JP5635222B2 - Antioxidant with medicinal properties - Google Patents

Description

The present invention relates to an anti-oxidant having medicinal properties, which is obtained by allowing a proteolytic enzyme (protease) to act on defatted sesame of black sesame.
Due to its excellent antioxidant activity, this antioxidant is used not only as an antioxidant, but also as a raw material or additive for health foods, and further as a pharmaceutical or additive for preventing or treating various diseases. It is highly probable and relates to health food preparation technology and pharmaceutical preparation technology.

Sesame seeds are very often used as a raw material for cooking oil.
In addition, it loves its flavor and is widely used not only in the food manufacturing industry and restaurants, but also in general households.
Furthermore, since pharmacological effects have been handed down to sesame and it is a safe material for the human body, various studies have been made on its components.

For example, Japanese Patent Application Laid-Open No. 08-231953 (Patent Document 1) includes lignans as characteristic compounds in sesame seeds, and studies on various physiologically active functions including their antioxidant activity have been made. In particular, it has been shown that phenolic lignans are present as sugar compounds (lignan glycosides) in sesame seeds or defatted cocoons thereof.
In addition, the sesame seed pulverized product is exemplified by the fact that a pinoresinol glycoside having an antioxidant effect on lipid oxidation can be obtained, and then a sesame seed humidified product or a germinated pulverized product or a defatted cocoon thereof is used. It discloses that a novel lignan glycoside obtained by extraction with a hydrous lower alcohol and removing a fat-soluble substance and a water-soluble substance from this extract is effective as a hydroxy radical scavenger.

  On the other hand, the applicant of the present application previously described in Japanese Patent No. 3108059 (Patent Document 2), as a method for producing a physiologically active peptide-containing material using a plant as a raw material, an incubation treatment with an acetic acid aqueous solution, A method for obtaining a peptide-containing product by protease treatment to reduce the molecular weight of a protein has been proposed.

  In Patent Document 2, a physiologically active peptide-containing material is obtained from polished white barley powder by a method for producing a physiologically active peptide-containing material. The obtained composition has an angiotensin I converting enzyme (ACE) inhibitory effect, tumor necrosis. In addition to having excellent physiological effects such as factor (TNF) -α production regulating action, active oxygen production inhibitory action, antitumor action, etc., it is disclosed that it is a natural product-derived composition that is extremely safe. .

  Furthermore, the applicant of the present application disclosed a method for obtaining a peptide-containing product by reducing the molecular weight of a protein, which can be applied to various plants.

That is, it is applied to all plants such as plant seeds, bark, fruit part, stem part, root part, leaf part, each part thereof or one part thereof, or a processed product thereof, food obtained therefrom, or a processed product thereof.
Plant seeds include pearl barley, rice, barley, wheat, oats, bare barley, buckwheat, soybeans, peas, broad beans, peanuts, green beans, corn, cucumbers, sesame seeds, millet, millet, sunflower, hemp seeds, vegetable seeds Others are mentioned.
The treated products include those obtained by threshing plant seeds, those obtained by pulverizing them, defatted products such as moss, yokoinin, hexane, water extraction residue, organic solvent extracts such as alcohol, and other various treatments of plant seeds. Things.
In addition, the processed product includes liquor cake, okara, shochu, soy sauce manufacturing cake, starch manufacturing cake, and other by-products when processing and treating plant seeds or processed products thereof to produce foods and drinks. The
JP-A-08-231953 (Claims, paragraph numbers 000 7-0009) Japanese Patent No. 3108059 (claims, paragraph numbers 0001, 0006)

  The inventor finds a peptide-containing product having an excellent function for a living body by reducing the molecular weight of the protein contained in various plant materials by a method of obtaining a peptide-containing product by reducing the molecular weight of the previously proposed protein. Therefore, a wide range of studies were conducted, and a study was conducted on the subject of finding a medicinal composition that can be used effectively as a raw material for health foods and pharmaceuticals.

As a result, the inventors of the present invention have developed a polypeptide, peptide, etc. by hydrolyzing the cocoon after extracting oil from black sesame seeds as a plant material, that is, defatted sesame of black sesame using a proteolytic enzyme. Preparation of low molecular weight protein and further containing bioactive peptide, especially that containing black polyphenol polymer, and further incubation in acetic acid solution in the previous method However, it was found that it is not necessary in the case of black sesame defatted lees, and the present invention was completed.

That is, the invention according to claim 1 of the present invention is
Using proteolytic enzymes, a shall such a degradation product obtained by the defatted lees black sesame hydrolyzed,
The decomposition product is an antioxidant having medicinal properties characterized by being insoluble in an aqueous ammonia solution having a pH of 9 .

The invention according to claim 2 of the present invention is
The use method for the manufacture of a pharmaceutical or health food of the antioxidant having the medicinal properties of claim 1 .

The antioxidant having the medicinal properties of the present invention exhibits the following excellent functions for living bodies in addition to the antioxidant function, and is effectively used as a raw material for health foods and as a raw material for pharmaceuticals. Is.
1. 1. Cholesterol lowering function 2. Prevention and healing function of fatty liver Improvement function of red blood cell deformability (improvement to so-called blood freezing condition)

  Since the antioxidant of the present invention can be used in a powder state or as an aqueous solution, weak acid or weak alkali aqueous solution, as an antioxidant and to express the above functions, It is used as a raw material for medicines, and at that time, it can be used efficiently and effectively.

  Such effects are surely present in antioxidants, such as lignan glycosides present in sesame, and low molecular weight proteins prepared by degradation, that is, oligopeptides and peptides, particularly bioactive peptides. Further, it is presumed to be achieved by the presence of polyphenol, which is a great and excellent feature of the antioxidant of the present invention.

In addition, the present invention effectively uses the defatted sesame meal of black sesame, which can be effectively used as an antioxidant for health foods and pharmaceuticals, and as a material for improving the function, as an antioxidant for defatted sesame meal of black sesame. It has an excellent effect of making it possible.

The anti-oxidant having medicinal properties of the present invention is obtained by using, as a raw material, a cocoon after extracting oil from black sesame, so-called sesame oil widely used for food, that is, defatted sesame of black sesame.
As will be described later, the defatted sesame seeds of black sesame contain a black polyphenol polymer having an excellent physiological function, and are essential as a raw material for the present invention.

In this invention, the antioxidant is dried or finely pulverized black sesame defatted soot, and then added to water, hot water or the like, and uniformly dispersed (homogenized) by stirring or the like. However, by adding a proteolytic enzyme to them and hydrolyzing the protein present in the defatted koji, it is used as an antioxidant having a medicinal effect including a low molecular weight protein.

As the proteolytic enzyme to be used, various proteases of animal, plant and microbial origin, including commercially available products, such as pepsin, papain, bioprase, promeline, thermolysin and the like can be used.
They can be used alone or in combination, and when used in combination, they may be used simultaneously or sequentially.
Preferred for the present invention is biolase (trademark; manufactured by Nagase ChemteX Corporation).

The decomposition temperature at the time of protein decomposition is determined by the activation temperature of the proteolytic enzyme used.
For example, in the case of protease, the temperature is about 45 to 60 ° C., and the decomposition time is determined according to the proteolytic ability and decomposition temperature of the proteolytic enzyme used, but is usually about 15 to 30 hours.

  The thus obtained antioxidant is presumed to be a composition containing a lignan glycoside, a low molecular weight protein such as a peptide, and a polyphenol polymer, and has various physiological functions described later. It can be effectively used as a raw material for health foods and as a raw material for pharmaceuticals.

In addition to the antioxidant function described above, the antioxidant of the present invention has a cholesterol-lowering function, a function of preventing and curing fatty liver, and a function of improving red blood cell deformability (so-called blood smoothness improvement). It has an action and is derived from natural food-related plants, and there is no problem in safety.
Therefore, it can be used as an antioxidant having medicinal properties in foods and drinks such as specified health foods and drinks, health foods, health drinks, and nutritional foods as well as normal foods and drinks. It is possible.

At this time, the antioxidant can be used in any of solid (powder, granule, etc.), concentrate, paste, liquid or suspension.
Furthermore, it can be used as tablets, capsules, granules, powders, syrups and the like by utilizing excipients, binders and the like used in health foods and pharmaceutical preparations.

This antioxidant is of natural origin and has been used as a traditional Chinese medicine and food for many years, so it has no or very low toxicity and exhibits excellent safety. There is no particular limitation, and even elderly, infants, and sick people can take it for a long time.

1) Preparation of anti-oxidant Deionized water was added to 1 kg of black sesame oil cake to make about 8 liters, adjusted to pH 9 with 5% ammonia solution, 10 g of biobrase was added, and the mixture was stirred in a thermostatic chamber at 55 ° C. The protein was hydrolyzed (incubated) using biolase (trademark: manufactured by Nagase ChemteX Corporation) for 20 hours while adjusting the pH to 9 with a 5% ammonia solution.
The obtained reaction solution was subjected to centrifugal filtration at 3,000 rpm for 10 minutes using an 80 mesh nylon filter cloth to separate the filtrate.
Further, about 4 liters of deionized water was added to the residue and stirred, and the filtrate was separated by centrifugal filtration as described above.
A combination of these filtrates is concentrated under reduced pressure using a rotary evaporator in a thermostatic bath at a temperature of 60 ° C., and further freeze-dried to produce an antioxidant (hereinafter referred to as “black P”) containing a low molecular weight protein. 525 g (yield: 52.5%) was obtained.
Moreover, according to the said procedure, the medicinal composition (henceforth "white P") was obtained with the yield of 44.5% using the white sesame oil cake for the comparison.
The analysis results of the components of the obtained black P are as shown in Table 1.

2) Analysis and fractionation by gel filtration Bio-Gel P-10 in which black P was dissolved in 0.1% aqueous ammonia solution and the supernatant obtained by centrifugation was washed with 0.1% aqueous ammonia solution in advance. The sample was applied to a (4 × 45 cm) column and developed with a 0.1% aqueous ammonia solution.
The eluate was collected in a fixed amount using a drop counter.
The components are detected by measuring the absorbance at 280 nm, and the results are shown in FIG. 1 (black P) and FIG. 3 (white P) as elution patterns in the gel filtration method.

3) Measurement of antioxidant activity 4.9 ml of linoleic acid-β-carotene solution was added to a spectrophotometer test tube cell into which 0.1 ml of each sample solution dispensed into 8 sections was dispensed, and stirred. The degree of fading of β-carotene when incubated in a thermostat at a temperature of 50 ° C. for a certain time was determined by absorbance at 470 nm, and the degree of oxidation converted to butylhydroxyanisole (BHA) concentration was determined.
The results are shown in FIG. 2 (black P) and FIG. 4 (white P).

4) Re-fractionation of black P To 10 g of black P, 200 ml of deionized water adjusted to pH 9 with ammonia was added and stirred to separate into dissolved and insoluble matters.
Insoluble matter (hereinafter, pH 9-P) was filtered and lyophilized to obtain 1.88 g.
The lysate was further adjusted to pH 5 with acetic acid and separated into lysate and insoluble matter.
Insoluble matter (hereinafter, pH 5-P) was filtered and freeze-dried to obtain 2.32 g.
Further, 5.80 g of a lysate (hereinafter, pH 5-S) was obtained by concentrating the solution under reduced pressure and freeze-drying.
The measurement results of their antioxidant activity are shown in FIG.
Moreover, the elution pattern by gel filtration is shown in FIG. 6 (pH5-S) and FIG. 7 (pH9-P).

5) Biological function test Thirteen adults aged 20 or older and under 65 years of age make tablets containing 75 mg of black P, and take 12 tablets a day (black P900 mg) in two divided doses for 4 weeks before ingestion In addition, erythrocyte deformability after ingestion and blood biochemical examination, as well as examination by doctors in charge of the examination, physical examination, and safety evaluation were performed.
The results of erythrocyte deformability and blood biochemical tests (fasting blood glucose, glycoalbumin, total cholesterol, LDL cholesterol, HDL cholesterol, etc.) are shown in Table 2.
In addition, the numerical value in each column of Table 2 is a value before ingestion in the upper row, and a value after ingestion in the lower row.

  The erythrocyte deformability uses a nickel mesh filter developed by the inventor as an alternative to the conventional method using a nucleipore filter, which is difficult to quantify and reproducibly. It was measured by the following method.

Nickel mesh is a nickel thin film filter made by combining a photoresist method and a special plating method. The number, shape and distribution of micropores are not only constant, but it is more than 100 times by ultrasonic cleaning for several seconds. Can be reused.
In addition, the edges of the nickel mesh micropores are smooth and tapered so that the leukocytes are not mechanically affected and the micropores have no fusion or branching.
Due to these characteristics, the following method maintains high quantitativeness and reproducibility.

Erythrocyte deformability measurement method (Nickel mesh filtration method)
In the test, a nickel mesh holder was connected to a vertically standing glass tube via a Tygon tube, and was prepared with physiological saline adjusted with a HEPES buffer, usually at a height of 15 cm (height: h). Filter the erythrocyte suspension.
Around the glass tube, constant temperature water is refluxed to keep the sample at a constant temperature.
The pressure drop during the filtration of the sample is continuously detected by a pressure (P) transducer installed at the zero level of the glass tube, and this is taken into a personal computer through an amplifier and an AD converter, and the flow rate (Q) ).
The flow rate is converted from pressure to height (P = ρgh), the differential value (dh / dt) of the height-time (ht) curve is taken, and this is multiplied by the cross-sectional area (a) of the glass tube. (Q = dh / dt · a).
The pressure-flow curve of the erythrocyte suspension was examined using the pressure-flow curve of a control solution that does not contain blood cells (HEPES buffer-adjusted saline: Newtonian fluid) as a control, and control at a certain constant pressure (usually 100 mm · H ₂ O). Erythrocyte deformability is evaluated based on the flow rate (%) of the erythrocyte suspension relative to the flow rate.

Of the data shown in Table 2, those having specificity or significance are illustrated in FIGS. 8 to 12.
FIG. 8 shows changes in total cholesterol before and after intake, similarly FIG. 9 shows changes in AST, FIG. 10 shows changes in ALT, FIG. 11 shows changes in γ-GTP, and FIG. 12 shows changes in erythrocyte deformability. Show.

From these figures, it was confirmed that by taking Black P, the values of total cholesterol, AST, ALT, and γ-GTP decrease and normalize in people with high values, and do not change in normal people. .
With regard to erythrocyte deformability, it can be seen that low values rise and normalize, and normal people do not change.

As is clear from these results, the antioxidant of the present invention has high antioxidant activity, cholesterol lowering function, fatty liver prevention and healing function, red blood cell deformation improving function, so-called blood smoothing function, etc. It has the following medicinal properties.

The anti-oxidant having medicinal properties of the present invention has the above-mentioned excellent characteristics and can be supplied as a powder or an aqueous solution, so that it is highly likely to be widely used in the health food industry and the pharmaceutical industry. It is.

It is an elution pattern figure in the gel filtration method of the antioxidant which used the defatted lees of black sesame of this invention as a raw material. It is a figure which shows the antioxidant activity of each fraction obtained by fractionating the antioxidant of this invention shown by the elution pattern figure of FIG. It is an elution pattern figure in the gel filtration method of the antioxidant which used the defatted lees of white sesame. It is a figure which shows the antioxidant activity of each fraction obtained by fractionating the antioxidant of this invention shown by the elution pattern figure of FIG. It is a figure which shows the antioxidant activity of each fraction obtained by fractionating the antioxidant of this invention shown by the elution pattern figure of FIG. 1 with an acid alkali. It is an elution pattern figure in the gel filtration method of fraction pH5-S. It is an elution pattern figure in the gel filtration method of fraction pH9-P. It is a figure which shows the change before intake after ingestion of the total cholesterol in Table 2. It is a figure which shows the change of AST in Table 2. FIG. It is a figure which shows the change of ALT in Table 2. FIG. It is a figure which shows the change of (gamma) -GTP in Table 2. It is a figure which shows the change of the erythrocyte deformability in Table 2.

    None

Claims (2)

Using proteolytic enzymes, a shall such a degradation product obtained by the defatted lees black sesame hydrolyzed,
An antioxidant having medicinal properties, wherein the decomposition product is insoluble in an aqueous ammonia solution having a pH of 9 . Use of the antioxidant having the medicinal properties according to claim 1 for the production of a medicine or a health food.