JP4112291B2 - Antioxidant - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a natural product-derived antioxidant that can be used in the fields of foods, cosmetics, quasi drugs, pharmaceuticals, and the like.
[0002]
[Prior art]
Fats and oils, especially fats and oils containing unsaturated fatty acids are easily oxidized, and are oxidized by various factors such as auto-oxidation or active oxygen to become lipid peroxides.
If this lipid peroxide is excessively generated and accumulated in the living body, lifestyle-related diseases (adult diseases) such as myocardial infarction, arteriosclerosis, diabetes, cancer, and stroke, and skin disorders such as stains, buckwheat, acne, and eczema In recent years, a number of reports have been reported. In food, cosmetics, quasi-drugs, pharmaceuticals, etc., lipid peroxide is produced during storage and use, which causes problems such as quality deterioration, nutritional deterioration, fading or browning, and toxicity to the human body. ing.
[0003]
As the action of such lipid peroxides becomes clear, antioxidants that suppress the formation and accumulation of lipid peroxides are used as preventive and therapeutic agents for the above-mentioned various diseases, or as foods, cosmetics, quasi drugs, and pharmaceuticals. It has been attracting attention as an oxidative degradation preventive agent for fats and oils contained in foods and the like, and is expected to be used practically.
For example, it is known that phenol derivatives such as BHA (butylhydroxyanisole) and BHT (butylhydroxytoluene) have extremely high antioxidant ability. However, since these are chemically synthesized, there are problems in safety such as side effects and mutagenicity on the human body, and there is a tendency to refrain from using them in foods, cosmetics, quasi drugs, pharmaceuticals, and the like.
[0004]
On the other hand, although antioxidants derived from natural products are evaluated for safety, they generally have a drawback that their antioxidant ability is inferior to that of synthetic antioxidants. Vitamin E (tocopherol), vitamin C (L-ascorbic acid), β-carotene, glutathione and the like are known as those having relatively excellent antioxidant ability. However, Vitamin E and Vitamin C have a problem in that they are poor in stability, particularly heat resistance, and are easily deactivated. Vitamin C and glutathione act as prooxidants (oxidation promoters) in the presence of metal ions, and may rather promote peroxidation of fats and oils depending on conditions. Since vitamin E and β-carotene are oil-soluble, they are difficult to dissolve in water and the application range is limited.
In recent years, attention has been paid to the antioxidant ability of polyphenol compounds such as catechin, quercetin and ellagic acid which are widely present in plants, and various searches have been conducted. However, these plant extracts themselves are often used as antioxidants, and as described above, not only have an antioxidant capacity, but also have a unique flavor and color, and their application range is limited. is the current situation.
[0005]
[Problems to be solved by the invention]
The present invention has been made in view of such a current situation, and the object of the present invention is to have an excellent lipid peroxide inhibiting effect, high safety to the human body, water-soluble and heat-added. However, it is to provide a natural antioxidant that does not deactivate, has no specific flavor and color, and can be used in the fields of food, cosmetics, quasi drugs, pharmaceuticals, and the like.
[0006]
[Means for Solving the Problems]
The present applicant has found that sericin extracted from silkworms or raw silk has a high lipid peroxide inhibitory effect and has filed a patent application (Japanese Patent Application No. 8-296015). As a result of further research based on such knowledge, the present inventor has found that sericin hydrolyzate extracted from silkworms or raw silk under specific conditions exhibits particularly excellent antioxidant ability, and completes the present invention. It came. That is, in the present invention, when silkworms or raw silk is extracted using an alkaline aqueous solution, and the resulting sericin hydrolyzate is isolated and purified, 0.5-10 mmol / L of sodium carbonate and sodium bicarbonate are added as the alkaline aqueous solution. The present invention relates to a method for producing a sericin hydrolyzate having excellent antioxidant ability, characterized by using a mixed aqueous solution containing 0.5 to 10 mmol / L. Moreover, the sericin hydrolyzate having excellent antioxidant ability of the present invention is usually used by adding to foods, cosmetics, quasi drugs and pharmaceuticals .
[0007]
DETAILED DESCRIPTION OF THE INVENTION
The sericin hydrolyzate used in the present invention is obtained by hydrolyzing a natural protein sericin present in silkworms or raw silk with a mixed aqueous solution of sodium carbonate and sodium hydrogen carbonate, and purifying it. Here, cocoon refers to cocoon, and raw silk refers to so-called cocoon yarn.
[0008]
In the present invention, the cocoon may be any of a rabbit that is raised and grown by a human and a wild pod that grows in a natural environment, and is not limited to a special type. Wrinkles are in a state in which wrinkles are contained, in a state in which a portion of the wrinkles are cut out, or in a state in which the wrinkles are taken out, or those that have been crushed, those that have also been made from raw silk, those that have been made into fabric by weaving, knitting, etc. Any of those that have been sewn or crushed can be used.
[0009]
For use in foods, cosmetics, quasi-drugs, pharmaceuticals, etc. that require particularly high safety, selection of silkworms and raw silk that do not require as much separation of impurities as possible is necessary because of the need to obtain high-purity sericin And use it. For example, there is a technique in which sericin is extracted from a cocoon that has been sterilized using an artificial feed that does not contain harmful substances such as heavy metals, and the cocoon has no cocoon.
The target sericin hydrolyzate is extracted from the above material using a mixed aqueous solution of sodium carbonate and sodium hydrogen carbonate as follows.
[0010]
The weight ratio of the cocoon or raw silk to the mixed aqueous solution is preferably the extraction solvent 20 to 30 with respect to the cocoon or raw silk 1. If the weight of the mixed aqueous solution is less than 20 with respect to the cocoon or raw silk 1, the volume of the cocoon or raw silk will be more than that of the extraction solvent, and the whole cocoon or raw silk will not be immersed or stirring operation will be impossible. As a result, the hydrolysis is insufficient, the water solubility of the sericin hydrolyzate is lowered, the antioxidant ability is not sufficiently exhibited, and the instability and reproducibility are reduced. When the weight of the mixed aqueous solution exceeds 30 with respect to the cocoon or raw silk 1, the load during refining is large and the efficiency becomes poor. The extraction temperature is preferably 80 to 100 ° C., and the time is preferably 30 minutes to 2 hours. When the extraction temperature is less than 80 ° C. or the extraction time is less than 30 minutes, the hydrolysis is insufficient, the water solubility of the sericin hydrolyzate is lowered, and the antioxidant ability is not fully exhibited. On the other hand, when the extraction temperature exceeds 100 ° C. or when the extraction time exceeds 2 hours, the damage to sericin is large, and the antioxidant capacity is lowered.
[0011]
Here, it is important that the concentration of sodium carbonate is 0.5 to 10 mmol / L, and the concentration of sodium bicarbonate is 0.5 to 10 mmol / L, more preferably 1 to 5 mmol / L. It is important that the concentration of sodium bicarbonate is 1 to 5 mmol / L, more preferably, both concentrations are equimolar. In the present invention, it is not impossible to use a sericin hydrolyzate extracted with an aqueous solution having a sodium carbonate concentration of 0.5 to 10 mmol / L and a sodium bicarbonate concentration of 0.5 to 10 mmol / L. However, in order to maximize the antioxidant ability of sericin and effectively achieve the object of the present invention, it is necessary that the concentration of the mixed aqueous solution has the above specific value. If either the concentration of sodium carbonate or sodium bicarbonate is less than 0.5 mmol / L, the water solubility decreases and it becomes difficult to effectively exhibit the antioxidant capacity inherent in sericin. On the other hand, when either the concentration of sodium carbonate or the concentration of sodium hydrogencarbonate exceeds 10 mmol / L, damage to sericin is large and excellent antioxidant ability cannot be obtained.
[0012]
The extraction is preferably performed at normal pressure. At normal pressure, there is no damage to sericin and it can be extracted stably and reproducibly. When it is performed under pressure, there is a possibility that problems such as damage to sericin are large and excellent antioxidant ability cannot be obtained, and unstable and poor reproducibility are generated. In addition, the cost for equipment and extraction increases, leading to an increase in cost. When it is performed under a low pressure, it is difficult to maintain the above-mentioned preferable extraction temperature of 80 to 100 ° C., and there is a possibility that the reproducibility is deteriorated because it is unstable. Similarly, the cost for equipment and extraction increases, leading to an increase in cost.
[0013]
The extract obtained here can recover the target sericin hydrolyzate by removing solids by filtration, centrifugation, etc., and further separating and purifying. The molecular weight of the sericin hydrolyzate in the extract is usually in the range of 5,000 to 100,000, and is broadly distributed. The separation and purification method used here is not particularly limited. For example, salting out, organic solvent precipitation, gel filtration chromatography, ion exchange chromatography, reverse phase chromatography, reverse osmosis, ultrafiltration, ultracentrifugation, A known method capable of fractionation by molecular weight such as electrodialysis, a known method for obtaining only a peptide with high purity, and a known method capable of efficiently removing salt can be used alone or in combination.
Through the process as described above, an aqueous solution containing a sericin hydrolyzate having particularly excellent antioxidant ability can be obtained. This may be used as it is or after adjusting to an appropriate concentration, or may be used after being dried by freeze drying, spray drying or the like.
[0014]
In the present invention, “excellent antioxidant ability” is compared with sericin hydrolyzate hydrolyzed in a solution other than the mixed aqueous solution of sodium carbonate and sodium hydrogen carbonate other than the present invention when measured according to the TBA method measurement test described later. It means that the amount of lipid peroxide produced is 1/10 or less and that the amount of lipid peroxide produced is 1/10 or less compared with vitamin C.
[0015]
When added to food, cosmetics, quasi-drugs, pharmaceuticals, etc., the sericin hydrolyzate as described above is more effective than sericin hydrolysates hydrolyzed with other than a mixed aqueous solution of sodium carbonate and sodium bicarbonate. Is 10 times or more, and if the same effect is expected, the addition amount can be less than 1/10. Since it is a hydrolyzate, it is highly hydrophilic and easily dissolved in cold water, the aqueous solution does not gel, and it is low in viscosity and easy to handle. Moreover, there is no special flavor and color, and even if it is added to foods, cosmetics, quasi-drugs, pharmaceuticals, etc., it does not affect the sensory characteristics, and the mixing with other raw materials is good.
[0016]
The antioxidant of the present invention can be applied to cosmetics, foods, quasi drugs, pharmaceuticals and the like in the same manner as conventional antioxidants. The antioxidant of the present invention is not toxic and does not cause any particular problems even when consumed in large amounts. What is necessary is just to determine suitably the addition amount to foodstuffs, cosmetics, a quasi-drug, a pharmaceutical, etc. from viewpoints, such as an effect and formulation stability.
[0017]
For example, when applied to cosmetics, quasi drugs, and pharmaceuticals for external use on the skin, the addition amount is usually 0.01 to 10% by weight, more preferably 0.1 to 2% by weight, still more preferably 0.5 to The applied amount is usually 1 to 100 mg, more preferably 10 to 50 mg, still more preferably 10 to 30 mg per 500 cm 2 of skin per day. Examples of forms to be applied externally include creams, emulsions, foundations, packs, lotions, powder oils, ointments and the like.
[0018]
In addition, when applied to foods to be taken orally, quasi-drugs, pharmaceuticals, etc., the addition amount is usually 0.01 to 100% by weight, more preferably 0.1 to 50% by weight, and still more preferably 0.5 to The dose to the human body is usually 1 to 2000 mg per kg body weight per day, more preferably 10 to 500 mg, and even more preferably 50 to 200 mg per day. Examples of forms in foods, quasi drugs, and pharmaceuticals include powders, solutions, gels, sticks, granules, capsules, and tablets.
[0019]
The scientific reason for the outstanding antioxidative performance compared with the sericin hydrolyzate similar to the sericin hydrolyzate used in the present invention is not yet clear, but the molecular weight including the molecular weight distribution etc. Presumed to be derived from sex.
[0020]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to a following example.
First, the production of the sericin hydrolyzate, an active ingredient of the antioxidant according to the present invention, will be described.
[0021]
Example 1
100 g of koji was added to 3000 g of distilled water, and heated at 95 ° C. for 1 hour using a mantle heater in a 5 L-volume eggplant flask equipped with a reflux. Next, sodium carbonate was added to 2 mmol / L and sodium hydrogen carbonate to 2 mmol / L, and the mixture was further hydrolyzed by heating at normal pressure at 80 ° C. for 1 hour. Undissolved soot residue was removed, and the extract was allowed to stand at room temperature, cooled, and then filtered through a 0.2 μm filter. This was purified by gel filtration chromatography and freeze-dried to obtain a sericin hydrolyzate solid. GPC analysis of this sericin hydrolyzate revealed that the weight average molecular weight was 40,000.
[0022]
Comparative Example 1
100 g of koji was added to 3000 g of distilled water, and heated at 95 ° C. for 1 hour using a mantle heater in a 5 L-volume eggplant flask equipped with a reflux. Next, sodium carbonate was added to 200 mmol / L, followed by hydrolysis by heating at 80 ° C. for 1 hour at normal pressure. Undissolved soot residue was removed, and the extract was allowed to stand at room temperature, cooled, and then filtered through a 0.2 μm filter. This was purified by gel filtration chromatography and freeze-dried to obtain a sericin hydrolyzate solid. GPC analysis of this sericin hydrolyzate revealed that the weight average molecular weight was 5000.
[0023]
Comparative Example 2
100 g of koji was added to 3000 g of distilled water, and heated at 95 ° C. for 1 hour using a mantle heater in a 5 L-volume eggplant flask equipped with a reflux. Next, sodium carbonate was added to a concentration of 0.05 mmol / L, followed by hydrolysis by heating at 80 ° C. for 1 hour at normal pressure. Undissolved soot residue was removed, and the extract was allowed to stand at room temperature, cooled, and then filtered through a 0.2 μm filter. This was purified by gel filtration chromatography and freeze-dried to obtain a sericin hydrolyzate solid. GPC analysis of this sericin hydrolyzate revealed that the weight average molecular weight was 80,000.
[0024]
Comparative Example 3
100 g of koji was added to 3000 g of distilled water, and heated at 95 ° C. for 1 hour using a mantle heater in a 5 L-volume eggplant flask equipped with a reflux. Next, citric acid was added to 0.1 mmol / L, and the mixture was further hydrolyzed by heating at normal pressure 95 ° C. for 2 hours. Undissolved soot residue was removed, and the extract was allowed to stand at room temperature, cooled, and then filtered through a 0.2 μm filter. This was purified by gel filtration chromatography and freeze-dried to obtain a sericin hydrolyzate solid. When GPC analysis of this sericin hydrolyzate was performed, it was found that the weight average molecular weight was 50,000.
[0025]
There are many reaction stages in the oxidation of fats and oils, and an antioxidant test has been established for each. Here, the antioxidant property was confirmed by two typical test methods.
[0026]
Antioxidant test 1 TBA method (1) Preparation of rat brain homogenate solution 9 g of 0.075 M phosphate buffer (pH 7.5) was added to 1 g of frozen rat brain and homogenized under ice-cooling (hereinafter referred to as homogenate solution).
[0027]
(2) Antioxidation test To 0.5 ml of this homogenate solution, 3.5 ml of 0.075M phosphate buffer (pH 7.5) containing the sericin hydrolyzate of Example 1 prepared to a final concentration of 0.1% was added, and the mixture was incubated at 37 ° C. Incubated for 2 hours. As a comparison, 3.5 ml of 0.075 M phosphate buffer (pH 7.5) containing sericin hydrolyzate of Comparative Examples 1 to 3 and vitamin C to a final concentration of 0.1% was similarly added to 0.5 ml of the homogenate solution. Incubated for 2 hours at ° C. 0.5 ml each of the liquid immediately before the incubation and after the incubation for 2 hours was collected, and TBARS (Thiobarbituric acid reactive substances), which is an index of lipid peroxide, was measured by the TBA (Thiobarbituric acid) method.
[0028]
(3) 2.5 ml of an 8% trichloroacetic acid solution and 2.0 ml of a 0.67% thiobarbituric acid solution were added to 0.5 ml of a TBA method sample, mixed well, and then heated in a boiling water bath for 15 minutes. Then, after centrifugation at 2000 G for 10 minutes, the absorbance of the supernatant at 535 nm was measured to determine the amount of TBARS. 1,1,3,3-tetraethoxypropane was used as a standard.
[0029]
The antioxidant capacity of each sample was calculated from the amount of TBARS when each sample was added and the amount of TBARS when not added according to the following equation 1, and was shown as the amount of lipid peroxide produced. The test was repeated three times and the results were shown as the average of the values obtained.
Formula 1 Lipid peroxide production (relative value when the control is 100) = A / B × 100
A: Increase value of TBARS amount when lipid peroxide inhibitor is added B; Increase value of TBARS amount when lipid peroxide inhibitor is not added (sample) (Liquid peroxide production amount)
Example 1) Sericin hydrolyzate 0.9
Comparative Example 1) Sericin hydrolyzate 25
Comparative Example 2) Sericin hydrolyzate 21
Comparative Example 3) Sericin hydrolyzate 16
Vitamin C 22
No additive (control) 100
The amount of lipid peroxide produced by the sericin hydrolyzate of Example 1 is 1/10 or less compared to the sericin hydrolyzate of Comparative Examples 1 to 3 and Vitamin C, and exhibits excellent antioxidant ability. Was confirmed.
[0030]
Antioxidation Test 2 Method by Quantification of Conjugated Diene (1) Preparation of Rat Brain Homogenate Solution The same procedure as in Antioxidation Test 1 was performed.
(2) Antioxidation test homogenate solution (0.5 ml) was added with 3.5 ml of 0.075 M phosphate buffer (pH 7.5) containing the sericin hydrolyzate of Example 1 prepared to a final concentration of 0.1%. Incubated for hours. As a comparison, 3.5 ml of 0.075M phosphate buffer (pH 7.5) containing each of the sericin hydrolyzate of Comparative Examples 1 to 3 and vitamin C to a final concentration of 0.1% was added to 0.5 ml of the homogenate solution in the same manner. Incubated for 2 hours at ° C. 0.5 ml of each of the solutions after the 2 hour incubation was collected, and 2.5 ml of ethyl ether / ethanol (1: 3) was added thereto and shaken vigorously for 1 minute. Subsequently, the mixture was centrifuged at 1000 G for 5 minutes, and the absorbance of the supernatant at 234 nm = the amount of conjugated diene was measured and used as an index of the amount of lipid peroxide produced.
[0031]
The antioxidant capacity of each sample was calculated from the amount of conjugated diene when each sample was added and the amount of conjugated diene when no sample was added according to the following equation 2, and was shown as the amount of lipid peroxide produced. The test was repeated three times and the results were shown as the average of the values obtained.
Formula 2 Lipid peroxide production (relative value when the control is 100) = C / D × 100
C: Increase value of conjugated diene amount when lipid peroxide inhibitor is added D: Increase value of conjugated diene amount when lipid peroxide inhibitor is not added (sample) (Liquid peroxide production amount)
Example 1) Sericin hydrolyzate 1.5
Comparative Example 1) Sericin hydrolyzate 29
Comparative Example 2) Sericin hydrolyzate 30
Comparative Example 3) Sericin hydrolyzate 30
Vitamin C 25
No additive (control) 100
Similar to the antioxidant test 1, the amount of lipid peroxide produced by the sericin hydrolyzate of Example 1 was 1/10 or less compared with the sericin hydrolyzate of Comparative Examples 1 to 3 and Vitamin C. It was confirmed that the antioxidant ability was exhibited.
In both different indexes, it was confirmed that the sericin hydrolyzate of Example 1 exhibits strong antioxidant ability. The same tendency was observed in the antioxidant tests 1 and 2, suggesting that it is more reliable and is not a phenomenon that can be seen only in a specific experimental system.
[0032]
【The invention's effect】
When the sericin hydrolyzate of the present invention is added to foods, cosmetics, pharmaceuticals, etc., it has an antioxidant effect of 10 times or more compared with conventional sericin hydrolysates, and if the same effect is expected. The amount added can be less than 1/10. Since it is a hydrolyzate, it is highly hydrophilic and easily dissolved in cold water, the aqueous solution does not gel, and it is low in viscosity and easy to handle. Moreover, there is no special flavor and color, and even if it is added to foods, cosmetics, quasi-drugs, pharmaceuticals, etc., it does not affect the sensory characteristics, and the mixing with other raw materials is good.

Claims (2)

When the cocoon or raw silk is extracted using an aqueous alkaline solution, and the resulting sericin hydrolyzate is isolated and purified , 0.5-10 mmol / L of sodium carbonate and 0.5-10 mmol of sodium bicarbonate are used as the aqueous alkaline solution. method for producing a sericin hydrolyzate having excellent antioxidant activity, characterized in Rukoto using a mixed aqueous solution containing / L. The method according to claim 1, wherein the extraction treatment is performed at 80 to 100 ° C for 30 minutes to 2 hours at normal pressure.