30 liters of water which contained 1.5 kilograms of ammonium chloride and 1.5 kilograms of hydrochloric acid. The pal of the cross section of fibrous protein containing material was about 7. The neutralized fibrous protein containing material was washed with 300 liters of running water and deposited in 600 liters of distilled water for 12 hours. The distilled water extracted residual salts from the fibrous protein containing material. The distilled water was drained and the fibrous protein containing material was charged to an auto-crave for 8 hours at a pressure of 2 kilograms/cm2. The treatment under heat and pressure hydrolyzed the palpated bonds within the collagen while leaving the elastic in a cross-linked condition. The oligopeptides formed of the hydrolyzed collagen were water soluble and had a molecular weight between 5,000 and 20,000. After treatment under heat and pressure, the oligopeptide solution was cooled to about 4C. Upon cooling a small amount of fat rose to the surface of the solution and cross-linked elastic along with other minor impurities precipitated. The collagen oligopeptides in solution were heated and filtered through filter paper. The filter khakis autoclave at 145C
for 4 hours to separate fats. The filter cake had 25.56 percent dry matter, 0.7 percent ash, and 13.8 to 14 percent cross-linked elastic -based upon the weight of the dry matter. Ten pounds of the raw elastic ~L2~23~:52 containing material (i.e. the autoclave filter cake) was washed with 300 percent by weight water at 60C for 1 hour to remove salt and other soluble impurities. The dispersion of the cross-linked elastic in water alas separated by filtration through a stainless steel sieve and washed in accordance with the procedure previously described. Ten liters of 0.5 percent by weight ammonium per sulfate and 0.5 percent hydrogen peroxide solution was prepared and the cross-iinked elastic containing material combined therewith. The material was then boiled for 3-1/2 hours at 100C. After boiling the raw elastic containing material in peroxide aqueous media was filtered and mixed with 10 liters of 6 percent hydrochloric acid solution and agitated there-with. The resulting elastic solution was reflexed for 6 hours to partially hydrolyze the elastic. The partially hydrolyzed solubilized elastic was filtered through filter paper and the filtrate dried lo under vacuum.
The dried material was redissolved in 2.5 kilograms of disk tilled water and the pi adjusted to I using a 0.1 percent aqueous sodium hydroxide solution.
Five pounds of the elastic solution was mixed under agitation with 0.05 pounds of activated carbon and boiled for one hour to de-colonize the elastic solution. The active carbon was removed by lit-traction.
The filtered elastic solution was treated with 0.05 percent of hydrogen peroxide based on the weight of the elastic overnight at room temperature to remove haze from the solution and thus impart ox-tree clarity to the solution. The solution was again filtered and the concentration of the partially hydrolyzed elastic adjusted to 10 to 30 percent.
The final solution was stabilized to impart shelf life thereto at a ratio of US kilograms of elastic solution to 2.5 grams sorbic acid and 5 grams of phenip.
The elastic solution so produced is useful in eschew cosmetic applications.

I

The elastic hydrolyzate prepared in accordance with Example 2 has substantially the same composition and the same chemical and pry-steal properties as the elastic prepared according to Example 1.

Example 2 was repeated except that the dried partially hydra-lazed soluble elastic was dissolved in 70 percent ethanol.

Example 2 was repeated except that the partially hydrolyzed soluble elastic was dissolved in propylene glycol.

Example 2 was repeated except that the partially hydrolyzed soluble elastic which was dried was redissolved in dipropylene glycol.
Thus, in accordance with the invention, a partial hydrolyzate of elastic which was water soluble was prepared having from 3 to 4 desmosine and isodesmosine residues/1,000 residues and having an average molecular weight of 8,000 to 15,000.
Although the process in accordance with the invention is spew civically set forth in exemplary fashion based upon a starting material of raw hide, untanned tannery wastes, limed splits, trimming scraps, and tendons, other sources of elastic may be used, such as blood Yes-sots, hearts, lungs and the like.
When these other materials are used as starting materials, it is necessary to treat them in the same manner when low in elastic as raw hides, untanned tannery wastes and the like in order to remove got-lager, globular proteins, fats and other impurities therefrom in order to have an elastin-rich starting material to produce the partially hydrolyzed soluble elastic in accordance with the invention.
Although the invention has been described with reference to specific materials and specific processes, it is only to be limited so far as is set forth in the accompanying claims.

Claims (11)

THE CLAIMS:

1. A method of preparing soluble partially hydrolyzed elastin comprising:
A. treating insoluble elastin with ammonium persulfate;
B. partially hydrolyzing the elastin; and C. recovering substantially pure soluble partially hydrolyzed elastin.

2. The method of Claim 1 wherein including treating the insoluble elastin with hydrogen peroxide.

3. The method of Claim 1 wherein said ammonium persulfate is present at a level of about 0.025 to 0.5 percent by weight based on the weight of insoluble elastin.

4. The method of Claim 3 wherein said ammonium persulfate is present at a level of about 0.1 to 0.25 percent by weight based upon the weight of insoluble elastin.

5. The method of Claim 1 wherein said insoluble elastin is treated with peroxide in an aqueous solution at reflux and ambient pressure.

6. The method of Claim 1 wherein said treated elastin is hydrolyzed by heating in the presence of acid.

7. The method of Claim 6 wherein said acid is aqueous acid.

8. A method of preparing partially hydrolyzed soluble elastin from fibrous protein containing material comprised of insoluble elastin and collagen comprising:
A. treating the fibrous protein containing material with an aqueous solution of a member selected from the group consisting of an alkali earth metal hydroxide and mixtures thereof in the presence of an agent which prevents overswelling of the fibrous protein, said treatment removing substantially all of the hair and fat from the fi-brous protein;
B. hydrolyzing the polypeptide chains of the collagen to form oligopeptides;
C. separating the oligopeptides formed of the collagen from the insoluble elastin;
D. treating the insoluble elastin with ammonium persulfate;
E. partially hydrolyzing the treated elastin; and F. recovering substantially pure partially hydrolyzed soluble elastin.

9. A soluble elastin partial hydrolyzate comprised of poly-peptides having from 3 to 4 desmosine and isodesmosine residues/1,000 residues.

10. The elastin partial hydrolyzate of Claim 9 wherein the polypeptides have a molecular weight range of 1 to 20,000.

11. The elastin partial hydrolyzate of Claim 1 having an average molecular weight of 8,000 to 15,000.