JP7592336B2 - Flavonoid Composition - Google Patents

Description

The present invention relates to an eye aging prevention agent and an eye aging prevention supplement that use myricetin and myricetin glycoside.

Age-related eye diseases and decreased vision significantly impair the quality of life of people with aging eyes. Age-related macular degeneration, a representative eye disease, is an incurable disease of unknown cause and is considered to be one of the major causes of blindness worldwide. The pathology is a functional abnormality of the retinal pigment epithelium cells, and the macular part of the retina degenerates, causing symptoms such as blurred or distorted vision in the central part of the visual field, loss of central vision, and darkness.
Since the abnormal function of retinal pigment epithelial cells is caused by aging, age-related macular degeneration is often seen in elderly people. There are still many unknowns about the mechanism of retinal pigment epithelial cell aging, and with current medical technology, there is no treatment other than slowing down the progression of the disease for patients who have already developed it, and once it has developed, there is no fundamental cure. Therefore, preventing the onset of age-related macular degeneration, that is, prevention, is important.
Age-related macular degeneration is also said to be an eye disease caused by ultraviolet light causing cell death of retinal pigment epithelial cells or promoting a decrease in retinal thickness. It is believed that certain wavelengths of ultraviolet light damage the lens and retina of the eye, resulting in eye diseases and aging of the eye.

Many drugs and treatments have been proposed for age-related macular degeneration. For example, Patent Document 1 describes that a compound having an isoquinoline skeleton inhibits spermidine-induced retinal pigment epithelial cell death, and proposes a retinal pigment epithelial cell protective agent containing the compound as an active ingredient. Furthermore, Patent Document 2 describes that S-allyl-L-cysteine has a function of inhibiting oxidation of a substance that accumulates in retinal pigment epithelial cells and is oxidized by light to damage the retinal pigment epithelial cells, and proposes a composition for preventing or treating eye diseases containing S-allyl-L-cysteine as an active ingredient.
On the other hand, because prevention is important for age-related macular degeneration, there is a demand for preventive agents that can be easily taken from a healthy state before the onset of unpleasant symptoms.

JP 2015-229671 A Special Publication No. 2015-528825

The present invention has been made in consideration of the above-mentioned circumstances, and aims to provide an anti-aging agent for the eyes that can prevent or suppress cell death of retinal pigment epithelial cells and reduction in retinal thickness due to aging, and can prevent, suppress or improve unpleasant conditions in the eyes.

As a result of extensive research to solve the above problems, the inventors discovered that myricetin and its glycosides have the effect of preventing, suppressing, or improving cell death of retinal pigment epithelial cells and reduction in retinal thickness caused by aging, and thus completed the present invention.

Specifically, the present invention relates to the following items.
1. An eye anti-aging agent comprising one or more active ingredients selected from myricetin and myricetin glycosides.
2. The eye anti-aging agent according to 1., characterized in that it contains myricetin and/or myricitrin as an active ingredient.
3. A supplement for preventing eye aging, comprising the eye aging preventive agent according to 1. or 2.

The eye aging prevention agent containing one or more selected from the myricetin and myricetin glycosides of the present invention as active ingredients can prevent or suppress retinal pigment epithelial cell death and retinal thickness reduction, and therefore exerts an effect of preventing eye diseases such as age-related macular degeneration. In addition, the eye aging prevention supplement of the present invention can prevent, suppress or improve subjective or objective eye discomfort caused by retinal pigment epithelial cell death and retinal thickness reduction due to ultraviolet rays passing through the cornea or crystalline lens.
In particular, in recent years, not only eye aging due to aging but also ultraviolet ray-induced eye damage caused by staring at the screens of devices that generate ultraviolet rays such as personal computers, smartphones, game consoles, etc. for a long time has become a problem. The supplement for preventing eye aging of the present invention can prevent, suppress, or improve subjective or objective eye discomfort caused by retinal pigment epithelial cell death and retinal thickness reduction due to ultraviolet rays that have passed through the cornea or lens.
On the other hand, myricetin and its glycoside, which are the active ingredients of the present invention, are recognized as food additives, are safe for humans, and are unlikely to cause any adverse effects even if taken for a long period of time.Therefore, they are useful as supplements that can be taken in daily life by healthy people, as well as by the elderly and young people regardless of their age.
Until now, it has not been known that myricetin or its glycosides are useful for preventing or suppressing human retinal pigment epithelial cell death or reduction in retinal thickness, and the present inventors are the first to discover this effect of myricetin and its glycosides, which is a particularly remarkable effect.

FIG. 1 is a diagram showing preliminary test results regarding the investigation of UV-B irradiation amount. FIG. 1 shows the results of a test (UV-B) to confirm the effect of inhibiting cell death in human retinal pigment epithelial cells. FIG. 1 is a diagram showing preliminary test results regarding the investigation of UV-A irradiation amount. FIG. 1 shows the results of a test (UV-A) to confirm the effect of inhibiting cell death in human retinal pigment epithelial cells.

The eye aging preventive agent and eye aging preventive supplement of the present invention will be described in detail below. In this specification, the term "eye" refers to the entire sensory organ that receives light.
In addition, in the present invention, "aging of the eye" refers to not only changes and functional decline of the entire light-receiving sensory organs with age, but also damage to retinal pigment epithelial cells caused by ultraviolet light regardless of age, so-called ultraviolet-induced aging of eye cells. As the regeneration and replenishment of cells decreases with age and frequency of exposure to ultraviolet light, abnormalities in visual function and a decrease in ability to maintain visual function occur. In particular, since optic nerve cells cannot regenerate, aging-related phenomena are prominent. Among "aging of the eye" in the present invention, cell death of retinal pigment epithelial cells and a decrease in retinal thickness occur prominently with aging of eye cells, and are one of the problems to be solved.
Ultraviolet rays are divided into three types according to wavelength: UV-A (ultraviolet A rays), UV-B (ultraviolet B rays), and UV-C (ultraviolet C rays), but it is known that UV-C is absorbed by the ozone layer and does not reach the earth's surface, and it is UV-A and UV-B that cause adverse effects on humans. Ultraviolet rays in this invention refer to UV-A and UV-B.

The eye aging prevention agent and eye aging prevention supplement of the present invention contain one or more selected from myricetin and myricetin glycosides as active ingredients.
Myricetin is a flavonoid found in fruits such as grapes and berries, vegetables, bayberry, wine, ginkgo leaves, tea, etc., and is a type of natural flavonol having the following chemical structure (A).
Myricetin is known to have a strong antioxidant effect, and is also reported to have various effects such as anti-cancer, prevention and treatment of Alzheimer's disease, anti-inflammation, and prevention and treatment of diabetes.
In the present invention, myricetin glycoside refers to a compound in which myricetin is bound to a sugar such as galactose, glucose, xylose, arabinose, rhamnose, fructose, mannose, or apiose in the form of a monosaccharide, or an oligosaccharide in which two or more sugars are bound in any combination, such as rutinose, or a compound in which an amino sugar having a nitrogen atom in part of the sugar molecule or a sugar acid such as glucuronic acid having an acid structure is bound.
In addition, as an active ingredient in the present invention, derivatives in which fatty acids, phosphoric acids, organic acids, polyphenols such as gallic acid, amino acids, etc. are bonded to myricetin or myricitrin via ester bonds, etc. are also useful.
As the myricetin glycoside in the present invention, myricetin 3-O-galactoside, myricetin 3-O-glucoside, myricetin 3-O-xylo-pyranoside, myricetin 3-O-arabino-pyranoside, myricetin 3-O-arabino-furanoside, myricetin 3-O-rhamnoside (myricitrin) are preferred, and among them, myricetin 3-O-rhamnoside (myricitrin) is more preferred.

Myricitrin is a rhamnose glycoside of myricetin and is a compound having the following chemical structure (B).
Myricitrin is contained in the bark, fruit, and leaves of Bayberry, and is extracted and purified from Bayberry bark and sold commercially as "Bayberry extract." It is listed as a food additive in the Japanese Food Additives Standards under the name "Bayberry extract," which contains myricitrin with a purity of 95.0 to 105.0% on an anhydrous basis.
The myricitrin in the present invention can be obtained by extracting Bayberry bark with water or a water-soluble solvent (e.g., ethanol, etc.) or an acidic/alkaline water-soluble solvent under a heated state from room temperature to about 100° C., and purifying the extracted extract. In addition, the myricitrin in the present invention may be a "bayberry extract" that is commercially available as a food additive.
Myricetin in the present invention can also be obtained by hydrolyzing myricitrin, which is a glycoside of myricetin, with hydrochloric acid or the like.

As will be described in detail in the examples below, it has been revealed that myricetin and its glycosides of the present invention have the effect of suppressing cell death of retinal pigment epithelial cells caused by ultraviolet rays UV-A and UV-B. On the other hand, ampelopsin (dihydromyricetin), which differs from myricetin only in that the 4-chromenone skeleton is a 4-chromanone skeleton, and taxifolin, in which one of the hydroxyl groups on the benzene ring of ampelopsin is replaced with hydrogen, have strong antioxidant power such as active oxygen scavenging ability, but no effect of suppressing cell death of retinal pigment epithelial cells caused by ultraviolet rays was observed. This difference in the inhibitory effect on cell death of retinal pigment epithelial cells between myricetin and ampelopsin (dihydromyricetin) or taxifolin due to slight differences in chemical structure has been revealed for the first time, and the inhibitory effect on cell death of retinal pigment epithelial cells caused by ultraviolet rays is a very interesting result that is not linked to the strength of antioxidant power.
In addition, as a reference example of the present invention, it was confirmed that blueberry extract, like myricetin and its glycosides, exhibits the effect of suppressing cell death of retinal pigment epithelial cells caused by ultraviolet rays UV-A and UV-B. Furthermore, it was confirmed that luteolinidin exhibits the effect of suppressing cell death of retinal pigment epithelial cells caused by ultraviolet rays UV-A.

The cell death of retinal pigment epithelial cells and the decrease in retinal thickness, which are one of the "aging of the eyes" in the present invention, cause an unpleasant state of impaired visual function, although it cannot be said to be a disease. The unpleasant state of impaired visual function includes eye fatigue due to aging, eye strain due to ultraviolet rays, etc., impaired depth perception, narrowing of the visible range, objects appearing dark, slow pupil reaction to changes in light, etc. If the cell death of retinal pigment epithelial cells and the decrease in retinal thickness worsen, they can also cause eye diseases such as glaucoma, age-related macular degeneration, retinal detachment, and blindness.
The eye aging prevention agent and eye aging prevention supplement of the present invention inhibit such cell death of retinal pigment epithelial cells and reduction in retinal thickness, and therefore exhibit the remarkable effect of not only preventing or improving the unpleasant state of impaired visual function caused by cell death of retinal pigment epithelial cells and reduction in retinal thickness as described above, but also preventing eye diseases such as glaucoma, age-related macular degeneration, retinal detachment, and sudden blindness.
Cell death of retinal pigment epithelial cells and reduction in retinal thickness can be induced by light stimuli such as ultraviolet light, and the eye aging prevention agent and eye aging prevention supplement of the present invention can suppress cell death of retinal pigment epithelial cells and reduction in retinal thickness caused by ultraviolet light.

The eye aging preventive agent of the present invention can exert its effect by administering or ingesting it to mammals including humans.For example, administration or ingestion of the eye aging preventive agent of the present invention can suppress cell death of retinal pigment epithelial cells caused by light stimuli such as ultraviolet rays.In addition, administration or ingestion of the eye aging preventive agent of the present invention can suppress the decrease in retinal thickness caused by light stimuli such as ultraviolet rays, and maintain an appropriate retinal thickness.
The eye aging prevention agent of the present invention is effective not only for humans but also for livestock such as cows, horses, sheep, and pigs, and further for pets such as dogs and cats.

The eye aging preventive agent of the present invention can be provided in the form of medicine, quasi-drug, food, feed (including pet food), etc. That is, the eye aging preventive agent of the present invention includes a pharmaceutical composition. The eye aging preventive agent of the present invention can be administered orally or by eye drops, but is preferably administered orally. Examples of dosage forms suitable for oral administration include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, and suspensions. These preparations can be formulated using pharmaceutical formulation assistants by methods commonly used in the field. Examples of pharmaceutical formulation assistants include excipients, binders, diluents, additives, flavors, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, and preservatives.
Furthermore, the eye aging preventive agent of the present invention includes a food composition, that is, a food or drink. The food composition can be used to prevent, suppress or improve unpleasant conditions of the eyes. The type of food or drink to be targeted is not particularly limited as long as the effect of the active ingredient of the eye aging preventive agent of the present invention is not inhibited. In addition, the food composition includes a supplement, and the formulation form of the supplement is not limited, and may also include other functional ingredients, such as xanthophylls such as lutein and zeaxanthin, carotenoids such as β-carotene, anthocyanins with cyanin and delphinidin as aglycons, green leaves containing chlorophyll, polyphenols such as gallic acid and catechin, flavonoids such as quercetin and luteolin, and oils and fats containing fatty acids such as oleic acid and linolenic acid as constituents. The eye aging preventive agent of the present invention is preferably in the form of a supplement.

The intake amount of the eye aging prevention supplement of the present invention is usually 1 to 1000 mg, preferably 5 to 600 mg, more preferably 20 to 250 mg of the active ingredient myricetin or its glycoside per person per day, and the intake can be increased or decreased as appropriate. It is usually suitable to continue for one day or more, preferably one week or more, more preferably one month or more.
The eye aging prevention supplement of the present invention is useful because it can prevent, suppress or improve subjective or objective eye discomfort over a period of time from the start of intake.

The present invention will be explained in more detail below with examples of preparation of the eye aging prevention agent of the present invention and evaluation of inhibition of cell death of retinal pigment epithelial cells by ultraviolet rays UV-B and UV-A, but the present invention is not limited to these examples.

<Adjustment of myricitrin>
1,000 mL of water was added to chipped bayberry bark (100 g), and extraction was performed for 1 hour with gentle stirring at 50°C, followed by solid-liquid separation by filtration to remove the water-soluble fraction. 500 mL of ethanol was added to the remaining bayberry bark, and extraction was performed at 70°C, and solid-liquid separation was performed by filtration while the extract was hot. The extract was concentrated, the ethanol was distilled off, and water was replaced, followed by addition of water. 200 mL of the aqueous extract was left to stand at a temperature of 20°C or less for 10 hours or more, and the insoluble precipitate was filtered off to obtain myricitrin (7.6 g, purity 98.7% (anhydrous equivalent)).

<Preliminary test: Consideration of UV-B irradiation amount>
A preliminary test was carried out to determine the amount of ultraviolet light to be irradiated onto retinal pigment epithelial cells.
Details of the cells and media used are as follows.
(1) Cells and culture medium used Cells: Human retinal pigment epithelial cell line (ARPE-19) (ATCC CRL-2302)
Growth medium: DMEM: F-12/10% FBS/1% PS
Test medium: DMEM: F-12/1% PS
(2) Preliminary test method The cells were seeded in a 96-well plate using a growth medium (10,000 cells/well/0.1 mL) and cultured in a carbon dioxide incubator (5% CO ₂ , 37°C) for one day. The medium was replaced with a new test medium and cultured for another 6 hours. Next, using a UV-B irradiation device, UV-B with a UV-B intensity of about 1500 μW/cm ² (intensity measured using an ultraviolet intensity meter) was irradiated for 0 seconds, 40 seconds, 80 seconds, 120 seconds, 160 seconds, 200 seconds, and 1200 seconds (UV-B irradiation amount = 0, 60, 120, 180, 240, 300, 1800 mJ). After culturing for 24 hours after irradiation, the test medium was replaced with a test medium containing 10% viable cell count measurement reagent (SF, manufactured by Nacalai Tesque) and cultured in a carbon dioxide incubator. The absorbance (measurement wavelength: 450 nm, reference wavelength: 630 nm) was measured with a plate reader after 30 minutes and 90 minutes, and the change in absorbance over 60 minutes was calculated to determine the relative cell viability (%). The test was performed six times, and the average absorbance was used.
(3) Preliminary Test Result Data The preliminary test results are shown in Table 1 below and FIG.

（４）予備試験結果からの結論
表１および図１の結果より、ＵＶ－Ｂ照射量０～１２０ｍＪの範囲では、ＵＶ－Ｂ照射量に比例して細胞生存率（％）が低下したが、ＵＶ－Ｂ照射量１８０ｍＪ以上では、細胞生存率（％）の減少率が鈍化することが確認された。この結果より、ヒト網膜色素上皮細胞の細胞死抑制確認試験におけるＵＶ－Ｂ照射量は、１８０ｍＪで実施することとした。

(4) Conclusions from preliminary test results From the results in Table 1 and Figure 1, it was confirmed that in the UV-B irradiation dose range of 0 to 120 mJ, the cell viability (%) decreased in proportion to the UV-B irradiation dose, but the rate of decrease in cell viability (%) slowed down at UV-B irradiation doses of 180 mJ or more. Based on these results, it was decided to use a UV-B irradiation dose of 180 mJ in the test to confirm cell death inhibition in human retinal pigment epithelial cells.

<Test to confirm the effect of inhibiting cell death in human retinal pigment epithelial cells: UV-B>
(1) Test Samples The following substances were used as test samples in the test to confirm the effect of inhibiting cell death in human retinal pigment epithelial cells.
Myricitrin: Uses the above-mentioned Bayberry bark extract Myricetin (a reagent manufactured by Tokyo Chemical Industry Co., Ltd.)
Taxifolin (Tokyo Chemical Industry Co., Ltd.)
Ampelopsin (reagent manufactured by Tokyo Chemical Industry Co., Ltd.)
Blueberry extract (Functional food ingredient grade)
(2) Test method The same human retinal pigment epithelial cells as those in the above preliminary test were seeded in a 96-well plate using a growth medium (10,000 cells/well/0.1 mL) and cultured in a carbon dioxide incubator (5% CO ₂ , 37°C) for one day. The above test specimen was dissolved in the test medium to a concentration of 1 mg/mL, filtered and sterilized using a 0.22 μm PVDF (polyvinylidene fluoride) filter, diluted with the test medium (3 concentrations = 0.001, 0.01, 0.1 mg/mL) and cultured for 6 hours after medium replacement to perform test specimen pretreatment. The cells not subjected to test specimen pretreatment were also cultured in the same manner after medium replacement with the test medium. Next, using a UV-B irradiation device, UV-B with a UV-B intensity of about 1500 μW/cm ² (intensity measured using an ultraviolet intensity meter) was irradiated for 120 seconds (UV-B irradiation amount: 180 mJ). The UV-B non-irradiation group was irradiated with UV-B for 0 seconds.
After 24 hours of incubation after irradiation, the test medium was replaced with a test medium containing 10% viable cell count measurement reagent (SF), and the medium was incubated in a carbon dioxide incubator. After 30 and 90 minutes, the absorbance (measurement wavelength: 450 nm, reference wavelength: 630 nm) was measured with a plate reader, and the change in absorbance over 60 minutes was calculated to measure the relative cell viability (%). The test was performed five times, and the average absorbance was used.
It was confirmed in a separate test that human retinal pigment epithelial cells did not undergo cell death in a medium containing the above test samples (three concentrations).

(3) Results of the Cell Death Inhibitory Effect Confirmation Test (UV-B) The results of the cell death inhibitory effect confirmation test are shown in Table 2 below and FIG.
The difference between the non-UV-B irradiated group and the UV-B irradiated group was confirmed to be significant by Student's t-test, which is shown in Figure 2 ("##": p<0.01). The difference between the non-test sample added group and the test sample added group in the UV-B irradiated group was shown in Table 2 and Figure 2, with p<0.05 marked as "*" and p<0.01 marked as "**" using Dunnett's test.

(4) Results of the cell death inhibition effect confirmation test (UV-B) From the results in Table 2 and Figure 2, it was revealed that the cell viability of human retinal pigment epithelial cells treated with 0.1 mg/mL myricetin and its glycoside myricitrin 0.1 mg/mL can be significantly improved by ultraviolet UV-B. In particular, it was confirmed that myricitrin showed the highest improvement in the cell viability of human retinal pigment epithelial cells.
On the other hand, no significant difference was observed between ampelopsin (dihydromyricetin) and taxifolin, whose chemical structure is very similar to myricetin, and the test sample non-added group, and it was confirmed that they cannot suppress the cell death of human retinal pigment epithelial cells caused by ultraviolet UV-B. As described above, ampelopsin (dihydromyricetin) and myricetin are compounds that differ only in that they have a 4-chromanone skeleton and a 4-chromenone skeleton, and taxifolin is a compound in which one of the hydroxyl groups on the benzene ring of ampelopsin is replaced with hydrogen, but the above test revealed that there is a large difference in the inhibitory effect on the cell death of human retinal pigment epithelial cells caused by ultraviolet light.
Furthermore, although this is a reference example of the present invention, it was confirmed that even in the group treated with 0.1 mg/mL of blueberry extract, the cell survival rate of human retinal pigment epithelial cells induced by ultraviolet UV-B was significantly improved, and cell death of retinal pigment epithelial cells and reduction in retinal thickness due to aging could be prevented or suppressed, thereby preventing, suppressing or ameliorating unpleasant eye conditions.

<Preliminary test: Consideration of UV-A irradiation amount>
A preliminary test was carried out to determine the amount of ultraviolet light to be irradiated onto retinal pigment epithelial cells.
The cells and medium used were the same as those used in the above "Study on UV-B irradiation dose."
(1) Preliminary test method Cells were seeded in a 96-well plate using growth medium (10,000 cells/well/0.1 mL) and cultured in a carbon dioxide incubator (5% CO ₂ , 37°C) for one day. The growth medium was replaced with new medium and cultured for 6 hours. After that, each well was replaced with DPBS (Dulbecco's Phosphate-Buffered Saline), and then UV-A with a UV-A intensity of about 30 mW/cm ² /sec (intensity measured using a UV intensity meter) was irradiated using a UV-A irradiation device for 0 minutes, 3 minutes, 6 minutes, 9 minutes, 12 minutes, 15 minutes, 18 minutes, and 21 minutes (cumulative UV-A irradiation amount = 0, 5.4, 10.8, 16.2, 21.6, 27.0, 32.4, 37.8 J). After irradiation, the medium was replaced with 100 μL of growth medium, and the cells were cultured in a carbon dioxide incubator for 24 hours.
The growth medium was replaced with 10% viable cell count reagent (SF) and cultured in a carbon dioxide incubator. After 30 and 90 minutes, the absorbance (measurement wavelength: 450 nm, reference wavelength: 630 nm) was measured with a plate reader, and the relative cell viability (%) was measured by calculating the change in absorbance over 60 minutes. The test was performed six times, and the average absorbance was used.
(2) Preliminary Test Result Data The preliminary test results are shown in Table 3 below and FIG.

（３）予備試験結果からの結論
表３および図３の結果より、ＵＶ－Ａ照射量０～１６．２Ｊの範囲では、ＵＶ－Ａ照射量に比例して細胞生存率（％）が低下し、ＵＶ－Ａ照射量２１．６Ｊ以上では、細胞がほぼ死滅した。この結果より、ヒト網膜色素上皮細胞の細胞死抑制確認試験におけるＵＶ－Ａ照射量は、５．４Ｊで実施することとした。

(3) Conclusions from preliminary test results From the results in Table 3 and Figure 3, in the range of UV-A irradiation doses of 0 to 16.2 J, the cell viability rate (%) decreased in proportion to the UV-A irradiation dose, and at UV-A irradiation doses of 21.6 J or more, the cells were almost completely killed. Based on these results, it was decided that the UV-A irradiation dose in the cell death inhibition confirmation test for human retinal pigment epithelial cells would be 5.4 J.

<Test to confirm the effect of inhibiting cell death in human retinal pigment epithelial cells: UV-A>
(1) Test Samples The following substances were used as test samples in the test to confirm the effect of inhibiting cell death in human retinal pigment epithelial cells.
Myricitrin: Uses the above-mentioned Bayberry bark extract Luteolinidin (Fujifilm Wako Pure Chemical Co., Ltd. reagent)
Blueberry extract (Functional food ingredient grade)
These test samples were dissolved in the same growth medium and DPBS as in the above "Preliminary test: Examination of UV-A irradiation dose" to a concentration of 1 mg/mL, sterilized by filtration using a 0.22 μm PVDF (polyvinylidene fluoride) filter, and then diluted with growth medium or DPBS to prepare three final concentrations of 0.001, 0.01, and 0.1 mg/mL.
(2) Test method The same human retinal pigment epithelial cells as those described in the above "Preliminary test: Examination of UV-A irradiation amount" were seeded in a 96-well plate using growth medium (10,000 cells/well/0.1 mL) and cultured in a carbon dioxide incubator (5% CO ₂ , 37°C) for one day. The medium was replaced with test specimen-added medium and cultured for 6 hours. Next, each well was replaced with test specimen-added DPBS, and UV-A with a UV-A intensity of about 30 mW/cm ² /sec (intensity measured using an ultraviolet intensity meter) was irradiated for 3 minutes (UV-A irradiation amount: 5.4 J) using a UV-A irradiation device. The UV-A non-irradiation group was UV-A irradiation time 0 seconds.
After irradiation, the medium was replaced with 100 μL of the medium containing the test substance, and the cells were cultured in a carbon dioxide incubator for 24 hours.
The medium was replaced with a growth medium containing 10% viable cell count reagent (SF), and the cells were grown in a carbon dioxide incubator. After 30 and 90 minutes, the absorbance (measurement wavelength: 450 nm, reference wavelength: 630 nm) was measured with a plate reader, and the change in absorbance over 60 minutes was calculated to measure the relative cell viability (%). The test was performed four times, and the average absorbance was used.
It was confirmed in a separate test that human retinal pigment epithelial cells did not undergo cell death in a medium containing the above test sample (three concentrations = 0.001, 0.01, 0.1 mg/mL).

(3) Results of the Cell Death Inhibitory Effect Confirmation Test (UV-A) The results of the cell death inhibitory effect confirmation test are shown in Table 4 below and FIG.
The difference between the non-UV-A irradiated group and the UV-A irradiated group was confirmed to be significant by Student's t-test, which is shown in Figure 4 ("##": p<0.01). The difference between the non-test sample added group and the test sample added group in the UV-A irradiated group was shown in Table 4 and Figure 4, with p<0.05 marked as "*" and p<0.01 marked as "**" using Dunnett's test.

(4) Results of the cell death inhibition effect confirmation test (UV-A) From the results in Table 4 and FIG. 4, it was revealed that the cell viability of human retinal pigment epithelial cells induced by ultraviolet light UV-A can be significantly improved in the group treated with 0.1 mg/mL myricitrin, a myricetin glycoside.
Furthermore, although this is a reference example of the present invention, it was confirmed that in both the luteolinidin 0.1 mg/mL treatment group and the blueberry extract 0.1 mg/mL treatment group, the cell survival rate of human retinal pigment epithelial cells induced by ultraviolet UV-A was significantly improved, and cell death of retinal pigment epithelial cells and reduction in retinal thickness due to aging can be prevented or suppressed, thereby preventing, suppressing or ameliorating unpleasant eye conditions.
Although UV-A has a lower energy content than UV-B, it is known that it reaches deep inside the human body, such as the retina, without being absorbed, and therefore has a large impact on ultraviolet damage in the human retina. The active ingredients of the present invention, myricetin and myricetin glycoside myricitrin, as well as luteolinidin and blueberry extract, can greatly suppress the death of retinal pigment epithelial cells and the decrease in retinal thickness caused by UV-B and UV-A on the human retina, and it has been confirmed by the above test that they contribute to preventing, suppressing or improving subjective or objective eye discomfort caused by these.

An eye anti-aging agent comprising blueberry extract as an active ingredient.
A supplement for preventing eye aging that contains blueberry extract as an active ingredient.
An agent for preventing stress on the eyes caused by ultraviolet rays UV-A, containing luteolinidin as an active ingredient.
A supplement containing luteolinidin as an active ingredient to prevent eye stress caused by UV-A ultraviolet rays.

The eye aging preventive agent and eye aging preventive supplement of the present invention can suppress cell death of retinal pigment epithelial cells and reduction in retinal thickness caused by ultraviolet rays UV-A and UV-B, and therefore exerts an effect of preventing eye diseases such as glaucoma, age-related macular degeneration, retinal detachment, blindness, etc. In addition, it is useful because it can prevent, suppress, or improve subjective or objective unpleasant conditions of the eyes.
The active ingredient of the present invention, myricetin and its glycoside, are recognized as food additives, are safe for humans, and are unlikely to cause any adverse effects even if taken for a long period of time.Therefore, they are useful as supplements that can be taken daily by healthy people, as well as by the elderly and young people, regardless of their age.

Claims (4)

An agent for preventing damage to retinal pigment epithelial cells caused by ultraviolet rays, containing myricetin as an active ingredient. An agent for inhibiting ultraviolet ray-induced retinal pigment epithelial cell death, containing myricetin as an active ingredient. A supplement for preventing eye diseases caused by damage to retinal pigment epithelial cells due to ultraviolet rays, comprising the preventive agent for preventing damage to retinal pigment epithelial cells caused by ultraviolet rays as described in claim 1. A supplement for preventing eye diseases caused by retinal pigment epithelial cell death induced by ultraviolet rays, comprising the ultraviolet ray-induced retinal pigment epithelial cell death inhibitor described in claim 2.