US2988485A - Depilatory composition - Google Patents

Description

United States Patent O'ice 2,988,485 DEPILATORY COMPOSITION Harry E. Mattin, New York, and Leon- M. Greenstein, Brooklyn, N.Y., assignors to The Mearl Corporation, Ossining, N.Y., a corporation of New Jersey No Drawing. Filed Apr. 11, 1958, Ser. No. 727,788 3 Claims. (Cl. 16789) This invention is a continuation-in-part of copending application Serial No. 655,520, filed April 29, 1957, now abandoned, and relates to a depilatory composition of relatively low alkalinity having as the active ingredient thereof, a proteolytic enzyme capable of acting on native, unaltered keratin.

Various depilatory compositions in the form of powders, creams, and liquids have been used for the removal of hair from human skin. Such compositions find particular application as cosmetics when it is desired to remove hair from the arms, legs and armpits. Of the more modern compositions broadly referred to as chemical depilatories, effective depilation is caused by splitting the disulfide linkages in the cystine component of the keratin molecule in hair. When these disulfide linkages of the cystine are broken, the combination of the free sulfhydryl degradation products with the alkali present in the depilatory causes swelling of the hair and ultimate disintegration.

Thus, the chemical depilatory compositions of the prior art required the presence of both a reducing agent for attacking the disulfide linkages of cystine and an alkaline agent to cause a swelling and dispersion of the reduced keratinaceous protein.

The most commonly used depilatories have heretofore involved the sulfides and hydrosulfides of alkaline earth metals, and in order for effective depilation to occur it has been necessary to provide a medium of high alkalinity, usually above a pH of 11. The effects of compositions of high alkalinity on the skin should be obvious and there has existed a considerable problem as to how to provide a more cosmetically acceptable composition.

In accordance with the present invention, it has been found that the problem of contacting the skin with a highly alkaline material is avoided through the formulation of a composition containing a proteolytic enzyme preparation elaborated as a result of the microbial activities of a number of Streptomyces fungal and bacterial cultures.

This proteolytic enzyme, which has been isolated and characterized, has been called keratinase because of its ability to digest native, unaltered keratin. This keratinase is substantially free of sulfhydryl groups (SH) as distinguished from depilating compositions commonly used in prior art procedures.

Accordingly, it is an object of this invention to provide a depilatory composition which is quick, effective and cosmetically acceptable, and does not require the highly alkaline media of depilatories of the prior art.

Another object of this invention is to provide an enzymatic depilatory composition in which the active ingredient is relatively free of sulfhydryl groups and .in which the medium therefor is maintained at relatively low pH values.

Another object of this invention is to provide a depilatory of good stability, low cost and rapid effectiveness which may be used in combination with various cosmetic vehicles.

These and other objects of this invention will become more apparent from the description which follows:

Essentially the present invention involves a depilatory composition in which the effective depilating agent is a keratinase enzyme.

In order to provide effective depilation using this com A 2,988,485 PatentedJune 13, 196

position the concentration of keratinase is such that the keratinase activity is maintained between 200,000 and 1,000,000 keratinase units per 10 grams of final depilatory composition. One unit of keratinase as herein defined is that amount of enzyme which will digest wool keratin to the extent of producing an increase in optical density at 280 millimicrons (2800 A.) of a .040 in three hours at a pH of 8.6. Thus, the weight of an enzyme preparation to produce this effect will vary with the purity and activity of the preparation. For example, in a dried broth preparation, one unit of keratinase is present in approximately ,ug. of material while with a preparation prepared by ammonium sulfate precipitation, 10 g. of the precipitate contains one unit of keratinase.

The activities as defined above may be readily converted into weight percentages. For example, if the particular keratinase has an activity of 200 keratinase units per mg., the concentration would be maintained between about 1.0 and 5.0 percent, by weight, in order that the final dipilatory composition may be of the requisite activity.

The keratinase herein referred to is active in the pH range of about 7.0 to 10.0 with a maximum activity in the pH range of about 8.5 to 9.5. However, the stability of the keratinase in solution is greatest at about a pH of 7.0 and above a pH of 8.0 its activity will begin to diminish within one or two days on storage at room temperature. Thus, if the dipilatory composition herein contemplated is to be stored for any length of time after the keratinase has been incorporated therein, it becomes important to store the aqueous mixture in a cool area and in a suitably buffered medium having a slightly alkaline pH in the range of about 7.0 to 8.0. Dry compositions containing keratinase may be stored for long periods at room temperature without significant loss of activity.

An example of a method for preparing a keratinase of the type herein contemplated is set forth below and in copending application of Nickerson et al., Serial No. 680,- 930, filed December 20, 1960. As stated in the aforesaid application the enzyme composition is one which is effective to digest a substantial amount of keratinaceous material including the trypsin-digestible protein of said material and a substantial amount of the keratin protein of said material that is indigestible by trypsin, said proteolytic enzyme being characterized (a) by being water soluble; (b) by being non-dialyzable; (c) by having its keratin-digestive-ability destroyed by heating to about 100 C. for five minutes; (d) by having its maximum keratin-digestive-ability at a pH in the range of about 8.5 to 9.5; (e) by being precipitable from an aqueous solution thereof by the addition of ammonium sulphate; and (f) by having its keratin-digestive-ability dependent on the presence of at least trace amounts of chelatable metal ions. Although the method for preparing the enzyme per se forms no part of the present invention, it is now briefly described in order to provide a full and complete disclosure of how this material may be prepared.

The keratinase-containing proteolytic enzyme mixture may be produced by the treatment of various keratinaceous materials with microorganisms under conditions such as to facilitate degradation, the degradation products being Water soluble. Such microorganisms include Streptomyces fradiae, Streptomyces griseus, Streptomyces griseoluteus, Streptomyces rimosas and Streptomyces parvus. These organisms are described on pages 97, 87, 52, 47 and 46, respectively, of Actin'o-mycetes and Their Antibiotics by Waksman and Lechevalier. Strains of these micro-organisms have been deposited in the culture collection of the Institute of Microbiology of Rutgers, the State University, New Brunswick, New Jersey, and all the numbers hereinafter recited refer to the numbers in this collection. A strain of Streptomyces fradiae which has been found especially useful in preparing the keratinase enzyme is No. 3739, which has been isolated from soil and which has been shown to be of the species Streptomyces jradiae.

, Kera-tinaceous materials which are available to be treated in this manner are, for example, animal hair, hoofs of slaughtered animals, chicken feathers, fish scales, etc. The treatment of this keratinaceous material for the pnoduction of keratinase is best carried out in an aqueous solution containing mineral salts, which for the best results during fermentation should have a pH in the range of about 6.5 and 8.8, and preferably between about 7.2 and 8.5. It is to be noted that the pH rises as the fermentation proceeds. The pH control may be attained by suitable buffering. In a period of from three to five days, after suitable aeration and agitation, the fermented enzyme-rich broth is ready for harvest and recovery by well known methods. The purified, concentrated enzyme powder would commonly have an activity of about 200 keratinase units per mg.

The resulting enzyme is a proteolytic enzyme capable of digesting native, unaltered keratin and keratinaceous material and liberating therefrom water soluble polypeptides, said enzyme being further characterized in that it is non-dialyzable, heat denaturable, and precipitable with alcohol, acetone, and ammonium sulfate, said enzyme not requiring pre-treatment of keratin by material containing sulfhydryl groups or by reducing agents for its operation as a keratinase-digesting agent.

The depilatory composition of the present invention comprises the combination of the keratinase produced in the above manner and a suitable cosmetic base. As stated above, the final composition will contain the keratinase in such an amount that there are present from the least useful concentration of 200,000 to as high as 1,000,000 units of keratinase activity per 100 grams of final depilatory composition. Generally it is desired that the activity be at least 200,000 units per 100 grams.

The cosmetic base may be in the form of a cream, paste, powder or liquid. Such a base would generally include a wetting agent, a filler or diluent, bodying or bonding agents, and preferably a suitable perfume. The keratinase may be mixed with the cosmetic base either immediately before use, or, if desired, a product containing the mixture of keratinase and base may be manufactured and sold as such so that no mixing operation is required to be performed just prior to use.

In the event that the keratinase is pre-mixed with the cosmetic base it is necessary that a suitable buffer be used so as to maintain the pH of the mixture below about 8.0 and thereby give it a long shelf life.

Various embodiments of the present invention would include depilatory compositions in the forms of creams, powders, pastes, liquids and also suitable shaving cream compositions in which the addition of the keratinase is found to greatly assist or even replace the shaving oporation. Such shaving cream compositions may be of the lather, brushless, or aerated types, the basic ingredients of which are well known to the art and are set forth in more detail in the examples hereinafter referred to.

When it is desired to provide a composition which is to be sold to the ultimate consumer in its final mixed form including the keratinase and the cosmetic base in intimate contact with each other, it is usually desirable to keep the pH of this composition in the range of about 7.0 to 8.0. If in solutiondry preparations, applied as a paste, may be buffered to give a pH of 8.0 to 9.5 upon the addition of water. It should be understood, however, that the keratinase per se is stable in more nearly neutral media, but for the convenience of the ultimate user it is highly desirable that the mixture as sold be of such a pH which will render the keratinase sufficiently active so that the depilatory can be applied directly to the skin Without any further mixing being necessary.

Various buffer compositions are suitable for use in the composition of the present invention, but it has been found that one highly desirable mixture would contain the keratinase in a cosmetic base which includes a dilute phosphate buffer such as potassium hydrogen phosphate (K i-IP0 Other suitable buffering compositions would include the following: glycine-sodium hydroxide, pyrophosphoric acid-sodium hydroxide, 'boric acid-sodium hydroxide and sodium veronal-hydrochloric acid. Inasmuch as the skin is quite acid, and has a tendency to continuously produce said substances, the buffer concentration should be high enough to maintain the desired alkaline pH. Buifer concentrations between 0.1 M and 1.0 M are in order with 0.5 M being a particularly suitable concentration. In the case of a K HPO buffer, a 0.5 M solution will contain 8.7 grams per 100 ml. of final solution.

The cosmetic base which preferably would be in the form of a cream or paste will contain a filling agent or diluent having hydrophilic properties. Examples of such materials would be calcium carbonate (precipitated chalk), calcium oxalate, starch, talc, gypsum, lithopone, kaolin, kieselguhr, bentonite, etc. All of these agents are stable, inert to the components of the depilatory, inexpensive and readily available.

The bodying or bonding agent as in the cream depilatory would include various fatty alcohols. Examples of such agents would be lauryl and cetyl alcohols, sodium stearate or myristate and soaps.

The perfuming agents used can be chosen over a wide variety of such agents and in view of the relatively low pH in the present composition no problem arises as in the depilatory of the prior art in selecting only those perfumes which are stable at relatively high pH values. Moreover, Since a sulfide is not used in this depilatory there is no need to use the quantities and types of perfume heretofore necessary to mask the odor of hydrogen sulfide. Thus, in accordance with the present invention relatively small amounts of light, fragrant, floral odors may be used and the amount of perfume generally need not exceed one percent.

The powder and liquid depilatories are similar to the cream and paste except that the consistency has been altered. Thus, a powder depilatory would contain an anhydrous mixture of fillers, diluents and bodying agents and no water would be added until the user actually applies the composition, while the liquid may, in accordance with the common cosmetic practice, include a suitable bodying agent such as polyvinylpyrrolidone.

A liquid depilatory composition would include relatively large quantities of water, generally of the order of 60 to percent rather than be limited to the relatively small amounts of water usually present in the paste or cream depilatory. In the latter case, the water content is ordinarily maintained between 15 and 60 percent, by weight. When the depilatory is to be stored in an aqueous medium, either as liquid or paste, a suitable cosmetic preservative, such as hexachlorophene, may be used.

Although the keratinase may be used in a composition adapted to replace shaving altogether, it is also contemplated to use the present depilatory in combination with various shaving soaps. The range of soap bases which may be used is quite wide in view of the fact that the soap ingredient will not react with the keratinase depilatory. The soap may be in the form of a cake, paste, powder or liquid and generally speaking would include an emulsifying agent for wetting the natural oil present on-the skin and for lubricating the face so as to allow the razor to pass over easily. Conventionally, such shaving creams would be of the brush or lather type and would be made from a fatty acid such as stearic acid with sufficient soap to emulsify the fatty acid in water. Waxes or paraffin may also be added to the composition which would act as a lubricant to prevent the razor from pulling or scratching.

A typical cream would consist, for the most part, of a solid fatty acid, i.e. stearic, palmitic acid, etc, with or without a solid fat like hydrogenated fat, lard, waxes, etc, or mixtures thereof. The quantity of fatty acid may range from 5 to 30 percent depending upon the hardness of the particular vehicle. A certain proportion of soap or other emulsifying or wetting agent may be used, ranging from the least useful amounts of about /2 to percent or more may also be included. I s Perfumes and other antiseptic agents may also be used in small amounts as required. The Water content preferably ranges between to 60 percent. The resulting product is a cream of whatever consistency may be desired. The cream is also such that when put on the face it immediately penetrates the grease layer and spreads until a very thin layer is obtained and washes off very easily with water by dissolving or dispersing in the water solution. The cream should also be of such consistency that it will not thin out on the face and fall oif the razor during shaving.

SPECIFIC EXAMPLES A keratinase was prepared in the manner previously indicated by the action of Streptomyces fradiae No. I. M. 3739 on autoclaved hoof meal or on hoof meal sterilized by contact with ethylene oxide for twenty-four hours. Other batches of enzyme were similarly prepared from chicken feathers, wool, etc.

Iii making up an aqueous medium,- 100 ml. of a sterile solution of buffer salts such as 0.01 molar phosphate barter containing KH PO or K HPO for establishing the pH in the aqueous medium of about 8.5, are added for each 200 mg. of the keratin. After fermentation, the keratinase was salted out, concentrated, and purified as described above. The keratinase as prepared above was found to have an activity of 200 units per mg. Using this keratinase, the following dipilatory compositions were prepared:

Example I A cream depilatory is prepared from the following ingredients:

The pH value of the above composition was adjusted to 7.5 with phosphoric acid solution. The keratinase is relatively stable at room temperature between a pH of from about 7.0 to 8.0 and sufiiciently active within this pH range. Thus, it is to be understood that the .pH adjustment may be made to any desired value within this range.

In a variation on the above formulation the cream is prepared complete except for the keratinase and is adjusted to a pH value of 8.5 (or elsewhere in the range of optimum activity, 8.09.5). The keratinase in the form of dry powder or as a solution in dilute phosphate buffer at a pH of 7.0 is added to the cream shortly before use. In this manner optimum shelf life and optimum activity are obtained simultaneously. Although an operable system can be formulated at a pH value above 9.5, the higher pH is generally not preferred since enzyme stability is decreased without any advantage in enzyme activity.

Just. prior to application the powder is moistened with warm. Water and is applied to the skin as a semi-fluid paste, with a pH of about 8.5. The dry mixture is stable, however, in the absence of water, thus making possible a product with good shelf life and optimum enzyme activity at the same time.

Example III A liquid depilatory is prepared by using relatively large quantities of water in combination with a glycol such as propylene glycol as in the following:

Propylene glycol 12.0 Polyvinylpyrrolidone 0.3 Water a 76.4 Perfume 1.0 Sodium veronal 10.3

The solution is adjusted to a pH value of 8.5 with bydrochloric acid. It is convenient to do this before all the water has been added. The quantity of water can then be adjusted to make the total weight grams without causing further dilution. The keratinase (1.0 gram dry powder with an activity of 200 units per mg.) is added to the solution just before use.

If desired, the pH of the solution can be adjusted to 7.0-8.0 instead of 8.5. The keratinase can then be in-v cluded in the formulation at the time the original solution is made up. In this case, however, it is desirable to use a higher concentration of keratinase because of the lower activity of the enzyme at the lower pH value. Thus from 2.0 to 3.0 grams is desirable in the place of the smaller quantity shown above.

Example IV A typical shaving composition in the form of a brushless shaving cream is as follows:

Before the full quanity of water is added the pH value is brought to the range 7.0 to 8.0 with pyrophosphoric acid. As indicated in previous examples, a higher pH value may be utilized with a consequent increase in activity if the keratinase is added shortly before use. It should be emphasized that the actual pH value and keratinase composition depend largely on the desired shelf life and that many combinations of these two factors are satisfactory depending onthe period of aging which is contemplated.

Example V In a further embodiment keratinase is admixed with an aerosol shaving cream base as follows:

Gm. Keratinase 2.5 Lanolin 1.5 Stearic acid 9.0 Mineral oil 1.0

After the addition of part of the water, enough triethanolamine is added to bring the pH value to about 7.5. The rest of the water is then added. After mixing the above formulation it is charged into an aerosol can so that the material in the can consists of 92 percent of the formulation and about 8 percent of propellant. This procedure, of course, is well known to the art, and, therefore, need not be described in any further detail. The resulting aerosol shaving cream contains 2.5 percent, by weight, of keratinase with an activity of 200 units per mg, thereby having an ultimate composition of an activity of about 500,000 units per hundred grams of the depilatory shaving composition. In the above examples, wherein the keratinase was utilized as a shaving aid, it is desirable that the keratinase activity be relatively high because of the short contact time involved between the application of the depilating shaving cream and the actual shaving operation. In this connection, it would be preferred that the keratinase activity be maintained above 1,000,000 units per hundred grams of shaving composition.

Each of the above examples showing specific modes of carrying out the present depil ating process, is also operative with keratinase enzymes derived through the action of the following strains of the Streptomyces species listed below:

It is to be understood that the keratinase herein contemplated may be used as a shaving cream substitute as well as a shaving cream additive, and in such case, it would be desired that the activity be kept relatively high and the keratinase concentration also be in the upper part of the range herein set forth, preferably such that the activity would be above about 500,000 units per 100 grams of final depilatory composition. In this connection it may, in some instances, be desired to increase the keratinase activity beyond the range herein specified, but such a procedure would be limited by' economic considerations.

As a shaving cream substitute, a shaving cream type of composition can be used, so long as the keratinase activity is high enough, or else the depilating mixture may be similar to the earlier examples cited for general depilatory purposes.

Although the examples which have been given describe the use of a purified keratinase, the fermentation liquor itself or the crude enzyme obtained by merely drying the fermentation liquor could be used directly. However, the activity of the crude preparation must be taken into account; since the activity will be lower than that of the purified material, higher concentrations would have to be used. Dried fermentation liquor, for example, may have an activity of 12 units per mg. It is also recognized that the activity of the keratinase may be enhanced in the examples cited by the inclusion of neutral activators such as NaCl or Na SO at concentrations from 0.1 to 1.0 percent and by the addition of various water-soluble magnesium salts so as to result in a concentration of magnesium ion in the range of l 10- to 1 10- M with a preferred concentration of 1 10- M.

If desired, sulfhydryl-containing salts such as NaSH, Na or Ca thioglycolate may be added to further promote depilating action. These compounds, if used at all, would be present in concentrations far less than those existing depilatories. Such additives in these concentrations would not require the presence of highly alkaline medium.

The application of the keratinase depilatory to the skin for the removal of hair therefrom will be greatly facilitated by utilizing the present compositions in that the keratinase may be applied from weakly alkaline media and will, therefore, not cause the skin irritation which so often has accompanied the use of alkaline sulfide or hydrosulfide compositions of the prior art. Generally, the keratinase may be pre-mixed with the cosmetic base and sold to the ultimate consumer in a buffered medium between 7.0 and 8.0. However, when more rapid depilating action is required, as in shaving, it may be desirable to supply the user with the keratinase powder or keratinase solution at a pH where the keratinase has highest stability and a separate package of the cosmetic or shaving cream base, which materials will be mixed by the user and then applied to the skin.

When relatively low keratinase concentrations are used it may be well to apply the depilatory to the skin and allow it to stay on several hours, e.g. overnight, before removal of the hair is attempted. However, when a more highly active composition is used the depilatory may be applied directly and the hair will be readily removable in a very short time, i.e. in less than an hour and as rapidly as within five to ten minutes of application or even less at very high concentrations.

The above are examples of the use of the keratinase as a cosmetic depilatory composition. As a shaving aid the keratinase would also find particular use for pre-operation hair removal and in general for other uses where it is desirable to easily and efliciently remove hair from the skin.

In the foregoing, this invention has been described in connection with preferred embodiments thereof. Many variations and modifications of the principles of this invention within the scope of the description herein are obvious. Accordingly, it is preferred that the scope of the invention be bound not by the specific disclosure herein, but only by the appending claims.

We claim:

1. The method of removing hair from living human skin which comprises applying thereto a cosmetic composition having incorporated therein a depilatory having as the active ingredient thereof, a proteolytic enzyme effective to digest a substantial amount of keratinaceous material including the trypsin-digestible protein of said material that is indigestible by trypsin, said proteolytic enzyme being characterized (a) by being water soluble; (b) by being non-dialyzable; (c) by having its keratindigestive-ability destroyed by heating to about C. for five minutes; (d) by having its maximum keratin-digestive-ability at a pH in the range of about 8.5 to 9.5; (e) by being precipitable from an aqueous solution thereof by the addition of ammonium sulphate; and (f) by having its keratin-digestive-ability dependent on the presence of at least trace amounts of chelatable metal ions, said composition having a keratinase activity of at least 200,000 units per 100 grams of the total composition, maintaining the depilatory in contact with the skin for a short period of time and subsequently removing the depilatory and the hair from the skin.

2. The method of removing hair from living human skin which comprises applying thereto a cosmetic depilatory composition having a pH not greater than 9.5, having incorporated therein a depilatory and, as the active ingredient thereof, a proteolytic enzyme in which the enzyme is characterized by its ability to digest native, unaltered keratin, said enzyme being derived from the treatment of keratinaceous material with a strain of Streptomyces fradiae, said composition having a keratinase activity of at least 200,000 units per 100 grams of the total composition, maintaining the depilatory in contact with the skin for a short period of time, less than one hour, and subsequently removing the depilatory and the hair from the skin.

3. The method of claim 2 in which the composition is bufiered so as to maintain the pH thereof between 7.0 and 8.0.

References Cited in the file of this patent UNITED STATES PATENTS Le Petit Aug. 5, 1930 Boidin July 7, 1931 OTHER REFERENCES

Claims (1)

1. THE METHOD OF REMOVING HAIR FROM LIVING HUMAN SKIN WHICH COMPRISES APPLYING THERETO A COSMETIC COMPOSITION HAVING INCORPORATED THEREIN A DEPILATORY HAVING AS THE ACTIVE INGREDIENT THEREOF, A PROTEOLYTIC ENZYME EFFECTIVE TO DIGEST A SUBSTANTIAL AMOUNT OF KERATINACEOUS MATERIAL INCLUDING THE TRYPSIN-DIGESTIBLE PROTEIN OF SAID MATERIAL THAT IS INDIGESTIBLE BY TRYPSIN, SAID PROTEOLYTIC ENZYME BEING CHARACTERIZED (A) BY BEING WATER SOLUBLE, (B) BY BEING NON-DIALYZABLE, (C) BY HAVING ITS KERATINDIGESTIVE-ABILITY DESTROYED BY HEATING TO ABOUT 100*C. FOR FIVE MINUTES, (D) BY HAVING ITS MAXIMUM KERATIN-DIGESTIVE-ABILITY AT A PH IN THE RANGE OF ABOUT 8.5 TO 9.5, (E) BY BEING PRECIPITABLE FROM AN AQUEOUS SOLUTION THEREOF BY THE ADDITION OF AMMONIUM SULPHATE, AND (F) BY HAVING ITS KERATIN-DIGESTIVE-ABILITY DEPENDENT ON THE PRESENCE OF AT LEAST TRACE AMOUNTS OF CHELATABLE METAL IONS, SAID COMPOSITION HAVING A KERATINASE ACTIVITY OF AT LEAST 200,000 UNITS PER 100 GRAMS OF THE TOTAL COMPOSITION, MAINTAINING THE DEPILATORY IN CONTACT WITH THE SKIN FOR A SHORT PERIOD OF TIME AND SUBSEQUENTLY REMOVING THE DEPILATORY AND THE HAIR FROM THE SKIN.