JP4268900B2 - Antioxidants, antibacterial agents, antitumor agents and food and drink - Google Patents

Description

  The present invention relates to an antioxidant, an antibacterial agent, an antitumor agent and a food and drink containing at least one flavanone compound selected from nymphaeol-A, B and C.

Conventionally, these flavanone compounds are disclosed in Non-Patent Documents 1 to 3. In other words, it is known that Nimpheol-A is isolated from the genus Hasnohagiri and Mizohozuki, Nimpheol-B is isolated from Hasunohagiri, and Nimfeol-C is isolated from Hasunohagiri and Mahang.
K. Yakushijin, K. Shibayama, H. Murata and H. Furukawa, New prenylflavanones from Hernandia nymphaefolia (presl) Kubitzki, Heterocycles, 14, 397-402, 1980. WRPhillips, NJBaj, AALGunatilaka and DGIKingston, C-Geranyl compounds from Mimulus clevelandii, J. Nat. Prod., 59, 495-497, 1996. MHTseng, CHChou, YMChen and YHKuo, Allelopathic prenylflavanones from the fallen leaves of Macaranga tanarius, J. Nat. Prod., 64, 827-828, 2001.

However, in each of the non-patent documents, the physiological activity of each flavanone compound is hardly elucidated.
This invention has been made by finding useful physiological activities exhibited by these flavanone compounds as a result of intensive studies by the present inventors. The object is to provide an antioxidant that exhibits a high antioxidant effect, an antibacterial agent that exhibits a high antibacterial effect, and an antitumor agent that exhibits a high antitumor effect. Another object is to provide a food or drink that is easy to improve the health promotion effect or preservation.

  In order to achieve the above object, the antioxidant of the invention described in claim 1 contains at least one flavanone compound selected from nymphaeol-A, nymphaeol-B and nymphaeol-C. The antibacterial agent of the invention described in claim 2 contains at least one flavanone compound selected from nymphaeol-A, nymphaeol-B and nymphaeol-C. The antitumor agent of the invention described in claim 3 contains at least one flavanone compound selected from nymphaeol-A, nymphaeol-B, and nymphaeol-C. The food-drinks of the invention described in claim 4 contain at least one flavanone compound selected from nymphaeol-A, nymphaeol-B, and nymphaeol-C.

According to the present invention, the following effects can be obtained.
According to the antioxidant of the invention described in claim 1, a high antioxidant action can be exhibited. According to the antibacterial agent of the invention described in claim 2, a high antibacterial action can be exhibited. According to the antitumor agent of the invention described in claim 3, a high antitumor action can be exhibited. According to the food / beverage product of invention of Claim 4, it becomes easy to aim at the improvement effect of health, or a preservability.

Hereinafter, embodiments embodying the present invention will be described in detail.
The first flavanone compound of the embodiment is nymphaeol-B (nymphaeol-B; 5,7,3 ′, 4′-tetrahydroxy-2′-C-geranylflavanone) having the structure shown in the following chemical formula 1.

この第１のフラバノン化合物は、分子式Ｃ₂₅Ｈ₂₈Ｏ₆、分子量４２４、融点（ＭＰ）８０〜８３℃である（図１参照）。このフラバノン化合物は、本発明者らによって沖縄産プロポリスから単離された天然物由来の有機化合物であり、エリオディクティオール（Eriodictyol）と類似した構造的特徴を有しているが、２’位にＣ−ゲラニル基を備えていることからエリオディクティオールよりも親油性（膜透過性）が高い。

This first flavanone compound has a molecular formula C ₂₅ H ₂₈ O ₆ , a molecular weight 424, and a melting point (MP) of 80 to 83 ° C. (see FIG. 1). This flavanone compound is a natural product-derived organic compound isolated from Okinawa propolis by the present inventors, and has structural features similar to Eriodictyol, but at the 2 ′ position. Since it has a C-geranyl group, it has higher lipophilicity (membrane permeability) than Eriodictiool.

  The second flavanone compound of the embodiment is nymphaeol-A (5,7,3 ′, 4′-tetrahydroxy-6-C-geranylflavanone) having the structure shown in the following chemical formula 2.

この第２のフラバノン化合物は、分子式Ｃ₂₅Ｈ₂₈Ｏ₆、分子量４２４、融点（ＭＰ）１７２〜１７５℃である（図３参照）。このフラバノン化合物は、本発明者らによって沖縄産プロポリスから単離された天然物由来の有機化合物であり、エリオディクティオールと類似した構造的特徴を有しているが、６位にＣ−ゲラニル基を備えていることからエリオディクティオールよりも親油性（膜透過性）が高い。

This second flavanone compound has a molecular formula of C ₂₅ H ₂₈ O ₆ , a molecular weight of 424, and a melting point (MP) of 172 to 175 ° C. (see FIG. 3). This flavanone compound is an organic compound derived from a natural product isolated from Okinawa propolis by the present inventors and has structural characteristics similar to Eriodictiool, but C-geranyl is located at the 6-position. Since it has a group, it is more lipophilic (membrane permeability) than Eriodictiool.

  The third flavanone compound of the embodiment is nymphaeol-C (5,7,3 ′, 4′-tetrahydroxy-6- (3 ′ ″, 3 ″) having the structure shown in the following chemical formula 3 '-dimetylallyl) -2'-C-geranylflavanone).

この第３のフラバノン化合物は、分子式Ｃ₃₀Ｈ₃₆Ｏ₆、分子量４９２である（図５参照）。このフラバノン化合物は、本発明者らによって沖縄産プロポリスから単離された天然物由来の有機化合物であり、エリオディクティオールと類似した構造的特徴を有しているが、６位に3''',3'''-ジメチルアリル基を備えるとともに２’位にＣ−ゲラニル基を備えている。このため、このフラバノン化合物は、エリオディクティオール、上記第１及び第２のフラバノン化合物よりも親油性（膜透過性）が高い。

This third flavanone compound has a molecular formula of C ₃₀ H ₃₆ O ₆ and a molecular weight of 492 (see FIG. 5). This flavanone compound is a natural product-derived organic compound isolated from Okinawa propolis by the present inventors, and has structural characteristics similar to Eriodictiool, but is 3 '' at the 6th position. It has a ', 3'''-dimethylallyl group and a C-geranyl group at the 2 'position. For this reason, this flavanone compound has higher lipophilicity (membrane permeability) than Eriodictiool and the first and second flavanone compounds.

  Each of the first to third flavanone compounds has a high antioxidant effect comparable to that of eriodictiool and α-tocopherol. In addition, any of these first to third flavanone compounds has an antitumor action that suppresses the growth of cancer cells such as breast cancer cells. Regarding the antitumor action, the second and third flavanone compounds are high, and the first flavanone compound has an antitumor action although it is slightly lower than both compounds. Furthermore, any of these first to third flavanone compounds removes unnecessary cells such as abnormal cells that are becoming cancerous or senescent cells that are reaching the end of their life due to physiological cell death (apoptosis) or the like. Has an effect. The first flavanone compound has an antibacterial action against gram-positive bacteria such as Staphylococcus aureus and spore bacteria such as Bacillus. The second and third flavanone compounds have antibacterial activity against gram-negative bacteria such as Escherichia coli, the gram-positive bacteria and spore bacteria.

  The antioxidant of the embodiment contains at least one selected from the first to third flavanone compounds as an active ingredient (antioxidant material). This antioxidant is used in foods and beverages as an anti-degradation agent for effectively suppressing deterioration of various products (mainly oxidative deterioration) such as oxidative degradation of fats and oils, perfume degradation, pigment degradation, and pigment fading. It can be used by adding. In addition, by using this antioxidant in health foods and other foods and drinks, it eliminates active oxygen in vivo when taken orally, enhances liver function, reduces acetaldehyde toxicity, It exerts health promoting effects such as antioxidant action of density cholesterol (LDL), growth inhibition of breast cancer cells, and improvement of immune function. In addition, this antioxidant can be used by being incorporated in cosmetics or quasi-drugs, and is useful for whitening effects such as skin and oral cavity and prevention of aging.

  The antibacterial agent of the embodiment contains at least one selected from the first to third flavanone compounds as an active ingredient. This antibacterial agent is mainly used by being added to food or drink, and prevents the food or drink from being spoiled. In addition, this antibacterial agent may be used by being contained in a pharmaceutical product, or may be used as a pharmaceutical product such as an infectious disease therapeutic agent or an antibacterial chemotherapy drug. Further, this antibacterial agent can be used by adding it to cosmetics or quasi-drugs, and helps to keep the skin, oral cavity, armpit, etc. clean. In this antibacterial agent, the active ingredient is preferably a concentration of 10 ppm or more, more preferably 15 ppm or more, still more preferably 30 to 10000 ppm, particularly preferably 50 to 50, in foods, beverages, pharmaceuticals, cosmetics or quasi drugs. It is added so as to be contained at a concentration of 1000 ppm. When the concentration of the active ingredient is less than 50 ppm, sufficient antibacterial action is not exhibited, and conversely, when it exceeds 10,000 ppm, it is uneconomical.

  The antitumor agent of the embodiment contains at least one selected from the first to third flavanone compounds as an active ingredient, and is mainly used as a pharmaceutical product. This antitumor agent is administered such that the active ingredient is exposed to cancer cells at a concentration of preferably 0.1 μM to 100 mM, more preferably 10 to 1000 μM. If the concentration of the active ingredient is less than 0.1 μM, the therapeutic effect is small, and conversely if it exceeds 100 mM, it is uneconomical. The dose of the antitumor agent is preferably such that 0.5 to 10 g, more preferably 2 to 5 g, of the active ingredient is administered per adult day. When the daily dose of this active ingredient is less than 0.5 g, the antitumor action cannot be sufficiently exerted, and conversely, when it exceeds 10 g, it is uneconomical. In the case of a dwarf, half the amount for an adult is a guide.

  On the other hand, when the active ingredient is contained in foods and drinks (health foods), cosmetics or quasi-drugs, it promotes physiological removal of unnecessary cells such as abnormal cells that are becoming cancerous, thereby preventing cancer. It can also be used as an effective product. At this time, the concentration of the active ingredient in the product is preferably 1 to 50%, more preferably about 10 to 30% in the case of the pharmaceutical product.

  The food / beverage products of embodiment contain at least 1 sort (s) chosen from the said 1st to 3rd flavanone compound, and may contain the said antioxidant or antibacterial agent. This food and drink can be utilized as a health food that exhibits various health promotion effects by eliminating active oxygen in vivo by sufficiently drawing out the antioxidant action of the flavanone compound. In addition, by sufficiently drawing out the deterioration preventing action of the flavanone compound, it is possible to prevent deterioration of food and drink, increase the storage stability, and maintain stable quality over a long period of time. Alternatively, by sufficiently drawing out the antibacterial action of the flavanone compound, it is possible to prevent rot of foods and drinks, improve the storage stability, and maintain stable quality over a long period of time. Similarly, by fully exploiting the anti-tumor action of the flavanone compound, it promotes the physiological removal of unwanted cells such as abnormal cells that are becoming cancerous and senescent cells that are reaching the end of their life, and promotes high health. It can also be used as a health food that is effective.

  In this food and drink, the daily intake of the flavanone compound is preferably 0.05 to 10 g, more preferably 0.2 to 5 g per day for an adult. If the daily intake of the active ingredient is less than 0.05 g, the antioxidant action by the active ingredient may not be exhibited effectively, and conversely if it exceeds 10 g, it is uneconomical. .

The effects exhibited by the above embodiment will be described below.
-Since the 1st to 3rd flavanone compounds of the embodiments all have an antioxidant action, an antitumor action and an antibacterial action, they are used for various types of applications including foods and drinks and pharmaceuticals. be able to. In particular, since each flavanone compound can simultaneously exhibit a plurality of types of multifaceted actions even though it is a single compound, it can be used as a multifunctional health food material typified by propolis. Is noteworthy. Each of these flavanone compounds can be used for various uses utilizing the fact that it is more lipophilic than Eriodictiool, in addition to being used for the same uses as Eriodictiool. Furthermore, since each of these flavanone compounds is contained in propolis used as a health food material, there is no problem in oral intake and transdermal administration.

  -Since the antioxidant of the embodiment contains a flavanone compound having a high antioxidant action as an active ingredient, it prevents deterioration of food, drink, cosmetics, or quasi-drugs to enhance storage stability, orally By ingestion or transdermal administration, health promotion effect and anti-aging effect can be exhibited. Since the antibacterial agent of the embodiment contains a flavanone compound having a high antibacterial action as an active ingredient, the antiseptic of foods, beverages, cosmetics or quasi-drugs is prevented to increase the storage stability, or the cosmetics or pharmacy Highly hygienic and deodorant effects can be achieved by transdermal administration as an external product, and it can also be used as a pharmaceutical product. Since the antitumor agent of the embodiment contains a flavanone compound having a high antitumor action as an active ingredient, it can exhibit a high therapeutic effect against cancer and can be expected to have a preventive effect.

  -Since the food / beverage products (beverage products or foodstuff) of embodiment contain the flavanone compound which has an antioxidant action, an antitumor action, an antibacterial action, etc., prevention of deterioration of food and drink itself, corruption prevention, health promotion effect Multifaceted and useful effects such as anti-aging, anti-aging effects, metabolic enhancement effects and cancer prevention effects can be exhibited simultaneously. Moreover, this food / beverage product is easy to raise only preservability using the deterioration prevention effect or anti-corrosion effect by setting content of a flavanone compound low.

Hereinafter, examples and comparative examples embodying the embodiment will be described.
<Isolation of compound>
An extraction operation was performed by adding 500 ml of ethanol to 50 g of the propolis raw material from Okinawa, sonicating for several minutes and stirring overnight at room temperature, and then removing the residue by filtration. The obtained extract was concentrated under reduced pressure to obtain 39.73 g of an ethanol extract. Next, the ethanol extract was fractionated into 11 fractions (1) to (11) by column chromatography under the following conditions.

Column tube: Glass column 5.0 x 45 cm
Filler: Silica gel about 590cm ³
Elution solvent: (1) Hexane: ethyl acetate = 90: 10 (350 ml)
(2) Hexane: ethyl acetate = 80: 20 (220 ml)
(3) Hexane: ethyl acetate = 70: 30 (250 ml)
(4) Hexane: ethyl acetate = 60: 40 (1000 ml)
(5) Hexane: ethyl acetate = 50: 50 (200 ml)
(6) Hexane: ethyl acetate = 40: 60 (100 ml)
(7) Hexane: ethyl acetate = 30:70 (100 ml)
(8) Hexane: ethyl acetate = 20: 80 (100 ml)
(9) Hexane: ethyl acetate = 10:90 (100 ml)
(10) Ethyl acetate (200ml)
(11) Methanol (700ml)
Next, when each fraction was analyzed under the following HPLC condition 1, it was confirmed that the fraction of (4) contained four main components.

HPLC condition 1
Column: YMC-Pack R & D ODS (4.6 × 250mm)
Solvent: A: Water (2% acetic acid), B: Acetonitrile (2% acetic acid)
Elution condition: 0-60min (gradient elution; A: B = 80: 20 → A: B = 20: 80)
Flow rate: 1ml / min
Detection: UV280nm
Next, the fraction (4) is subjected to fractionation and purification under the following HPLC condition 2, whereby compound 1 (yield 61.1 mg), compound 2 (yield 65.7 mg) and compound 3 (yield 99.99). 8 mg) was isolated. Further, the fraction (4) was used for fractionation / purification under the following HPLC condition 3 to isolate compound 4 (yield 20.0 mg).

HPLC condition 2
Column: YMC-Pack R & D ODS (20 × 250mm)
Solvent: Water (0.1% TFA): Acetonitrile (0.1% TFA) = 40:60
Flow rate: 9ml / min
Detection: UV280nm
HPLC condition 3
Column: YMC-Pack R & D ODS (20 × 250mm)
Solvent: Water (0.1% TFA): Acetonitrile (0.1% TFA) = 20:80
Flow rate: 9ml / min
Detection: UV280nm
<Identification of each compound>
For each of the compounds 1, 3 and 4, structural analysis was performed by measuring ¹ H-NMR, ¹³ C-NMR, MS, IR, UV spectrum and the like. Details are described below. The structural analysis of compound 2 was conducted in the same manner, and it was found to be 5,7,3 ′, 4′-tetrahydroxy-5′-C-geranylflavanone.

(Compound 1: Compound 1)
The physicochemical properties are shown in FIG. Molecular weight from measurement by ESI-MS is identified as 424, a hydroxyl group, the presence of carbonyl group 1680 cm ^-1 was suggested to 3420cm ^-1 from the IR spectrum. In addition, in the UV spectrum, a characteristic spectrum of flavanone or flavanol was shown, so that it was presumed to have a flavanone skeleton. ^From the integrated value of the ¹ H-NMR spectrum, 28 protons were confirmed, and in the ¹³ C-NMR spectrum, 25 signals were observed. From the DEPT spectrum, 3 methyls, 4 methylenes, 7 methines, and 11 quaternary carbons were confirmed. From these information, the molecular formula was estimated as C ₂₅ H ₂₈ O ₆ . In addition to these, NMR data such as HSQC spectrum, ¹ H- ¹ H COSY, HMBC spectrum, and CD spectrum are summarized in FIG. Based on the above results, this compound had the structure shown in Chemical Formula 1 above, and was identified as nymphaeol-B (5,7,3 ′, 4′-tetrahydroxy-2′-C-geranylflavanone). Although this compound has already been reported to be isolated from Hasunohagiri, this study is the first to be isolated from propolis.

(Compound 3: Compound 3)
The physicochemical properties are shown in FIG. Molecular weight from measurement by ESI-MS is identified as 424, a hydroxyl group, the presence of carbonyl group 1680 cm ^-1 was suggested in 3380 cm ^-1 from the IR spectrum. In addition, in the UV spectrum, a characteristic spectrum of flavanone or flavanol was shown, so that it was presumed to have a flavanone skeleton. ^From the integrated value of the ¹ H-NMR spectrum, 28 protons were confirmed, and in the ¹³ C-NMR spectrum, 25 signals were observed. From the DEPT spectrum, 3 methyls, 4 methylenes, 7 methines, and 11 quaternary carbons were confirmed. From these information, the molecular formula was estimated as C ₂₅ H ₂₈ O ₆ . In addition to these, NMR data such as HSQC spectrum, ¹ H- ¹ H COSY, HMBC spectrum, and CD spectrum are summarized in FIG. Based on the above results, this compound had the structure shown in Chemical Formula 2 above, and was identified as nymphaeol-A (5,7,3 ′, 4′-tetrahydroxy-6-C-geranylflavanone). Although this compound has already been reported to be isolated from the genus Hasunohagiri and Mizohozuki, this study is the first to be isolated from propolis.

(Compound 4: Compound 4)
The physicochemical properties are shown in FIG. Molecular weight from measurement by FAB-MS was identified as 492, a hydroxyl group, the presence of carbonyl group 1640 cm ^-1 was suggested in 3400 cm ^-1 from the IR spectrum. In addition, in the UV spectrum, a characteristic spectrum of flavanone or flavanol was shown, so that it was presumed to have a flavanone skeleton. ^From the integrated value of the ¹ H-NMR spectrum, 36 protons were confirmed, and in the ¹³ C-NMR spectrum, the presence of 30 carbons was estimated. From the DEPT spectrum, 5 methyls, 5 methylenes, 7 methylenes and 13 quaternary carbons were confirmed. From these information, the molecular formula was estimated as C ₃₀ H ₃₆ O ₆ . In addition to these, NMR data such as HSQC spectrum, ¹ H- ¹ H COSY, HMBC spectrum, and CD spectrum are summarized in FIG. From the above results, this compound has the structure shown in Chemical Formula 3 above, and nymphaeol-C (5,7,3 ', 4'-tetrahydroxy-6- (3''', 3 '''-dimetylallyl ) -2'-C-geranylflavanone). Although this compound has already been reported to be isolated from Sunflower and Macaranga tanarius, this study is the first to be isolated from propolis.

<Measurement of content in propolis>
The amount of the compounds 1 to 4 contained in Okinawa propolis was analyzed under the following HPLC condition 4 to determine the content. As a result, it was confirmed that Compound 1 contained 12.7 g, Compound 2 contained 10.5 g, Compound 3 contained 13.5 g, and Compound 4 contained 9.1 g in 100 g Okinawa propolis raw material.

HPLC condition 4
Column: YMC-Pack R & D ODS (4.6 × 250mm)
Solvent: A: Water (0.1% TFA), B: Acetonitrile (0.1% TFA)
Elution condition: 0-50min (gradient elution; A: B = 65: 35 → A: B = 0: 100)
Flow rate: 1ml / min
Detection: UV280nm
<DPPH radical scavenging activity test>
DPPH (α, α-diphenyl-β-picrylhydradil) is a purple stable radical having a maximum absorption at 517 nm, and turns into colorless hydrazine by obtaining hydrogen. Utilizing this color reaction, radical scavenging activity was measured by the following method. That is, 3 ml of a sample solution having a concentration of 25 μM was prepared by dissolving the compound 1, 3 or 4 as a sample in ethanol. Subsequently, 0.75 ml of 0.5 mM DPPH solution (solvent is ethanol) was added to each sample solution, stirred, and allowed to react in the dark for 1 hour, and then the absorbance at 517 nm was measured. On the other hand, as a comparative control, the same test was conducted using BHT (butylated hydroxytoluene), α-tocopherol or eriodictiool as a sample instead of the compound. Moreover, the test was similarly implemented using the thing which has not added the said sample as a control. The radical scavenging activity (%) was calculated by the following formula 1. The results are shown in Table 1 below (all tests were performed in triplicate, showing the mean and standard deviation).

表１より、これら３種類の化合物全てが比較対照としてのＢＨＴを上回っており、さらにα−トコフェロールとほぼ同程度の高いラジカル捕捉活性を示すことが明らかとなった。中でも化合物３は最も高い活性を示し、その活性はエリオディクティオールとほぼ同等であった。化合物１，３，４は既知物質であるが、これらの化合物のラジカル捕捉活性に関する検討は従来なされておらず、本研究において、これら３種の天然由来化合物は非常に高いラジカル捕捉活性を有していることが初めて確認された。

From Table 1, it was clarified that all of these three types of compounds exceeded BHT as a comparative control, and also showed a radical scavenging activity almost as high as α-tocopherol. Among them, compound 3 showed the highest activity, and the activity was almost equivalent to that of Eriodictiool. Compounds 1, 3, and 4 are known substances, but no studies have been made on the radical scavenging activity of these compounds. In this study, these three naturally derived compounds have very high radical scavenging activity. It was confirmed for the first time.

<Β-Carotene fading test>
This method utilizes a phenomenon in which the color of β-carotene disappears by the reaction of the linoleic acid peroxide generated with auto-oxidation of linoleic acid with the double bond of β-carotene. Antioxidant activity was measured by the method described above. That is, first, 2 ml of 200 mg / ml Tween 40 chloroform solution, 0.4 ml of 100 mg / ml chloroform solution of linoleic acid, and 3 ml of 0.1 mg / ml β-carotene chloroform solution were mixed, and then the solvent was removed using nitrogen gas. Removed. Subsequently, 100 ml of distilled water was added and stirred sufficiently to obtain an emulsion. Ethanol was added to 3 ml of this emulsion to completely dissolve the solute in the solvent, and then a reaction solution was prepared by mixing 50 μl of each 1.2 mM sample solution, and the reaction solution was incubated at 60 ° C. for 60 minutes. The absorbance at 470 nm was measured for the reaction solution before incubation (sample reaction solution of 0 minutes) and the reaction solution after incubation (sample reaction solution of 60 minutes).

  The sample solution was prepared by dissolving the compound 1, 3 or 4 as a sample in ethanol to a concentration of 1.2 mM. The sample concentration in the reaction solution was adjusted to 20 μM. On the other hand, as a control for comparison, a reaction solution using BHT, α-tocopherol or eriodictiool as a sample instead of the compound was prepared and similarly tested. Moreover, the control reaction liquid which did not add the said sample was prepared, and the test was implemented similarly. Antioxidant activity (%) was determined by the following formula 2. The results are shown in Table 1 above (all tests were performed in triplicate, showing the mean and standard deviation).

但し、上記コントロールの退色速度は、（０分のコントロール反応液の吸光度／６０分のコントロール反応液の吸光度）／６０の値の自然対数で表され、上記試料の退色速度は、（０分の試料反応液の吸光度／６０分の試料反応液の吸光度）／６０の値の自然対数で表される。その結果、表１より、化合物３は若干活性が弱いものの、３種類の化合物とも高い抗酸化活性を示し、それらの活性はＢＨＴ、α−トコフェロール及びエリオディクティオールとほぼ同等であったことが確認された。化合物１，３及び４は既知物質であったが、これらの化合物の抗酸化活性に関する検討は従来なされておらず、本研究において、これら３種類の天然由来化合物は非常に高い抗酸化活性を有していることが初めて確認された。

However, the color fading rate of the control is expressed by the natural logarithm of the value of (absorbance of the control reaction solution of 0 minutes / absorbance of the control reaction solution of 60 minutes) / 60. It is expressed by the natural logarithm of the value of absorbance of the sample reaction solution / absorbance of the sample reaction solution of 60 minutes / 60. As a result, it was found from Table 1 that although compound 3 was slightly weak in activity, all three compounds showed high antioxidant activity, and their activities were almost equivalent to BHT, α-tocopherol and eriodictiool. confirmed. Compounds 1, 3 and 4 were known substances, but no investigation has been made on the antioxidant activity of these compounds. In this study, these three naturally derived compounds have very high antioxidant activity. It was confirmed for the first time.

<Breast cancer cell growth inhibition test>
By culturing breast cancer cells (MCF-7), estradiol (17β-Estradiol) having a cell growth promoting action can be added to facilitate the growth of breast cancer cells in a short period of time. Utilizing this, the growth inhibitory effect of each compound on breast cancer cells was measured by the following method. That is, first, the compound 1, 3 or 4 as a sample was diluted with dimethyl sulfoxide to prepare a sample solution. Next, 2 × 10 ³ breast cancer cells (MCF-7) were seeded in each well of a 96-well plate, and estradiol and a sample solution were added after 4 hours. Estradiol was added at a final concentration of 0.1 nM, and the sample solution was added at a final concentration of 0.2 μM, 2 μM, or 20 μM. After a predetermined culture period has elapsed (3, 5 days), the medium is changed, and an enzyme solution (Cell counting Kit-8: Wako) is added in an amount of 10% of the medium, followed by heating in a 37 ° C. incubator for 2 hours. Then, the absorbance at a wavelength of 450 nm (reference wavelength: 630 nm) was measured with a spectrophotometer, and the number of living cells in each section was quantified.

  In addition, the culture period was divided into 5 days, after the medium was changed on the 3rd day of culture, estradiol and the sample were added and the culture was continued until the 5th day. On the 0th day, the enzyme solution was added 4 hours after seeding the cells, and after heating according to the above method, the absorbance was measured. As a comparative control, the same test was performed using eriodictiool as a sample instead of the compound. Moreover, the test was similarly carried out using estradiol and the sample to which no sample was added as control 1 and using the sample to which no sample was added as control 2. On the other hand, the quantification of the number of viable cells is expressed as the relative cell number when the absorbance on the 0th day of Control 1 is 1, and the average value of the measured values and the standard deviation is obtained by performing the test for each well in 8 wells. Was used as data. The results are shown in Table 2.

表２より、化合物１，３，４とも、効果に若干の強弱はあるものの乳癌細胞の増殖を抑制し、さらには濃度依存的に乳癌細胞の増殖抑制効果が強まる傾向が認められた。特に化合物３，４を２０μＭ添加した区分では、コントロール１よりも強い乳癌細胞増殖抑制効果（乳癌細胞に対する細胞毒性）が認められた。化合物１，３及び４は既知物質であったが、これらの化合物の癌細胞増殖抑制作用に関する検討は従来なされておらず、本研究において、これら３種の天然由来化合物は非常に高い抗腫瘍活性を有していることが初めて確認された。

From Table 2, it was recognized that although the compounds 1, 3 and 4 had some effects, they suppressed the growth of breast cancer cells, and further, the growth-inhibiting effect of breast cancer cells increased in a concentration-dependent manner. In particular, in a group to which 20 μM of compounds 3 and 4 were added, a stronger breast cancer cell proliferation inhibitory effect (cytotoxicity against breast cancer cells) than control 1 was observed. Although compounds 1, 3 and 4 were known substances, studies on the inhibitory effect of these compounds on cancer cell proliferation have not been made so far. In this study, these three naturally-derived compounds have very high antitumor activity. For the first time.

<Antimicrobial activity test for ethanol extract>
In order to evaluate antibacterial activity, E.coli (IFO3366) is used as an indicator for Gram-negative bacteria, Staphylococcus aureus (IFO15035) is used as an indicator for Gram-positive bacteria, and an indicator for spore bacteria that is resistant to heat sterilization and other processes. Antibacterial activity of the above compounds was evaluated using Bacillus cereus (IFO15305T) and Bacillus coagulans isolated from degraded canned foods as indicator bacteria for fungi causing degradation in canned foods. That is, first, the ethanol extract in the above <Isolation of Compound> was dissolved in 70% ethanol, and then the standard was prepared so that the extract concentration was 0 ppm, 12.5 ppm, 25 ppm, 50 ppm, 100 ppm or 150 ppm. An agar medium was prepared. Subsequently, these standard agar media were sterilized by autoclaving to prepare evaluation media, and each of the above-described bacteria was inoculated into these evaluation media to confirm their growth. As a result, it was confirmed that the antibacterial effect was enhanced against all the above indicator bacteria depending on the concentration of the ethanol extract. Further, it was confirmed that all the indicator bacteria were not detected when the ethanol extract concentration was 30 ppm or more, preferably 50 ppm or more. In addition, the said 70% ethanol had little influence on said each indicator microbe.

<Antimicrobial activity test for compounds 1, 3 and 4>
In order to evaluate antibacterial activity, the above-mentioned E. coli and Salmonella enteritidis (S.en; NBRC3313) are used as indicator bacteria for Gram-negative bacteria, and the above-mentioned Staphylococcus aureus (Sta.) And indicator bacteria for Spore bacteria are used as indicator bacteria for Gram-positive bacteria. The antibacterial activity of compounds 1, 3 and 4 was evaluated using the above Bacillus cereus (B.ce). That is, except that Compound 1, 3 or 4 as a sample was dissolved in 70% ethanol, and then a standard agar medium was prepared so that the sample concentration was 0 ppm, 5 ppm, 10 ppm, 15 ppm, 20 ppm or 50 ppm. In the same manner as in the above <Antimicrobial activity test on ethanol extract>, each of the above bacteria was inoculated into an evaluation medium. Subsequently, after culturing each bacterium, the number of bacteria per petri dish (CFU / petri dish) was measured. The results for compound 1 are shown in Table 3, and the results for compounds 3 and 4 are shown in Table 4.

表３より、化合物１は、上記グラム陽性菌及び芽胞菌に対し、濃度に依存して抗菌効果が高められる傾向が認められた。また、化合物１は、上記 E.coli 及び S.en に対し上記全ての濃度において抗菌効果がなかった。表３より、抗菌剤が化合物１（第１のフラバノン化合物）を有効成分として含有するときには、該抗菌剤は有効成分が上記飲食品等中に好ましくは１０ｐｐｍ以上の濃度、より好ましくは１５ｐｐｍ以上の濃度で含有されるように添加される。

From Table 3, the tendency for the antibacterial effect of Compound 1 to be enhanced depending on the concentration of the Gram-positive bacteria and spore bacteria was observed. Compound 1 had no antibacterial effect on all of the above concentrations against E. coli and S.en. From Table 3, when the antibacterial agent contains Compound 1 (first flavanone compound) as an active ingredient, the antibacterial agent preferably has a concentration of 10 ppm or more, more preferably 15 ppm or more in the food or drink. It is added so as to be contained at a concentration.

  On the other hand, from Table 4, compounds 3 and 4 have antibacterial activity against all the above indicator bacteria, and in particular have high antibacterial activity against E.coli, Sta. And B.ce. Was recognized. From Table 4, when the antibacterial agent contains Compound 3 (second flavanone compound) as an active ingredient, the antibacterial agent preferably has a concentration of 10 ppm or more, more preferably 15 ppm or more in the food or drink. It is added so as to be contained at a concentration. In addition, when the antibacterial agent contains compound 4 (third flavanone compound) as an active ingredient, the antibacterial agent preferably has a concentration of 10 ppm or more, more preferably 50 ppm or more in the food or drink. Added to contain. In addition, the said 70% ethanol had little influence on said each indicator microbe.

Further, the technical idea that can be grasped from the embodiment will be described below.
-The food-drinks of Claim 4 containing 30 ppm or more of flavanone compounds. The food / beverage product according to claim 4, comprising 30 to 10,000 ppm of a flavanone compound. When comprised in this way, the preservability of food-drinks can be improved easily.

  -Cosmetics containing at least one flavanone compound selected from nymphaeol-A, nymphaeol-B and nymphaeol-C. When comprised in this way, it becomes easy to aim at the improvement of a whitening effect, a skin care effect, or preservability. A quasi-drug containing at least one flavanone compound selected from nymphaeol-A, nymphaeol-B and nymphaeol-C. When comprised in this way, it becomes easy to aim at the improvement of a hygiene effect, a deodorizing effect, or preservability. A pharmaceutical comprising at least one flavanone compound selected from nymphaeol-A, nymphaeol-B and nymphaeol-C. When comprised in this way, useful drug effects, such as an antitumor effect and an antibacterial effect, can be exhibited easily.

The physicochemical properties of Compound 1 of the Example are summarized. The NMR data of the compound 1 of an Example are shown. The physicochemical properties of Example 3 were summarized. The NMR data of the compound 3 of an Example are shown. The physicochemical properties of Example 4 were summarized. The NMR data of the compound 4 of an Example are shown.

Claims (4)

An antioxidant containing at least one flavanone compound selected from nymphaeol-A, nymphaeol-B and nymphaeol-C. An antibacterial agent containing at least one flavanone compound selected from nymphaeol-A, nymphaeol-B and nymphaeol-C. An antitumor agent comprising at least one flavanone compound selected from nymphaeol-A, nymphaeol-B and nymphaeol-C. A food or drink containing at least one flavanone compound selected from Nimpheol-A, Nimpheol-B and Nimpheol-C.

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