JP3521155B2 - Method for producing fruit polyphenols - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] FIELD OF THE INVENTION The present invention relates to a fruit polyphenol.LeMade
RecipeTo the lawRelated. [0002] BACKGROUND OF THE INVENTION Edible fruits of the family Rosaceae, especially apples, pears,
In the process of cultivating fruit trees such as moss, usually from mid-May to mid-July
During the season, a process called “mining” was performed.
Is The content of the work is that the fruit that has set in a tuft after pollination is still
During the immature stage, leave some fruits and discard others
However, due to this work, a lot of immature fruits
Has been disposed of unused. This immature fruit
Extremely bitter compared to ripe fruit, and easily cut fruit
Browns. This fact indicates that polyphenols
It indicates that the compound is present in large quantities. [0003] Polyphenol compounds are produced by secondary metabolism in plants.
That they are universal, diverse, and abundant in the plant kingdom
But some of these are very diverse
In the field of pharmacy for a long time
Has attracted attention in the field of food chemistry for years. [0004] In particular, research has been concentrated recently.
In tea polyphenols (catechins), antibacterial,
Antiviral, antioxidant, antimutant, anticancer, platelet aggregation suppression
Control, blood pressure rise suppression, blood sugar rise suppression, blood cholesterol
Decrease, anti-carious, anti-allergic, intestinal flora improvement, deodorant
It is recognized that it has a very wide range of physiological effects
is there. (JP-A-63-214183, JP-A-2-64)
No. 99, JP-A-4-178320, etc.) [0005] As described above, for example, tea
From polyphenols have a wide range of physiological effects
However, the present inventor has considered from another viewpoint.
To make polyphenols more efficient and economical
Search of raw materials for manufacturing and manufacturing method,
The physiological effects of polyphenols were examined and confirmed. The result
Fruits, rose fruits, especially immature fruits such as apples, pears and peaches
Various materials can be obtained by using
Efficient production of polyphenols with physiological effects
The inventors have found that the present invention has been achieved. [0006] [0007] That is, according to the present invention, there is provided:
IfCollected when harvesting is performedApple, pear
Or squeezing and / or extracting immature fruits of peaches,
This squeezed fruit juice and / or extract is converted to styrene-
Divinylbenzene synthetic adsorption resin, anion exchange tree
Fatty acid or octadecyl group chemically bonded silica gel
Through the adsorbent to purify the polyphenol fraction.
And its component composition is a simple polyphenol compound.
Caffeic acid derivatives, p-coumaric acid derivatives, flavan-
3-ols, flavonols, dihydrochalcones,
And catechins as high molecular weight polyphenol compounds
Polypheno Containing Condensed Tannins, Regular Polymers
Polyphenol mixture characterized by obtaining a mixture of polyphenols
And a method of manufacturing the product. [0008] [0009] [0010] [0011] Hereinafter, the present invention will be described in detail. In the present invention
As mentioned above, the fruit polyphenols
Juice of the fruits that belong, especially immature fruits of apples, pears and peaches
From the polyphenol fraction purified from fruit juice and extract
However, the purification of this polyphenol fraction
Performed by treating juice and extract with an adsorbent
To the fraction adsorbed by the adsorbent (hereinafter referred to as adsorbed fraction)
Polyphenols are contained. And the adsorbed fraction
By eluting with water alcohol (such as ethanol)
Thus, the polyphenol fraction is purified. The polyphenol fraction is then concentrated.
By processing, a liquid preparation can be obtained. Further
Spray dry or freeze the concentrate of the polyphenol fraction
Powder preparation can be obtained by sintering
You. The raw material used in the present invention belongs to the family Rosaceae.
FruitRuriNgo, pearOr peachListed
Apples are preferred. Also, as a fruit,More
Contains polyphenolic compounds and has a wide range of raw materials
Contains a large amount of various active ingredients that haveRe
Coral, pear or peachImmature fruitUse. [0015] In the squeezing method, the raw materials are washed, and
Or crushed while adding sulfurous acid, squeezed by pressing
The juice is obtained and preferably a pectin-degrading enzyme is added. Next
Method of obtaining clear juice by means such as centrifugation and filtration
Can be mentioned. As an extraction method, wash
Mixed with alcohol (ethanol, methanol, etc.)
Crush and combine, immerse and squeeze as is, or heat reflux
Extraction, and then concentrate the alcohol under reduced pressure.
After removal, centrifugation and filtration, or organic solvent (hex
, Chloroform, etc.) and filtration.
A method for obtaining a clear extract can be mentioned. As a purification method, polyphenols are selected.
Adsorbents that can be selectively adsorbed and eluted, such as styrene-di
Vinylbenzene synthetic adsorption resin, anion exchange resin,
Filled with octadecyl group chemically bonded silica gel (ODS)
Pass the above clear juice or clear extract through the packed column
This allows the polyphenol fraction to be adsorbed. Then
After washing by passing distilled water, 20 to 100%
Alcohol (eg ethanol) solution, preferably about 50
% Alcohol solution through the column
Fraction can be eluted and collected. Polyphenol obtained
The alcohol was distilled off by concentrating the solution under reduced pressure.
Real polyphenol liquid formulation (preferably with malic acid, etc.)
Acid). In addition, liquid formulations
As it is or by adding a powder auxiliary such as dextrin,
Spray dry or freeze dry, fruit polyphenol powder
A powder formulation can be obtained. A set of fruit polyphenols obtained by the present invention
As a result, caffeine is used as a simple polyphenol compound.
Acid derivative, p-coumaric acid derivative, flavan-3-ol
(Catechins), flavonols (quercetin glycosides)
), Dihydrochalcones (phloretin glycosides)
Condensed tan
The present inventor believes that the majority is
Has confirmed. Therefore, the fruit polypheno obtained by the present invention
Is considered to have various physiological functions
However, as a result of the inventor's intensive studies, firstly,
Phenol prevents oxidation of linoleic acid, a vegetable oil
That there is a large amount of components that
Issued. That is, the fruit polyphenol obtained in the present invention is
It is extremely effective as an antioxidant. Second, this polyphenol contains blood.
Angiotensin I converting enzyme which is an enzyme related to pressure increase
Has the effect of inhibiting the action of element (hereinafter referred to as ACE)
Component was found to be contained in large amounts. Therefore,
The fruit polyphenol obtained in the present invention is used as a hypotensive agent.
However, it is extremely effective. Further, the present inventor further studied and
Detect ACE inhibitory substances in fruit polyphenols
As a result, the condensed tannin represented by the structural formula in FIG.
I confirmed that In addition, many recent research results
Higher correlation between carcinogens and mutagens
And that carcinogenicity and mutagenicity are closely related.
You. That is, substances that suppress mutagenicity prevent carcinogenicity
Can be expected. Therefore, the fruit obtained by the present invention
Find out if polyphenols have antimutagenic effects
As a result, it was found that there was an effect. Follow
Therefore, the fruit polyphenol obtained in the present invention is anti-mutagenic.
It is also extremely effective as an agent. In addition, histamine is used for various allergic diseases.
Known as an in vivo chemical mediator
Degranulation mainly from mast cells and basophils.
Release. Hyaluronidase is involved in connective tissue remodeling
Enzymes that are thought to form a coherence in cellular activity
However, recent studies (Chem. Pharm. Bu)
ll.33.p642.1985, ibid.33.198
5, ibid.33.p3787.1985, ibid.33.p5
079.1985, ibid.40.1439.1992)
More hyaluronidase inhibitory activity and His from mast cells
Tamine release inhibitory activity is a synthetic antiallergic agent (Cromogli
Sodium citrate and tranilast)
By measuring these activities, several types of
Anti-allergic substances are being searched. Therefore, in the present invention
Hyaluronidase inhibitory activity of the resulting fruit polyphenols
Whether it has histamine release inhibitory activity
When it was confirmed, it was found that the effect was obtained. Subordinate
Therefore, the fruit polyphenols obtained in the present invention are allergic
-Very effective as an inhibitor. Furthermore, today, caries (cavities) are St.
reptococcus mutans
It has been confirmed that this is a kind of bacterial infection,
Plaque formation is considered to be a particularly important factor in the life process
I have. In other words, sucrose contained in food
Glucosyltransferase (hereinafter referred to as GT)
abbreviated as "ase") to form sticky insoluble glucan
Synthesized, through which bacteria attach to the tooth surface and form
Plaque causes caries. This inhibits GTase activity
The substance is expected to be applicable as an effective anti-caries agent
(Appl. Env. Microbiol., 59 (4), 968-973, 1993,
Biosci. Biotech. Biochem., 56 (5), 766-768, 1992, Agric.
Biol.Chem., 54 (11), 2925-2929,1990, Chem.Pharm.Bul
l., 38 (3), 717-720, 1990). Therefore, in the present invention,
S. sobrinus, one of the representative species of cariogenic bacteria, produces
Fruit polypheno against insoluble glucan-producing GTase
Investigating the inhibitory ability of
It turned out to be. Therefore, the fruit po
Liphenol is an extremely effective anti-cariogenic agent.
You. Further, among the malodorous ingredients derived from foods,
The intensity of bad breath is proportional to the content of hydrogen sulfide, methyl
Volatile sulfur compounds such as mercaptan and dimethyl sulfide
(Journal of Periodontology, 17,233-245,1975). in
Also showed a strong correlation with methyl mercaptan concentration.
(Journal of Periodontology, 18, 1-12, 1976 and Dental Society, 76, 1
923,1976). Therefore, the fruit polypheno obtained by the present invention
Odor of methyl mercaptan and dimethyl sulfide
When the effect is measured, it is found that it has the effect
did. In addition, the fruit polyphenol methyl mercapta
, Dimethyl sulfide and dimethyl disulfide
The effect was measured and found to be effective.
Revealed. Therefore, the fruit polyphenol obtained by the present invention
Is extremely effective as a deodorant. [0025] Next, the present invention will be described in more detail based on examples.
Explain, but the invention is not limited to these examples
is not. (Example 1) [Ingredient analysis of immature apple juice]
General component analysis was performed. -Sample apple (Fuji) mature fruit: commercial product (bagless cultivation product) Apple (Fuji) immature fruit: collected in mid-June -Sample processing method Suitable amount of metabisulfite as an antioxidant for fruit samples
While adding the lithium, the mixture was crushed and squeezed with a mixer. Squeezing
The juice is centrifuged and filtered to obtain clear juice,
The test was performed. Measurement items (and methods) -Average weight per individual (n = 50) -Juice ratio (%) -PH -Acidity (g / l in terms of malic acid) -Brix -Total phenol (chlorogenic acid equivalent, ppm) -Total ascorbic acid -Organic acid composition -Sugar composition -Metal ion composition -Free amino acid composition Table 1 shows the measurement results. [0028] [Table 1] As can be seen from Table 1, apple immature fruit juice
Has a large difference in composition when compared with mature fruit juice
Was. Of particular emphasis are phenolic components, Asco
Rubic acid, organic acids (especially quinic acid), metal ions, free
Amino acids (particularly asparagine, phenylalanine, γ-a
(Minobutyric acid). On the other hand, sugars are very
There were few. Next, a polyphenol component group of immature apple fruit
The composition was examined by high performance liquid chromatography.
The measurement sample was a solid phase extraction of polyphenol components from the fruit extract.
Using the product purified by the above method (see Example 2).
Was. FIG. 1 shows the measurement results. From this, simple polypheno of immature apple fruit
The composition of the cholesterol compound is caffeic acid derivative (chlorogenic acid)
Other), p-coumaric acid derivatives, flavan-3-ols
(Catechin, epicatechin, etc.), flavonols (ke
Lucetin glycosides), dihydrochalcone (phloretin)
Glycosides, especially phlorizin).
Was. Quantitatively, chlorogenic acid, catechin, epicatechin,
The phlorizin was assumed to be high. (Embodiment 2) [Antioxidant action of immature fruit polyphenols]
The following fruits were used as ingredients. Apple mature fruit: commercial product (bagless product) Apple immature fruit: collected in mid-June         "Fuji"-average weight 4.97 g (n = 50)         "Tsugaru"-average weight 7.80 g (n = 50)         "Jona Gold"-average weight 3.86 g (n = 50)         "Hokuto"-average weight 3.32 g (n = 50)         "Wang Lin"-average weight 10.34 g (n = 50) Pear immature fruit: collected in early June "Hosui"-average weight 8.98 g (n = 50) Peach immature fruit: collected in early June. "AKATSUKI"-average weight 5.03 g (n = 50) Sample preparation Juice sample-Obtain clear juice by the same treatment as in Example 1.
Was. Extracted sample-As an extraction method, 400 g of raw material was
After homogenizing with acidic methanol, heat to reflux
While extracting (3 times), concentrating the extract under reduced pressure
After distilling off the solvent, chloroform was added and partitioned (twice).
Collect the layers, filter, and make up to 200 ml with distilled water.
To obtain an extracted sample. If necessary, extract the juice sample and extract
The polyphenol component was collected from the sample using Sep-pak C18.
Purified by phase extraction. -Antioxidant test method The antioxidant test method was as follows. That is, the substrate solution (4
5% linoleic acid in ethanol) and phosphate buffer (p
H7.0) 4 ml and 1 ml of test solution were mixed in a sealed test tube.
Then, it was stored in a constant temperature bath at 50 ° C. protected from light. At this time,
Ethanol was added as a roll instead of substrate
Add sample solvent instead of sample as blank
This was stored under the same conditions. During the storage period, the reaction solution was
And the resulting peroxide is purified by the isothiocyanate method (Roda
(Iron method). -Measurement results Anti-acid on extracts of immature and mature fruits of apple (Fuji)
The presence or absence of chemical conversion was examined. "Fuji" extract of immature and mature fruits
When 1 was added to the reaction system in the range of 1-1000 μl / gLA, 1
The amount of lipid peroxide produced after a week (absorbance 500nm value)
FIG. 2 shows a graph of the additive concentration. From now on, "Fuji"
Linoleic acid in both immature and mature fruit extracts
Antioxidant effect, but especially immature fruit extract
Had a strong effect. Subsequently, what is this powerful active ingredient
Extract the immature and mature fruits in order to determine if they are substances.
It was roughly fractionated into two by p-pak C18 (fraction A:
Sep-pak C18 non-adsorbed fraction. Fraction B: Sep-pak C18 adsorption image
(Polyphenol fraction)). Extract and each fraction
1 When added to the reaction system in the range of -1000 μl / gLA, 1
The amount of lipid peroxide produced after a week (absorbance 500nm value)
FIG. 3 shows a graph of the additive concentration. As a result, the antioxidant activity of the mature fruit extract was
It was found to be derived from the component B. Immature fruit
In the extract, antioxidant activity was observed in both fractions A and B.
B showed a stronger effect. From the above results, both extractions
The antioxidant properties observed in the effluent are mainly due to the formation of fraction B
Minute, that is, due to the presence of polyphenol compounds.
Was strongly suggested. Furthermore, simple polyphenols contained in apples
Antioxidant for pure compounds that can be obtained
The presence or absence of sex was examined. Add 2 μmol / gLA of each substance to the reaction system
1 week after addition, the amount of lipid peroxide
FIG. 4 shows a comparison graph of the light intensity (500 nm value). In FIG.
In addition to polyphenol compounds, known anti-
About three kinds of oxidizing agents, BHA, BHT and Vitamin E
Quinic acid, malic acid, γ
-Aminobutyric acid (GABA) was also studied. As a result, among the polyphenol compounds,
Faic acid, chlorogenic acid, (+)-catechin, (-)-epicate
Kin, quercetin, rutin (quercetin-3-glu-rha) 6
The species had antioxidant power comparable to BHA and BHT.
However, even with the same polyphenol compound, p-coumaric acid and
Loretin and phlorizin have no antioxidant effect
Was. The components in fraction A also have antioxidant effects.
Was not seen. The content of each component in apple
From the comparison of the amount and antioxidant power, the extract of apple immature fruit and
The strong antioxidant effect exerted by the juice is mainly due to
Acid, (+)-catechin, (-)-epicatechin
It was estimated that From the results so far, it was found that apple (fuji)
Ripe fruit has strong antioxidant activity and its activity
Revealed minutes. Next, immature apples other than Fuji
It was examined whether the fruits had the same effect. each
Seed apple immature fruits (Fuji, Tsugaru, Jonagold)
Fraction B (polyphenol) of the extract of "Hokuto" and "Wang Lin"
For each fraction, react each fraction in the range of 1-500 μl / gLA.
Lipid peroxide produced after one week when added to the system
A graph of the amount (absorbance 500 nm value) and the concentration added is shown in FIG.
Was. FIG. 5 shows the results of the mature fruit of “Fuji” as a control.
Fruits are also shown. As a result, any apple variety
Strong antioxidant properties, and anti-acid
Almost no difference in the chemical strength was observed. Further, antioxidant activity of immature fruits other than apples
The effect was also studied. Immature fruits (apple
Extract, pear “Hosui”, peach “Akatsuki”)
For fraction B (polyphenol fraction), each fraction was 1-500
One week after adding to the reaction in the range of μl / gLA
Of lipid peroxide (absorbance 500nm value) and added concentration
Is shown in FIG. As a result, immature peaches are almost the same as immature apples.
It has almost the same antioxidant power and immature pear has phosphorus
It turns out to have quite strong antioxidant power, although not as good as Go
did. Example 3 [Immature Fruit Polyphenol]
Angiotensin I converting enzyme (ACE) inhibitory action]
  The following fruits were used as raw materials. Unripe apples: same as in Example 2 -Sample preparation: "Extracted sample" and "Polyf
An enol fraction "was prepared. -ACE inhibition test method The ACE inhibition test was performed according to a conventional method. Ie commercially available
Add sample solution to ACE solution and pre-incubate
Thereafter, Bz-Gly-His-Leu was added as a substrate and reacted. Anti
The resulting His fragment was treated with orthophthaldialdehyde.
After fluorescent labeling, fluorescence intensity (Ex.360nm, Em.490nm)
Was measured. At this time, the fluorescence intensity of the test solution was S,
Instead, the values when water is added are C and S enzyme blanks (yeast
Value with water added instead of elementary)_B, C enzyme bra
Link value to C_B And ｛1- (SS)_B) / (C-C_B)} × 100
The ACE inhibitory activity was expressed as (%). -Measurement results- Extracts of immature and mature apples (both "Fuji")
Is separated from the non-adsorbed fraction (→ fraction A) using Sep-pak C18.
It was fractionated into a polyphenol fraction (→ fraction B). Each fraction
ACE inhibitory activity was examined. The results are shown in FIG. The result
A, which is strong in fraction B in all of the fruits, immature fruits and mature fruits
CE inhibitory activity was observed. Especially for fraction B of immature fruit,
Although 0% inhibition was observed, the strength of both inhibitory activities was further increased.
For a detailed comparison of the two fractions added to the reaction system
Changes in ACE inhibitory activity when the amount of B added was changed
Examined. The results are shown in FIG. As a result, the ACE of the fraction B of the mature fruit was not
50% inhibitory concentration (IC₅₀) Was about 3ul,
Ripe fruit fraction B shows a very strong activity of less than 0.1 ul.
Was. This strong inhibitory activity on immature fruits
The same was observed in cultivars. The above results
And immature apples have potent inhibitors of ACE
Exists and its active substance is a polyphenol compound.
Was strongly suggested. In addition, ACE of pear and peach immature fruit
The inhibitory activity was weaker than that of apple. Next, it is found in the immature apples, and
For simple polyphenol compounds that are available in pure form,
The presence or absence of ACE inhibitory activity was examined. Specifically, substances
To the reaction system so that the intensity of activity between the two can be compared
ACE inhibitory activity when the addition concentration of each substance was changed
The variation was examined. The results are shown in FIG. In FIG. 9, for comparison, the ACE inhibitory activity has already been obtained.
Tea catechins that are known to have
A measurement was made. As a result, (-)-epigallocatechingare
IC for (ECC) and (-)-epicatechin gallate (ECg)₅₀value
Was 0.3 mM and 2 mM, respectively, indicating strong inhibitory activity.
In addition, GABARON in tea leaves with increased GABA concentration by processing
Tea "lowers blood pressure in an experiment using spontaneously hypertensive rats
It was reported that GABA was used in this experiment.
Did not show any ACE inhibitory activity. The essential apple phenols are all high.
Although it had inhibitory activity at the concentration, IC₅₀The value is low,
Even the most active quercetin is about 5 mM, and EGCg
The inhibitory activity was much weaker than that of ECg. Here, polypheno in the apple immature fruit fraction B
Assuming that all the tools are EGCg,
When calculating the total phenol content in Min. B, about 15000 ppm
And the polyphenol concentration of fraction B was 33.8% in terms of EGCg.
It was calculated to be equivalent to 2 mM. Using this calculated value,
When the activity curve of fraction B was plotted on
g (IC₅₀= 0.2 mM) Strong inhibitory activity
Sex was observed. EGCg and ECg actually exist in fraction B
And other simple polyphenols as described above
Do not show significant inhibitory activity. Observed in FIG.
The strong ACE inhibitory activity of immature fraction B is mixed
Due to the presence of other unidentified polyphenols
Was strongly suggested. (Embodiment 4) [Identification of ACE inhibitors in immature apple polyphenols]
Fixed] -Sample As in Example 3, from immature apple "Fuji"
An extraction sample was obtained, and polyphenol
Fraction was purified to give the following samples. -Experimental method The sample solution was loaded on a Sephadex LH-20 column. Column
After washing with distilled water, 20% to 60% methanol (hydrochloric acid
), 70% acetone (acidic with hydrochloric acid)
Phenol was eluted and fractionated. About the obtained fraction,
Perform HPLC composition analysis, total phenol measurement, ACE inhibition test
Was. If necessary, measure the absorption spectrum and gel
Permeation chromatography was performed. -Experimental results ACE inhibitory activity in sample from other polyphenols
Sephadex LH-20 colum as a specific means for isolation
Tried fractionation by n. The elution fraction obtained from the experiment
HPLC analysis of simple polyphenols and ACE inhibition
When the test was performed, the total
Check that all simple polyphenols were completely eluted.
I accepted. The ACE inhibitory activity was as follows:
% Acetone eluted fraction. The isolated fraction is
After the solvent was distilled off, the residue was freeze-dried and powdered. Active body in water
It is easily soluble (aqueous solution is orange).
0.7 g was obtained. Next, the obtained powder is dissolved in water,
The absorption spectrum was measured with a photometer. Figure 1 shows the results
0. The spectrum obtained has a maximum absorption at 280 nm.
And its shape is (+)-catechin or (-)-epicatechin
It was very similar. Also, apply the aqueous solution at 120 ° C for 10 minutes.
After crepe treatment, the solution turned red and
It was thought that anidine was formed. From these results
The active body is a procyanid known as a condensed tannin.
Strongly gins (regular polymers of catechins)
Was suggested. However, elution with Sephadex LH-20
Behavior and elution behavior of silica gel HPTLC
The substance is procyanidin B_Two About dimer like
Not a degree of polymerization but a higher order polymer substance
Estimated. Therefore, the molecular weight of this substance was measured by GPC.
Tried to determine. Generally, polyphenol compounds have hydrophobic bonds and
Strong affinity for column packing due to hydrogen bonding
GFC (Gel Filtrati) in aqueous systems used for ordinary proteins
on Chromatography) is considered difficult.
Therefore, the phenolic hydroxyl group of the active substance is converted to pyridine / anhydrous
After acetylation with acetic acid to make it soluble in organic solvents,
GPC analysis in organic solvent (THF) was attempted. Fig. 11 shows the results.
Indicated. As a result, a broad
A single peak appears, due to the polystyrene
According to the molecular weight calibration curve, the average molecular weight of this substance is about 2000.
It was calculated. Although not shown here, other experiments
(Partial decomposition with toluene-α-thiol, FAB-MS measurement
Etc.)
The structural formula of the ACE inhibitory active substance is shown in FIG. This is it
This substance was determined to be a type of condensed tannin. The purified polyphenol fraction was separated from Sephadex
 When fractionating with LH-20, the fraction before fractionation (that is,
After the fractionation (70% acetone eluted fraction: sand)
Total phenol content of the condensed tannin fraction)
And compared the values. The results are shown in Table 2. [0062] [Table 2] From this, it was found that all polypheno in immature apple fruit
About half of the tannins are condensed tannins,
It turned out that it was a large component. The ACE of this substance
IC against₅₀Was calculated, the IC of EGCg was determined by weight comparison.₅₀
Is very low, about 1/10 or less of the
It was confirmed that it was a very powerful ACE inhibitory active substance.
Was. From the above results, the apple immature fruit polyphenols are strong.
It could be judged that it could be a powerful ACE inhibitor. (Embodiment 5) [Production example of immature fruit polyphenol] immature apple fruit
(5-10g / piece) SO in about 50kg_Two While adding an appropriate amount
Crushed with a crusher and squeezed with an oil press. Obtained
Pectin-degrading enzyme was added to the fruit juice at about 50 ppm and centrifuged.
Separation or diatomaceous earth filtration followed by microfiltration
Thereby, 35 L of clear juice was obtained. Then, this clarification
Industrial synthesis adsorption of fruit juice with styrene-divinylbenzene system
The solution was passed through a column filled with resin (6 L). Furthermore, 0.
After removing sugars by passing 6 L of 1% HCl-containing water, the solution was added with 0.1% HCl.
Elution with 50% ethanol containing 1% HCl
3 L of the liphenol fraction was obtained. The obtained fraction was concentrated under reduced pressure using an evaporator.
To a concentrated fraction 1.5L, dried with a spray dryer,
228.2g of immature apple polyphenol powder formulation
Obtained. The data on recovery are described below. Column recovery rate: 95.6% Spray drying recovery rate: 93.0% Recovery rate from fruit juice; 0.65% Powder recovery from polyphenols in fruit juice; 88.9% (Embodiment 6) [Anti-mutagenic effect of immature fruit polyphenol]
In the examples, the antimutagenicity was determined by the Ames method (Muta
Tion Research, vol. 31, p34
7, 1975). -Raw materials and sample preparation Same as in Examples 2, 3, and 4. However, it was obtained in Example 4.
The “condensed tannin” is hereinafter abbreviated as “apple tannin”. -Method for measuring antimutagenicity Mutagenic substances (benzo (a) pyrene, Trp-P-2)
Phosphate buffer, sample solution, S9-mix, salmo
Salmonella typhimurium TA98 or TA
100) After mixing the overnight culture and culturing at 37 ° C for 2 days,
The number of colonies that appeared was counted. Each polyphenol
Is the exponential concentration gradient between 1 and 300 (μg / plate).
And the mutagenic suppression activity rate is calculated by the following formula.
Was. [0069]Mutagenic suppression activity rate = ｛(CB) − (S
−B)｝ / (CB) × 100 (%) At this time, the number of colonies in the test solution was S,
Controls with water added instead were C, samples and mutagens
Instead, the blank to which water was added was designated as B. -Result Regarding various polyphenols and apple tannin, Trp
FIG. 13 shows the rates of mutagenicity inhibitory activity against
FIG. 14 shows the mutagenicity inhibitory activity against zo (a) pyrene.
Indicated. The vertical axis shows the mutagenicity inhibitory activity rate,
100% was suppressed to the same level as that of the ink. side
The axis indicates the amount of sample per plate. As a result, apple tannin was found to bind to each mutagen.
Inhibits its mutagenicity in a concentration-dependent manner
Inhibiting effect in the lower or higher concentration range than techin gallate
did. Under the conditions of this experiment, Trp-P-2 and 1 μg
About 17 μg of apple tannin (epigallocatechin gale)
About 51 μg of benzo (a) pyrene
About 37 μg (about 56 μg), 50% mutagenic
It showed inhibitory activity. In other words, this polyphenol
When applied to mutagens (carcinogens),
It contained a large amount of components that strongly inhibited metagenicity. Follow
Therefore, the fruit polyphenol obtained in the present invention is anti-mutagenic.
It is also very effective as an agent. (Embodiment 7) [Immature fruit polyphenols inhibit hyaluronidase
Effect and histamine release inhibitory effect]
The allergic inhibitory activity is a hyaluronidase inhibitory activity
And histamine release inhibitory effect using animal cells
It was measured as an index. -Ingredients: immature apple, unripe pear, immature peach,
Coral mature fruit -Sample preparation Each fruit extract was prepared in the same manner as in Example 2,
Phenol fraction '' with Sephadex LH-20
The purified product was used as apple tannin. Method for measuring hyaluronidase inhibitory activity The method for measuring hyaluronidase inhibitory activity is basically described in J. Bio
l. , Vol. 250, p. 79, performed by the modified method of 1975.
Was. That is, a sample solution is added to a commercially available hyaluronidase solution.
After pre-incubation,
Add a 48/80 compound solution and add hyaluronic acid at 37 ° C.
Activate dase and add hyaluronic acid solution as substrate
It was added and reacted. N-acetylglucosa produced by the reaction
Min is developed by Morgan-Elson method and absorbance is measured.
(586 nm), and the hyaluronidase is calculated by the following formula.
The inhibitory activity rate was calculated. Hyaluronidase inhibitory activity rate = ｛(CC)
_B)-(S-S_B)｝ / (CC)_B) × 100 (%) At this time, the absorbance of the test solution is S,
C and S enzyme blanks
S_B, C enzyme blank to C_BAnd Method for measuring histamine release inhibitory activity Histamine release inhibitory activity is measured by J. Food Hyg. Soc. Japa
n, vol. 35, p497, 1994. That is, rats
10% FBS, α-basophil leukemia cell RBL-2H3
Cultivation in MEM medium (37 ° C, 5% CO_Two) And antibody DN
P-lgE was allowed to act for 90 minutes, and HEPES buff
After washing with er, the antigen DNP-BSA acts for 35 minutes
Let it. Apple ingredients are added during the action of antibodies or antigens
I do. Use ice-cold HEPES buffer to stop the reaction
Add extracellular solution, add perchloric acid, cool, and centrifuge.
After separation, 0.45μ filtered and analyzed by HPLC
Then, the amount of histamine released from the cells was measured. Hiss
Tamine release inhibitory activity was calculated by the following equation. Histamine release inhibitory activity rate = ｛(CB)
− (S−B)｝ / (C−B) × 100 (%) At this time, the amount of histamine in the test liquid was S,
Instead of adding a HEPES buffer
C, B where no antibody or antigen was added was designated as B. -Result Each fruit extract and sodium cromoglycate and apple
The hyaluronidase inhibitory activity rate for tannin is shown in FIG.
And FIG. As a result, each fruit extract and
Apple tannin inhibits hyaluronidase in a concentration-dependent manner
Its activity was observed. Synthetic anti-allergy as control
Compared to sodium cromoglycate
% For the concentration that inhibits the activity
Sodium molycate is about 0.051mg / ml
On the other hand, apple tannin is about 0.086 mg / ml
The values were close. In addition, each fruit extract and apple tannin
Antigen DNP-lgE during the action of antigen DNP-BSA
17 and FIG. 1 show the histamine release inhibitory activity rate during the action.
8 is shown. As is evident from these results,
Apple tannin in the enol fraction
Histamine release inhibitory activity was observed. Is it more than
Have shown that this polyphenol has
Has the effect of inhibiting the action of the enzyme hyaluronidase.
And histamine release inhibitory component
Minutes. Therefore, the fruits obtained by the present invention
Real polyphenols are extremely useful as allergy suppressants
It is effective. (Embodiment 8) [Glucosyltransfer of immature fruit polyphenols
Rase Inhibiting Action] In this example, representative species of cariogenic bacteria
Insoluble glucan produced by S. sobrinus
Inhibition ability of fruit polyphenols on generated GTase
I examined it. -Raw materials and sample preparation Same as Examples 6 and 7 -Used cells: Streptococcus sobrinus ATCC 334
78 Preparation of GTase; GT of S. sobrinus
se was prepared by the following method. S. sobrinus was transformed into a TTY medium (Agric. Biol. Chem., 54 (11), 29
25-2929, 1990) and incubated at 37 ° C for 18 hours, followed by centrifugation.
The supernatant was obtained by removing the cells. 50% saturation in this supernatant
Generated by centrifugation after addition of ammonium sulphate
The precipitate was collected. The precipitate was prepared in 0.05M phosphate buffer (pH
After redissolving in 6.5), the mixture was dialyzed against the same buffer. Dialysate
GTase is hydroxylapatite chromatograph
Purified and recovered by the method described above. At this time, about 0.4M phosphorus
Insoluble glucan-producing GTase elutes at the acid buffer concentration.
Was. The eluted fraction was collected and subjected to the following experiment. Method for measuring GTase inhibitory activity 1 ml of substrate solution (2% sucrose, 0.1% sodium azide
, 0.1 M phosphoric acid containing 40 μM dextran T10
Buffer solution (pH 6.5)) and purified GTase solution and sample solution
The solution was added and diluted to a total volume of 2 ml with water.
The reaction was performed for 8 hours. Insoluble glucan produced by the reaction
Was measured as turbidity, indicated by absorbance at 550 nm, and
The production rate was calculated from the above equation. Insoluble glucan production rate = (SS-SB) / (CS-C
B) × 100 (%) SS: Sample SB: enzyme blank of sample CS: Control CB: Control enzyme blank -Result The polyphenol fraction of each fruit extract and each polyphenol
The GTase inhibitory activity of the enol component is shown in FIGS.
did. The vertical axis shows the insoluble glucan generation rate, with no sample added.
Calculate the amount of production in the control as 100%
Was. The horizontal axis is the addition of the sample or polyphenol to the reaction system.
The amount added was indicated by volume or concentration. As shown in FIG.
Of the polyphenol fractions of various fruit extracts,
Polyphenol fraction of "Fuji" immature and "Shintaka" immature fruits
Showed remarkable GTase inhibitory activity. At this time,
50% inhibition of GTase of mature fruit by about 50%
μl, whereas that of immature "Fuji" is about 0.7
μl, immature apple fruit is about
It is found to have 70 times stronger GTase inhibitory activity.
Revealed. [0086] Next, various po
The GTase inhibitory activity of the liphenol component was examined.
Was. As shown in FIG. 20, among the components in the apple,
GTase inhibitory activity of lorogenic acid and phlorizin is very weak
It was a good thing. On the other hand, among various catechins,
While the inhibitory activity of the techin monomer was weak,
Polymeric epicatechin weight represented by 12 putative chemical structural formulas
Merging (indicated as apple tannin in FIG. 20) is a strong GT
It was found to have ase inhibitory activity. In FIG.
Is already known as a GTase inhibitor as a control.
Epicatechin, a typical catechin of green tea
Gallate (Agric. Biol. Chem., 5 (11), 2925-2929, 1990, Ch.
em.Pharm.Bull., 38 (39, 717-720, 1990)
However, the epicatechin gallate had a 5
0% inhibitory concentration is about 200 ppm, whereas phosphorus
Gotannin is about 2ppm and about 100 times stronger
It was calculated to be. From these results, see also FIG.
The powerful GTase inhibitory activity of immature fruits of "Fuji"
It was presumed to be due to the presence of the substance. based on the above results,
The fruit polyphenol obtained in the present invention is a powerful GTas
e Since it has an inhibitory active ingredient, it is extremely
It is considered effective. (Embodiment 9) [Deodorizing effect of immature fruit polyphenol]
The deodorizing effect test is performed by the method of Ui et al. (Nissho Kogaku, 38,109
8-1102, 1991). -Sample Polypurified from immature fruit juice of apple described in Example 2
Spray dried product of phenol fraction (apple polyphenol powder)
Powder formulation). For further comparison, known deodorant
Green tea polyphenol preparation and copper chlorophyll
Phosphorus sodium was used. -Deodorizing effect test method 1 ppm methyl mercaptan solution and 1
A ppm dimethyl sulfide solution was prepared. 30ml content
0.1 M phosphate buffer containing samples of each concentration in vials
1 ml of liquid (pH 7.5)
1), 1 ml of a 1 ppm methyl mercaptan solution,
Or add 1 ml of 1 ppm dimethyl sulfide solution and immediately
Plugged and stirred. Incubate this at 37 ° C for 5 minutes.
After that, inject 5ml of headspace gas into GC,
Increase the peak height of methyl mercaptan or dimethyl sulfide
It was measured. Measurement results Investigate whether each sample has a deodorizing effect on malodorous components
Was. 0.05-0.5% or 0.05-5% of sample
In the headspace gas when added to the reaction system in the range of
Of methyl mercaptan or dimethyl sulfide in water and sample
The relationship with the additive concentration is shown in FIGS. As a result
As is evident from the above, apple polyphenol powder formulation is
Concentration dependent on methyl mercaptan and dimethyl sulfide
It was found to have a deodorizing effect. (Embodiment 10) [Effects of immature fruit polyphenol on production of malodorous substances]
The test for suppressing the production of offensive odors in this example was performed by Ui et al.
The method was modified using the method described above (Nisshoku Kogaku, 38, 1098-1102, 1991).
Was. -Sample Apple polyphenol powder formulation as in Example 9, commercially available
Green tea polyphenol preparation and copper chlorophyllin sodium
Um was used. Test method for effect of suppressing production of malodorous substances Natural flow from those who prohibited eating and drinking for 2 hours before saliva collection
Saliva was collected. Each in a 30 ml vial
Concentration of sample and 40 mM L-methionine.
Add 1 ml of 1M phosphate buffer (pH 7.5)
1 ml of saliva collected in advance was added to this.
Then, it was immediately stoppered and stirred. 24 hours at 37 ° C
After incubation, add 5 ml of headspace gas to G
C, methyl mercaptan produced by the reaction,
Measure the peak height of dimethyl sulfide and dimethyl disulfide.
Was. Measurement results Each sample was examined for the presence of the effect of suppressing production of malodorous substances.
Add the sample to the reaction system in the range of 0.0015-0.5%
Methyl mercaptan in the headspace gas when
Dimethyl sulfide and dimethyl disulfide concentrations and sample addition concentrations
23 to 25 are shown in FIGS. The result clearly shows
As expected, apple polyphenol powder formulation is concentration dependent
Methyl mercaptan, dimethyl sulfide, dimethyl disulfide
Has a production inhibitory effect on the [0093] As described above, according to the present invention,,
ReNgo, pearOr peachMade from unripe fruits
Due to the constant treatment, various physiological effects, especially anti-acid
Polyphenol with fruiting ability and ACE inhibitory activity
Can be provided. [0094] [0095]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing a comparison between a wavelength-variable chromatogram of apple immature fruit polyphenol and an ultraviolet absorption spectrum of each peak. FIG. 2: The amount of lipid peroxide produced (absorbance 500 nm) one week after apple “Fuji” extract was added to the reaction system
2) is a graph showing the relationship between the value and the added concentration. FIG. 3 shows the amount of lipid peroxide produced (absorbance 500 nm) after one week when each fraction of apple “Fuji” was added to the reaction system.
2) is a graph showing the relationship between the value and the added concentration. FIG. 4 shows the amount of generated lipid peroxide (absorbance 500 nm) one week after the addition of the polyphenol compound to the reaction system.
Value). FIG. 5 shows the amount of lipid peroxide produced (absorbance) after one week when each apple polyphenol fraction was added to the reaction system.
A graph showing the relationship between the addition concentration (500 nm value) and the addition concentration. FIG. 6 shows the amount of generated lipid peroxide (absorbance) after one week when each immature fruit polyphenol fraction was added to the reaction system.
A graph showing the relationship between the addition concentration (500 nm value) and the addition concentration. FIG. 7 is a graph showing the ACE inhibitory activity of each fraction. FIG. 8 is a graph showing ACE inhibitory activity when the amount of Fraction B added was changed. FIG. 9 is a graph showing ACE inhibitory activity when the concentration of each substance added to the reaction system was changed. FIG. 10 is a graph showing an absorption spectrum of an isolated fraction measured by a spectrophotometer. FIG. 11 is a graph showing the results of measuring the molecular weight by GPC. FIG. 12 is a structural formula of a putative ACE inhibitory active substance. FIG. 13 is a graph showing the mutagenicity-suppressing activity of polyphenols against Trp-P-2. FIG. 14 is a graph showing the mutagenicity inhibitory activity of polyphenols on benzo (a) pyrene. FIG. 15 is a graph showing the hyaluronidase inhibitory activity of various fruit extracts. FIG. 16 is a graph showing the hyaluronidase inhibitory activities of an antiallergic agent and apple tannin. FIG. 17 is a graph showing the histamine release inhibitory activity rate of each fruit extract and apple tannin when the antigen DNP-BSA acts and when the antibody DNP-lgE acts. FIG. 18. Antigen DNP-BSA for apple tannin
The graph which shows the histamine release inhibitory activity rate at the time of action | operation, and the antibody DNP-lgE action. FIG. 19 is a graph showing the relationship between the amount of insoluble glucan produced and the amount added when each fruit polyphenol fraction was added to the reaction system. FIG. 20 is a graph showing the relationship between the amount of insoluble glucan produced and the concentration added when each polyphenol compound was added to the reaction system. FIG. 21 is a graph showing the residual amount of methyl mercaptan when apple polyphenol, green tea polyphenol, and sodium copper chlorophyllin were added to the reaction system. FIG. 22 is a graph showing the residual amount of dimethyl sulfide when apple polyphenol, green tea polyphenol, and sodium copper chlorophyllin were added to the reaction system. FIG. 23 is a graph showing the amount of methyl mercaptan generated when apple polyphenol, green tea polyphenol, and sodium copper chlorophyllin were added to the reaction system. FIG. 24 is a graph showing the amount of dimethyl sulfide generated when apple polyphenol, green tea polyphenol, and sodium copper chlorophyllin were added to the reaction system. FIG. 25 is a graph showing the amount of dimethyl disulfide generated when apple polyphenol, green tea polyphenol, and sodium copper chlorophyllin were added to the reaction system.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI A61K 35/78 ADZ A61K 35/78 ADZ AED AED (72) Inventor Shunji Shimoda 4-1-5 Higashinaka Shinjuku, Kashiwa-shi, Chiba Nikka Whiskey Corporation Kashiwa-ryo (56) References JP-A-4-193834 (JP, A) JP-A-61-122219 (JP, A) JP-A-2-102299 (JP, A) JP-A-6-183985 (JP, A) JP-A-1-265010 (JP, A) JP-A-2-25413 (JP, A) JP-A-3-77817 (JP, A) JP-A-59-196884 (JP, A) JP-A-6-340532 ( JP, A) JP-A-59-216810 (JP, A) Kanayoshi Akamatsu, “Revised Wakan Yaku”, Medical and Dental Medicine Publishing Co., Ltd. S55.10.5 p.359-360, 364-365, 371-372 Nakabayashi Toshiro, "Tannin Components of Fruits and Vegetables", Journal of the Japan Food Industry Association (1968), 15 (2) P73-78 N. Prabha et al. , "Polyphenol oxidase and peroxidase sezyme activities and the irisozyme mepatterns in ripening fruits" (Gran, 1991, Acta Alimentaria), Acta Alimentaria (1986). No. 6, p. 270-274 (58) Fields surveyed (Int. Cl. ⁷ , DB name) A61K 35/78

Claims (1)

(57) [Claim 1] Juvenile and / or extracted unripe fruits of lingo, pear or peach which are collected at the time of the fruiting operation , and then extract the juice Juice and / or extract
The polyphenol fraction is purified by passing through a styrene-divinylbenzene-based synthetic adsorption resin, an anion exchange resin, or an octadecyl group chemically bonded silica gel adsorbent. Obtaining a polyphenol mixture containing an acid derivative, a p-coumaric acid derivative, flavan-3-ols, flavonols, dihydrochalcones, and a condensed tannin which is a regular polymer of catechins as a high molecular weight polyphenol compound. A method for producing a polyphenol mixture.