JPH0967570A - Antioxidant and active-oxygen eliminator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To effectively extract and obtain at a low cost an antioxidant and an active-oxygen eliminator by concentrating an org.-solvent extract soln. of prune fruits. SOLUTION: The antioxidant is obtd. by extracting prune fruits with a hydrophilic org. solvent, partitioning the extract between water and a fat-sol. org. solvent, and concentrating the resultant org. solvent layer or by extracting the fruits with water, extracting the extraction residue with a fat-sol. org. solvent, and concentrating the resultant extract. The eliminator is obtd. by extracting the fruits with water and removing fibers and saccharides from the extract or by extracting the fruits with a hydrophilic org. solvent and removing saccharides from the extract. Since the major part of the active antioxidant component is fat-sol., an antioxidant having a strong activity similar to DL-α- tocopherol, BHT, or BHA is obtd. by extracting the fruits with a fat-sol. solvent followed by concentration. Since the major part of the active-oxygen-eliminating component is water-sol., the eliminator is obtd. by the extraction with water or a hydrophilic org. solvent followed by the removal of fibers and saccharides from the extract.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antioxidant and an active oxygen scavenger which are produced from natural prune and can be used in the fields of foods, food additives, cosmetics, pharmaceuticals and the like.

[0002]

2. Description of the Related Art Oxygen is indispensable to living organisms, but on the other hand, it produces active oxygen which is a factor causing harm to living organisms. This active oxygen is reduced by a metal catalyst such as iron and copper to become hydrogen peroxide, and further becomes a hydroxyl radical, which cleaves DNA and oxidizes lipids, which is a peroxidation factor which is an aging promoting factor. It produces lipids.

In the living body, this active oxygen is usually SO
Although it is erased by an enzyme called D (superoxide dismutase), this SOD decreases due to stress, aging, etc., and lipid peroxide accumulates,
It causes cancer, diabetes, myocardial infarction, arteriosclerosis, stroke, cataract, stiff shoulders, coldness, spots, freckles, wrinkles, etc., and adversely affects health.

Chemical substances such as dl-α-tocopherol, BHT (butylhydroxytoluene), and BHA are used to suppress the oxidation of lipids that have such adverse effects.
Antioxidants such as (butylhydroxyanisole) are known. In addition, as a substance that eliminates active oxygen itself, SOD, which is the above-mentioned in-vivo enzyme, has been found.

However, dl-α-tocopherol, BH
Since T and BHA are chemically synthesized products, in view of safety and health consciousness, the recent consumer demand for antioxidants derived from natural products is not met, and SOD is difficult to purify, and further Since it is stable and easily deactivated, it is extremely expensive. Therefore, there is a demand for an antioxidant and an active oxygen scavenger that are derived from natural products and can be obtained at low cost.

Therefore, the natural product Prune
domestica L. ). This prune is a plant of the genus Prunus rosacea, a kind of western plum,
The fruit has been called "miracle fruit" for a long time, and it is well known that its origin is the longevity region of the world, the Caucasus region. The fruits of this prune (hereinafter referred to as "prune") are generally eaten as foods that can be expected to promote health, such as dried dry prune and prune extract extracted from this dry prune with hot water.

However, for prune, potassium,
Other than the nutritional component characteristics that it is rich in minerals such as calcium and iron and dietary fiber, the details are not well known, and as a specific functional component, prune polysaccharide that has an activating effect on the human immune system. Body (Tokuhei 7-264
9) is only found.

As a result of extensive studies on the functionality of prune, the present invention found a strong antioxidative activity and active oxygen scavenging activity (SOD-like activity) in the prune extract, and the antioxidants and active oxygen as its components. The purpose is to effectively extract the erasing agent and provide it at a low cost.

[0009]

Means for Solving the Problems The present invention is to achieve the above object, wherein after extraction with an organic solvent which is more hydrophilic than the fruit of prunes, the extract is distributed between water and a fat-soluble organic solvent, Method of concentrating the solvent layer, or after extracting from the fruit of prunes with water, extract from the residue with a fat-soluble organic solvent,
Antioxidant obtained by the method of concentration and extraction with water from pruned fruits, followed by defibrination and desugarization of the extract, or extraction from pruned fruits with a hydrophilic organic solvent, followed by desugarization of the extract It is characterized by an active oxygen scavenger obtained by the method.

[0010]

The main component of the antioxidative active ingredient is fat-soluble, and it is extracted with a fat-soluble solvent from prunes and concentrated to obtain the currently used dl-α-tocopherol, BHT, BH.
It is possible to obtain an antioxidant having the same level of activity as that of A. Furthermore, a large amount of this antioxidant active component remains in the residue after the production of pruned extract by water extraction. It turns out that it can be manufactured very cheaply.

The main component of the SOD-like active ingredient is water-soluble, and it can be obtained by extracting with water or a hydrophilic organic solvent, defibering and desugaring the extract.

[0012]

EXAMPLES The present invention will be described more specifically below with reference to examples and test examples. However, the invention is not limited to the examples shown here.

Example 1 [Preparation of antioxidative active ingredient from pruned methanol extract] Dry prune pulp part 400 g to methanol 1000 m
Extraction was performed four times with 1 l, and the extracts were combined and concentrated under reduced pressure to obtain 297.6 g of an extract. After partitioning this extract with distilled water and methylene chloride, concentrate each under reduced pressure to give 285.1 g of water layer concentrate and 1.6 g of methylene chloride layer concentrate.
I got

Test Example 1 [Method for measuring antioxidative activity (TBA method)] (1) 2 mg of a sample was precisely weighed and dissolved in 1 ml of DMSO, 9 ml of ethanol was added, and 2 ml of this was collected, 2.51% 2 ml of ethanolic linoleic acid solution in ethanol, 4 ml of 0.05M phosphate buffer (pH 7.0) and 2 ml of water were added to a brown vial with a screw cap (φ = 35 mm, H = 75 mm) and mixed, Seal tightly and store in a 40 ° C incubator. After 1 week, 2 ml of this solution was collected, 2 ml of 20% trichloroacetic acid aqueous solution and 1 ml of 0.67% thiobarbituric acid aqueous solution were added, and this was heated in a boiling water bath for 10 minutes, cooled, and then centrifuged. Separate the supernatant (3000 rpm) and measure the absorbance at 532 nm. (2) Use the control that performs the same processing without adding the sample.

[Evaluation method] The absorbance value of the control is 100
The absorbance value of the sample is expressed as a relative percentage.

The antioxidant activity of the aqueous layer concentrate and the methylene chloride layer concentrate obtained in Example 1 was measured by the method of Test Example 1 as shown in FIG. Had a much higher antioxidant activity than the aqueous concentrate.

Example 2 [Preparation of antioxidative active ingredient from prune water extract residue]
Extracted from 200g of dry prunes twice with 500ml of hot water and once with 300ml of water, and concentrate the extracts together under reduced pressure to obtain 181.2g of prune extract (Bx70 °).
I got At this time, 50 g of an extraction residue was produced. This residue 5
Extraction was performed from 0 g with 150 ml of ethyl acetate, and the extract was concentrated under reduced pressure to obtain an ethyl acetate extract of 214 mg.

The antioxidant activity of the ethyl acetate extract obtained in Example 2 was measured by the method of Test Example 1, and dl-α-tocopherol, BHT, BH were used as comparative controls.
The results using A are shown in FIG. 2, and the ethyl acetate extract showed a strong antioxidant activity. Also, as a general antioxidant, dl-α-tocopherol, BHT,
Even when compared with BHA, it showed almost the same strong antioxidant activity.

Example 3 [Preparation of Antioxidative Active Ingredient from Prune Ethanol Extract] Ethanol (99.5%) 30 from 100 g of dry prune
Extraction was performed twice with 0 ml and 200 ml, and the extracts were combined and concentrated under reduced pressure to obtain 55.6 g of an extract. The extract was distributed between distilled water and methylene chloride, and then concentrated under reduced pressure. Layer concentrate 25.3 g and methylene chloride layer concentrate 445 mg were obtained.

Test Example 2 [Method for measuring antioxidative activity (iron iron method)] 2 mg of a sample was precisely weighed and dissolved in 1 ml of DMSO, 9 ml of ethanol was added, and 2 ml of this was taken, and 2.51% of linole was added. Acid ethanol solution 2 ml, 0.05M phosphate buffer (pH 7.0) 4 ml, water 2
ml, add to a brown vial with a screw cap (φ = 35 mm, H = 75 mm), mix, and stopper tightly.
Store in a 40 ° C incubator. Collect 0.1 ml of this solution every day, add 9.7 ml of 75% ethanol and 0.1 ml of 30% ammonium thiocyanate aqueous solution, respectively, and add 0.1 ml of 0.02 M ferric chloride 3.5% hydrochloric acid solution to make exactly 3 After a minute, measure the absorbance value at 500 nm. This operation is 1
~ 2 weeks, to measure the degree of deterioration of linoleic acid over time.

The antioxidant activity of the methylene chloride layer concentrate obtained in Example 3 was measured by the method of Test Example 2. As a comparative control, dl-α-tocopherol,
And BHT were used. As a result, as shown in FIG.
The methylene chloride layer concentrate was found to have a strong antioxidant activity higher than that of dl-α-tocopherol and equivalent to that of BHT.

Example 4 [Preparation of Antioxidant Active Ingredient from Prune Acetone Extract] From 100 g of dry prune to 300 ml of acetone, and further
Extraction was performed twice with 200 ml, and the extracts were combined and concentrated under reduced pressure to obtain 15.5 g of an extract. This extract was partitioned with distilled water and methylene chloride, and then concentrated under reduced pressure to obtain 15.2 g of the aqueous layer concentrate and 383 mg of the methylene chloride layer concentrate.
I got

Test Example 3 [Method for measuring antioxidant activity of perilla oil by preventing oxidation] After adding a sample to perilla oil at a rate of 0.02%, the sample is stored at 40 ° C, and the POV of perilla oil is measured with time. As a control, use perilla oil stored at 40 ° C without adding a sample.

The antioxidant effect of the methylene chloride layer concentrate obtained in Example 4 was measured by the method of Test Example 3. BHT was used as a comparative control. as a result,
As shown in FIG. 4, the methylene chloride layer concentrate has a remarkable antioxidant effect on perilla oil as compared with the control, and is superior in antioxidant property to BHT, which is widely used as an antioxidant. The effect was recognized.

Example 5 [Extraction of antioxidative active ingredient from residue after production of prune extract] When a prune extract was produced by extracting water from 1000 kg of dry prune, 24.3 kg of extraction residue was generated. The residue was extracted with 80 l of ethyl acetate, and the extract was concentrated under reduced pressure to obtain 1.99 kg of an ethyl acetate extract.

With respect to the ethyl acetate extract obtained in Example 5, the antioxidant effect was measured by the method of Test Example 3.
In addition, dl-α-tocopherol was used as a comparative control. As a result, as shown in FIG. 5, the ethyl acetate extract remarkably prevented the oxidation of perilla oil as compared with the control.
Further, even when compared with dl-α-tocopherol widely used as an antioxidant, an excellent antioxidant effect was recognized.

Example 6 [Preparation of SOD-like active ingredient from prune water extract] 100 g of the prune extract obtained in Example 2 was collected and water 1
After dissolving in 00 ml, add 300 ml of ethanol (99.5%),
The precipitate generated at this time was removed by suction filtration to remove fibers. The obtained filtrate was concentrated under reduced pressure and then MCI gel (manufactured by Mitsubishi Kasei Co., Ltd., product number: CHP20P, 75-150).
μ) Glass column filled with 100 ml (φ3 cm x 50 cm)
Was adsorbed on the column, about 3 l of water was used as a moving layer, and the effluent was removed to remove sugar. Next, about 1 liter of methanol was used as a mobile phase, and the effluent was collected and concentrated under reduced pressure to obtain about 1.2 g of SO 2.
A D-like active ingredient was obtained.

Example 7 [Preparation of SOD-like active ingredient from prune hydrophilic organic solvent extract] From 200 g of dry prune, ethanol (99.5
%) 500 ml twice, and the extracts were combined and concentrated under reduced pressure to obtain 103.7 g of an extract. This extract was adsorbed on a glass column filled with the above-mentioned MCI gel, and about 5 liters of water was used as a moving layer to remove the effluent, thereby saccharification. Next, about 2 liters of methanol was used as a mobile phase, and the effluent was collected and concentrated under reduced pressure to obtain about 2.1 g of SOD-like active ingredient.

Test Example 4 [Method of measuring SOD-like activity (NBT method)] A: A coloring solution (0.1 M phosphate buffer pH 8.0, xanthine 0.4 mM / l, nitroblue tetrazolium) was added to 0.1 ml of a 0.4 to 2% sample aqueous solution. 0.24mM / l) 1.0ml, enzyme solution (xanthine oxidase 0.049unit / ml, 0.1M phosphate buffer)
After adding 1.0 ml of pH 8.0) and heating accurately in a 37 ° C water bath for 20 minutes, sodium dodecyl sulfate solution (69 mM / l) 2.
0 ml was added to stop the reaction, and then 560 nm with a spectrophotometer.
The absorbance value of is measured. B: Performed using distilled water instead of the sample aqueous solution in A,
Measure the absorbance value at 560 nm. C: 1.0 M phosphate buffer 1.0 ml instead of the enzyme solution in A
And the absorbance value at 560 nm is measured. D: Distilled water is used in place of the sample aqueous solution in A, and 1.0 ml of 0.1 M phosphate buffer is used in place of the enzyme solution, and the absorbance value at 560 nm is measured. The inhibition rate was calculated according to the following formula using the values of A to D.

[0030]

[Equation 1]

SODs obtained in Examples 6 and 7
The SOD-like activity of the active ingredient was measured according to the method of Test Example 4. As a result, as shown in Table 1, for the SOD-like active ingredient of Example 6, the sample concentration in the reaction solution was
Inhibition rate 100% at 5000ppm 86.8% at 1000ppm, 200p
46.3% at pm, 7.0% at 40 ppm, and for the SOD-like active ingredient of Example 7, the inhibition rate was 100% when the sample concentration in the reaction solution was 5000 ppm, and 92.
49.6% at 5% and 200ppm, 9.2% at 40ppm,
It was revealed that both components exhibit SOD-like activity.

[0032]

[Table 1]

[0033]

INDUSTRIAL APPLICABILITY According to the present invention, d, which has been frequently used as an antioxidant from the natural product of prunes.
It is possible to obtain an antioxidant active ingredient having a strong activity similar to that of l-α-tocopherol, BHT, BHA and the like, and an SOD-like active ingredient having an active oxygen scavenging action. Particularly, the antioxidant active ingredient can be obtained inexpensively and easily from the residue after the production of pruned extract by water extraction which is generally performed, and the industrial advantage is very great.

These components are useful in the fields of foods, food additives, cosmetics, pharmaceuticals, etc., and antioxidative activity and SOD-like activity can be expected to have effects such as aging prevention and health promotion.

[Brief description of drawings]

FIG. 1 is a diagram comparing the antioxidant activity of a water layer concentrate and a methylene chloride layer concentrate obtained by partitioning and concentrating a dry prune methanol extract with distilled water and methylene chloride.

FIG. 2 is a diagram comparing the antioxidative activity of an ethyl acetate extract, which is the residue after extraction with pruned extract, with an antioxidant that has been conventionally used.

FIG. 3 is a diagram comparing the antioxidant activity of an extract of dry prunes with distilled water and methylene chloride, and comparing the concentration of a methylene chloride layer with an antioxidant that has been conventionally used.

FIG. 4 is a view showing the antioxidant effect of an acetone extract of dry prune on perilla oil.

FIG. 5 is a view showing an antioxidant effect of an ethyl acetate extract of a residue after extraction of prune extract on perilla oil.

Claims (4)

[Claims] 1. A pruned fruit is extracted with a hydrophilic organic solvent, and the extract is distributed between water and a fat-soluble organic solvent.
An antioxidant obtained by concentrating the organic solvent layer. 2. An antioxidant obtained by extracting pruned fruits with water and then extracting and concentrating the residue with a fat-soluble organic solvent. 3. An active oxygen scavenger obtained by extracting water from pruned fruits, and then defiberizing and desugaring the extract. 4. An active oxygen scavenger obtained by extracting pruned fruits with a hydrophilic organic solvent and desugaring the extract.