JP7526576B2 - New gamma ray irradiated material - Google Patents

Description

This technology relates to novel gamma ray irradiated products and compositions containing the same.

Hyaluronic acid is a type of polysaccharide that maintains affinity for body tissues, classified as glycosaminoglycan, and is an acidic polysaccharide in which N-acetylglucosamine and D-glucuronic acid (-3) GlcNAcβ (1-4) GlcAβ (1-) are linked in a linear chain. This hyaluronic acid is widely present in connective tissues such as the vitreous body of the eye, the umbilical cord, the skin, and synovial fluid, and is also involved in vascular permeability. In particular, hyaluronic acid is abundant in the skin, where it plays an important role as an extracellular matrix. For this reason, hyaluronic acid is deeply involved in the firmness, elasticity, and moisture of the skin, and if the production of hyaluronic acid decreases due to aging or sunburn, it can lead to symptoms such as dry skin, rough skin, age spots, and wrinkles.

For example, Patent Document 1 proposes a composition for promoting hyaluronic acid production, which contains a levan composition as an active ingredient. Levan is a polysaccharide contained in the roots of plants of the Gramineae and Liliaceae families, and is a homopolysaccharide composed only of D-fructofuranose residues.
For example, Patent Document 2 proposes a composition for promoting hyaluronic acid production that contains hinesol as an active ingredient. Hinesol is an essential oil component (sesquiterpenoid) contained in plants, and is known to have an anticholinergic effect.
For example, Patent Document 3 proposes a composition for promoting hyaluronic acid production, the active ingredient of which is a lactic acid bacteria fermentation product obtained by fermenting tomato juice with lactic acid bacteria.

JP 2019-006725 A JP 2018-52843 A JP 2017-128537 A JP 2014-25035 A

The main purpose of this technology is to provide a new material that promotes hyaluronic acid production.

The present inventors have focused on a polysaccharide derived from unicellular green algae of the genus Parachlorella and have investigated the hyaluronic acid production promoting effect of this polysaccharide.
The polysaccharide obtained by culturing the unicellular green algae Parachlorella had no effect of promoting hyaluronic acid production, and the acid hydrolysate of the polysaccharide did not have any effect of promoting hyaluronic acid production.

However, as a result of further intensive research, the present inventors have newly discovered that a novel gamma-ray irradiated polysaccharide derived from unicellular green algae of the genus Parachlorella can be obtained by irradiating the polysaccharide derived from unicellular green algae of the genus Parachlorella with gamma rays, and that the gamma-ray irradiated polysaccharide exhibits a hyaluronic acid production promoting effect, thus completing the present invention. That is, the present invention is as follows.

The present technology provides a polysaccharide derived from unicellular green algae of the genus Parachlorella that has been gamma irradiated.
The present technology provides a composition comprising a polysaccharide derived from unicellular green algae of the genus Parachlorella that has been gamma irradiated.
The polysaccharide that has been irradiated with gamma rays may have a molecular weight distribution with a weight average molecular weight of less than 10,000, and the proportion of this distribution region may be 50% or more.
The gamma ray irradiated polysaccharide may have at least a molecular weight distribution of 1,000 or more but less than 10,000 and a molecular weight distribution of less than 1,000, with the proportions of the respective distribution regions being 45 to 65% and 10 to 30%.
The gamma ray irradiation may be performed at an irradiation intensity of 1 to 1000 kGy.
The polysaccharide derived from the unicellular green algae of the genus Parachlorella may be a polysaccharide in which the sugar residues in the basic structure of the polysaccharide are composed of at least galactose and mannose, and furanose type is present in the galactose.The unicellular green algae of the genus Parachlorella may be Parachlorella kesseri.
The composition may be a composition for promoting hyaluronic acid production or an external preparation for skin.

This technology can provide a new material that has the effect of promoting hyaluronic acid production. Note that the effects described here are not necessarily limited to those described herein, and may be any of the effects described in this specification.

FIG. 1 shows the results of GPC analysis of polysaccharides derived from unicellular green algae of the genus Parachlorella that were irradiated with gamma rays.

The following describes a preferred embodiment for implementing the present technology. Note that the embodiment described below is an example of a representative embodiment of the present technology, and is not intended to narrow the scope of the present technology. Note that percentages in this specification are expressed by mass unless otherwise specified. Furthermore, the upper and lower limits of each numerical range can be combined in any desired way.

<1. Gamma-ray irradiated polysaccharides derived from Parachlorella unicellular green algae in this technology>
The polysaccharide derived from unicellular green algae of the genus Parachlorella in the present technology is hereinafter also referred to as "Parachlorella polysaccharide of the present technology."
The gamma ray irradiated polysaccharide derived from unicellular green algae of the genus Parachlorella in the present technology is also referred to as the "gamma ray irradiated product of the present technology."

The gamma-ray irradiated polysaccharides derived from Parachlorella unicellular green algae in the present technology are processed using edible unicellular green algae. For this reason, the gamma-ray irradiated polysaccharides of the present technology are highly safe and highly useful, even when administered continuously for a long period of time, as there is little need to worry about side effects. Furthermore, they are highly safe when used in combination with other ingredients or other compositions.
The form of the processed product of the present technology may be liquid, fluid, solid, powder, etc., but is not particularly limited.

In the present technology, the gamma-ray irradiated product of the Parachlorella polysaccharide has a molecular weight distribution of 10,000 or more in weight-average molecular weight in GPC analysis (pullulan standard), and the proportion of the distribution region is not particularly limited, but the preferred lower limit is preferably 1% or more, more preferably 5% or more, and even more preferably 10% or more, which is suitable from the viewpoint of protecting the skin surface with the polymeric polysaccharide. The preferred upper limit is preferably 50% or less, more preferably 20% or less, even more preferably 10% or less, and even more preferably 5% or less, which is suitable from the viewpoint of penetration action and hyaluronic acid production promotion action.

In the present technology, the gamma ray irradiated product of the Parachlorella polysaccharide has a molecular weight distribution of less than 10,000 weight average molecular weight in GPC analysis (pullulan standard), and although the proportion of this distribution region is not particularly limited, the preferred lower limit is preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, even more preferably 75% or more, even more preferably 80% or more, and the preferred upper limit is preferably 95% or less, more preferably 90% or less, even more preferably 85% or less. This makes it easier for the product to exhibit skin penetration effects and hyaluronic acid production promotion effects, and the efficacy of the present technology can be demonstrated.

In addition, the gamma ray irradiated product of the Parachlorella polysaccharide in this technology has a molecular weight distribution with a weight average molecular weight of less than 3,000 in GPC analysis (standard pullulan), and although the proportion of this distribution region is not particularly limited, the preferred lower limit is preferably 20% or more, more preferably 30% or more, even more preferably 40% or more, and even more preferably 45% or more, and the preferred upper limit is preferably 70% or less, more preferably 65% or less, and even more preferably 60% or less.

In addition, the gamma ray irradiated product of the Parachlorella polysaccharide in this technology has a molecular weight distribution with a weight average molecular weight of less than 1,000 in GPC analysis (standard pullulan), and although the proportion of this distribution region is not particularly limited, the preferred lower limit is preferably 5% or more, more preferably 10% or more, even more preferably 15% or more, and even more preferably 20% or more, and the preferred upper limit is preferably 40% or less, more preferably 35% or less, and even more preferably 30% or less.

In addition, the gamma ray irradiated product of the Parachlorella polysaccharide in this technology has a molecular weight distribution with a weight average molecular weight of less than 500 in GPC analysis (standard pullulan), and although the proportion of this distribution region is not particularly limited, the preferred lower limit is preferably 1% or more, more preferably 3% or more, even more preferably 5% or more, and even more preferably 8% or more, and the preferred upper limit is preferably 25% or less, more preferably 20% or less, and even more preferably 15% or less.

In addition, the gamma-ray irradiated product of the Parachlorella polysaccharide in this technology has a molecular weight distribution of 3,000 or more and less than 10,000 weight average molecular weight in GPC analysis (standard pullulan), and the proportion of this distribution region is not particularly limited, but the preferred lower limit is preferably 5% or more, more preferably 10% or more, even more preferably 15% or more, and even more preferably 20% or more, and the preferred upper limit is preferably 45% or less, more preferably 40% or less, and even more preferably 35% or less.

In addition, the gamma-ray irradiated product of the Parachlorella polysaccharide in this technology has a molecular weight distribution of a weight average molecular weight of 1,000 or more and less than 3,000 in GPC analysis (standard pullulan), and although the proportion of this distribution region is not particularly limited, the preferred lower limit is preferably 5% or more, more preferably 10% or more, even more preferably 15% or more, and even more preferably 20% or more, and the preferred upper limit is preferably 45% or less, more preferably 40% or less, and even more preferably 35% or less.

In addition, the gamma ray irradiated product of the Parachlorella polysaccharide in this technology has a molecular weight distribution of 500 or more and less than 1,000 weight average molecular weight in GPC analysis (standard pullulan), and although the proportion of this distribution region is not particularly limited, the preferred lower limit is preferably 1% or more, more preferably 3% or more, even more preferably 5% or more, and even more preferably 8% or more, and the preferred upper limit is preferably 25% or less, more preferably 20% or less, and even more preferably 15% or less.

In the present technology, the proportions of the distribution regions of the respective weight average molecular weights and the proportions of the distribution regions of the respective weight average molecular weights can be appropriately combined.
The gamma-ray irradiated polysaccharide has, in GPC analysis (pullulan specimen), at least a molecular weight distribution with a weight molecular weight of 1,000 or more and less than 10,000 and a molecular weight distribution with a weight molecular weight of less than 1,000, and the proportions of the respective distribution regions are preferably 40 to 70% and 10 to 30%, and more preferably 55 to 65% and 15 to 25%. The processed product with a distribution region of a weight molecular weight of less than 1,000 in the present technology has the advantage of being easily absorbed percutaneously and easily penetrating into the dermis region.

<2. Manufacturing method of gamma ray irradiation-treated Parachlorella polysaccharide in the present technology>
An example of a method for producing a gamma-ray irradiated polysaccharide derived from unicellular green algae of the genus Parachlorella in the present technology is shown below, but the present technology is not limited to the following production method.
The gamma ray irradiation treatment product of the present technology can be obtained by subjecting the parachlorella polysaccharide of the present technology to gamma ray irradiation treatment. This makes it possible to reduce the molecular weight of the parachlorella polysaccharide. Furthermore, this can provide the effect of promoting hyaluronic acid production and improving transdermal absorbability.

<2-1. Gamma ray irradiation treatment of Parachlorella polysaccharides using this technology>

<2-1-1. Parachlorella polysaccharides used in the gamma ray irradiation treatment of this technology>
The Parachlorella polysaccharide used in the present technology is not particularly limited as long as it is a polysaccharide derived from a unicellular green alga of the genus Parachlorella. For example, it can be obtained by the production method described below (for example, see Patent Document 4: JP 2014-25035 A), or a commercially available Parachlorella extract (manufactured by PANAC Corporation) may be used.

The form of the parachlorella polysaccharide is not particularly limited and may be either a solution or a powder, but a solution is preferred, and an aqueous solution is more preferred.

The solvent of the solution is not particularly limited, but is preferably an organic solvent and/or an aqueous solvent, and more preferably a water-soluble organic solvent or an aqueous solvent. Examples of the water-soluble organic solvent include lower alcohols (having about 1 to 3 carbon atoms), polyhydric alcohols, and acetone. Examples of the lower alcohol include methanol, ethanol, and n-butanol. Examples of the polyhydric alcohol include dihydric alcohols such as 1,3-butylene glycol and ethylene glycol; and trihydric alcohols such as glycerin. Of the solvents, one or more selected from the group consisting of these can be used. The content of water in the solvent is preferably 95% by mass or more, more preferably 99% by mass or more, and even more preferably 100% by mass (i.e., only water).

The concentration of Parachlorella polysaccharide in the solution is not particularly limited, but the preferred lower limit is preferably 0.01% by mass or more, more preferably 0.05% by mass or more, and even more preferably 0.1% by mass or more, and the preferred upper limit is preferably 10% by mass or less, more preferably 5% by mass or less, and even more preferably 3% by mass or less. The preferred numerical range is more preferably 0.05 to 5% by mass, and even more preferably 0.1 to 1.0% by mass.

<2-1-2. Gamma ray irradiation treatment conditions of this technology>
The conditions for the gamma ray irradiation treatment in the present technology are not particularly limited, and the polysaccharides can be depolymerized by appropriately adjusting the gamma ray irradiation amount (e.g., gamma ray irradiation intensity, gamma ray irradiation dose rate, irradiation time, etc.), temperature, etc. In this manner, the gamma ray irradiation treatment product of the present technology can be obtained by irradiating the Parachlorella polysaccharide of the present technology with gamma rays.

The gamma ray irradiation intensity (kGy) of the present technology is not particularly limited, but the preferred lower limit is preferably 1 kGy or more, more preferably 10 kGy or more, even more preferably 30 kGy or more, more preferably 50 kGy or more, and more preferably 60 kGy or more, and the preferred upper limit is preferably 1000 kGy or less, more preferably 500 kGy or less, even more preferably 200 kGy or less, more preferably 150 kGy or less, more preferably 100 kGy or less, more preferably 90 kGy or less, more preferably 85 kGy or less, and more preferably 80 kGy or less. The preferred numerical range is more preferably 1 to 1000 kGy, even more preferably 10 to 500 kGy, more preferably 60 to 100 kGy, and more preferably 50 to 90 kGy.

The gamma ray exposure dose rate (kGy/hr) of the present technology is not particularly limited, but the preferred lower limit is preferably 0.1 (kGy/hr) or more, more preferably 1 (kGy/hr) or more, even more preferably 3 (kGy/hr) or more, and even more preferably 5 (kGy/hr) or more, and the preferred upper limit is preferably 50 (kGy/hr) or less, more preferably 30 (kGy/hr) or less, even more preferably 20 (kGy/hr) or less, and even more preferably 15 (kGy/hr) or less. The preferred range is more preferably 0.1 to 50 (kGy/hr), even more preferably 1 to 30 (kGy/hr), and even more preferably 5 to 15 (kGy/hr).

The gamma ray irradiation time (hr) of the present technology is not particularly limited, but the preferred lower limit is preferably 1 hour or more, more preferably 3 hours or more, and even more preferably 5 hours or more, and the preferred upper limit is preferably 100 hours or less, preferably 50 hours or less, preferably 40 hours or less, preferably 20 hours or less, more preferably 15 hours or less, and even more preferably 10 hours or less. The preferred range is more preferably 1 to 20 hours, more preferably 5 to 10 hours, and even more preferably 7 to 8 hours.

The type of atmosphere during gamma ray irradiation in the present technology is not particularly limited, but examples include inert gases such as nitrogen and rare gases; air, carbon dioxide, and the like.
The temperature of the atmosphere during gamma ray irradiation in the present technology is not particularly limited, but is preferably room temperature (about 20 to 30°C). In the case of a solution, the water temperature is preferably room temperature of about 20 to 30°C.

The gamma-ray irradiated product of the Parachlorella polysaccharide of the present technology may be appropriately separated and purified using known separation and purification techniques, such as liquid-liquid separation, solid-liquid separation, filtration membrane, activated carbon, adsorption resin, ion exchange resin, etc. This allows inactive impurities to be removed from the gamma-ray irradiated product, or the gamma-ray irradiated product may be further separated and purified into fractions in the desired molecular weight range.

<2-2. Parachlorella polysaccharides of this technology>
The Parachlorella polysaccharide used in the gamma ray irradiation treatment of the present technology is not particularly limited as long as it is a polysaccharide derived from a unicellular green alga of the genus Parachlorella. The Parachlorella polysaccharide is preferably one in which the sugar residues in the basic structure of the polysaccharide are mainly composed of at least galactose (hereinafter also referred to as "Gal") and mannose (hereinafter also referred to as "Man"). The galactose is preferably D-Gal, and the mannose is preferably D-mannose. It is more preferable that the galactose is at least of the furanose type, and the main part (90% or more) of the galactose in the polysaccharide is preferably D-galactofuranose. It is more preferable that the polysaccharide has a 1,5-β-D-galactofuranose structure.

The ratio of the sugar residues of mannose and galactose is not particularly limited, but is preferably Man 1:Gal 2-4, and more preferably Man 1:Gal 2.5-3.5, when mannose is taken as 1.

The galactose content is preferably 50 to 79% by mass, and more preferably 60 to 79% by mass, when the total amount of the neutral monosaccharide composition of the polysaccharide is taken as 100% by mass (hereinafter referred to as "total neutral sugars").
The neutral monosaccharide composition of the polysaccharide of the present disclosure may include, in addition to mannose and galactose, arabinose (hereinafter also referred to as "Ara"), rhamnose (hereinafter also referred to as "Rha"), and xylose (hereinafter also referred to as "Xyl").

The content of these is preferably 0 to 16% by mass, more preferably 5 to 16% by mass, when the total amount of the neutral monosaccharide composition of the polysaccharide is taken as 100% by mass (hereinafter referred to as "total neutral sugars"). Furthermore, the contents of arabinose, rhamnose and xylose are preferably 3 to 7% by mass for arabinose, 2 to 6% by mass for rhamnose and 0 to 3% by mass for xylose, respectively, in the total neutral sugars.
In this case, the contents of mannose and galactose in the total neutral sugars are preferably 84 to 100% by mass, the mannose content is preferably 19 to 25% by mass, and the galactose content is preferably 65 to 75% by mass in the total neutral sugars.

The molecular weight of the Parachlorella polysaccharide of the present technology is not particularly limited, but is preferably 10 ⁴ to 10 ⁶ , more preferably 3 to 50×10 ⁴ , and more preferably 4 to 20×10 ⁴ , as measured by GPC.
In addition, the parachlorella polysaccharide of the present technology is preferable because it is water-soluble and can be easily used in a variety of fields.
In addition, the Parachlorella polysaccharide of the present technology is preferably a polysaccharide that does not belong to the classification of acidic polysaccharides (for example, a neutral polysaccharide).

The Parachlorella polysaccharide of the present technology can be obtained by using unicellular algae of the genus Parachlorella.
More preferably, unicellular green algae of the genus Parachlorella are subjected to subordinate culture in a culture medium containing a carbon source under aerobic conditions to produce polysaccharides, and more preferably, the polysaccharides are recovered. The green algae may be modified.
It is preferable that the dissolved oxygen concentration in the culture is 3 to 13 ppm.
After the cultivation, the algae are removed by centrifugation and filtration, either alone or in combination, and then the mixture is freeze-dried and further dissolved in an aqueous solution, and the water-soluble fraction is preferably recovered.

<2-3. Method for producing Parachlorella polysaccharides according to the present invention>
The method for producing Parachlorella polysaccharides of the present technology is not particularly limited as long as it is possible to produce Parachlorella polysaccharides, but it is preferable to use microorganisms such as bacteria and algae, or modified forms thereof. The modified forms are genetically modified organisms or mutants of microorganisms that have the function of producing Parachlorella polysaccharides of the present technology, and may be modified to improve productivity, durability, etc., as necessary.

The unicellular green algae of the genus Parachlorella are not particularly limited, but among the Parachlorella genus, Parachlorella kessreli is preferred.
The Parachlorella kessleri is not particularly limited, but among the Parachlorella kessleri, the Parachlorella kessleri-PNC1 strain (FERM BP-11493) is more preferable.
In the present technology, the algae strain used may be substantially the same as the algae strain used. A substantially identical algae strain has 99.5% or more (preferably 99.8% or more, more preferably 100%) identical base sequences of its 18S rRNA gene, and preferably has the same phycological properties (morphological observation and extracellular polysaccharide production) as the above-mentioned algae strain. Furthermore, the algae strain of the present technology may be an algae strain bred from the above-mentioned algae strain or an algae strain substantially the same as the above-mentioned algae strain by mutation treatment, genetic recombination, selection of natural mutants, or the like, as long as it has the same or higher degree of the effect of the present technology (particularly the ability to produce Parachlorella polysaccharides of the present technology).

As a medium used for culturing the unicellular green algae of the genus Parachlorella to produce polysaccharides, a carbon source-containing culture medium in which a carbon source such as glucose is added to a basal medium in which general unicellular green algae can be cultured is suitable. Examples of basal media in which unicellular green algae can be cultured include the media described in [Appl Microbiol Biotechnol. 2011 Jul;91(1):31-46. Best practices in heterotrophic high-cell-density microalgal processes: achievements, potential and possible limitations. Bumbak F, Cook S, Zachleder V, Hauser S, Kovar K].

Inorganic salts in the basal medium include trace inorganic components such as _KH2PO4 and _MgSO4 , _and nitrogen sources include ammonium sulfate, urea, etc. As this basal medium, a medium composition shown in Table 1 is suitable for culturing the unicellular green algae of the genus Parachlorella of the present technology, and the content of each component is preferably within the range of ±10%.
The carbon source may be monosaccharides such as glucose, fructose, etc.; oligosaccharides such as sucrose, etc. One or more selected from the group consisting of these may be used and added to the basal medium.
The concentration of the carbon source is not particularly limited, but is preferably 0.1 to 30% by mass, more preferably 1 to 10% by mass in the culture medium.

In the culture for producing the Parachlorella polysaccharide of the present technology, it is preferable to culture under aerobic conditions since the polysaccharide can be stably produced. Cultivation under aerobic conditions is preferable in terms of improving the productivity of the polysaccharide. Examples of aeration means include stirring, shaking, aeration, and bubbling. One or more types selected from the group consisting of these can be used. This allows an appropriate amount of gas to be mixed into the culture medium.
The dissolved oxygen (DO) in the culture medium may be adjusted to provide aerobic conditions, preferably at 3 ppm or more, more preferably at 5 to 13 ppm.

The culture temperature is not particularly limited, but may be about room temperature, between 5 and 40°C.
The pH of the culture medium (at 20° C.) is preferably 4-9, more preferably 5-8.
The culture period is not particularly limited, but it is preferable to have one cycle of about 4 days to 2 weeks in order to produce polysaccharides. When performing preculture and main culture, the period of main culture may be about 4 days to 1 week. During this culture, light irradiation may be performed, but polysaccharides can be produced without light irradiation.
In addition, when increasing the number of algal bodies under autotrophic culture conditions, light such as sunlight, plant cultivation lamps, and artificial light sources such as LEDs can be used, and it is preferable to supply carbon dioxide gas and stir the culture.

The method for producing Parachlorella polysaccharides of the present technology may be either a batch system or a continuous system, but a continuous system is preferred for improving productivity. In addition, either an open system culture or a closed system culture may be used, such as a sealed culture in a culture tank or an open-air culture. In terms of controlling culture conditions, a closed system culture is preferred.

After the Parachlorella polysaccharides of the present technology are produced in the unicellular green algae, it is preferable to separate the polysaccharides from the algae by physical and chemical means such as washing with an aqueous solution, ultrasonic waves, centrifugation, and filtration, and obtain a supernatant containing the Parachlorella polysaccharides of the present technology.
The obtained Parachlorella polysaccharide of the present technology may be appropriately adjusted to a state such as a diluted solution, a concentrated solution, a dried product, etc. Freeze-drying is preferred as a drying method.

The Parachlorella polysaccharides of the present technology may be further purified by removing inactive impurities using known separation and purification techniques, such as liquid-liquid separation, solid-liquid separation, filtration membranes, activated carbon, adsorption resins, and ion exchange resins.

<2. Use of the gamma ray irradiated polysaccharide derived from Parachlorella unicellular green algae in this technology>
As shown in the examples below, the gamma-ray irradiated polysaccharides derived from Parachlorella unicellular green algae in the present technology or compositions containing the same have a hyaluronic acid production promoting effect and a skin penetrating effect, and are effective against symptoms or diseases that can be prevented, improved, or treated by exerting the hyaluronic acid production promoting effect and the skin penetrating effect. The symptoms or diseases include one or more selected from the group consisting of a decrease in hyaluronic acid production ability caused by aging, exposure to ultraviolet rays, etc., dry skin, rough skin, age spots, wrinkles, etc. In addition, the gamma-ray irradiated product of the present technology may be an injection-based administration agent that is injected into cells or tissues, or an oral agent that is taken orally, but since it has a skin penetrating effect, it is preferable to use an external skin agent that is contacted with the skin or mucosa by spraying, applying, or attaching.
Therefore, the composition of the present technology, the composition for promoting hyaluronic acid production, or the skin external preparation, etc., contains at least the polysaccharide derived from Parachlorella unicellular green algae gamma ray irradiation treatment product. The polysaccharide derived from Parachlorella unicellular green algae gamma ray irradiation treatment product in the present technology can be used by being contained in the composition, the composition for promoting hyaluronic acid production, or the skin external preparation as an active ingredient.

In this technology, the polysaccharides derived from Parachlorella unicellular green algae that have been gamma ray irradiated can be used as is, or can be mixed with physiologically or pharma- ceutical acceptable ordinary solvents or diluents.

In addition, the present technology can provide a gamma-ray irradiated polysaccharide derived from unicellular green algae of the genus Parachlorella, or a composition containing the same, or use of the same, which is used for an effect or purpose that the gamma-ray irradiated polysaccharide derived from unicellular green algae of the genus Parachlorella can exert.
In addition, the gamma-ray irradiated polysaccharide derived from unicellular green algae of the genus Parachlorella in the present technology or a composition containing the same can be used as an active ingredient in a method in which the treated product can be exerted.
In addition, the gamma ray irradiated polysaccharide derived from unicellular green algae of the genus Parachlorella in the present technology or a composition containing the same can be used for producing various preparations or compositions having the effect that the treated product can exert or for the purpose of use.
Examples of the effects or purposes that the gamma ray irradiated product can exert include, but are not limited to, the effect of promoting hyaluronic acid production; the effect of skin penetration; the prevention, improvement, inhibition or treatment of a decrease in hyaluronic acid production ability caused by aging, exposure to ultraviolet rays, etc.; and the prevention, improvement, inhibition or treatment of symptoms such as dry skin, rough skin, age spots, and wrinkles.

In addition, the gamma-ray irradiated polysaccharide derived from the unicellular green algae of the genus Rapala Chlorella or a composition containing the same in the present technology can be used for various purposes and compositions such as skin external preparations, cosmetics, medicines, quasi-drugs, foods, and functional foods (e.g., foods for specified health uses, etc.). The purpose or form of the composition is not particularly limited, and may be any form such as liquid, paste, gel, solid, powder, etc.
In the present technology, from the viewpoint of high skin penetration and/or hyaluronic acid production, it is preferable to use it in topical skin preparations, cosmetics, and quasi-drugs, and preparations that are applied to the skin or brought into contact with the skin are suitable.

In the present technology, the gamma-ray irradiated polysaccharides derived from unicellular green algae of the genus Parachlorella or a composition containing the same may be used in combination with an optional ingredient as appropriate. As the optional ingredient, ingredients acceptable for use in pharmaceuticals, topical skin preparations, foods, beverages, feeds, etc. may be used as appropriate. Examples of optional ingredients include cell activators, antioxidants, moisturizers, UV protection agents, solvents (water, alcohols, etc.), oils, surfactants, thickeners, powders, chelating agents, pH adjusters, emulsifiers, stabilizers, colorants, gloss agents, flavorings, odorants, excipients, binders, disintegrants, lubricants, diluents, osmotic pressure adjusters, and fragrances, which may be blended according to the intended formulation or composition.

The gamma ray irradiated polysaccharides derived from unicellular green algae of the genus Parachlorella according to the present technology may be used for humans or non-human animals (preferably primates), with humans and pets being preferred, and humans being more preferred.

Additionally, the technology may be for therapeutic or non-therapeutic uses.
"Non-therapeutic purposes" is a concept that does not include medical procedures, i.e., procedures performed by doctors on the human body. Examples of non-therapeutic purposes include health promotion and prevention of lifestyle-related diseases (e.g., borderline pre-existing diseases).
"Prevention" refers to preventing or delaying the onset of a disease or condition in a subject, or reducing the risk of a disease or condition in a subject.
"Amelioration" refers to reversing a disease, symptom or condition; preventing or slowing its progression; or reversing, preventing or slowing its progression.

The amount of gamma-ray irradiated polysaccharides derived from unicellular green algae of the genus Parachlorella contained in the composition of the present technology is not particularly limited, but is preferably 0.0001 to 50% by mass, more preferably 0.001 to 1% by mass, and even more preferably 0.001 to 0.01% by mass.

The amount of the gamma ray irradiated polysaccharide derived from unicellular green algae of the genus Parachlorella of the present technology used in the composition of the present technology may be appropriately determined.
The amount of gamma ray irradiated polysaccharide derived from unicellular green algae of the genus Parachlorella of the present technology per administration may be appropriately determined depending on the gender, age, condition, and severity of the disease of the person to be administered or ingested.
The amount of the gamma ray irradiated polysaccharide derived from a unicellular green alga of the genus Parachlorella used in the present technology is, for example, preferably 0.1 to 10,000 mg/day, more preferably 1 to 2,000 mg/day, and even more preferably 10 to 1,000 mg/day. The amount per kg of body weight is preferably 0.01 to 1,000 mg/kg body weight/day, more preferably 0.1 to 200 mg/kg body weight/day, and even more preferably 1 to 100 mg/kg body weight/day.

In addition, the method of use (administration or ingestion method, number of times and duration) of the gamma-ray irradiated polysaccharide derived from Parachlorella unicellular green algae of the present technology is not particularly limited. Even when the present technology is for use in medicines, food and drink, or feed, it is preferable to use the same method as the present technology.
The number of times the gamma-ray irradiated polysaccharide derived from unicellular green algae of the genus Parachlorella of the present technology is administered or ingested per day is not particularly limited, and can be determined appropriately depending on the amount of the gamma-ray irradiated polysaccharide derived from unicellular green algae of the genus Parachlorella of the present technology administered or ingested per day.

The present technology can also employ the following configuration.
[1] A polysaccharide derived from a unicellular green alga belonging to the genus Parachlorella that has been irradiated with gamma rays, or a composition containing said irradiated product. The gamma irradiated product or said composition is preferably used for promoting hyaluronic acid production, for skin penetration, or for external application to the skin.
[2] The gamma ray irradiated polysaccharide product according to [1] above, wherein the gamma ray irradiated polysaccharide product has a molecular weight distribution with a weight average molecular weight of less than 10,000, and the proportion of said distribution region is 50% or more.
[3] The gamma ray irradiated polysaccharide according to [1] or [2] above, wherein the gamma ray irradiated polysaccharide has at least a molecular weight distribution of 1,000 or more but less than 10,000 and a molecular weight distribution of less than 1,000, with the proportions of the respective distribution regions being 45 to 65% and 10 to 30%, or a composition containing the treated product.
[4] The gamma ray irradiated product according to any one of [1] to [3] above, or a composition containing the product, wherein the gamma ray irradiation is 1 to 1000 kGy.
[5] The polysaccharide derived from Parachlorella unicellular green algae is a polysaccharide in which the sugar residues in the basic structure of the polysaccharide are composed of at least galactose and mannose, and the galactose has a furanose form. The gamma ray irradiation treatment product according to any one of [1] to [4] above, or a composition containing the treatment product.
[6] The gamma ray irradiation treated product according to any one of [1] to [5] above, or a composition containing the treated product, wherein the unicellular green algae of the genus Parachlorella is Parachlorella serrata.

[7] It is preferable that the polysaccharide derived from a unicellular green alga of the genus Parachlorella according to any one of [1] to [6] above is a polysaccharide in which the sugar residues in the basic structure of the polysaccharide are composed of at least galactose and mannose, and the galactose has a furanose form.
It is more preferable that the content of galactose is 50 to 79% by mass in the total neutral sugars.
[8] It is preferable that the weight-weight molecular weight of the polysaccharide according to any one of [1] to [7] above is on the order of 10 to the 8th power.
[9] It is preferable that the ratio of sugar residues of galactose to mannose in the polysaccharide according to any one of [1] to [8] above is 2.5 to 3.5:1.
[10] The polysaccharide according to any one of [1] to [9] above is a polysaccharide derived from Parachlorella kessreli, and more preferably the polysaccharide is water-soluble.

[11] The polysaccharide according to any one of [1] to [10] above is preferably obtained by a polysaccharide production method comprising subculturing a unicellular alga of the genus Parachlorella or a modified alga in a carbon source-containing culture medium under aerobic conditions and recovering the produced polysaccharide.
[12] The polysaccharide according to any one of [1] to [11] above is preferably obtained by a polysaccharide production method using Parachlorella kessreli, which is one of unicellular algae of the genus Parachlorella.

[13] A gamma-ray irradiated product of the polysaccharide derived from the unicellular green algae of the genus Parachlorella according to any one of [1] to [12] above, or a composition containing the treated product.
[14] The gamma ray irradiated product or a composition containing the treated product according to any one of [1] to [13] above, wherein the gamma ray irradiated product or a composition containing the treated product is used for promoting hyaluronic acid production, for skin penetration, or for external use on the skin.

[15] A method for promoting hyaluronic acid production, a method for promoting skin penetration, or a method for applying to the skin, comprising administering the gamma ray irradiated product according to any one of [1] to [14] above, or a composition containing the treated product.
[16] The gamma-ray irradiated product according to any one of [1] to [14] above, or a composition containing the treated product, for use in medicine, food and drink, feeding, skin external preparations, promotion of hyaluronic acid production, promotion of skin permeation, or skin application, or use thereof. Use for non-therapeutic purposes is also possible.
[17] The gamma-ray irradiated product or a composition containing the treated product according to any one of [1] to [14] above, or use thereof, for producing a composition, a pharmaceutical product, a food or drink, a feed, an external preparation for skin, a composition for promoting hyaluronic acid production, a composition for promoting skin penetration, or a composition for application to skin. This may be used for non-therapeutic purposes.

The present technology will be described in more detail below based on test examples, examples, etc. Note that the test examples, examples, etc. described below are merely examples of representative examples of the present technology, and should not be construed as narrowing the scope of the present technology.

<Test Example 1: Method for producing polysaccharides derived from Parachlorella unicellular green algae>
Polysaccharides derived from unicellular green algae of the genus Parachlorella were produced using Parachlorella kessleri-PNC1 strain (FERM BP-11493) as the unicellular green algae of the genus Parachlorella.
The production method of polysaccharides and the analysis method of polysaccharides (hydrolysis, methylation analysis, NMR analysis, uronic acid quantification, etc.) in this test example were performed with reference to Patent Document 4: JP 2014-25035 A.
(1) Parachlorella kessleri-PNC1 strain (FERM BP-11493) (Deposit date: July 19, 2012) Deposit address: Tsukuba Center Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki Prefecture, 305-8566, Japan, National Institute of Technology and Evaluation, Patent Organism Depositary (NITE-IPOD).

A slant medium (50 mL) containing Parachlorella kessleri-PNC1 strain (FERM BP-11493) was added to a 3 L baffled Erlenmeyer flask containing 250 mL of glucose-containing culture medium, and pre-cultured for 4 days at 160 rpm, 28°C, and illuminance of 8000 to 10000 lux. Note that the number of algae may be increased preliminarily before pre-culture.
The glucose-containing culture medium was a basal medium shown in Table 1 containing 10 g of glucose per 1 L.

240 mL of the preculture was transferred to a 30 L jar fermenter and cultured for 12 days to obtain the main culture. The culture conditions were aerobic at 30° C., 230 rpm, aeration of 0.56 vvm, internal pressure of 0.3 kgf/cm ² , and pH 7.2, and no light irradiation was performed.
The main culture was centrifuged (7000 rpm (6500 G), 25° C.) to separate the supernatant containing the polysaccharide of interest from the algae. The stock solution was then concentrated to 2 L using a UF membrane (SIP1013) with a molecular weight cutoff of 6000. 200 mL of anion exchange resin was added to the concentrate (2 L), and the concentrate was recovered to a Brix of 0.1, concentrated to 500 mL by UF membrane filtration, and freeze-dried to obtain polysaccharides derived from Parachlorella (hereinafter also referred to as "Parachlorella polysaccharides"). The yield was 0.2 to 0.4 g per 1 L of culture solution.

40 mL of distilled water was added to 1 g of polysaccharide derived from Parachlorella, and the mixture was stirred to obtain a suspension. This suspension was left overnight at 4°C, and then centrifuged (7000 rpm, 6500 g, 5 minutes) to separate the supernatant and precipitate. The supernatant was freeze-dried to obtain water-soluble Parachlorella polysaccharide (hereinafter also referred to as "water-soluble Parachlorella polysaccharide") (0.4-0.5 g).

<Test Example 2: Structural analysis of Parachlorella polysaccharide>
Water-soluble parachlorella polysaccharide was hydrolyzed with 2M-TFA (trifluoroacetic acid) at 120°C for 2 hours. The hydrolyzate was reduced with _NaBH4 at room temperature and further acetated with (acetic anhydride:1-methylimidazole 9:1) to obtain an alditol acetate derivative. The alditol acetate derivative was analyzed by GC.
GC measurements were performed on a GL Science GC-353 gas chromatograph (column SP-2330 (Spelco); detector FID; isothermal analysis at 220° C.).
As a result of this GC, arabinose (4.7%), rhamnose (4.0%), xylose (1.7%), mannose (22.5%) and galactose (67.1%) were found in the water-soluble Parachlorella polysaccharide.

Methylation analysis was also performed on water-soluble parachlorella polysaccharides. Polysaccharide samples were methylated to obtain methylated polysaccharides. The methylated polysaccharides were hydrolyzed in 2M TFA at 120°C for 2 hours, reduced with _NaBD4 , and acetylated with acetic anhydride and 1-methylimidazole. The resulting derivatives were analyzed by GC-MS.
GC-MS was performed using a Shimadzu QP-5000 GC-MS gas chromatograph mass spectrometer (column: SPB-50 (0.32 x 30 m)).
The mannose residues of this polysaccharide were found to be non-reducing terminal, 1,2-linked, and 1,3-linked residues, and the majority of the galactose residues in this polysaccharide were furanose, and the galactose residues in this polysaccharide were found to be non-reducing terminal, 1,2-linked furanose, 1,5-linked furanose, 1,6-linked furanose, 1,2,6-linked furanose, and 1,3-linked pyranose.

Furthermore, ^1H -NMR analysis (Varian Unity plus 500, 1H: 500 MHz, 13C: 125 MHz, [solvent: heavy water ( _D2O ), measurement temperature: 30°C, internal standard: DSS]) confirmed that most of the galactose residues in the water-soluble Parachlorella polysaccharide exist in the furanose form.
Furthermore, the protein content (Lowry method) and uronic acid content (carbazole-sulfuric acid method) of the water-soluble parachlorella polysaccharide were measured, and the presence of protein and uronic acid was hardly detected.

The quaternary ammonium salt precipitation method is commonly used to recover acidic polysaccharides. Using this method, the presence or absence of polysaccharide precipitation was confirmed using cetylpyridinium chloride, a type of quaternary ammonium salt. 20 mL of an aqueous solution (w/v) containing 0.2% polysaccharide was placed in a test tube, 10 g of CPC was added to this aqueous solution, mixed, and left for 12 hours. After centrifugation, the presence or absence of precipitation was confirmed visually. As a result, the water-soluble parachlorella polysaccharide did not precipitate into the quaternary ammonium salt. From this, it was considered that the water-soluble parachlorella polysaccharide does not fall under the category of acidic polysaccharides that contain a large amount of uronic acid or ester sulfate.

The DL of galactose and mannose present in water-soluble parachlorella polysaccharides were hydrolyzed and trimethylsilylated, and the resulting derivatives were analyzed by GC. As a result, D-galactose and D-mannose were confirmed.

Based on this, it was presumed that the sugar residues in the basic structure of the polysaccharide derived from Parachlorella are composed mainly of at least galactose and mannose, and that most of the galactose exists in the furanose form. It was also presumed that this polysaccharide has a 1,5-β-D-galactofuranose structure.

<Test Example 3: Gamma-ray irradiated polysaccharide derived from Parachlorella unicellular green algae>
[Production Example 1: Production method and weight average molecular weight of parachlorella polysaccharide]
Polysaccharides were obtained in the same manner as in Test Example 1, except that the culture period in the main culture was changed from 4 days to 1 week.

The weight average molecular weight (Mw) of the polysaccharides was measured by GPC analysis for the parachlorella polysaccharide and the water-soluble parachlorella polysaccharide obtained in Production Example 1. As a result of the GPC analysis, the parachlorella polysaccharide had the highest peak at a retention time of 27.2 minutes and a shoulder peak at a retention time of 26.2 minutes, and the weight average molecular weight of each was calculated from these retention times (peaks) and the standard (pullulan). The weight average molecular weights (Mw) were 49,000 to 54,000 and 170,000 to 190,000, respectively, and the overall weight average molecular weight (Mw) was about 5 to 20 x 10 ^4. No peaks were observed in the region of weight average molecular weights less than 100,000.
The weight-average molecular weight of the Parachlorella polysaccharide before gamma-ray irradiation was in the range of 10 to the power of 4 or 5 in GPC analysis. When the Parachlorella polysaccharide was further dissolved by ultrasonic treatment, the weight-average molecular weight was in the range of 10 to the power of 4.

<GPC analysis>
Measurement column: Two TSKgel GMPW _XL (7.8mmID x 300 mm: Tosoh Corporation) in series Measurement temperature: 50°C
Mobile phase: 0.1 M _NaNO3
HPLC system: Shimadzu Shodex GPC-101 system Detector: Differential refractometer RI-71S
Standard substance: Pullulan (Shodex standard P-82)
Software used (GPC calculation): System Instruments 480II Data Station GPC program. Molecular weight and elution time for pullulan (P-82): 100,000/12.2 min, 30,000/13.7 min, 10,000/15.0 min, 3,000/16.4 min, 1,000/17.7 min, 500/18.6 min

[Production Example 2: Gamma-ray irradiated product and its production method]
The parachlorella polysaccharide obtained in the above [Production Example 1] was dissolved in ion-exchanged water to obtain a 0.1% by mass aqueous solution and a 1.0% by mass aqueous solution. The 0.1% by mass aqueous solution was irradiated with gamma rays at a dose rate of 10 kGy/hr, an irradiation time of 7 to 8 hr, and an irradiation intensity of 70 kGy to obtain a gamma-ray irradiation treatment product 1. The 1.0% by mass aqueous solution (20 to 30° C.) was also irradiated with gamma rays under the same conditions (irradiation intensity of 70 kGy) as for the 0.1% by mass aqueous solution to obtain a gamma-ray irradiation decomposition product 2.

The gamma-ray irradiated product 1 of parachlorella polysaccharide (0.1% by weight aqueous solution) was measured using the above-mentioned <GPC analysis> (see the lower part of FIG. 1). The molecular weight distribution results are shown in Table 2.
As shown in FIG. 1 and Table 2, the parachlorella polysaccharide was decomposed and broken down into smaller molecules by irradiating the parachlorella polysaccharide with gamma rays.

<Test Example 4: Hyaluronic acid production promotion test (Example 1 and Comparative Examples 1 and 2)>
The hyaluronic acid production promotion test of each sample is carried out by quantitative PCR using fibroblasts.As samples, the polysaccharide of Production Example 1 before gamma ray irradiation (Comparative Example 1), the gamma ray irradiation treatment product 1 of Production Example 2 (Example 1), and the acid hydrolyzate of the polysaccharide of Production Example 1 (Comparative Example 2) are used.
Table 3 shows the relative expression levels of hyaluronic acid synthase HAS2 in the polysaccharide decomposition product by gamma irradiation (Example 1) and the polysaccharide decomposition product after acid hydrolysis (Comparative Example 2). The relative expression levels are shown when the negative control is set to 1.
The acid hydrolysis was carried out by hydrolyzing the Parachlorella polysaccharide of Production Example 1 (0.1% by mass aqueous solution) in 2M-TFA (trifluoroacetic acid) at 100° C. for 3 hours.
The hyaluronic acid production promotion test in Test Example 4 is described in more detail below, and the results are shown in Table 3.

<Test for evaluating HAS2 mRNA expression promoting effect>
The sample was tested for its ability to promote expression of hyaluronan synthase mRNA as follows.

(1) Cell culture and sample addition
Normal human dermal fibroblasts (NHDF, Kuraray Industries, Inc.) were seeded at 2 x 105 cells/well on a collagen-coated 12-well plate using D-MEM (Dulbecco's minimal essential medium (containing low glucose, L-glutamine, and phenol red), Fujifilm Wako Pure Chemical Industries, Ltd.) medium containing 10% FBS (fetal bovine serum, SIGMA Corporation) and ¹ % penicillin-streptomycin (penicillin-streptomycin-L-glutamine solution (x100), Fujifilm Wako Pure Chemical Industries, Ltd.), and cultured at 37°C and 5% _CO2 for 24 hours.

Gamma ray treated product 1 was dissolved in D-MEM medium containing 1% FBS and 1% penicillin-streptomycin to give concentrations of 0.01 and 0.1 mg/mL, respectively, and these were used as the test samples (test samples 1 and 2) of Examples 1 and 2, respectively. In addition, D-MEM medium containing 10% FBS and 1% penicillin-streptomycin (control sample 1) was prepared as control example 1.

24 hours after the seeding of the normal human dermal fibroblasts, 1 mL each of test sample 1, test sample 2, and control sample 1 was replaced in the wells seeded with the normal human dermal fibroblasts, and cultured at 37° C. under 5% _CO2 for an additional 72 hours.

After the incubation, total RNA was extracted. After removing the medium from the wells by aspiration, total RNA was extracted using an RNeasy Mini Kit (QIAGEN) according to the operating instructions for the kit.

(2) Synthesis of cDNA cDNA was synthesized using a cDNA synthesis kit (PrimeScript ^™ RT reagent Kit, manufactured by Takara Bio Inc.) and a master cycler (TaKaRa PCR Thermal Cycler Dice ^R Touch, manufactured by Takara Bio Inc.). Specifically, using the total RNA (about 250 ng/μL) of each of the test samples 1 and 2 and the control sample 1 obtained by the above extraction, a reaction solution was prepared according to the instruction manual for the cDNA synthesis kit, and the reaction was carried out using a master cycler at 37° C. for 15 minutes and at 85° C. for 5 seconds to synthesize cDNA to be used as a template for real-time PCR to measure the expression level of HAS2 mRNA.

(3) Real-time PCR reaction using the intercalation method As primers for amplifying the HAS2 gene, a sense primer and an antisense primer having the following sequences were used.
Sense primer: 5'-ACCGGGGTAAAATTTGGAAC-3' (SEQ ID NO: 1)
Antisense primer: 5'-TAAGGCAGCTGGCAAAAGAT-3' (SEQ ID NO: 2)
As primers for amplifying the GAPDH gene as an internal standard, a sense primer and an antisense primer having the following sequences were used.
Sense primer: 5'-CAGCCTCAAGATCATCAGCA-3' (SEQ ID NO: 3)
Antisense primer: 5'-TGTGGTCATGAGTCCTTCCA-3' (SEQ ID NO: 4)

For each of the test samples (Example 1, Comparative Examples 1 and 2), real-time PCR reactions were carried out on cDNA prepared from cultured cells using the above primer set with a real-time PCR device (Eco ^™ Real-Time PCR System, AS ONE Corporation) and a real-time PCR kit (TB ^Green® Premix Ex Taq ^™ II (Tli RNaseH Plus) and its instruction manual, Takara Bio Inc.). The reaction was incubated at 95° C. for 30 seconds, followed by 40 cycles of 95° C. for 5 seconds and 60° C. for 30 seconds, and the amount of luminescence from TB Green was measured for each cycle.

(4) Analysis Amplification curves of DNA fragments encoding HAS2 and GAPDH were prepared based on the amount of luminescence from TB Green in each cycle, and the amount of luminescence was relatively quantified using the comparative Ct method (ΔΔCt method) with GAPDH as an internal standard gene.

<Results of Test Example 4>
The expression level of hyaluronic acid synthase HAS2 was not increased by the parachlorella polysaccharide in Comparative Example 1. Thus, the parachlorella polysaccharide could not exert the effect of promoting hyaluronic acid production.
In the acid hydrolyzate (acid decomposition product) of Comparative Example 2, which was obtained by acid hydrolyzing parachlorella polysaccharide, the expression level of hyaluronic acid synthase HAS2 did not increase, even though it was reduced in molecular weight. Thus, even if parachlorella polysaccharide was reduced in molecular weight by acid hydrolysis, it could not exhibit the effect of promoting hyaluronic acid production.

In contrast, when parachlorella polysaccharides were decomposed into smaller molecules by gamma ray irradiation (Example 1), the expression level of the hyaluronic acid synthase HAS2 increased significantly by approximately 1.4 to 1.5 times. In this way, by decomposing parachlorella polysaccharides into smaller molecules by gamma ray irradiation, a new effect of promoting hyaluronic acid production that was not possible with parachlorella polysaccharides can be exerted. In this way, it was found that the new gamma ray degradation products of parachlorella polysaccharides can exert a new and remarkable effect of promoting hyaluronic acid production.

<Test Example 5: In-vitro skin permeation test using pig skin>
The skin permeation test was performed by an in-vitro skin permeation test using pig skin. As a specimen, an aqueous solution of the gamma-ray-treated product 1 prepared in the above-mentioned Production Example 2 to a concentration of 1% by mass was used. The skin permeation test of Test Example 5 is described in more detail below, and the results are shown in Table 3.

Pig skin was cut into a circle with a diameter of 3.0 cm and placed in a Franz diffusion cell (opening diameter 2.0 cm, receptor volume approximately 2.4 mL) with the stratum corneum facing the donor side. After placement, the donor side and receptor side were filled with PBS (pH 7.4) and the electrical resistance was measured to check for the presence or absence of skin damage. After confirming that the skin was in a normal condition, excess water on the stratum corneum side was lightly wiped off with cotton, and 1 mL of the sample was administered to the stratum corneum side. After administration of the sample, the skin permeation test was performed with the temperature of the buffer solution on the receptor side kept at 32°C. 6 hours after the start of the test, 1 mL of the receptor solution was sampled and polysaccharide quantification was performed.

The polysaccharide quantitative value (mg/mL) was measured using the phenol-sulfuric acid method (standard Glc aqueous solution, measured absorbance at 490 nm).
The transmittance concentration was calculated by subtracting the blank value from the active value to obtain the transmittance concentration (mg/mL).
The transmittance was calculated as follows: "Transmittance (%) = (transmittance concentration ÷ quantitative polysaccharide value of 1% by mass gamma-ray treated product before treatment) × 100." For example, (transmittance concentration of active epidermis/dermis extract "0.037 mg/mL" ÷ quantitative polysaccharide value of gamma-ray treated product before treatment "0.63 mg/mL") × 100 = 5.87%.

[Amount of permeation into the stratum corneum after 6 hours]
After the above-mentioned permeation test was completed, the remaining liquid on the skin surface was removed, the skin surface was wiped with wet cotton, the skin was removed from the cell, and the stratum corneum was peeled off 15 times with cellophane tape (the first peeling tape was discarded because it may have contained the sample attached to the skin surface). 14 sheets of cellophane tape were placed in a 50 mL centrifuge tube, 15 mL of a mixture of pure water:ethanol = 1:1 (v/v) was added, and extraction was performed by ultrasonic treatment for 1 hour. The extract was transferred to a 100 mL beaker, evaporated in a thermostatic bath at 70 ° C, re-dissolved by adding 1 mL of pure water, and centrifuged (12,000 rpm, 5 minutes). The supernatant was collected in a microtube, and polysaccharide quantification was performed.

[Epidermal/dermal penetration amount after 6 hours]
The epidermis and dermis layers, excluding the stratum corneum, were finely cut and placed in two crushing tubes, 0.5 mL of pure water was added to each tube, and the mixture was crushed with a crusher for 20 minutes. After that, the mixture was collected from the two tubes into a 50 mL centrifuge tube, and 10 mL of ethanol was added to each crushing tube, mixed well, and centrifuged (5000 rpm, 5 minutes). After centrifugation, the supernatant was transferred to a 100 mL beaker and evaporated in a thermostatic bath at 70°C. After evaporation, 1 mL of pure water was added to the residue to redissolve it, and centrifuged (12,000 rpm, 5 minutes). The supernatant was collected in a microtube, and polysaccharide quantification was performed.

<Results of Test Example 5>
It was confirmed that the gamma-ray irradiated parachlorella polysaccharide penetrated into the dermis where the hair papilla cells are present. This means that the molecular weight can be reduced by gamma-ray irradiation, improving the transdermal permeability. Furthermore, since it was confirmed that the gamma-ray irradiated parachlorella polysaccharide penetrated into the dermis where the fibroblasts are present, it is expected that the gamma-ray irradiated parachlorella polysaccharide of this technology will promote the production of hyaluronic acid when applied to actual skin.

Generally, the skin barrier prevents the invasion of bacteria, but conversely, this skin barrier makes it difficult for active ingredients to penetrate. For this reason, even if an active ingredient is usually found to be beneficial in in vitro tests, if it has low skin permeability, it may not be suitable for use as an external skin agent.
On the other hand, the gamma ray irradiation treatment product of the present technology has high skin permeability, and as mentioned above, also has the effect of promoting hyaluronic acid production.In this way, the present technology has the very excellent advantage that it can efficiently exert various effects related to the promotion of hyaluronic acid production even by percutaneous administration.In addition, since the present technology is low molecular weight, it is believed that it can efficiently exert its effects even by oral ingestion or injection administration.

Claims (8)

Polysaccharides derived from Parachlorella unicellular green algae, treated with gamma rays. The gamma ray irradiated polysaccharide according to claim 1, wherein the gamma ray irradiated polysaccharide has a molecular weight distribution with a weight average molecular weight of less than 10,000, and the proportion of said distribution region is 50% or more. The gamma ray irradiated polysaccharide according to claim 1 or 2, wherein the gamma ray irradiated polysaccharide has at least a molecular weight distribution of 1,000 or more but less than 10,000 and a molecular weight distribution of less than 1,000, with the proportions of the respective distribution regions being 45-65% and 10-30%. The gamma ray irradiated product according to any one of claims 1 to 3, wherein the gamma ray irradiation is performed with an irradiation intensity of 1 to 1000 kGy. The gamma ray irradiated product according to any one of claims 1 to 4, wherein the polysaccharide derived from Parachlorella unicellular green algae is a polysaccharide in which the sugar residues in the basic structure of the polysaccharide are composed of at least galactose and mannose, and the galactose has a furanose form. The gamma ray irradiated product according to any one of claims 1 to 5, wherein the unicellular green algae of the genus Parachlorella is Parachlorella kesseri. A composition comprising a polysaccharide derived from a unicellular green alga belonging to the genus Parachlorella that has been gamma-irradiated according to any one of claims 1 to 6. The composition according to claim 7, which is a composition for promoting hyaluronic acid production or a skin topical agent.