TWI845736B - Antioxidant composition - Google Patents

Abstract

An objective of the present invention is to provide an antioxidant composition containing an apple extract and a natural extract.

As a solution, provided is an antioxidant composition containing the following components A and B as active ingredients.

A component: apple extract

B component: one or more selected from pomegranate flower extract, cherry flower extract, hawthorn extract, chamomile flower extract, and white peach flower extract.

Description

Antioxidant composition

The present invention relates to a composition that exerts an antioxidant effect.

Apple extracts (apple extracts) used as food additives or in health foods are rich in polyphenols and have a variety of physiological effects, including promoting collagen production (patent document 1), promoting blood circulation (patent document 2), and promoting neutral fat metabolism (patent document 3).

In addition, it is known to exert a strong antioxidant effect, reduce the accumulation of free radicals and superoxides in the body, and inhibit aging (patent document 4). In recent years, the antioxidant effect and physiological effects of apple extracts have attracted much attention.

In addition, the antioxidant effects of natural extracts such as natural compositions and apple extracts, which are not chemically synthesized compounds, have attracted attention. In addition, many natural compounds, extracts, and compositions are known to exhibit antioxidant effects.

Patent document 5 lists various substances as such naturally derived antioxidants or extracts. For example, it describes: vitamin A, vitamin C, vitamin E, ubiquinone, inorganic selenium, manganese, melatonin, α-carotene, β-carotene, lycopene, lutein, zeanthin, cryptoxanthin, resveratrol, eugenol, quercetin, catechin, gossypol, hesperetin, curcumin, ferulic acid, acid), rutin, hydroxytyrosol, turmeric, thyme, olive oil, lipoic acid, glutathione, glutamine, oxalic acid, tocopherol, pectin, tocotrienols, coenzyme Q10, zeaxanthin, astaxanthin, canthaxanthin, saponins, limonoids, kaempferol, cypermethrin, isorhamnetin, proanthocyanidins, rutin, luteolin, apigenin, tangeretin, naringin, ingin), 3',4',5,7-tetrahydroxydihydroflavonoids (eriodictyol), flavan-3-ols (e.g., anthocyanidins), gallocatechins, epicatechin and its gallate forms, epigallocatechin and its gallate forms (ECGC), theaflavins and its gallate forms, thearubigins, isoflavone phytoestrogens, genistein, daidzein, glycitein, anthocyanidins, cyanidin, delphinidin, malvaccin, pelargonidin, peony pigment, petunidin, ellagic acid acid), gallic acid, salicylic acid, rosmarinic acid, cinnamic acid and its derivatives (e.g., ferulic acid), chlorogenic acid, chicoric acid, gallic acid tannins, ellagitannins, anthoxanthins, betacyanins and other plant pigments, silymarin, citric acid, lignan, anti-nutrient absorption agents, bilirubin, uric acid, R-α-lipoic acid, N-acetylcysteine, emblicanin, apple extract, apple peel extract (apple polyphenols), Rooibos extract red, Rooibos extract green, hawthorn berry extract, red raspberry extract, green coffee antioxidant (GCA), wild cherry extract 20%, grape seed extract (VinOseed), cocoa extract, hops extract, mangosteen extract, mangosteen shell extract, cranberry extract, pomegranate extract, pomegranate peel extract, pomegranate seed extract, Pomella pomegranate extract, cinnamon extract, grape skin extract, bilberry extract, pine bark extract, pycnogenol, elderberry extract, mulberry root extract, wolfberry extract, blackberry extract, blueberry extract, blueberry leaf extract, raspberry extract, turmeric extract, citrus bioflavonoids, black currant, ginger, acai berry (Acai berry) powder, green coffee bean extract, green tea extract, rose extract and phytic acid.

The antioxidant effect of these substances showing antioxidant properties when used together is considered to be a simple additive effect of the antioxidant effects of each substance.

[Prior Art Literature]

[Patent Literature]

Patent document 1: Japanese Patent Publication No. 2012-62278

Patent document 2: Japanese Patent Publication No. 2006-265220

Patent document 3: Japanese Patent Publication No. 2006-151944

Patent document 4: Japanese Patent Publication No. 10-75740

Patent document 5: Japanese Patent Publication No. 2009-523407

As mentioned above, although it is believed that only additive antioxidant effects can be exerted, the inventors of the present invention conducted preliminary tests on the combination of these natural antioxidant substances and plant extracts and found that only the combination of apple extract and specific natural extracts can exert a strong synergistic antioxidant effect. Further in-depth research resulted in the completion of the present invention.

That is, the subject of the present invention is to provide an antioxidant composition containing apple extract and specific natural extracts.

The main components of the present invention are as follows.

(1) An antioxidant composition comprising the following components A and B as active ingredients:

Ingredient A: Apple extract;

Ingredient B: one or more selected from pomegranate flower extract, cherry flower extract, hawthorn extract, golden chrysanthemum extract, and white peach flower extract.

(2) The antioxidant composition as described in (1), wherein the composition contains 1 to 30 parts by weight of any component selected as component B, based on a dry weight, relative to 1 part by weight of component A.

(3) The antioxidant composition as described in (1) or (2), wherein component A contains polyphenol.

(4) The antioxidant composition as described in (3), wherein the polyphenol content per dry mass of component A is 50% by mass or more.

According to the present invention, a novel antioxidant composition is provided.

The composition of the present invention can inhibit the production of oxidative substances such as superoxide, because the antioxidant effects of the apple extract and the specific flower extract work synergistically.

Therefore, it is possible to prevent the occurrence of aging or diseases caused by superoxide.

Figure 1 is a graph showing the antioxidant effect of pomegranate flower extract combined with 1μg/mL apple extract.

Figure 2 is a graph showing the antioxidant effect of pomegranate flower extract combined with 2μg/mL apple extract.

Figure 3 is a graph showing the antioxidant effect of cherry blossom extract combined with apple extract at 1 μg/mL.

Figure 4 is a graph showing the antioxidant effect of hawthorn extract combined with apple extract at 1μg/mL.

Figure 5 is a graph showing the antioxidant effect of golden chrysanthemum extract when it is combined with apple extract at 1μg/mL.

Figure 6 is a graph showing the antioxidant effect of white peach flower extract when used in combination with 1μg/mL apple extract.

The present invention is an invention of an antioxidant composition, which contains component A: apple extract and component B: one or more selected from pomegranate flower extract, cherry flower extract, hawthorn extract, golden chrysanthemum flower extract and white peach flower extract as active ingredients.

The composition of the present invention can be used as a medicine, health food, beverage, or food for oral administration or ingestion, and can also be used as a food additive.

The following is an explanation of the ingredients contained in the composition of the present invention.

Ingredient A:

The varieties of apple (Malus pumila) used as raw materials for apple extracts include, for example, Fuji, Guoguang, Wanglin, Hongyu, Jonagold, Delicious, Sansa, Qianqiu, etc., without any particular restrictions. The extraction parts of apples are not particularly limited, for example, fruits, leaves, branches, flowers, etc., preferably fruits. The fruits may be, for example, immature fruits (young fruits) or ripe fruits, without any particular restrictions. The parts of the fruits used for extraction are not particularly limited, for example, the whole fruit, pulp, peel, seeds, etc. may be listed. For apple extracts, these parts may be extracted individually or two or more parts may be extracted in combination.

There is no particular limitation on the method of extracting apple extract, and a conventionally known method may be used. A specific example of the extraction method may be as shown below.

First, the whole apple fruit is washed with water and then crushed by a grinder. The crushed product can be subjected to pectinase treatment, centrifuged, and then filtered by an extraction solvent to prepare an apple extract. There is no particular limitation on the pectinase treatment, and for example, 10 to 50 ppm of pectinase can be added at a temperature of 20 to 60°C. There is no particular limitation on the extraction solvent, and for example, organic solvents such as water, alcohol, hexane, and chloroform can be listed. The extraction solvent is removed to obtain an apple extract.

Apple extracts can be commercially available apple extracts or prepared by extracting from apple fruits. There are no special restrictions.

The content of the apple extract in the composition of the present invention is not particularly limited, but the apple extract is preferably an apple extract containing more than 50% by mass of polyphenols from apples.

Apple extracts may also be substances containing high concentrations of polyphenols. There is no particular limitation on the fractionation method used to concentrate polyphenols, and conventionally known methods may be used. Polyphenols can be fractionated by, for example, feeding the apple extract to a column, flushing the adsorbent from the column, and decompressing and distilling the flushed fraction to concentrate it. In addition, a powdered auxiliary agent may be further added to the concentrated liquid, and freeze-dried or spray-dried to prepare a powder containing high concentrations of polyphenols.

When a substance containing high concentration of polyphenols from apple extract is used as the apple extract of the present invention, for example, a commercially available substance sold as polyphenols from apple can be used. Such commercial products include, for example, "APPL'IN AFPOMM 9051" or "APPL'IN-POLYPHENOLIC APPLE EXTRACT POWDER" sold by UNITEC FOODS Co., Ltd.

Ingredient B:

Pomegranate flower extract is an extract of pomegranate (Punica granatum) flowers. In the present invention, there is no particular limitation on the variety of pomegranate.

There is no particular limitation on the extraction method of pomegranate flower extract, and a conventionally known method can be used. As a specific example of the extraction method, the following method can be used. First, the collected pomegranate flowers are washed with water and then crushed by a grinder or the like. The obtained crushed material is used as the extraction raw material. There is no particular limitation on the extraction solvent, and examples thereof include water and organic solvents such as alcohol, hexane, and chloroform. Then, the extraction solvent is removed to obtain the pomegranate flower extract.

As the pomegranate flower extract, for example, commercially available pomegranate flower extract can be used. Such flower extracts can be exemplified by "Pomegranate flower extract powder: manufactured by Xiangrong Industrial Co., Ltd."

In the composition of the present invention, 1 to 30 parts by weight of pomegranate flower extract are mixed in terms of dry weight relative to 1 part by weight of apple extract, preferably 2 to 18 parts by weight, and particularly preferably 2 to 10 parts by weight.

The cherry blossom flower extract is an extract of the flowers of the genus Prunus (Prunus, Cerasus, Japanese cherry, Sakura) of the subfamily Prune of the Rosaceae family. In the present invention, there is no particular restriction on the variety of cherry blossoms.

There is no particular restriction on the extraction method of cherry blossom flower extract, and a conventionally known method can be used. As a specific example of the extraction method, the following method can be used. First, the collected cherry blossoms are washed with water and then crushed by a grinder or the like. The obtained crushed material is used as the extraction raw material. There is no particular restriction on the extraction solvent, and examples thereof include water and organic solvents such as alcohol, hexane, and chloroform. Then, the extraction solvent is removed to obtain the cherry blossom flower extract.

As the cherry blossom flower extract, for example, commercially available cherry blossom flower extracts can be used. As such flower extracts, "Sakura Flower Extract-P: manufactured by Oryza Petrochemical Co., Ltd." can be exemplified.

In the composition of the present invention, 1 to 30 parts by mass of cherry blossom extract are mixed with 1 part by mass of apple extract in dry mass, preferably 2 to 18 parts by mass, and particularly preferably 4 to 10 parts by mass.

Western hawthorn extract is an extract of Crataegus laevigata or C. monogyna, also known as hawthorn (English: Hawthorn). The present invention uses an extract extracted from the whole plant.

There is no particular limitation on the method of extracting hawthorn extract, and a conventionally known method can be used. As a specific example of the extraction method, it can be implemented as shown below. First, the whole hawthorn plant collected is washed with water and then crushed by a grinder. The obtained crushed material is used as the extraction raw material. There is no particular limitation on the extraction solvent, and examples include water and organic solvents such as alcohol, hexane, and chloroform. Then, the extraction solvent is removed to obtain the hawthorn extract.

As the hawthorn extract, for example, commercially available hawthorn extracts can be used. As such an extract, "Hawthorn dried extract GMP133: manufactured by ASK Pharmaceutical Co., Ltd." can be exemplified.

In the composition of the present invention, 1 to 30 parts by weight of hawthorn extract are mixed in terms of dry weight relative to 1 part by weight of apple extract, preferably 2 to 18 parts by weight, and particularly preferably 2 to 4 parts by weight.

Golden chrysanthemum flower extract is an extract of Matricaria recutita, which is also known as chamomile. The present invention uses an extract extracted from the flowers of Golden chrysanthemum.

There is no particular restriction on the extraction method of golden chrysanthemum flower extract, and a conventionally known method can be used. As a specific example of the extraction method, it can be implemented as shown below. First, the collected golden chrysanthemum flowers are washed with water and then crushed by a grinder or the like. The obtained crushed material is used as the extraction raw material. There is no particular restriction on the extraction solvent, and examples thereof include water and organic solvents such as alcohol, hexane, and chloroform. Then, the extraction solvent is removed to obtain the golden chrysanthemum flower extract.

The golden chrysanthemum flower extract can also be used, for example, a commercially available golden chrysanthemum flower extract. Such an extract can be exemplified by "Golden Chrysanthemum Extract Powder H: manufactured by Nippon Powder Pharmaceutical Co., Ltd." obtained by hot water extraction of golden chrysanthemum flower bodies, or "Golden Chrysanthemum Dry Extract GMP742: manufactured by ASK Pharmaceutical Co., Ltd." obtained by hydrous ethanol extraction of golden chrysanthemum flower bodies.

In the composition of the present invention, 1 to 30 parts by weight of golden chrysanthemum extract are mixed with 1 part by weight of apple extract, preferably 2 to 20 parts by weight, and particularly preferably 5 to 20 parts by weight.

White peach flower extract is an extract from the flowers of white peach (Prunus persica Batsch).

There is no particular restriction on the extraction method of white peach flower extract, and a conventionally known method can be used. As a specific example of the extraction method, the following method can be used. First, the collected white peach flowers are washed with water and then crushed by a grinder or the like. The obtained crushed material is used as the extraction raw material. There is no particular restriction on the extraction solvent, and examples thereof include water and organic solvents such as alcohol, hexane, and chloroform. Then, the extraction solvent is removed to obtain the white peach flower extract.

White peach flower extracts, for example, commercially available white peach flower extracts can also be used. As such extracts, "White peach flower extract powder N: manufactured by Nippon Powder Pharmaceutical Co., Ltd." can be exemplified.

In the composition of the present invention, 1 to 30 parts by weight of white peach flower extract are mixed in terms of dry weight relative to 1 part by weight of apple extract, preferably 2 to 18 parts by weight, and particularly preferably 4 to 10 parts by weight.

When the composition of the present invention is formulated into medicines, supplements, beverages, or jelly and other food for ingestion, 1 mg to 10 g, preferably 1 mg to 5 g, and particularly preferably 1 mg to 3 g per day can be taken orally to achieve an antioxidant effect in vivo.

When making a drink, its form is not particularly limited. In addition, when making a drink, there is no particular limitation on other additive ingredients formulated in the composition of the present invention.

[Implementation example]

<Antioxidant effect measurement>

The in vivo antioxidant effect of the composition of the present invention was confirmed according to the ORAC method. ORAC is an evaluation method developed by the National Institute on Aging in 1992.

ORAC is a method that uses fluorescein, a fluorescent substance, as a fluorescent probe, and measures the fluorescence intensity of fluorescein that is decomposed by the free radical initiator 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) over time, and uses the change as an indicator to measure antioxidant power. If an antioxidant substance is present in the reaction system at the same time, the rate of decrease in the fluorescence intensity of fluorescein will be delayed, so the degree of delay in the rate of decrease of fluorescein in the presence of a standard substance can be compared to calculate the antioxidant power of the sample converted to the standard substance. Therefore, when the ORAC test shows a high value, it can be said that the stability against oxidation is high.

In addition, ORAC is considered to be useful as an indicator for evaluating the antioxidant capacity of food in vivo.

1. Antioxidant effect determination method

The principle of ORAC measurement is to use fluorescein, which is a fluorescent substance, as a fluorescent probe as described above, measure the fluorescence intensity of fluorescein decomposed by AAPH, a free radical initiator, over time, and use the change in the fluorescence intensity as an indicator to measure antioxidant power. Specifically, the fluorescence intensity (RFU) is measured in the absence of antioxidant substances, and a fluorescence intensity change curve (Blank) is prepared. Then, a fluorescence intensity change curve in the presence of Trolox standard solution or sample is prepared (Antioxidant), and the AUC of each curve is calculated. The AUC obtained by subtracting the AUC value in the absence of antioxidant substances from the AUC value in the presence of Trolox standard solution or sample is used as the Net AUC. Based on the calibration curve prepared by the determination of Trolox standard solution, the value converted from the Net AUC of the sample to Trolox is calculated, that is, the Trolox equivalent (TE), and the antioxidant capacity is evaluated based on this value.

2. Test samples

The composition to be measured was prepared from commercially available extract products listed in Table 1 that were judged to have a synergistic antioxidant effect in a preliminary screening.

[Table 1]

In addition, the polyphenol content per dry mass in the apple extract shown in Table 1 is 80 mass %.

Among the reagents used in the test, Trolox [(±)-6-hydroxy-2,5,7,8-tetramethyldihydrobenzopyran-2-carboxylic acid] and fluorescein were purchased from SIGMA-ALDRICH, AAPH was purchased from Tokyo Chemical Industry Co., Ltd., and methyl-β-cyclodextrin was purchased from Junsei Chemical Co., Ltd. In addition, potassium dihydrogen phosphate, potassium dihydrogen phosphate, dimethyl sulfoxide (DMSO), ethanol, and acetone were purchased from Fuji Film and Koshun Chemical Co., Ltd.

The experimental equipment used: microplate reader (INFINITE 200 PRO, TECAN Japan Co., Ltd.), 96-well plate (3881-096, IWAKI), multi-well plate sealing film (FG-BC100PCR, Japan Genetics Co., Ltd.).

3. Measurement method

(1) Preparation of test samples

The samples in Table 1 above were dissolved in DMSO at 100 mg/mL and stored at -20°C. Then, 990 μL of phosphate buffer was added to 10 μL of the solution and dissolved to prepare a 1 mg/mL (1% DMSO) solution. Further, the solution was diluted with 1% DMSO phosphate buffer solution to prepare solutions of the following concentrations as test samples for measurement.

Ingredient A: Apple extract 1μg/mL and 2μg/mL

Ingredient B:

Pomegranate flower extract 8μg/mL

Cherry blossom extract 8μg/mL

Hawthorn extract 2μg/mL and 4μg/mL

Golden chrysanthemum extract is golden chrysanthemum extract (E) 8μg/mL

Golden chrysanthemum extract (W) 18μg/mL

White peach flower extract 8μg/mL

In addition, the above concentrations are based on the dry mass of the samples.

(2) Preparation of reagents

1)Trolox standard solution

Prepare a 500μM Trolox solution by adding 5μL of 50mM Trolox, 45μL of DMSO, and 450μL of phosphate buffer and dissolving. Prepare a 160μM Trolox solution by adding 340μL of 1% DMSO phosphate buffer solution to 160μL of the aforementioned 500μM Trolox. Below, a series of two-fold dilutions with 1% DMSO phosphate buffer solution (0, 6.25, 12.5, 25, 50μM, 1% DMSO phosphate buffer solution) is prepared as a standard solution for making a calibration curve.

2) Fluorescence

Add 100 mL of phosphate buffer to 39.8 mg of luciferin to prepare a 1.2 mM solution. Then, add 2 mL of phosphate buffer to 10 μL of the 1.2 mM luciferin to prepare a 6.0 μM stock solution.

3) 110.7nM luciferin solution

Prepare by adding 25 mL of phosphate buffer to 470 μL of 6.0 μM luciferin stock solution to dilute and dissolve.

‧31.7mM AAPH solution (prepared before use)

Add 15 mL of phosphate buffer to 129 mg of AAPH and dissolve.

(3) Determine the operation process

1) Prepare a 96-well plate and inject the following test solutions containing the test samples prepared above.

Trolox standard solution: 35μL

Assay solution for component A: A mixed solution of 17.5μL of the apple extract solution used as the test sample for component A and 17.5μL of 1% DMSO/phosphate buffer solution

B component test solution: a mixed solution of 17.5μL of any one of the pomegranate flower extract, cherry flower extract, hawthorn extract, golden chrysanthemum extract, and white peach flower extract solutions used as the test samples of the above B component and 17.5μL of 1% DMSO/phosphate buffer solution

The measurement solution of component A + component B: a mixed solution of 17.5μL of the apple extract solution as the test sample of component A and 17.5μL of any one of the pomegranate flower extract, cherry flower extract, hawthorn extract, golden chrysanthemum extract, and white peach flower extract solutions as the test sample of component B.

2) Add 115μL of 110.7nM luciferin solution and apply the multi-well plate seal film.

3) Place the 96-well plate into a microplate reader, measure the fluorescence intensity at Ex485nm and Em538nm, and incubate at 37°C for 10 minutes.

4) Use the microplate reader to measure again at Ex485nm and Em538nm, and take out the 96-well plate.

5) Add 50μL of 31.7mM AAPH solution, seal the multi-well plate again, and place in the microplate reader. Measure for 90 minutes at 2-minute intervals. The measurement conditions are as follows.

Multiwell plate settings: Temperature: 37°C,

Wavelength: Ex485nm/Em538nm,

Stirring: 10 seconds,

Cycle: 45 times,

Interval: 2 minutes,

Gain: Manual 100%.

4. Determination and evaluation results of antioxidant effects

The measurement results of Trolox equivalent as an antioxidant effect and the multiplication effect are shown in Figures 1 to 6 and Tables 2 and 3 below. In addition, the names shown in the graphs shown in Figures 1 to 6 are recorded with the abbreviations enclosed in parentheses in Tables 2 and 3.

In the synergistic effect judgment, the calculated value of (measured value/theoretical value)×100% is 120% or more, which is considered "yes", and it is judged that there is a synergistic effect brought about by the combined use of components A and B. In addition, the theoretical value refers to the total value of the Trolox equivalent measurement results of component A alone and the Trolox equivalent measurement results of component B alone, which is a value showing an additive effect.

That is, the measured value = the measured value of the solution in which component A is mixed with component B

Theoretical value = measured value of component A alone + measured value of component B alone.

[Table 2]

[table 3]

As shown above, it is clear that when any one of the B ingredients of pomegranate flower extract, cherry flower extract, hawthorn extract, golden chrysanthemum flower extract, and white peach flower extract is used in combination with the A ingredient: apple extract, the antioxidant effect is synergistically enhanced.

Claims (4)

An antioxidant composition, which contains the following components A and B as active ingredients: Component A: apple extract extracted from the pulp, peel and seeds with water and/or alcohol; Component B: at least one selected from pomegranate flower extract, cherry flower extract, hawthorn extract, golden chrysanthemum flower extract, and white peach flower extract, each extracted with water and/or alcohol. The antioxidant composition as described in claim 1, wherein, relative to 1 part by weight of the dry weight of the component A, it contains 1 to 30 parts by weight of any component selected as the component B on a dry weight basis. The antioxidant composition as described in claim 1 or 2, wherein component A contains polyphenol. The antioxidant composition as described in claim 3, wherein the polyphenol content per dry mass of component A is 50% by mass or more.

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