WO2015163443A1 - Agent for delaying appearance of physical symptoms associated with aging - Google Patents

Abstract

　Provided is an agent for delaying the appearance of physical symptoms that develop in association with aging, such as drooping eyelids, curvature of the spine, and the like. The agent for delaying the appearance of physical symptoms associated with aging has, as an active ingredient, kale or a processed form thereof.

Description

Delayed onset of physical symptoms associated with aging

The present invention relates to an agent that delays the onset of various physical symptoms that occur with aging.

Known physical symptoms associated with aging include skin symptoms such as wrinkles and sagging, eye symptoms such as presbyopia and eyelid droop, hair symptoms such as thinning hair, and bone symptoms such as osteoporosis. Guanidine derivatives have been used as drugs for improving these symptoms (Patent Document 1).

Also, as a physical symptom that accompanies aging, there is a curvature of the spine, which is considered to be affected by normal posture.

Japanese Patent Laid-Open No. 9-202710

However, conventional drugs improve symptoms already developed and do not delay the onset of physical symptoms associated with aging.
The subject of this invention is providing the agent which delays the onset of the physical symptom which arises with aging.

Therefore, the present inventor has ingested various components using an aging-promoting mouse and has studied the action of delaying the onset of physical symptoms caused by aging. The present invention was completed by finding that the onset of physical symptoms represented by drooping eyelids and spinal curvature caused by aging is delayed.

That is, the present invention provides the following [1] to [24].

[1] An agent for delaying onset of physical symptoms associated with aging, comprising kale or a processed product thereof as an active ingredient.
[2] The onset delay agent according to [1], wherein the physical symptoms associated with aging are eye and / or bone symptoms.
[3] The onset delay agent according to [2], wherein the eye and / or bone symptom is drooping eyelids and / or spine curvature.
[4] Delayed onset of physical symptoms accompanying aging is caused by increased expression of one or more genes selected from EGF gene, Sirt2 gene and Sirt3 gene by ingesting kale or processed product thereof [ [1] The expression retarder according to any one of [3].
[5] Any one of [1] to [4], wherein the processed kale product is a processed kale product obtained by branching a kale and having a glucoraphanin content of 1 mg or more per 1 g of kale-derived solid content. The expression retarder described in 1.
[6] The expression retarder according to any one of [1] to [5], wherein the kale is Hyperl (Ministry of Agriculture, Forestry and Fisheries, Variety Registration No. 20555).
[7] Use of kale or a processed product thereof for producing an agent for delaying onset of physical symptoms associated with aging.
[8] The use according to [7], wherein the physical symptoms associated with aging are eye and / or bone symptoms.
[9] The use according to [8], wherein the eye and / or bone symptom is drooping eyelids and / or spine curvature.
[10] Delayed onset of physical symptoms accompanying aging is caused by increased expression of one or more genes selected from EGF gene, Sirt2 gene and Sirt3 gene by ingesting kale or processed product thereof [ 7] to [9].
[11] Any of [7] to [10], wherein the processed kale product is obtained by blanching kale and has a glucoraphanin content of 1 mg or more per 1 g of kale-derived solid content. Use as described in.
[12] The use according to any one of [7] to [11], wherein the kale is hyperle (Ministry of Agriculture, Forestry and Fisheries Variety Registration No. 20555).
[13] Kale or a processed product thereof for delaying the onset of physical symptoms associated with aging.
[14] The kale according to [13] or a processed product thereof, wherein the physical symptoms associated with aging are eye and / or bone symptoms.
[15] The kale according to [14] or a processed product thereof, wherein the eye and / or bone symptom is drooping eyelid and / or spine curvature.
[16] Delayed onset of physical symptoms accompanying aging is caused by increased expression of one or more genes selected from EGF gene, Sirt2 gene and Sirt3 gene by ingesting kale or processed product thereof [ [13] The kale according to any one of [15] or a processed product thereof.
[17] Any one of [13] to [16], wherein the processed kale product is obtained by blanching kale and has a glucoraphanin content of 1 mg or more per 1 g of kale-derived solid content. Or a processed product thereof.
[18] The kale according to any one of [13] to [17] or a processed product thereof, wherein the kale is hyperle (Ministry of Agriculture, Forestry and Fisheries, Variety Registration No. 20555).
[19] A method for delaying the onset of physical symptoms associated with aging, comprising administering an effective amount of kale or a processed product thereof.
[20] The method according to [19], wherein the physical symptoms associated with aging are eye and / or bone symptoms.
[21] The method of [20], wherein the eye and / or bone symptom is drooping eyelids and / or spine curvature.
[22] Delayed onset of physical symptoms accompanying aging is caused by increased expression of one or more genes selected from EGF gene, Sirt2 gene and Sirt3 gene by ingesting kale or processed product thereof [ [19] The method according to any one of [21] to [21].
[23] Any one of [19] to [22], wherein the processed kale product is obtained by blanching kale and has a glucoraphanin content of 1 mg or more per 1 g of kale-derived solid content. The method described in 1.
[24] The method according to any one of [19] to [23], wherein the kale is hyperle (Ministry of Agriculture, Forestry and Fisheries Variety Registration No. 20555).

According to the present invention, the onset of physical symptoms that occur with aging can be delayed.

It is a photograph which shows the physical symptom accompanying aging of an aging promotion mouse | mouth. The mouse | mouth which the eyelid drooping has arisen, and the mouse | mouth which the curvature of the spine has arisen are shown. It is a photograph which shows the symptom of the aging promotion mouse | mouth which administered the kale processed material. The mouse | mouth which has not produced the eyelid droop, and the mouse | mouth which has not produced the curvature of the spine are shown. It is a figure which shows the expression level of an EGF gene. It is a figure which shows the expression level of Sirt2 gene. It is a figure which shows the expression level of Sirt3 gene.

The active ingredient of the agent for retarding the onset of physical symptoms accompanying aging according to the present invention is kale or a processed product thereof.

Kale is a cruciferous plant that is a kind of cabbage but has the property of not forming a head. Kale contains glucoraphanin (sulforaphane glucosinolate), and when ingested, glucoraphanin is decomposed into sulforaphane by intestinal bacteria. Sulforaphane has been reported to inhibit carcinogenesis, induce phase II detoxification enzyme, inhibit H. pylori infection, and the like. However, no action is known to delay the onset of physical symptoms associated with aging.

There are a plurality of varieties of kale, and in the present invention, any varieties may be used, but Hyperl (Ministry of Agriculture, Forestry and Fisheries, Variety Registration No. 20555) is used from the viewpoint of the delayed action of physical symptoms accompanying aging. Is preferred. High pearl is a kale variant (Brassica oleracea L.convar. Acephala (DC.) Alef. Var. Sabellica L.). In addition, the whole plant of kale may be used, or a part thereof, such as leaves, stems, roots, flower buds, etc., may be used. From the viewpoint of the delayed action of physical symptoms accompanying aging, the stems And / or leaves are preferred.

Examples of the form of the processed kale include shredded compositions, crushed compositions, juiced compositions, and the like, but juiced compositions are preferred from the viewpoint of ease of drinking. The shredded composition, crushed composition, squeezed composition, etc. may be in the form as they are, but those pulverized by means such as hot air drying, spray drying, freeze drying and the like are preferred in terms of storage stability. In particular, spray drying is preferable as a drying method because the alteration due to heat is smaller than that of hot air drying and the drying treatment time is shorter than that of freeze drying.
Among the kale processed products, a blanched processed (heated) processed product is preferable from the viewpoint of the delayed action of physical symptoms accompanying aging. Furthermore, in terms of delayed action of physical symptoms accompanying aging, kale is obtained by blanching treatment, and a processed kale product having a glucoraphanin content of 1 mg or more per 1 g of kale-derived solid content (hereinafter referred to as this) It is preferable to use a blanched kale product.

The content of glucoraphanin per 1 g of kale-derived solid content of the blanched kale processed product is 1 mg or more, preferably 2 mg or more, more preferably 3 mg or more, 7 mg or more from the viewpoint of delayed action of physical symptoms accompanying aging. The above is more preferable. The content of glucoraphanin per gram of Kale-derived solid content of a normal kale processed product is about 0.1 mg to 0.6 mg, and the glucoraphanin content of a blanched kale processed product is higher than that of a normal kale processed product It is.
The content of glucoraphanin in the processed kale is measured by HPLC according to the method described in Examples below.

The content of sinigrin per gram of the kale-derived solid content of the blanched kale processed product is not particularly limited, but is preferably less than 1 mg from the viewpoint of the delayed action of physical symptoms accompanying aging. More preferably, it is less than 1 mg, and even more preferably less than 0.02 mg. The signiglin content per gram of the kale-derived solid content of a normal kale product is 2 mg or more, and the signiglin content of the blanched kale product is low.
The content of sinigrin in the processed kale is measured by HPLC according to the method described in Examples below.

The progoitrin content per gram of the kale-derived solid content of the blanched kale processed product is not particularly limited, but is preferably less than 0.1 mg from the viewpoint of delayed action of physical symptoms associated with aging. More preferably, it is less than 0.02 mg. The progoitrin content per 1 g of the kale-derived processed product of the normal kale processed product is 0.2 mg or more, and the progoitrin content in the blanched kale processed product is lower than that of the normal kale processed product. Progoitrin has been reported to be associated with the development of thyroid hypertrophy and goiter, and a lower content is desirable.
The progoitrin content in the processed kale is measured by HPLC according to the method described in the examples below.

The blanching process may be performed in a kale state or in a kale processed state, but is preferably performed in a kale state. The blanching conditions are preferably performed at 60 to 100 ° C. for 1 to 10 minutes from the viewpoint of glucoraphanin content.

The blanching method is not particularly limited, and examples thereof include steaming and underwater heat treatment (boiled treatment). In water heating treatment is more preferable, since it is likely to cause variation and so-called “steaming unevenness” may occur and uniform blanching may be difficult. When heating the kale in water, the amount of kale to be added to the weight of water is not particularly limited, and any ratio may be used as long as it can be uniformly heated. In the heat treatment in water, an alkaline agent such as sodium citrate, sodium ascorbate, sodium lactate, calcium hydroxide, sodium hydroxide, sodium carbonate, sodium hydrogen carbonate and the like can be added to water as a pH adjuster. Further, the pH of the water for the underwater heat treatment is not particularly limited, but decomposition of chlorophyll in the kale is promoted under strongly acidic conditions, and the color tone of the processed kale deteriorates. 9 is preferred.

It is preferable in terms of color tone that the kale after the blanching treatment is quickly cooled. Examples of the cooling means include natural cooling, a method using a refrigerant such as ice and water, and the like.

The kale or the processed kale has an excellent onset delaying action on the physical symptoms associated with aging, as shown in Examples below. Here, physical symptoms associated with aging include skin symptoms such as wrinkles and sagging, presbyopia, eye symptoms such as eyelid drooping, hair symptoms such as thinning hair, spine curvature, osteoporosis, osteoarthritis, etc. Examples include bone symptoms. Examples of the curvature of the spine include degenerative spondylosis or scoliosis. Of these, the kale or the processed kale has an excellent onset delaying action on eye and / or bone symptoms, particularly on the drooping of the eyelids and / or the curvature of the spine. The action of delaying the onset of physical symptoms accompanying aging is an action different from the action of improving the physical symptoms. That is, the onset delaying action is an action that delays the onset of the physical symptom, whereas the improving action is an action that restores a state in which the physical symptom has already developed to a state in which it does not occur. Therefore, the expression delaying action and the improving action are clearly different. It has never been known at all that kale or a processed kale has a delayed action of physical symptoms associated with aging.
In addition, kale or processed kale has an action of increasing the expression of one or more genes selected from EGF (epidermal growth factor) gene, Sirt2 (Sirtuin2) gene, and Sirt3 (Sirtuin3) gene. Since these genes are genes related to aging, it is considered that the action of delaying the expression of physical symptoms accompanying aging according to the present invention is caused by an increase in the expression of these genes.

As the method for administering the agent for delaying the onset of physical symptoms accompanying aging according to the present invention, either oral administration or parenteral administration can be used, but oral administration is preferred. In administration, the active ingredient can be mixed with a solid or liquid non-toxic pharmaceutical carrier suitable for administration methods such as oral administration, rectal administration and injection, and administered in the form of a conventional pharmaceutical preparation.
Examples of such preparations include solid agents such as tablets, granules, powders and capsules, solutions such as solutions, suspensions and emulsions, and lyophilization agents. These preparations can be prepared by conventional means on the preparation. Examples of the non-toxic pharmaceutical carrier include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin , Water, physiological saline and the like. In addition, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, isotonic agents, excipients and the like can be appropriately added as necessary.

The kale which is an active ingredient of the agent for delaying the onset of physical symptoms according to the present invention or a processed product thereof has been used conventionally and its safety has been confirmed. Although there is no strict limitation on the dosage when used as an agent for delaying the onset of symptom symptoms, the preferred dosage is 1 to 50 g per day as a solid content derived from kale, and 1 g to 50 g for a human with a body weight of 70 kg, particularly 10 g to 40 g is preferred.

EXAMPLES Hereinafter, although this invention is demonstrated concretely using an Example, this invention is not limited to these Examples.
In addition, the measurement of the glucosinolates in this Example (hereinafter sometimes referred to as GSL, specifically, glucoraphanin, progoitrin, and synigrin) was performed by the following method.
(Measurement of glucosinolates)
Specifically, GSL was quantitatively determined as follows. 1 mL (around 1000 mg) of a sample (Kale juice or a dried product thereof dissolved in water) is accurately weighed and dispensed into a Falcon tube (15 mL). Add 9 mL of 0.1% formic acid solution (mixed with 1 mL of formic acid in 80% aqueous methanol solution to a total volume of 1 L) and suspend by vortexing. The suspension was centrifuged (2,100 g, 15 minutes), and the supernatant was collected to obtain a sample.
A solid phase extraction column (Oasis ^™ WAX 1 cc Cartridge 30 mg Sorbent per Cartridge 30 μm Particle size, Waters Co., Ltd.) was activated with 1 mL of methanol, and 0.1% formic acid water (1 mL of formic acid was mixed with water to make the total volume 1 L) ) Equilibrate with 1 mL, adsorb 100 μL of the sample prepared above, and perform column washing with 1 mL of 0.1% formic acid and then 1 mL of methanol. The fraction adsorbed on the column is eluted with 1 mL of 5% ammonia solution (28% aqueous ammonia diluted with methanol), concentrated under reduced pressure with an evaporator, dried under reduced pressure in a desiccator and dried. Add 300 μL of 0.1% aqueous formic acid to the dried product, re-dissolve, transfer to a vial with a filter, and filter the sample for analysis.
Analysis is performed using HPLC-Prominence (Shimadzu Corporation) under the following conditions. The injection amount is 10 μL.
In addition, the kale origin solid content of squeezed liquid is calculated | required by measuring the mass after drying, after drying squeezed liquid and evaporating a water | moisture content.
<Analysis conditions>
Column: Develosil RPAQUEOUS AR-5 (Nomura Chemical, φ4.6 × 250mm)
Mobile phase A: 10 mM ammonium formate water (pH 3.75)
Mobile phase B: 10 mM ammonium formate in 90% (v / v) acetonitrile / water (pH 3.75)
Flow rate: 1 mL / min Column oven temperature: 40 ° C
Mobile phase gradient: min AB
0 100 0
10 100 0
12 0 100
17 0 100
17.1 100 0
30 100 0
Detector: UV detector Detector voltage: 100V
Wavelength: 226nm
Measurement time: 30 minutes In addition, the quantitative determination of GSL was performed by comparing the peak area area corresponding to GSL in the analysis data with the peak area area of the standard product GSL.

Production Example 1
The raw leaves of Kale (Hyperl: Ministry of Agriculture, Forestry and Fisheries, Variety Registration No. 20555, March sowing, harvested in November) were washed with water, heat-treated in a 98 ° C. warm bath for 2 minutes, and then cooled in the water bath. The cooled kale was pulverized with a crushing pump (Sekiguchi Seisakusho: SS-001) and then squeezed with a screw press (VETTER: DV) to obtain a squeezed liquid. This squeezed liquid was sterilized at 117 ° C. for 35 seconds using a plate heat exchanger. To this juice, dextrin was added in an amount of 1.5 parts by weight based on 1 part by weight of the kale-derived solid, and spray-dried with a spray dryer (Sanyo Co., Ltd .: SC-600) to obtain a powder sample.
The obtained powder sample was dissolved in ion-exchanged water so that its concentration was 4% w / w, and the glucosinolates were quantified by the above method. The results are shown in Table 1.

Hyperl had a glucoraphanin amount of 14.5 mg, and progoitrin and sinigrin were not detected.

Example 1 (Effect of hyperle intake on mouse appearance)
(animal)
Male SAMP8 mice (Japan SLC) were used.
All mice are individually housed at a temperature of 20-23 ° C., a humidity of 40-70%, and a light / dark cycle of 12h / 12h.

(Experimental plan)
Sixteen-week-old mice were divided into two groups of 12 mice.
The control group was bred with AIN-93M feed (Oriental Yeast Co., Ltd.), and the hyperle intake group was 1% (w / w) of the hyperl powder prepared in Production Example 1 (0.4% in terms of hyperl-derived solid content). It was raised with the AIN-93M feed contained.
Feed and water were ad libitum. There was no significant difference in feed intake between the control group and the hyperl intake group.
After 26 weeks, the appearance of the mice was evaluated. (In the control group, 3 mice died during breeding, so the appearance of 9 mice was evaluated.)

(result)
In the control group, 7 out of 9 animals (78%) had smaller eyes compared to the hyperle intake group, and symptoms of eyelid droop due to aging were confirmed (FIG. 1). In addition, the spine was curved in the mouse, and symptoms of osteopathic spondylosis were observed (FIG. 1). Of the mice in which these symptoms were confirmed, 2 were severe symptoms and 5 were moderate symptoms. On the other hand, in the hyperl intake group, 3 of 12 animals (25%) were confirmed to have eyelid droop or spondylotic spondylosis as in the control group, but in 9 of 12 animals (75%) No symptoms of eyelid drooping or osteopathic spondylosis were observed (FIG. 2). All mice with symptoms confirmed in the high pearl intake group had moderate symptoms.
From these results, it was considered that in the hyperl intake group, the onset of physical symptoms associated with aging such as eyelid drooping and spine curvature was delayed by hyperl intake. Severe symptoms refer to the symptoms shown in FIG. 1, and moderate symptoms refer to those with less spinal curvature or eyelid drooping than the symptoms shown in FIG. .

Example 2 (Gene expression analysis)
Of the control group and hyperle intake group of Example 1, 4 randomly selected animals were dissected and the skin tissue was collected.

(Extraction of Total RNA from skin)
The back of the mouse was shaved and the epidermis to the dermis were collected in 1 cm squares and cut into pieces with scissors. Homogenization was performed with polytron homogenizer PT3100 (KINEMATICA), and total RNA was extracted with TRI Reagent (Molecular Research Center).

(Preparation of sample solution)
CDNA was synthesized from the total RNA obtained above using MessageAmp ^™ II-Biotin Enhanced Kit (Life technologies) and purified using MinElute PCR Purification Kit (Qiagen).
Then, aRNA (amplified RNA) labeled with biotin was synthesized using MessageAmp ^™ II-Biotin Enhanced Kit, and aRNA was purified using RNeasy® MinElute ^™ Cleanup Kit (Qiagen).
Next, aRNA was fragmented to an appropriate chain length using MessageAmp ^™ II-Biotin Enhanced Kit, and a sample solution to be used in an automatic hybridization washing apparatus was prepared.

(Hybridization)
The sample solution is subjected to an automatic hybridization washing apparatus (Mitsubishi Rayon: AHF-200), and a skin chip of a DNA chip “Genopal (registered trademark)” (a DNA chip carrying a gene relating to skin formation and turnover) is used. Hybridization and washing were performed.

(detection)
The fluorescence signal intensity of the DNA chip was measured using a biochip reader (Yokogawa: MB-V1). The results are as shown in Tables 2 to 5 and FIGS.

From this result, the expression level of EGF gene, Sirt2 gene, Sirt3 gene in the hyperle intake group is 1.2 times or more compared to the control group, and the P value is less than 0.05. It was found that there was a statistically significant increase relative to the control group. Due to the increased expression of these genes, it is considered that in the hyperl ingestion group, delayed onset of physical symptoms accompanying aging occurs (EGF: Table 4, FIG. 3, Sirt2: Table 5, FIG. 4, Sirt3: Table 5, FIG. 5).
In the hyperle intake group, the expression of Acer2 gene (alkaline ceramide degrading enzyme) was increased (Table 4). Ceramide is a kind of skin constituent protein and has the role of keeping the skin firm. Therefore, it is considered that the decomposition of ceramide is promoted in the hyperle intake group, the skin tension is eliminated, and the possibility of symptom of eyelid drooping increases. Onset was delayed.
In Tables 2 to 5 and FIGS. 3 to 5, Control is a control group, and Sample is a hyperle intake group. The table shows the average value of the signal intensity of gene expression and the P value of t test, and the graph shows the average value of the signal intensity of gene expression.

Claims (24)

1. An agent that delays the onset of physical symptoms associated with aging, using kale or its processed product as an active ingredient.
2. 2. The onset delay agent according to claim 1, wherein the physical symptoms associated with aging are eye and / or bone symptoms.
3. 3. The expression delaying agent according to claim 2, wherein the eye and / or bone symptom is drooping eyelids and / or spine curvature.
4. The delayed onset of physical symptoms associated with aging is caused by increased expression of one or more genes selected from EGF gene, Sirt2 gene, and Sirt3 gene by ingesting kale or a processed product thereof. 4. The expression retarder according to any one of 3 above.
5. The kale processed product according to any one of claims 1 to 4, wherein the processed kale product is obtained by blanching kale and has a glucoraphanin content of 1 mg or more per 1 g of kale-derived solid content. Onset retarder.
6. The expression retarder according to any one of claims 1 to 5, wherein the kale is hyperle (Ministry of Agriculture, Forestry and Fisheries Variety Registration No. 20555).
7. Use of kale or its processed products for the production of agents that delay the onset of physical symptoms associated with aging.
8. The use according to claim 7, wherein the physical symptoms associated with aging are eye and / or bone symptoms.
9. Use according to claim 8, wherein the eye and / or bone symptom is drooping eyelids and / or spine curvature.
10. The delayed onset of physical symptoms associated with aging is caused by increased expression of one or more genes selected from EGF gene, Sirt2 gene and Sirt3 gene by ingesting kale or a processed product thereof. The use according to any one of 9 above.
11. The kale processed product according to any one of claims 7 to 10, wherein the processed kale product is a processed kale product obtained by blanching kale and having a glucoraphanin content of 1 mg or more per 1 g of the kale-derived solid content. use.
12. The use according to any one of claims 7 to 11, wherein the kale is a hyperle (Ministry of Agriculture, Forestry and Fisheries Variety Registration No. 20555).
13. Kale or a processed product to delay the onset of physical symptoms associated with aging.
14. The kale according to claim 13 or a processed product thereof, wherein the physical symptoms accompanying aging are eye and / or bone symptoms.
15. 15. The kale according to claim 14 or a processed product thereof, wherein the eye and / or bone symptom is drooping eyelids and / or spine curvature.
16. The delayed onset of physical symptoms associated with aging is caused by increased expression of one or more genes selected from the EGF gene, Sirt2 gene and Sirt3 gene by ingesting kale or a processed product thereof. 15. The kale according to any one of 15 or a processed product thereof.
17. The processed kale product is a processed kale product obtained by blanching kale and having a glucoraphanin content of 1 mg or more per 1 g of the kale-derived solid content. Kale or its processed product.
18. The kale according to any one of claims 13 to 17, or a processed product thereof, wherein the kale is hyperle (Ministry of Agriculture, Forestry and Fisheries Variety Registration No. 20555).
19. A method for delaying the onset of physical symptoms associated with aging, comprising administering an effective amount of kale or a processed product thereof.
20. The method according to claim 19, wherein the physical symptom associated with aging is an ocular and / or bone symptom.
21. 21. The method according to claim 20, wherein the eye and / or bone symptom is drooping eyelids and / or spine curvature.
22. The delayed onset of physical symptoms associated with aging is caused by increased expression of one or more genes selected from EGF gene, Sirt2 gene and Sirt3 gene by ingesting kale or a processed product thereof. 22. The method according to any one of 21.
23. The processed kale product according to any one of claims 19 to 22, wherein the processed kale product is obtained by blanching kale and has a glucoraphanin content of 1 mg or more per 1 g of kale-derived solid content. Method.
24. The method according to any one of claims 19 to 23, wherein the kale is hyperle (Ministry of Agriculture, Forestry and Fisheries Variety Registration No. 20555).

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