Lactoferrin hydrolyzate for use as a tyrosinase inhibition agent

Abstract: Lactoferrin hydrolyzates, having a decomposition rate between 4-50% as measured by formol titration, for use as a tyrosinase inhibition agent, which are obtainable by the conventional method for hydrolysis with acid or enzyme, and which are stable to heating.

Description: The present invention relates to the use of lactoferrin hydrolyzate for the manufacture of a tyrosinase inhibition agent. BACKGROUND OF THE INVENTION Lactoferrin is known as an iron-binding protein occurring in lacrima, saliva, peripheral blood, milk and the like. It has not been known that lactoferrin and its hydrolyzates have potent tyrosinase inhibition activity. Tyrosinase is known as an enzyme which may act as a catalyst for oxidization of tyrosin and other monohydric phenols and corresponding dihydric orthophenols with molecular oxygen and which widely occurrs in plants such as mushrooms, potatoes, apples as well as in animal tissues. It has been also known that tyrosinase is related to darkening phenomena at an injured portion of plant tissue, and also related to formation of melanine pigment in various tissues of animals, especially in epidermal cells (Editorial Committee of Encyclopedia Chimica, Encyclopedia Chimica, Vol. 5, page 976, Kyohritsu Shuppan; 1960). It has been also known that pigmentation of melanin in epidermal cells or mucous membrane in Addison disease is resulted from decrease in secretion of adrenal cortex hormones which antagonize melanotropin which in turn promote tyrosinase activity (Editorial Committee of Encyclopedia Chimica, Encyclopedia Chimica, Vol. 1, Page 65, Kyohritsu Shuppan; 1960) Therefore, it has been strongly desired, in the industrial fields of pharmaceutical, cosmetics, food and the like, to develop a tyrosinase inhibition agent for therapy of symptoms resulted from undesirable effects of tyrosinase activity and prevention therefrom. Especially in the cosmetics industry, researches have been actively made on cosmetics or medicines for external use for effective inhibition of melanin-formation and for whitening of skin, and many products containing tyrosinase inhibition agents have been successively developed. There has been known many tyrosinase inhibition agents, for example, cystein and vitamin C (Yutaka Mishima et al, Fundamental Dermatology, page 258, Asakura Shoten; 1973), kojic acid (Nikkei Sangyo Newspaper, May 24th 1988), arbutin (Ken-ichi Tomita, Preliminary Text for 20th F.J. Seminar, page 21, Fragrance Journal Company, March 14, 1990), products of microorganism belonging to the genus of Trichoderma (Unexamined Japanese Patent Application Gazette No. 2(1990)-145189). The conventional tyrosinase inhibition agents, however, had more or less defects that they are unstable in the products, they have excessively potent function to melanocyte which produce melanin pigment, and they are too expensive due to difficulty to obtain their raw materials, and they were not satisfiable to use as cosmetics or medicines for external use in the view points of safety, economics, preservability, reliability for whitening effect and so on. The inventors of the present invention found that lactoferrin, especially its hydrolyzate have potent tyrosinase-inhibition activity. The present invention is also based on this discovery. SUMMARY OF THE INVENTION In accordance with the present invention there is provided the use of lactoferrin hydrolyzate for the manufacture of tyrosinase inhibition agent characterized in that the lactoferrin hydrolyzate is obtainable by hydrolysis of lactoferrin. Preferable range in decomposition rate of lactoferrin hydrolyzate for use as tyrosinase inhibition agent is 4-50%, especially 6-40%, as measured by formol titration method (percentage of formol nitrogen to total nitrogen). It should be noted that an additional effect of bacteriostatic action can be expected when 0.05% or more of lactoferrin hydrolyzate having a decomposition rate of 6-20% is included in the tyrosinase inhibition agent. This provision is the subject-matter of European Publication No. 0 438 750. Lactoferrin hydrolyzate can be prepared by the conventional method (for example, hydrolysis of lactoferrin by organic or inorganic acids or by enzymes). Any lactoferrin substance can be used as the starting material for preparation of lactoferrin hydrolyzate, for example, lactoferrin obtainable in the market, native lactoferrin just isolated by the conventional method (for example, ion-exchange chromatography) from mammalian milk, apolactoferrin obtainable by removing iron from native lactoferrin with hydrochloric acid, citric acid and the like, metal saturated lactoferrin obtainable by chelating apolactoferrin with iron, copper, zinc, manganese and the like, or a mixture thereof (hereinafter these lactoferrin substances are abbreviated as LF). Any mammalian milk (for example, human breast milk as well as cow's, sheep's, goat's, horse's milk and the like) at any lactation stage (for example, colostrum, transitional milk, matured milk, milk in later lactation) can be used as the source of LF. Furthermore, processed milk or byproducts in milk-processing such as skim milk, whey and the like can be used as the source of lactoferrin (hereinafter they are referred to as milk and the like). Acid hydrolysis can be performed in accordance with the conventional methods, for example, LF is dissolved into water or purified water in a concentration within the range of 0.1 - 20 % (by weight, the same will be applied otherwise indicated), preferably 5 - 15 %, followed by pH adjustment of the resultant solution to 1 - 4, preferably to 2 - 3, and hydrolysis reaction at a proper temperature depending upon the pH of the solution. For instance, when the pH is adjusted to 1 - 2, the solution is heated at 80 - 130 °C, preferably at 90 - 120 °C; when pH is adjusted to 2 - 4, the solution is heated at 100 - 130 °C, preferably at 100 - 120 °C ; respectively for 1 - 120 minutes, preferably 5 - 60 minutes until decomposition rate (by formol titration) of LF hydrolyzate is reached to 6 - 20 %, preferably to 7 - 15 %. Enzymatic hydrolysis can be performed in accordance with the conventional method, for example, LF is dissolved into water or purified water in a concentration between 0.5 - 20 %, preferably 5 - 15 %, followed by pH adjustment of the resultant solution into the optimum pH range, and enzymatic hydrolysis under proper conditions, for example, a temperature between 15 - 55 °C, preferably between 30 - 50 °C for 30 - 600 minutes, preferably for 60 - 300 minutes. The reacted mixture is neutralized, followed by deactivation of the enzyme in accordance with the conventional method. In the preparation of tyrosinase inhibition agent, there is no limitation to the enzymes to be used, for example, Amano A (trademark, by Amano Seiyaku; optimum pH: 7.0), trypsin, exopeptidase originated from lactic acid bacteria recited in Japanese Examined Patent Application Gazette No. 48(1973)-43878 and commercial Shohyu-enzyme (soy sauce enzyme) comprising exopeptidase (by Tanabe Seiyaku) can be used, in addition to any acidic enzymes such as Molsin F (trademark; by Seishin Seiyaku; optimum pH.: 2.5 - 3.0), swine pepsin (by Wakoh Junyaku Kogyo; optimum pH: 2 - 3), Sumizyme AP (trademark; by Shin Nihon Kagaku; optimum pH: 3.0), Amano M (trademark; by Amano Seiyaku; optimum pH: 3.0) and the like, individually or in any combination thereof. The quantity of enzymes to be used may be 0.1 - 5.0 %, preferably 0.5 - 3.0 % to the substrate used. Regardless of the method of hydrolyzation, the resultant solution containing LF hydrolyzate is cooled by the conventional method, if necessary, followed by neutralization, demineralization, and decolorization. The resultant solution can be used as a liquid product as it is or if required, the solution is further concentrated and/or dried to obtain a concentrated liquid product or a powdery product. The conditions of hydrolysis referred in the above are not critical, but can be modified depending upon the temperature, the period of time, the pressure as well as the kind and quantity of the acid or enzyme used. LF hydrolyzate in the present invention is a mixture of the decomposed substances having different molecular weights. Thus obtained LF hydrolyzate is highly stable to heating and has tyrosinase inhibition activity. DETAILED DESCRIPTION OF THE INVENTION Now some tests will be described hereunder for exemplifying the utility of LF hydrolyzate as a tyrosinase inhibition agent. [TEST 1] The purpose of this test is to exemplify tyrosinase inhibition activity of LF hydrolyzate resulted from acid hydrolysis of LF. 1) PREPARATION OF SAMPLES Native LF sold in the market (by Oleofina, Belgium) was dissolved into purified water in 5 % concentration. The resultant solution was divided into several lots which were adjusted into different pH values, 1, 2, 3 and 4 by adding 1 M hydrochloric acid thereto. The resultant solutions having different pH values were subjected to hydrolysis reaction under different conditions in a combination of a temperature between 60 - 130 °C and a reaction time between 5 - 60 minutes, to thereby prepare 8 kinds of solutions of LF hydrolyzate having different decomposition rates as shown in Table 1. The resultant solutions were adjusted to pH 7 with 1 M sodium hydroxide, then lyophilized to thereby obtain 8 kinds of powdery LF hydrolyzate in different decomposition rates between 4 - 30 %. 2) METHOD 2-1) DETERMINATION OF DECOMPOSITION RATE Decomposition rate (%) of the resultant LF hydrolyzate was determined in such a manner that the quantity of formol nitrogen in the respective samples was measured by formol titration method, then the resultant values were applied to the following formula. Decomposition rate = 100 × (A / B) (wherein A denotes quantity of formol nitrogen, and B denotes quantity of total nitrogen) 2-2) MEASUREMENT OF TYROSINASE INHIBITION ACTIVITY 2-2-1) PREPARATION OF VARIOUS SOLUTIONS 2-2-1-1) PREPARATION OF SUBSTRATE SOLUTION L-tyrosin as guaranteed grade reagent (by Wakoh Junyaku Kogyoh) was dissolved into 0.1 M phosphate buffer solution in 0.045 % (W/V) concentration. 2-2-1-2) PREPARATION OF ENZYME SOLUTION Tyrosinase derived from mushroom (by Sigma, 3,000 unit/mg) was dissolved into 0.1 M phosphate buffer solution (pH 7.0) in 0.1 % (W/V) concentration. 2-2-1-3) PREPARATION OF COPPER ION SOLUTION Copper sulfate as guaranteed grade reagent (by Wakoh Junyaku Kogyoh) was dissolved into purified water in 1 % (W/V) concentration. 2-2-1-4) PREPARATION OF SAMPLE SOLUTION Each of the solutions of LF hydrolyzate previously prepared in item 1) in different decomposition rates was respectively dissolved into 0.1 M phosphate buffer solution (pH 7.0) to prepare sample solutions in 8 different concentrations, so that when the sample solutions were added in the proportions described in the next item 2-2-2), 8 lots of test samples in different concentrations as shown in Table 1 were prepared for each LF hydrolyzate having different decomposition rates. 2-2-2) ENZYMATIC REACTION Each of the resultant sample solutions, previously prepared substrate solution and the copper ion solution, which are previously heated to 37 °C, were mixed in a test tube in the proportion as follows: sample solution 1.0 mℓ substrate solution 0.9 mℓ copper ion solution 0.02 mℓ To each of the mixtures, 0.08 mℓ of enzyme solution which was previously heated to the same temperature was added (in total 2.0 mℓ), and enzymatic reaction was performed at 37 °C for 3 minutes. After the reaction, 2 mℓ of 30 % acetic acid solution was added to each of the reaction mixture to terminate enzymatic reaction, then absorbancy of the respective reaction mixtures was measured with a spectrophotometer at the wave length of 640 nm (the values of the absorbancy are denoted as B). As a control, a solution was prepared substituting 1.0 mℓ of 0.1 M phosphate buffer solution for sample solution, and the resultant solution was subjected to enzymatic reaction and measurement of absorbancy in the same manner as test samples No. 1 - No. 8 (the value of the absorbancy was denoted as A). In case of that the sample solution was muddy, absorbancy of a corresponding mixture which has the same composition as the muddy sample solution in question except that the enzyme solution was substituted with 0.08 mℓ of 0.1 M phosphate buffer solution was measured (the value of the absorbancy are denoted as C), and the value of C is reduced from the value B to obtain the difference of absorbancy of the reaction mixture before and after enzymatic reaction. The measured values were applied to the following formula to obtain tyrosinase inhibition rate (%). inhibition rate = 100 × [1 - (B - C) / A] 3) RESULTS The results are shown in Table 1. sample No. decomposition rate (%) concentration of LF hydrolyzate (%) 0.02 0.05 0.1 0.3 0.4 0.5 1.0 2.0 control 0 0 0 0 0 0 0 0 0 1 4 - - 3 11 - 19 30 50 2 6 11 29 59 80 - 100 - - 3 8 9 33 61 79 - 100 - - 4 10 16 33 63 85 - 100 - - 5 15 10 32 61 81 - 100 - - 6 20 9 30 62 77 - 100 - - 7 25 9 28 60 82 - 100 - - 8 30 13 31 58 81 - 100 - - Note: tyrosinase inhibition rate (%) Sample No. 1 which had 4 % decomposition rate showed about 10 % tyrosinase inhibition rate with addition of 0.3 % of the LF hydrolyzate. The inhibition activity increased as the concentration of LF hydrolyzate increased, 30 % inhibition rate with addition of 1 % LF hydrolyzate, and 50 % inhibition rate with addition of 2 % of the LF hydrolyzate. On the other hand, Samples No. 2 - 8 having decomposition rates 6 - 30 % showed about 30 % inhibition rate with addition of LF hydrolyzate only in 0.05 % concentration. The inhibition rate was increased as the concentration of LF hydrolyzate was increased, about 60 % with 0. 1 % concentration, 80 % with 0.3 % concentration, and 100 % with 0.5 % concentration. It was also confirmed that acid hydrolyzate of other LF substances such as apolactoferrin, zinc-, copper-, and iron-LF showed similar tyrosinase inhibition rate. [TEST 2] The purpose of this test is to exemplify tyrosinase inhibitory efficacy of LF hydrolyzate resulted from enzymatic hydrolysis of LF. 1) PREPARATION OF SAMPLE LF sold in the market (by Oleofina) was dissolved into purified water in 5 % concentration. The resultant LF solution was distributed into 8 lots to which different combination of enzymes comprising swine pepsin, Amano A and soy source enzyme containing peptidase sold in the market was added in different concentration between 0.1 - 6 % to prepare samples Nos. 9 - 16. The resultant mixtures were kept at 37 °C for different reaction times between 10 minutes - 24 hours for enzymatic reaction, the reacted solutions were adjusted to pH 7, heated to 80 °C for 10 minutes for deactivation of enzymes, then lyophilized to obtain LF hydrolyzate having different decomposition rates between 6 - 50 %. 2) METHOD Decomposition rates and tyrosinase inhibition rates of the respective LF hydrolyzate were measured in the same manner as in Test 1. 3) RESULTS The results are shown in Table 2. sample No. decomposition rate (%) concentration of LF hydrolyzate (%) 0.02 0.05 0.1 0.3 0.5 1.0 2.0 control 0 0 0 0 0 0 0 0 9 6 10 28 55 82 100 - - 10 10 8 32 60 79 100 - - 11 15 9 33 58 80 100 - - 12 20 12 31 62 78 100 - - 13 30 9 30 61 83 100 - - 14 40 11 28 60 80 100 - - 15 45 - - 2 11 23 35 54 16 50 - - 3 10 27 33 52 Note: tyrosinase inhibition rate (%) Sample Nos. 9 - 14 which had a decomposition rate between 6 - 40 % showed about 30 % tyrosinase inhibition rate with addition of 0.05 % LF hydrolyzate. The inhibition activity increased as the concentration of LF hydrolyzate increased, about 60 % inhibition rate was achieved with addition of 0.1 % LF hydrolyzate, about 80 % inhibition rate with addition of 0.3 %, and 100 % inhibition rate with addition of 0.5 % LF hydrolyzate. On the other hand, Sample Nos. 15 and 16 having decomposition rates of 45 and 50 % respectively showed about 10 % inhibition rate at 0.3 % concentration of LF hydrolyzate. The inhibition rate was increased as the concentration of LF hydrolyzate was increased, about 30 % inhibition rate with 1 % concentration, about 50 % inhibition with 2 % concentration. It was also confirmed that enzymatic hydrolyzate of other LF substance, that is apolactoferrin, metal saturated LF chelated with metals such as zinc, copper, iron and the like showed substantially the same tyrosinase inhibition rate. More specifically it was found that tyrosinase inhibition rate mainly depends upon decomposition rate of LF hydrolyzate. Now, some examples will be described for better understanding of the present invention. Example 1 LF solution was prepared by dissolving 80 g of LF sold in the market (by Oleofina) into 1000 mℓ of purified water, then pH of the resultant LF solution was adjusted to 2 with 1 M hydrochloric acid. The resultant solution was heated to 115 °C for 10 minutes, the solution was adjusted to pH 7 with 1 M sodium hydroxide solution, then lyophilized to thereby obtain about 77 g of LF hydrolyzate powder having 15 % decomposition rate as measured by the same method as in Test 1. Having homogeneously mixed 50 g of the resultant LF hydrolyzate, 900 g of glycine (by Wakoh Junyaku Kogyo), and 50 g of lysozyme (by Wakoh Junyaku Kogyo), about 1000 g of a tyrosinase inhibition agent to be used for keeping freshness of food was prepared. Tyrosinase inhibition rate of 20 % aqueous solution of the resultant tyrosinase inhibition agent was 100 % measured in the same method as in Test 1. EXAMPLE 2 After dissolving 270 g of LF sold in the market (by Oleofina) into 6300 mℓ of purified water, the resultant solution was adjusted its pH to 2.5 with 10 % aqueous solution of citric acid, and kept at room temperature for 1 hour for iron removing reaction. The reacted solution was subjected to ultrafiltration, and the resultant concentrate was lyophilized to thereby obtain about 260 g of apolactoferrin. Sixty g of the resultant apolactoferrin was dissolved into 1000 mℓ of water, the resultant solution was adjusted to pH 3 with 2 M phosphoric acid solution. The resultant solution was heated to 121 °C for 25 minutes, adjusted its pH to 7 with 1 M sodium hydroxide solution, then lyophilized to thereby obtain about 55 g of powdery LF hydrolyzate having 23 % decomposition rate measured by the same method as in Test 1. Having homogeneously mixed 20 g of the resultant LF hydrolyzate, 400 g of propylene glycol (by Wakoh Junyaku Kogyo), 4 g of oleyl alcohol (by Wakoh Junyaku Kogyo), 200 g of ethanol (by Wakoh Junyaku Kogyo) and 3376 g of purified water, about 4000 g of tyrosinase inhibition agent for use in cosmetics was obtained. Apparent inhibition rate against tyrosinase of the resultant agent was 75 %, but substantial inhibition rate against tyrosinase was calculated 100 % in consideration of LF hydrolyzate concentration. EXAMPLE 3 Having homogeneously mixed 100 g of LF sold in the market (by Oleofina) and 1000 mℓ of purified water, pH of the resultant solution was adjusted to 2 with 1 M hydrochloric acid. To the resultant solution, 5 g of swine pepsin sold in the market (by Wakoh Junyaku Kogyo) was added and reacted at 37 °C for 60 minutes. The reacted solution was heated to 80 °C for 10 minutes for deactivation of the enzyme, then lyophilized to thereby obtain about 95 g of LF hydrolyzate having 12 % decomposition rate measured by the same method as in Test 1. About 2000 g of tyrosinase inhibition agent for cosmetic use was prepared by homogeneously mixing 30 g of the resultant LF hydrolyzate, 200 g of propylene glycol (by Wakoh Junyaku Kogyo), 2 g of oleyl alcohol (by Wakoh Junyaku Kogyo), 100 g of ethanol (by Wakoh Junyaku Kogyo) and 1668 g of purified water. Tyrosinase inhibition rate of the resultant tyrosinase inhibition agent was 100 % as measured by the same method as in Test 1. EXAMPLE 4 One hundred and fifty g of LF sold in the market (by Oleofina) was dissolved into 150 g of purified water, pH of the resultant solution was adjusted to 6 with 1 M sodium hydroxide solution, then pasteurized at 60 °C for 10 minutes. The resultant solution was cooled to 50 °C, and 15 g of trypsin (by Nobo) and 30 g of soy sauce enzyme containing peptidase sold in the market (by Tanabe Seiyaku) were added to react at 50 °C for 5 hours. The reacted solution was heated at 80 °C for 10 minutes for deactivation of the enzymes, lyophilized to thereby obtain about 145 g of powdery LF hydrolyzate having 38 % decomposition rate as measured by the same method as in Test 1. Having homogeneously mixed 12 g of sodium hyaluronate (by Wakoh Junyaku Kogyo), 15 g of placenta extract (by Botoger, West Germany), 10 g of glycerin (by Wakoh Junyaku Kogyo) and 962 g of purified water, about 1000 g of tyrosinase inhibition agent was obtained. Tyrosinase inhibition rate of the resultant agent was 100 % measured by the same method as in Test 1. EXAMPLE 5 About 78 g of Fe-LF was prepared by dissolving 90 g of LF sold in the market (by Oleofina) into 2100 mℓ of purified water, the resultant solution was reacted with 755 mℓ of 2.6 mM aqueous solution of ferrous sulfate at room temperature for 24 hours, the resultant reacted solution was subjected to ultrafiltration, then the resultant concentrate was lyophilized. Forty g of the resultant Fe-LF was dissolved into 500 mℓ of purified water, pH of the resultant solution was adjusted to 3 with 1 M hydrochloric acid solution. To the resultant solution, 5 g of Sumizyme AP (by Shin Nihon Kagaku) was added and the resultant mixture was subjected to hydrolysis at 30 °C for 3 hours. The reacted mixture was heated at 80 °C for 10 minutes for deactivation of the enzyme, then lyophilized to thereby obtain about 35 g of LF hydrolyzate having 18 % decomposition rate measured by the same method as in Test 1. About 500 g of tyrosinase inhibition agent to be used for keeping freshness of food was prepared by homogeneously mixing 30 g of the resultant LF hydrolyzate, 450 g of glycine (by Wakoh Junyaku Kogyo), 20 g of lysozyme (by Wakoh Junyaku Kogyo). Tyrosinase inhibition rate of 20 % aqueous solution of the resultant agent was 100 % measured by the same method as in Test 1. In summary, it can be concluded that: 1) LF hydrolyzate for use as a tyrosinase inhibition agent is safe for human and animals, since it is a natural tyrosinase inhibitory substance derived from hydrolysis of milk components and the like. 2) LF hydrolyzate for use as a tyrosinase inhibition agent has remarkably potent tyrosinase inhibition activity. 3) Lf hydrolyzate for use as a tyrosinase inhibition agent is stable to heating, and can be provided in liquid and powdery forms, thus it has wider application. 4) The tyrosinase inhibition agent comprising LF hydrolyzate can be prepared by mixing them with one or more of excipients or other medicines, inclusive of other tyrosinase inhibition agents. 5) The tyrosinase inhibition agent consisting of or comprising LF hydrolyzate can be utilized as a component of various products such as cosmetics, foods, feeds and other products which are desirable to be prevented or inhibited from undesirable effects of tyrosinase activity. It is specifically noted that inclusion of the tyrosinase inhibition agent consisting of or comprising LF hydrolyzate in such products is effective not only for preservation of the products, but also effective for therapy of pigmentation of melanine or prevention there from when the products are given to human and other animals or applied to the body surface thereof. 6) The tyrosinase inhibition agent consisting of or comprising LF hydrolyzates can be utilized for treatment of various materials, for example washing or dipping the materials in a solution of the agent so that the agent is sticked to or coated onto or impregnated into those materials, for maintenance of sanitary conditions and for prevention from deterioration in freshness thereof. It is specifically noted that the materials treated are also effective for therapy against pigmentation of melanine or prevention there from when they are taken into or applied onto the body surface of human or animals subject to that effective amount of LF hydrolyzate is accompanied therewith (or remained therein).

Claims: Use of lactoferrin hydrolyzate for the manufacture of tyrosinase inhibition agent characterized in that the lactoferrin hydrolyzate is obtainable by hydrolysis of lactoferrin. The use according to claim 1 wherein decomposition rate of said hydrolyzate is 4-50%. The use according to claim 1 or 2 wherein at least 0.05% of said hydrolyzate is included in said agent.

Inventors: KOBAYASHI SUSUMU, TOMITA MAMORU, KOBAYASHI, SUSUMU, TOMITA, MAMORU
Applicants: MORINAGA MILK INDUSTRY CO LTD, MORINAGA MILK INDUSTRY CO., LTD.
Application year: 1990
Main classification: A61K38
IPC codes: A61K38