JP2014125485A - Flavanone compounds, antioxidants containing the same, and production methods thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide novel flavanone compounds which exert high antioxidative effect, and production methods thereof.SOLUTION: The present invention provides, e.g., flavanone compounds having a structure represented by the specified general formula (1). These flavanone compounds are preferably used as antioxidants.

Description

  The present invention relates to a novel flavanone compound, an antioxidant containing the flavanone compound, and a method for producing the flavanone compound.

  Conventionally, as this type of flavanone compound, nymphaeol-A, B, C is known (see Non-Patent Document 1). These compounds can be extracted from grasshopper as described in Patent Document 1 in addition to Hasunohagiri. Further, these compounds are known to exhibit useful effects such as an antioxidant effect and an antibacterial effect (see Patent Document 2).

JP 2005-272374 A Japanese Patent Laying-Open No. 2005-29778

K. Yakushijin, K. Shibayama, H. Murata and H. Furukawa, New prenylflavanones from Hernandia nymphaefolia (presl) Kubitzki, Heterocycles, 14, 397-402, 1980.

  The present invention has been made by synthesizing a novel flavanone compound and finding useful physiological activity as a result of intensive studies by the present inventors. The object is to provide a novel flavanone compound that can be used for various uses such as pharmaceuticals and a method for producing the same. Another object is to provide an antioxidant that exhibits a high antioxidant effect.

  In order to achieve the above object, the flavanone compound of the present invention has a structure represented by the following general formula (1).

In order to achieve the above object, the flavanone compound of the present invention has a structure represented by the following general formula (2).

The antioxidant of this invention contains the flavanone compound as described in the said General formula (1) or (2), It is characterized by the above-mentioned.

Preferably, the antioxidant is used as a lipid antioxidant.
The method for producing a flavanone compound of the present invention is a method for producing a flavanone compound having a structure represented by the above general formula (1), wherein eriodictiool as a raw material and prenyl bromine as a prenyl group donor in the presence of a catalyst. It has the process made to react, It is characterized by the above-mentioned.

  The method for producing a flavanone compound of the present invention is a method for producing a flavanone compound having a structure represented by the above general formula (2), in which eriodictiool as a raw material and linalool as a prenyl group donor are reacted in the presence of a catalyst. It is characterized by providing the process to make.

  The novel flavanone compound of the present invention can be used for various uses such as pharmaceuticals. Another object is that the novel flavanone compound of the present invention can exhibit a high antioxidant effect.

Hereinafter, embodiments embodying the present invention will be described in detail.
The first flavanone compound of this embodiment has a structure represented by the following general formula (1).

The flavanone compound represented by the general formula (1) is 6′-prenyleriodictiool, has a molecular formula of C ₂₀ H ₂₀ O ₆ and a molecular weight of 356. This flavanone compound has structural characteristics similar to those of Eriodictyol, but is more hydrophobic than Eriodictiol because it has a C-prenyl group at the 6 ′ position. I think that the.

This flavanone compound has a higher antioxidant effect than eriodictiool and α-tocopherol.
The second flavanone compound of the present embodiment has a structure represented by the following general formula (2).

The flavanone compound represented by the general formula (2) is 6′-geranyl eriodictiool, has a molecular formula of C ₂₅ H ₂₈ O ₆ and a molecular weight of 424. This flavanone compound has structural characteristics similar to those of Eriodictyol, but is more hydrophobic than Eriodictiool because it has a C-geranyl group at the 6 ′ position. I think that the.

This flavanone compound has a higher antioxidant effect than eriodictiool and α-tocopherol.
When using each 1st or 2nd flavanone compound of this embodiment as an antioxidant, those flavanone compounds are used as an active ingredient (antioxidant material) of an antioxidant. Antioxidants are food / beverage products (beverages) that effectively prevent deterioration of various products (mainly oxidative deterioration) such as oxidative degradation of fats and oils, perfume degradation, pigment degradation, and pigment fading. Alternatively, it can be used by being added in food). Antioxidants can also be used in health foods and other foods and drinks to eliminate active oxygen in vivo when taken orally, enhance liver function, reduce acetaldehyde toxicity, and lipids. It exerts a health promoting action by inhibiting the oxidation of the fatty acid, for example, by inhibiting the oxidation of unsaturated fatty acids and the antioxidant action of low density cholesterol (LDL). Furthermore, the antioxidant can be used by being incorporated in cosmetics, pharmaceuticals or quasi-drugs, and is useful for the whitening effect of skin, oral cavity, etc., prevention of aging, and the like.

  In the food and drink, the daily intake of each flavanone compound is preferably 0.001 to 10 g, more preferably 0.01 to 1 g per adult day. When the daily intake of each flavanone compound is 0.001 g or more, the antioxidant effect can be exhibited more effectively. Conversely, if it is 10 g or less, it can be taken economically. In the case of a dwarf, half the amount for an adult is a guide. As said food-drinks, as other components, for example, gelling agent-containing foods, sugars, fragrances, sweeteners, fats and oils, base materials, excipients, food additives, auxiliary materials, bulking agents and the like may be appropriately blended. Good.

  When applying an antioxidant to cosmetics, it can apply by mix | blending with a cosmetics base material. The form of the cosmetic may be any of emulsion, cream, powder and the like. Cosmetic bases are generally blended in common with cosmetics, and include, for example, oil, purified water and alcohol as main components, surfactants, moisturizers, other antioxidants, thickeners, At least one selected from an antiseborrheic agent, a blood circulation promoter, a whitening agent, a pH adjuster, a pigment, a preservative, and a fragrance is appropriately blended.

  When the antioxidant is used as a pharmaceutical or quasi drug, the dosage form is not particularly limited, and examples thereof include ointments, liquids, sprays, sheets, powders, and powders. Moreover, as an additive, you may mix | blend an excipient | filler, a base, an emulsifier, a solvent, a stabilizer etc., for example. In addition to administration by administration (oral intake), any administration method such as subcutaneous injection, intravascular administration, and transdermal administration can be employed. Although it does not specifically limit as a dosage form, For example, a powder, a powder agent, a granule, a tablet, a capsule, a pill, a suppository, a liquid agent, an injection, etc. are mentioned. Moreover, as an additive, you may mix | blend an excipient | filler, a base, an emulsifier, a solvent, a stabilizer etc., for example.

Next, the action of the novel flavanone compound configured as described above will be described.
The novel flavanone compounds of this embodiment are 6′-prenyl eriodictiool of the above general formula (1) and 6′-geranyl eriodictiool of the above general formula (2). Since these flavanone compounds have an excellent antioxidant action, they can be used for various types of applications including pharmaceuticals.

Next, the manufacturing method of the novel flavanone compound comprised as mentioned above is demonstrated.
The first flavanone compound of the present embodiment can be synthesized, for example, by mixing eriodictiool as a raw material and prenyl bromine as a prenyl group donor in a solvent and reacting in the presence of a catalyst.

As the raw material Eriodictiool, any of biosynthetic products, chemically synthesized products, crude extracts extracted from natural materials using water / hydrophilic organic solvents, or purified products can be used. Examples of the catalyst include boron trifluoride-ethyl ether complex (BF ₃ -etherate). Although it does not specifically limit as a solvent, For example, a dioxane is mentioned. The reaction temperature and time are not particularly limited. For example, the target product can be produced by performing the reaction at room temperature (25 ° C.) for several hours. In addition, it is preferable that reaction is performed under nitrogen filling. Examples of the prenyl bromine used as the prenyl group donor include 3,3-dimethylallyl bromide.

  The first flavanone compound obtained by the above reaction may be further purified using a known method. As a purification method, the reaction product is isolated by purifying it using one or more chromatography. As the chromatography, known chromatography such as liquid chromatography, supercritical fluid chromatography, and thin layer chromatography can be used. As liquid chromatography, column chromatography can be used, for example, and more specifically, high performance liquid chromatography (HPLC) and open column chromatography can be mentioned. Examples of the chromatography carrier include ion exchange chromatography, partition chromatography (normal phase / reverse phase chromatography), adsorption chromatography, and molecular exclusion chromatography. For example, in a method using silica gel column chromatography, separation can be performed by using a water / acetonitrile gradient system as a mobile phase. This purification step can be performed using the already purified components as an index or the antioxidant action described above as an index.

  The second flavanone compound of this embodiment can be synthesized, for example, by dissolving eriodictiool as a raw material and linalool as a geranyl group donor in a solvent and reacting in the presence of a catalyst. The same raw material, catalyst, solvent, and reaction conditions as those for the first method for producing a flavanone compound can be employed.

According to the novel flavanone compound of the above embodiment, the following effects can be obtained.
(1) The 1st flavanone compound of this embodiment is 6'- prenyleriodictiool of the said General formula (1). Since this flavanone compound has an excellent antioxidant action, it can be used for various types of applications including pharmaceuticals. Moreover, this flavanone compound can be used for various uses utilizing the fact that it is more hydrophobic than Eriodictiool, in addition to being used for the same applications as Eriodictiool.

  (2) The 2nd flavanone compound of this embodiment is 6'-geranyl eriodictiool of the said General formula (2). Since this flavanone compound has a particularly excellent antioxidant action, it can be used for various kinds of uses including pharmaceuticals. Moreover, this flavanone compound can be used for various uses utilizing the fact that it is more hydrophobic than Eriodictiool, in addition to being used for the same applications as Eriodictiool.

  (3) Since the antioxidant containing the flavanone compound of the present embodiment contains a flavanone compound having a high antioxidant action as an active ingredient, it prevents deterioration of food, drink, cosmetics or quasi drugs. Thus, the effect of promoting health and the effect of preventing aging can be exhibited by improving the storage stability, or taking orally or dermally.

  (4) The antioxidant containing the flavanone compound of this embodiment is particularly excellent in the effect of preventing lipid oxidation. Therefore, it is expected that it can be applied to the treatment or prevention of various diseases caused by the oxidation of lipids, and the preservability of raw materials such as foods and drinks, pharmaceuticals and cosmetics containing lipids.

  (5) The flavanone compound of this embodiment is excellent in the effect which suppresses the oxidation of saturated fatty acid and LDL. Therefore, it is expected to be applicable to treatment and prevention of various diseases such as arteriosclerosis caused by lipid peroxide and oxidized LDL.

In addition, you may change the said embodiment as follows.
-The novel flavanone compound of the above embodiment is a non-human animal, for example, domestic animals such as horses, cows and pigs (non-human mammals), poultry such as chickens, or pets such as dogs, cats, rats and mice ( You may apply to various domestic animals.

  -The flavanone compound of the said embodiment has the outstanding antioxidant effect | action. You may apply the antioxidant containing the flavanone compound of the said embodiment as a reagent for experiment and research. It can be used for the purpose of elucidating the mechanism of physiological action related to antioxidant action.

Next, the embodiment will be described more specifically with reference to examples and comparative examples.
<Production of new flavanone compounds>
(1) Manufacture of 6′-prenyl eriodictiol First, eriodictiool (crude) (1.00 g, 3.47 mmol) was transferred to a 50 mL branch eggplant flask and dried, and then dioxane (10 mL). Dissolved in. BF ₃ OEt ₂ (1.2 mL, 9.52 mmol, 2.1 eq.) Distilled with stirring under ice cooling was added dropwise and prenyl bromine (3,3-dimethylallyl bromide) (1.1 mL, 9.54 mmol). 2.6 eq.) Was slowly added dropwise. Then, it stirred at room temperature for 3 hours. The reaction was dissolved in Et ₂ O (50 mL) and washed 3 times with RO water (30 mL). Then, after dehydrating with Na ₂ SO ₄ , the solvent was removed to obtain a fraction AK-36-1 containing a flavanone compound, and then purified by silica gel column chromatography (CH ₂ Cl ₂ : MeOH = 20: 1), Fraction AK-36-5 (crude) (154.3 mg) and fraction AK-36-6 (242.9 mg) (raw material recovery) were obtained. The fraction AK-36-5 was analyzed under the following condition 1 using reverse phase high performance liquid chromatography (RP-HPLC).

<Condition 1 of HPLC>
Column: InertSustain C18 (4.6 × 150 mm)
Gradient: 0.1% TFA mQ: 0.1% TFA CH ₃ CN (0 min / 99: 1, 5 min / 99: 1, 35 min / 0: 100, 45 min / 0: 100, 55 min / 99: 1)
Detection: 214 nm
Thereafter, their structures were determined by ¹ H-NMR was fractionated. The results of structural analysis are shown below.

¹ H NMR (400MHz, ACETN) δ7.06 (s, 1H), 6.73 (s, 1H), 5.95 (s, 2H), 5.62 (dd, 1H, J = 10.8, 2.7 Hz), 5.24-5.19 (m , 1H), 3.41-3.27 (m, 2H), 3.13 (dd, 1H, J = 17.2, 13.2 Hz), 2.65 (dd, 1H, J = 14.4, 2.7 Hz), 1.70 (s, 3H), 1.65 ( s, 3H).

From the ¹ H-NMR data, it was confirmed to be 6′-prenyleriodictiool having the structure of the general formula (1).
Based on the obtained structural analysis data, 6′-prenyleriodictyol was purified from fraction AK-36-5 under the following HPLC condition 2.

<Condition 2 of HPLC>
Column: Inertsil ODS-3 20 × 250 mm
Gradient: 0.1% TFA mQ: 0.1% TFA CH ₃ CN (0 min / 40: 60, 30 min / 25: 75)
Detection: 214 nm
The peak with a retention time of 10.383 minutes was collected to obtain fraction AK-38-2 (14.7 mg, y (yield) 1.2%) (6′-prenyleriodictiool).

(2) Production of 6′-geranyl eriodictiool First, eriodictiool (crude) (2.2 g, 7.63 mmol) was transferred to a 50 mL branch eggplant flask and dried, followed by dioxane (10 mL). Dissolved in. BF ₃ OEt ₂ (2.6 mL, 20.6 mmol, 2.1 eq.) Distilled with stirring under ice cooling was added dropwise, and linalool (4.4 mL, 24.8 mmol, 2.6 eq.) Was added. Slowly dripped. Then, it stirred at room temperature for 3 hours. The reaction was dissolved in Et ₂ O (50 mL) and washed 3 times with RO water (30 mL). Thereafter, it was dehydrated with Na ₂ SO ₄ , and the solvent was removed to obtain a fraction RT-2-27-1, which was then purified by silica gel column chromatography (CH ₂ Cl ₂ : MeOH = 20: 1), and RT-2- 27-4 (crude) (1.62 g), fraction RT-2-27-5 (440 mg) (raw material recovery) was obtained. Fraction RT-2-27-4 was analyzed by RP-HPLC under the following HPLC condition 3.

<HPLC conditions 3>
Column: InertSustain C18 (4.6 × 150 mm)
Gradient: 0.1% TFA mQ: 0.1% TFA CH ₃ CN (0 min / 99: 1, 5 min / 99: 1, 35 min / 0: 100, 45 min / 0: 100, 55 min / 99: 1)
Detection: 214 nm
Thereafter, the fraction RT-2-27-4 and the 99.5% ethanol extract of the grasshopper were analyzed by RP-HPLC under the same conditions, and three matched peaks (retention time: 33.892 minutes, 34. (893 minutes, 35.417 minutes), and they were collected and subjected to structure determination using ¹ H-NMR. In addition, the 99.5% ethanol extract of the grasshopper was first chopped dry leaves of the grass, then crushed in a mortar, and after adding 1000 mL of 99.5% ethanol to 100 g of the dried leaves, Extraction was performed in the dark at room temperature (25 ° C.) for 1 week. Subsequently, after purification by solid-liquid separation using a filtering agent such as activated carbon, the extract was dried to obtain an ethanol extract of a wolffish.

Components with a retention time of 34.893 minutes: ¹ H NMR (400 MHz, ACETN) δ6.91 (d, 1H, J = 1.8 Hz), 6.81 (d, 1H, J = 1.8 Hz), 5.95 (s, 2H), 5.40-5.33 (m, 1H), 5.12 (t, 1H, J = 5.4 Hz), 3.37 (d, 2H, J = 7.7 Hz), 3.15-3.08 (m, 1H), 2.73-2.68 (m, 1H) , 1.72 (s, 3H), 1.63 (s, 3H), 1.57 (s, 3H).

The ¹ H-NMR data was consistent with Nymphaeol-B.
Components with a retention time of 35.417 minutes: ¹ H NMR (400 MHz, ACETN) δ7.02 (s, 1H), 6.87 (d, 2H, J = 1.0), 6.04 (s, 1H), 5.37 (dd, 1H, J = 3.2, J = 12.7), 5.26 (t, 1H, J = 7.1), 5.08 (t, 1H, J = 7.1), 3.26 (d, 2H, J = 7.1), 3.12 (dd, 1H, J = 13.0, J = 17.1), 2.66 (dd, 1H, J = 3.2, J = 17.1), 1.77 (s, 3H), 1.61 (s, 3H), 1.56 (s, 3H).

The ¹ H-NMR data was consistent with Nymphaeol-A.
On the other hand, from the result of ¹ H-NMR, 33.892 minutes (RT-2-33-3) is a structure in which the 5′-position or 6′-position is geranylated, and isonympheol-B (Isonymphaeol- B). However, since it could not be completely identified, fraction RT-2-27-4 was purified under different conditions (HPLC condition 4 below).

<Condition 4 of HPLC>
Column: Inertsil ODS-3 (20 × 250 mm)
Gradient: 0.1% TFA mQ: 0.1% CH ₃ CN (0 min / 35: 65, 30 min / 20: 80)
Detection: 214 nm
The peak with a retention time of 21.567 minutes was collected to obtain a fraction RT-2-33-4 (Nimpheol-B) (10.9 mg, y (yield) 0.3%). Next, a peak with a retention time of 23.758 minutes was collected to obtain a fraction RT-2-33-5 (Nimpheol-A) (53.3 mg, y (yield) 1.6%).

Moreover, since the peak of the retention time of 17.242 minutes was a mixture of two kinds of compounds (fraction RT-2-3-3-3) as a result of ¹ H-NMR analysis, it was further subjected to another condition (Condition 5 below). Attempted purification.

<Condition 5 of HPLC>
Column: Inertsil ODS-3 (20 × 250 mm)
Gradient: 0.1% TFA mQ: 0.1% CH ₃ CN (0 min / 50: 50, 60 min / 40: 60)
Detection: 214 nm
A peak with a retention time of 50.467 minutes (fraction RT-2-43-1) and a peak with a retention time of 53.725 minutes (fraction RT-2-43-2) were collected, and ¹ H-NMR was used. The structure was determined.

Components of fraction RT-2-43-1: ¹ H NMR (400 MHz, ACETN) δ7.05 (s, 1H), 6.73 (s, 1H), 5.94 (s, 2H), 5.62 (dd, 1H, J = 13.2, 2.8Hz), 5.23 (t, 1H, J = 7.8Hz), 5.09-5.05 (m, 1H), 3.42-3.26 (m, 2H), 3.12 (dd, 1H, J = 17.6, 13.6Hz) 2.64 (dd, 1H, J = 14.7, 2.8 Hz), 1.65 (s, 3H), 1.62 (s, 3H), 1.56 (s, 3H).

From the ¹ H-NMR data, it was confirmed to be 6′-geranyl eriodictiool having the structure of the general formula (2).
Components of fraction RT-2-43-2: ¹ H NMR (400 MHz, ACETN) δ6.96 (d, 1H, J = 8.3 Hz), 6.82 (d, 1H, J = 8.3 Hz), 5.96 (s, 2H), 5.61 (dd, 1H, J = 2.7, 13.4 Hz), 5.18 (t, 1H, J = 7.1), 5.07 (t, 1H, J = 7.1 Hz), 3.54 (d, 2H, J = 6.6 Hz ), 3.17 (dd, 1H, J = 13.4, 17.1 Hz), 2.66 (dd, 1H, J = 2.7, 17.1 Hz), 1.69 (s, 3H), 1.61 (s, 3H), 1.55 (s, 3H) .

The ¹ H-NMR data was consistent with Isonymphaeol-B.
The fraction RT-2-43-1 (6′-geranyl eriodictyol) was 17.4 mg, and y (yield) 0.5%. Fraction RT-2-43-2 (isonimphele-B) was 4.5 mg, y (yield) 0.1%.

<Antioxidation test of new flavanone compounds>
(1) Radical scavenging ability test DPPH radical scavenging activity was measured in order to evaluate the radical scavenging ability in an aqueous system of the antioxidant activity which is one of the physiologically active actions of the new flavanone compound. This test utilizes the fact that DPPH (1,1-Diphenyl-2-picrylhydrazyl) having a maximum absorption of 517 nm in a radical state is reduced by an antioxidant and fades. As samples used in this test, 6′-prenyleriodictiool of Example 1 and 6′-geranyleriodictiool of Example 2, and as controls, Eriodictiool, Nimpheol-A, Nimpheol-B , And isonimpheol-B were used.

In a quartz cell, 1.8 mL of ethanol and 1.2 mL of 40 mM MES (2- [N-Morpholino] ethanonesulfonic acid) buffer (final concentration 1.6 mM) are added and zeroed. As a control, 1170 μL of ethanol, 1200 μL of 40 mM MES buffer (final concentration of 1.6 mM), and 600 μL of 500 μM DPPH (final concentration of 100 mM) were added to the quartz cell to perform zero adjustment. Thereafter, 30 μL of ethanol was added and the absorbance was measured every 5 minutes to examine the change in DPPH absorbance for 30 minutes. Next, 30 μL of each sample was added and the same measurement was performed to determine the concentration EC ₅₀ when the absorbance (517 nm) after 30 minutes was reduced to 50%. The measurement was performed three times at 25 ° C., and the absorbance was measured using a HITACHI U-2000 Spectrophotometer as a plate reader. The results are shown in Table 1. Data were expressed as mean ± standard error.

As shown in Table 1, it was confirmed that the 6′-prenyl eriodictyol of Example 1 and the 6′-geranyl eriodictyol of Example 2 have excellent antioxidant activity.

(2) Test of linoleic acid conjugation formation inhibitory activity Antioxidant activity was compared by measuring conjugated dienes generated in the process of oxidative modification of linoleic acid. 2.81 mL of 50 mM phosphate buffer (pH 7.4) that had been set to 40 ° C. in advance was placed in a quartz cell. Then, 10 μL of each sample prepared, 30 μL of a solution containing 16 mM linoleic acid (final concentration 0.16 mM), and finally 150 μL of 40 mM AAPH (2,2′-Azobis (2-methylpropionamidine) dihydrochloride) (final concentration) 2 mM) was added to make the total volume 3 mL. Absorbance measurement was started with the time when AAPH was added as 0 minutes. The absorbance of the conjugated diene produced in the process of linoleic acid oxidation was measured for 180 minutes at 37 ° C. for 180 minutes at a peak 234 nm. The time (min) until linoleic acid per 1 μM sample was oxidized was determined. The measurement was performed three times, and the absorbance was measured using a HITACHI U-3300 Spectrophotometer as a plate reader. The results are shown in Table 2. Data were expressed as mean ± standard error.

As shown in Table 2, it was confirmed that the 6′-prenyl eriodictyol of Example 1 and the 6′-geranyl eriodictyol of Example 2 have excellent antioxidant activity. In particular, it was confirmed that the 6′-geranyl eriodictyol of Example 2 has an extremely high effect of suppressing oxidative modification of linoleic acid.

(3) Test of LDL antioxidant activity The antioxidant action against LDL (low density lipoprotein), which is a major cholesterol transport protein in blood and has been correlated with arteriosclerosis, was tested.

(A) Isolation / Purification of LDL First, human blood was collected, allowed to stand at room temperature for about 30 minutes, and then centrifuged at 3000 rpm and 4 ° C. for 10 minutes. After centrifugation, the supernatant (serum) was collected with Pipetteman and transferred to a 15 mL centrifuge tube. All subsequent experimental operations are performed at low temperatures. Next, KBr is dissolved in serum so as to be 0.325 g / mL. After confirming that KBr was completely dissolved, the serum was put into an ultracentrifuge tube using a pipetteman. Next, an aqueous NaCl solution (d = 1.006) having a concentration of 8.5 g / L was gently poured into a volume ratio of (NaCl layer) :( serum layer) = 2: 3 to form two layers.

  Next, centrifugation was performed for 60 minutes under the conditions of 80000 rpm and 4 ° C. in an ultracentrifuge. After centrifugation, the centrifuge tube was carefully and promptly taken out, and the LDL layer fractionated by the concentration gradient was collected with a Pipetman and transferred to a tube (Eppendorf).

  A sample was collected using a 1 mL syringe not equipped with a needle, and then a 22 μm microfilter was attached, followed by filtration into another new tube (Eppendorf). Next, after sucking the sample with a 10 mL syringe equipped with a needle, the needle is inserted into the dialysis membrane and the sample is injected. The dialysis membrane was floated, dialyzed for 30 minutes at 4 ° C. in 1 × PBS buffer while stirring with a stirrer, exchanged Buffer, and dialyzed for another 30 minutes. Finally, the buffer was changed and dialyzed overnight. All three dialysis were performed in a cold room. In addition, 1 × PBS buffer for dialysis was used after sufficiently aerated with nitrogen and cooled. The purified LDL after dialysis was collected with a 10 mL syringe and stored in a tube (Eppendorf) under light shielding at 4 ° C. under nitrogen filling.

(B) Quantification of LDL (BCA method)
In order to prepare a BSA calibration curve, standard BSA was adjusted so that the final concentrations were 0, 0.125, 0.25, 0.5, and 1 mg / mL, respectively. The LDL sample scored 3 points by dilution of 4 times, 6 times and 8 times. The sample was diluted with 1 × PBS buffer. 25 μL each of the diluted sample and standard BSA was applied to a 96-well plate for a microplate reader, and 200 μL of (BCA / A solution 3000 μL) + (BCA / B solution 60 μL) was applied. After incubation at 37 ° C. for 30 minutes, OD ₅₇₀ measurement was performed using a microplate reader, and the LDL concentration and the isolated LDL amount were calculated from a calibration curve.

(C) TBARS assay In the thiobarbituric acid reaction product (TBARS) assay, a final reaction product (TBARS) produced by oxidation of lipid was measured, and an experiment was conducted by a method for measuring a lipid oxidation inhibitory effect.

First, 1 × PBS buffer, 10 μL of each sample sample, LDL, and 10 μL of 500 μM copper sulfate (oxidant) (final concentration 5 μM) were mixed in this order so as to be LDL (50 μg / 100 μm) to a total volume of 100 μL. After incubation at 37 ° C. for 4 hours, the entire sample was transferred to a 15 mL centrifuge tube to which 1 mL of 1% phosphoric acid aqueous solution was added. Further, 50 μL of 10 mM BHT was added to stop the oxidation. Finally, 1 mL of 0.67% TBA was added, and then vortexed for about 5 seconds and treated for 30 minutes (100 ° C.). After completion of the reaction, the sample was cooled in ice and returned to room temperature, 2 mL of butanol was added, and vortexed for about 30 seconds. With sufficient agitation, TBARS dissolves in butanol. Using a centrifuge (KUBOTA 5910), the mixture is centrifuged for 10 minutes at 3000 rpm and 4 ° C., and separated into two layers. After centrifugation, the butanol layer (upper layer) was collected, and the absorbance at 532 nm was measured using a HITACHI U-2000 Spectrophotometer. The concentration IC ₅₀ when the oxidation of LDL was suppressed by 50% was determined. The results are shown in Table 3. The measurement was performed three times, and the data was expressed as mean ± standard error.

As shown in Table 3, it was confirmed that the 6′-prenyl eriodictyol of Example 1 and the 6′-geranyl eriodictyol of Example 2 have an excellent LDL oxidation inhibitory action. .

(D) LDL-conjugated diene production inhibitory activity Measures the end product of lipid oxidation (c) In contrast to the TBARS assay, LDL-conjugated diene production inhibitory activity is known as a method for measuring the initial degree of oxidation of lipids. ing.

For measurement, 30 μL (final concentration: 1 μM) of each compound described in Table 4 in a quartz cell, LDL (final concentration: 50 μg / mL), 30 μL of 500 μM CuSO ₄ (final concentration: 5 μM) were added in this order, and the whole volume was obtained with PBS. Was adjusted to 3 mL. Then, the time change of the absorbance at 234 nm derived from the conjugated diene structure generated by the LDL peroxidation reaction was measured with the time of adding CuSO ₄ as 0 minute. Absorbance was measured using a HITACHI U-2000 Spectrophotometer. The time (time lag (minutes)) from when CuSO ₄ was added to when oxidation was started was determined. The results are shown in Table 4. The measurement was performed three times, and the data was expressed as mean ± standard error.

A compound having a larger lag time has a higher effect of delaying the formation of the conjugated diene, which is an initial oxidation reaction product, and has a strong LDL oxidation inhibitory activity. As shown in Table 4, it was confirmed that the 6′-prenyl eriodictyol of Example 1 and the 6′-geranyl eriodictyol of Example 2 have an excellent LDL oxidation inhibitory action. .

  As described above, LDL is responsible for cholesterol transport in the blood. It is known that oxidized LDL is formed when lipids in LDL are oxidized in vivo by free radicals or the like. Normally, LDL is taken into the liver through the LDL receptor, but oxidized LDL is taken into macrophages through the scavenger receptor, not the LDL receptor. Macrophages that have taken up a large amount of this oxidized LDL are foamed, causing vascular endothelial cell damage and atherosclerosis. Therefore, the novel flavanone compound of the present invention, which is excellent in the effect of suppressing LDL oxidation, is expected to suppress arteriosclerosis caused by oxidized LDL.

  As described above, it has been clarified that the novel flavanone compound of the present invention has an excellent antioxidant action with respect to known flavanone compounds. In addition, in the catechin which is flavan-3-ol having a different basic skeleton from the present flavanone compound, the radical scavenging activity is enhanced by the structure of the planar catechin in which an electron-donating isopropyl group is introduced at the 6′-position of the B ring. (K. Fukuhara et.al .; J. Am. Chem. Soc., 124, 5952-5953 (2002)). On the other hand, the flavanone compound does not have a hydroxyl group at the 3-position of the C ring and cannot take a structure like a planar catechin. In other words, in the flavanone compound, it is predicted that the functional effect is improved as compared with the conventional compound, and a novel compound is obtained by adding a prenyl group composed of a carbon number 5 isoprene unit to the B ring 6′-position. There is no motion to try. Therefore, it is thought that it is surprising that the novel flavanone compound of the present invention as described above has an excellent antioxidant action with respect to known flavanone compounds.

Next, the technical idea that can be grasped from the above embodiment and other examples will be described below.
(A) A food, beverage, cosmetic or pharmaceutical comprising the novel flavanone compound represented by the general formula (1) or (2).

  (B) An anti-arteriosclerotic agent comprising the novel flavanone compound of the general formula (1) or the general formula (2) as an active ingredient.

Claims (6)

A flavanone compound having a structure represented by the following general formula (1).
A flavanone compound having a structure represented by the following general formula (2).
  An antioxidant, comprising the flavanone compound according to claim 1 or 2.   The antioxidant according to claim 3, which is used as an antioxidant for lipids.   The process for producing a flavanone compound according to claim 1, comprising a step of reacting eriodictiool as a raw material and prenyl bromine as a prenyl group donor in the presence of a catalyst.   The method for producing a flavanone compound according to claim 2, comprising a step of reacting eriodictiool as a raw material and linalool as a geranyl group donor in the presence of a catalyst.