JP2001354958A - Antioxidant and its manufacturing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antioxidant which has no uneasiness of the side effect of synthetic antioxidants when used in the fields of foods, cosmetics, and drugs and has anti-oxidant capacity withstanding the use in the above fields. SOLUTION: The antioxidant contains an extract of Zanthoxylum piperitum seeds as the effective component.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an antioxidant which can be used in the fields of foods, cosmetics, pharmaceuticals, etc. More specifically, the present invention relates to a novel antioxidant containing an extract from Sansho as an active ingredient. The present invention relates to the manufacturing method.

[0002]

BACKGROUND ART Conventionally, chemically synthesized substances such as BHT (2,6-di-t-butyl-4-methylphenol) and BHA (butylhydroxyanisole) have extremely high antioxidant properties. Is known, but in recent years,
Since there is a concern about side effects on humans and the possibility of mutagenicity, there is a tendency to refrain from using it in foods, cosmetics, pharmaceuticals, and the like.

[0003] On the other hand, as an antioxidant which can be industrially obtained from natural products, vitamin E is conventionally known and used in foods, cosmetics, pharmaceuticals and the like. Furthermore, in recent years, Japanese Patent Application Laid-Open Nos.
As described in JP-A-000-73057 and the like,
In addition to rosemary and leaf tobacco, natural antioxidants derived from plants such as tea leaves and sesame have also been proposed, and are expected to have low side effects on the human body. .

However, among such conventional natural antioxidants, an antioxidant obtained from one plant generally has extreme oil-solubility or water-solubility, so that its application range is limited. There was a problem that would be done.
That is, it has been extremely difficult to obtain from a single plant a natural antioxidant capable of exhibiting an effective effect regardless of whether the target is aqueous or oily.

It has been experimentally and theoretically confirmed that Vitamin E is extremely oil-soluble but can exert a large antioxidant property. Since Vitamin E is often dissolved, expectations for an increase in the antioxidant capacity by the new addition of Vitamin E are not so large in reality.

[0006]

Here, the present invention has been made in view of the above-mentioned circumstances, and the object of the present invention is to provide a lipophilic or hydrophilic property as required. An object of the present invention is to provide a novel antioxidant derived from a natural product, which is different from vitamin E, in which an oxidized substance can be appropriately obtained, and an advantageous method for producing the same.

[0007]

An embodiment of the present invention which has been made to solve such a problem will be described below. The components employed in each of the embodiments described below can be employed in any combination as possible. In addition, aspects or technical features of the present invention are not limited to those described below, but are described in the entire specification and drawings, or based on the invention ideas that can be understood by those skilled in the art from the descriptions. It should be understood that it is recognized on the basis of.

That is, in order to solve the above-mentioned problems,
The inventors screened a large number of natural products widely used as foods for a substance having antioxidant ability, and as a result, sansho (Zanthoxylum piperitum DC.)
The extract has a new antioxidant ability different from the known plant for the collection of conventional antioxidants, especially natural substances derived from antioxidants having both lipophilic and hydrophilic properties. A novel antioxidant has been found, and as a result of intensive studies based on such knowledge, the present invention has been completed.

The feature of the present invention relating to the antioxidant is that the antioxidant contains a pepper extract as an active ingredient.

Another feature of the present invention relating to the method for producing an antioxidant is that a material containing at least one of a seed, a seed coat and a leaf of a pepper is extracted using an organic solvent to obtain a pepper extract. After that, an antioxidant is obtained by extracting and separating a vegetable oil from such a pepper extract.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION
Conventionally known citrus family salamander (Zanthoxylum pipe
ritum DC.) and its congeners. As a congener plant, Asakusan show (Zanthoxylum piperium DC. F
orma inerme Makino), Yamasa cherry tree show (Zant)
hoxylum piperium DC.forma brevispinosum Makino
), Pepper (Zanthoxylum bungeanum Sieb. Et Zuc
c.), Zanthoxylum schinifolium Sie
b. et Zucc.), Zanthoxylum avicen
nae DC., Zanthoxylum simulans Hance, Zanthoxylum p
lanispinum Sieb. et Zucc.) and the like. In particular,
Any kind of pepper that is used for food can be suitably used.

In the present invention, the portion of the pepper used to obtain the antioxidant, that is, the extracted portion, is the trunk of the pepper,
Any site such as branches, roots, stems, epidermis, etc. can be targeted,
In particular, the seeds, seed coats and leaves of the pepper are preferred, and the seeds and seed coat of the pepper are more preferred from the viewpoints of collection efficiency of the antioxidant and workability.

Further, as a method for obtaining an extract which can be an antioxidant from the pepper, for example, it is possible to employ exploitation or the like. Preferably, a material comprising an appropriate portion of the pepper is used, if necessary. After pretreatment by cutting, crushing, pulverizing, or the like, the target extract can be advantageously obtained by extracting the components with an organic solvent, a water-containing organic solvent, water, or the like.

More specifically, for example, ethanol, methanol, 1-propanol, 2-propanol, 1-propanol,
Butanol, alcohols such as 2-butanol, ethers, ethers such as tetrahydrofuran, esters such as ethyl acetate, ketones such as acetone, nitriles such as acetonitrile, aromatic hydrocarbons such as benzenetoluene, methylene chloride, By appropriately selecting and using halogenated aliphatic hydrocarbons such as chloroform, n-hexane, water, and the like, it is possible to extract the target component from the pepper, particularly preferably after extraction. From the viewpoints of safety, operability, handleability, and the like in use in foods and the like, alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, and 2-butanol, and n-hexane are used. .

The thus-obtained pepper extract can be used as it is. If necessary, a solvent such as water or alcohol is removed to obtain an antioxidant. Furthermore, it is also possible to add a decolorizing or deodorizing treatment to the extract. Specifically, the decolorization treatment is performed by, for example, mixing activated carbon or bleached soil, heating and filtering the mixture. The deodorizing treatment is performed by, for example, vacuum distillation.

Furthermore, the obtained pepper extract can be provided as a dried product by removing the solvent by a freeze-drying method or a spray-drying method using a freeze dryer or a spray dryer. Further, it can be provided in a state of being dissolved in an appropriate solvent or in the form of an emulsion. Furthermore,
If necessary, various surfactants are added to prepare a lipophilic or hydrophilic milk, whereby the lipophilicity or hydrophilicity is appropriately adjusted, and thus the anti-microbial property of the specific target used is reduced. An antioxidant that can more effectively and continuously exhibit an oxidizing ability can also be used.

Further, for example, a reversed phase system, a polyamide system,
It can also be used as a fraction obtained by treating the obtained pepper extract using chromatography using a hydrophobic chromatography resin or the like. By performing such a fractionation, it is possible to extract only specific antioxidants having different degrees of lipophilicity or hydrophilicity from the pepper extract, for example, depending on the target to be used, It is also possible to selectively provide an antioxidant having a specific property or to adjust the property.

The antioxidant fractionated by chromatography or the like is similarly subjected to deodorization and decolorization treatments as necessary, and is also used in a dry state, an emulsion,
It is provided in a suitable form such as a solubilized state.

Furthermore, the pepper extract according to the present invention can be used as an antioxidant, but can also be used as a hydroxy radical scavenger. That is, by adding the pepper extract as described above, it is possible to suppress the radical reaction upon oxidation or the like, or to slow down the rate of the radical reaction generated in a chain. Applications to fields other than the category of antioxidants, such as erasing agents, are also possible.

[0020]

EXAMPLES In order to clarify the present invention more specifically, antioxidants as examples of the present invention will be described below.
The present invention is not limited to only these examples.

Example 1 500 g of methanol was added as an organic solvent to 100 g of pepper seed powder, and the mixture was shaken for 24 hours at room temperature protected from light. An extract was obtained by filtration through a membrane filter. Further, 500 ml of methanol was added to the residue after the filtration, and the same extraction operation as described above was repeated six times, and a total of 3 liters of filtrate (extract) was obtained by the six extraction operations.

Subsequently, distilled water was added to the obtained extract to make the whole an 80% methanol solution, and 500 ml of the methanol solution was added to a 2,000 ml separatory funnel, and n-
After adding 125 ml of hexane and shaking, the aqueous layer and n-
To make the boundary of the hexane layer clear, several ml of a saturated saline solution was added to remove the n-hexane layer. After repeating the same operation twice, the aqueous layer was collected and dried under reduced pressure at 40 ° C. or lower. Further, the obtained dried product is desalted by adding methanol, filtered through a 0.45 μm membrane filter, and the obtained filtrate is dried at 40 ° C. or lower under reduced pressure to obtain about 10 g of a target. An antioxidant (Example 1) derived from a Japanese pepper seed extract was obtained.

"Test 1" Using the accumulation of lipid peroxide due to the autoxidation of unsaturated fatty acids as an index, the antioxidant ability of the above "Example 1" by adding the antioxidant was examined according to the test method described below. .

(Preparation of sample) 0.13 ml of linoleic acid was added to a 50 ml Erlenmeyer flask with a stopper, and ethanol was added.
Dissolved in ml. Subsequently, 10 ml of a 50 mM phosphate buffer (pH 7.0) and 5 ml of distilled water were added. Samples were prepared as Examples and Comparative Examples by adding antioxidants and the like as in Example 1 and Comparative Examples 1 and 2 below. Example sample (a): 100 μm of the pepper extract obtained in Example 1
Comparative sample (a): 100 μg of methanol was added Comparative sample (b): 100 μg of natural vitamin E was added

Each of the three samples obtained as described above was incubated at 40 ° C. in the dark.
Then, lipid peroxide produced by autoxidation of linoleic acid was analyzed by a thiocyanic acid method described below. In addition, the measurement for such an analysis is based on the Abs. The process was performed until the value at 500 nm became approximately 1.0, and the process was completed. "Thiocyanic acid method" The accumulation of lipid peroxide produced by oxidizing linoleic acid is measured according to the following principle. That is, lipid peroxide deprives electrons of divalent iron added as a coloring reagent and oxidizes it to trivalent iron. This iron oxide reacts with ammonium thiocyanate to give a red color and is measured. I do. More specifically, the antioxidant ability test sample was placed at 0.
Transfer only 1 ml to a test tube, add 5 ml of 75% ethanol, and stir well. Then, 0.1 ml of 30% ammonium thiocyanate was added to 0.02 mM FeCl ₂ (1N
HCl solution), and after 3 minutes Abs. 5
The absorption at 00 nm was measured. The result is shown in FIG.

As shown in FIG. 1, the antioxidant ability of the sample of Example (a) to which the pepper extract was added was comparable to that of Comparative Example (b) to which natural vitamin E was added. Was observed.

Examples 2 to 8 The antioxidants obtained in Example 1 were separated and purified by high performance liquid chromatography (HPLC). The HPLC conditions are shown in Table 1 below, and the results of separation and purification are shown in FIG. Note that the straight line in FIG. 2 shows the methanol concentration (% by mass) of the eluted solution.

[0028]

[Table 1]

The fractions 1 to 7 of the pepper extract obtained as described above were each dried under reduced pressure by an evaporator to obtain antioxidants of Examples 2 to 8.
The correspondence between each fraction number and the example number is shown in Table 2 below.

[0030]

[Table 2]

Using the above Examples 2 to 8, the following Examples (b) to (h) were prepared, and the antioxidant ability of each of the Examples was measured in the same manner as in Test Method 1. The result is shown in FIG. Example sample (b): 100 μm of the pepper extract obtained in Example 2
Example sample (c): 100 μm of the pepper extract obtained in Example 3
Example sample (d): 100 μm of the pepper extract obtained in Example 4
Example sample (e): 100 μm of the pepper extract obtained in Example 5
Example sample (f): 100 μm of the pepper extract obtained in Example 6
Example sample (g): 100 μm of the pepper extract obtained in Example 7
Example Sample (h): 100 μm of the pepper extract obtained in Example 8
Comparative sample (a): 100 μg of methanol was added Comparative sample (b): 100 μg of natural vitamin E was added

As shown in FIG.
It is recognized that the antioxidants Nos. To 8 have the same antioxidant ability as the comparative sample (b) to which natural vitamin E was added. That is, it was confirmed that the antioxidant comprising the pepper extract had a plurality of partially purified fractions, each of which had a strong antioxidant ability. The fraction eluted at a low methanol concentration has a strong hydrophilicity, while the fraction eluted at a high methanol concentration has a relatively strong lipophilicity. In short, the partially purified fractions are substances with various polarities and each have a strong antioxidant ability, so when applying to food etc., use the fractions corresponding to hydrophilic and lipophilic substances respectively. It is possible to

Example 9 The pepper extract obtained in Example 5 was diluted with a 50 mM phosphate buffer (pH 5.1) to obtain an antioxidant containing 4.8 ng of the extract in 1 ml. Was.

Example 10 The pepper extract obtained in Example 5 was diluted with a 50 mM phosphate buffer (pH 5.1) to obtain an antioxidant containing 48 ng of the extract in 1 ml.

Example 11 The pepper extract obtained in Example 5 was diluted with a 50 mM phosphate buffer (pH 5.1) to obtain an antioxidant containing 480 ng of the extract in 1 ml.

"Test 2"
Spin trap method using electrospin resonance (ESR) (Cohen et al., FEBS Lett., Cohen G. et al.,
FEBS Lett., 138 (2), 258-260, 1982). The measurement conditions for Test 2 are shown in Table 3 below.

[0037]

[Table 3]

Subsequently, 4.6 mM 5,5-dimethyl-1-
20 μg of pyrroline-N-oxide (DMPO) and 1.0 m
M iron sulfate-diethylenetriaminepentaacetic acid (DETAPA
C), 75 μl of any of the above Examples 9 to 11 and 75 μl of 9.8 mM hydrogen peroxide were added to prepare the following Examples (i) to (k), and the reaction was carried out. Started. And after exactly 45 seconds, ESR
Sweep started. All solutions were dissolved in 50 mM phosphate buffer (pH 5.1). Example sample (i): The extract of Example 9 was added. Example sample (j): The extract of Example 10 was added. Example sample (k): The extract of Example 11 was added.

The study of the active oxygen scavenging ability in each of the sample samples (i) to (k) was carried out by examining the ratio of the signal height (H) derived from DMPO-OH to the signal height (S) derived from Mn ^2+. Take
The active oxygen scavenging ability was determined by the following equation.

[0040]

(Equation 1)

Hstand: High signal of control (without adding antioxidant) Sstand: High signal of internal standard of control (without adding antioxidant) Hsamp: High signal when adding antioxidant Ssamp: Addition of antioxidant Internal standard signal height at the time

As is clear from the experimental measurement results shown in FIG. 4, the active oxygen scavenging ability of the sample samples (i) to (k) to which the antioxidants of Examples 9 to 11 were added in a concentration-dependent manner. Was confirmed. In particular, the effect was remarkable in the example sample (k).

[0043]

As is apparent from the above description, the antioxidant according to the present invention is a novel substance extracted from Sansho, which is widely used for edible use, and is therefore a conventionally known synthetic antioxidant. Less anxiety such as side effects than oxidants,
It is safe and has a strong antioxidant effect when used in the cosmetics and pharmaceutical fields.

Further, according to the method of the present invention, the antioxidant of the present invention can be easily collected with particularly excellent efficiency.

[Brief description of the drawings]

FIG. 1 is a graph showing the measurement results of the antioxidant ability in Example 1 of an antioxidant derived from a pepper seed methanol extract together with Comparative Examples.

FIG. 2 is a graph showing measurement results of characteristics of a product obtained by partially purifying the antioxidant of Example 1 by HPLC.

FIG. 3 is a graph showing measurement results of antioxidant capacity in Examples 2 to 8 together with Comparative Examples.

FIG. 4 is a graph showing measurement results of active oxygen scavenging ability in Examples 9 to 11.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Minoru Inagaki 1515 Uehama-cho, Tsu-shi, Mie Pref. NA14 ZC41 4H025 BA02

Claims (4)

[Claims] 1. An antioxidant comprising a pepper extract as an active ingredient. 2. The antioxidant according to claim 1, which comprises a fraction of the pepper extract as an active ingredient. 3. Extraction of a material containing at least one of the seeds, seed coat and leaves of pepper by using an organic solvent to obtain a pepper extract, followed by extracting and separating vegetable oil from the pepper extract. A method for producing an antioxidant, comprising obtaining an antioxidant. 4. A hydroxy radical scavenger comprising a pepper extract as an active ingredient.