JP2014105202A - Immunostimulator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunostimulator for enhancement of antiviral antibody production.SOLUTION: An immunostimulator contains a polysaccharide, wherein sugar residues in the basic structure comprise galactose and mannose at least and the galactose exists in a furanose form. The content of galactose is preferably 50-79 mass%, when the total amount of the neutral monosaccharide composition is considered to be 100 mass%. A polysaccharide is preferably an extracellular polysaccharide derived from unicellular algae of the Parachlorella group. The immunostimulator significantly reduces the amount of type A influenza viruses both in lung and respiratory tract lavage fluid as compared to a negative control group.

Description

  The present invention relates to an immunostimulator.

The background art of the present invention is the same as that of the prior application described later.
[Prior art documents]

  Prior art documents of the present invention are the same as those of the prior application described later.

  An object of the present invention is to provide an immunostimulator that promotes the production of anti-pathogen antibodies.

  The immunostimulant of the present invention is characterized in that the sugar residue of the basic structure of the polysaccharide is composed of at least galactose and mannose, and the galactose contains a polysaccharide having a furanose type.

  According to this aspect, the resistance against pathogens in the administration subject can be easily increased.

  According to the present invention, it is possible to easily increase resistance to pathogens in administration subjects.

It is a figure which shows the result of having visualized the polysaccharide outside a cell (algae) by Indian ink stain. It is a molecular phylogenetic tree based on the 18S rRNA gene sequence of the algal PNC1 strain. The result of the GPC analysis of the disclosed polysaccharide is shown. It is a figure which shows the time-dependent change of the symptom of the mouse | mouth infected with the herpes simplex virus type 2 (HSV-2). FIG. 4 (A) is a graph showing changes over time in the onset score of mice infected with HSV-2. FIG. 4 (B) is a table showing the death status of mice infected with HSV-2. It is a graph which shows the proliferation amount of HSV-2 in the mouse | mouth infected with HSV-2 3 days after the infection. It is a graph which shows the time-dependent change of the body weight in the mouse | mouth infected with the influenza A virus. It is a graph which shows the proliferation amount of the influenza A virus in the mouse | mouth infected with the influenza A virus 3 days after the infection. It is a graph which shows the amount of neutralizing antibodies 14 days after the infection in the mouse | mouth infected with the influenza A virus. FIG. 8A is a graph showing the amount of neutralizing antibody in serum. FIG. 8B is a graph showing the amount of neutralizing antibody in the airway washing solution. It is a graph which shows the virus specific secretory immunoglobulin A (IgA) amount 14 days after the infection in the mouse | mouth infected with the influenza A virus. FIG. 8 (A) is a graph showing the amount of IgA antibody in the airway washing solution. FIG. 8 (B) is a graph showing the amount of IgA antibody in excreta.

In order to describe the embodiments of the present invention, first, the description of Japanese Patent Application No. 2012-168776 (hereinafter referred to as the prior application) which is the basis of the present invention will be described, and then the embodiments of the present invention will be described. In the description related to the prior application (the part up to [the embodiment of the present invention]), “the present invention”, “the present disclosure”, and “the present technology” are respectively referred to as “the invention of the prior application” and “the prior application”. "Disclosure of" and "technology of prior application".
[Technical Field of Prior Application]

The present invention relates to a novel polysaccharide, a method for producing the same, and a skin external preparation, a hair-restoring agent, a whitening agent and the like containing the novel polysaccharide.
[Background of prior application]

Polysaccharides are used in a wide range of fields such as pharmaceuticals, foods, and industrial products. Examples of acidic polysaccharides well known in the pharmaceutical and food fields include glycosaminoglycans such as chondroitin sulfate and hyaluronic acid, xanthan gum, and the like, and β1,3 glucan and cyclodextrin as neutral polysaccharides. It is known that chondroitin sulfate contained in shark cartilage, chicken fowl, etc. and hyaluronic acid capable of producing microorganisms have an effect of improving knee joint pain and the like. Β1,3 glucan is known to have an immunostimulatory action and the like.
Xanthan gum is a polysaccharide produced by Xanthomonas campestris bacteria, and its sugar composition is a branched polysaccharide having D-glucose, D-mannose, and D-glucuronic acid as constituent sugars, and can now be industrially mass-produced. Therefore, it is used around the world as a thickener and the like (for example, Patent Document 1).
Moreover, it is described that the polysaccharide containing 81.4 to 91.9% galactose produced extracellularly by Chlorella unicellular green algae has an antitumor effect on mouse sarcoma cell lines (for example, Patent Document 2). ).
Furthermore, in reality, new polysaccharides that can be used in various applications are desired in the industry.
[Prior Art Documents of Prior Applications]
[Patent Literature]

Japanese Patent Publication No. 61-11596 Japanese Patent Laid-Open No. 6-248003 [Summary of Invention of Prior Application] [Problems to be Solved by Invention of Prior Application]

Therefore, in view of such a situation, the present invention intends to provide a novel polysaccharide that can be used for various applications.
[Means for solving the problems of the prior application]

As a result of intensive studies on novel polysaccharides that can be used for various applications, the present inventors have found novel polysaccharides containing galactose and mannose as main constituent sugars. Furthermore, it has also been found that the novel polysaccharide of the present invention has an excellent hair growth effect and a whitening effect. It has been found that the novel polysaccharide of the present invention can also be used for external preparations for skin, cosmetics, foods, pharmaceuticals and the like. Moreover, since the novel polysaccharide of the present invention is a polysaccharide that can be produced extracellularly by Parachlorella unicellular green algae, it can be industrially mass-produced and can be expected to be used in a wide range of fields. It is.

By the way, in recent years, due to various factors such as increased stress and changes in dietary habits, men and women who suffer from thinning hair and hair loss have been on the rise, and expectations for hair restorers are increasing.
For example, Japanese Patent Application Laid-Open No. 2010-150203 discloses that a hair restoration composition containing soybean isoflavone glycoside selected from daidzin, gestinin and flavosterone SE and 6-benzylaminopyrine reduces hair loss and bristle hair. Is disclosed.
JP 2012-097008 A discloses that a sugar-free pineapple extract obtained by extracting a pineapple edible portion with 90% by volume ethanol at 80 ° C. for 2 hours has an effect of promoting proliferation of hair papilla cells. Has been.
JP 2010-1000053 discloses that a compound having an angular furocoumarin skeleton having a predetermined structure contained in an extract of Angelica atropurpurea exhibited hair elongation of pig hair follicles. Yes.
Thus, the actual condition is that studies on effective hair-growth agents from various action mechanisms by a wide range of compounds are being conducted. However, it is completely unexpected that the polysaccharide of the present invention has a high hair-growth action even though it is a polymer system, and it is also possible to provide an excellent hair-growth agent.

  Therefore, in order to solve the above problems, the present invention provides a polysaccharide in which the sugar residue of the basic structure of the polysaccharide is composed of at least galactose and mannose, and the galactose has a furanose type.

  In the present invention, the sugar residue of the basic structure of the polysaccharide is composed of at least galactose and mannose, and contains a polysaccharide having a furanose type in the galactose and / or an extracellular polysaccharide derived from parachlorella. The present invention provides hair matrix cell growth activators, FGF-7 production promoters, VEGF production promoters, hair restorers, melanin production inhibitors, whitening agents, and skin external preparations.

The present invention also provides a method for producing a polysaccharide by subculturing a Parachlorella unicellular algae or a modified form in a carbon source-containing culture medium under aerobic conditions and recovering the produced polysaccharide. is there.
The present invention also provides an algal body named Parachlorella kessleri-PNC1 and deposited as FERM BP-11493.
[Effect of invention of prior application]

ADVANTAGE OF THE INVENTION According to this invention, the novel polysaccharide which can be utilized for various uses can be provided.
[Mode for carrying out the invention of the prior application]

1. 1. Polysaccharides of the present disclosure 2. Production method of polysaccharide of the present disclosure Uses of polysaccharides of the present disclosure

1. Polysaccharide of the Present Disclosure In the polysaccharide of the present disclosure, the sugar residue of the basic structure of the polysaccharide is composed of at least galactose (hereinafter also referred to as “Gal”) and mannose (hereinafter also referred to as “Man”). The galactose preferably has at least a furanose type.
The ratio of the sugar residues of the mannose and galactose is preferably Man 1: Gal 2-4, more preferably Man 1: Gal 2.5-3.5, where mannose is 1. It is.

The content of the galactose is preferably 50 to 79% by mass when the total amount of the neutral monosaccharide composition of the polysaccharide is 100% by mass (hereinafter referred to as “in all neutral sugars”). Preferably it is 60-79 mass%.
In addition to mannose and galactose, the neutral monosaccharide composition of the polysaccharide of the present disclosure includes arabinose (hereinafter also referred to as “Ara”), rhamnose (hereinafter also referred to as “Rha”) and xylol (hereinafter referred to as “Xyl”). May be included).
These contents are preferably 0 to 16% by mass, more preferably 5% when the total amount of the neutral monosaccharide composition of the polysaccharide is 100% by mass (hereinafter referred to as “in total neutral sugar”). ~ 16% by weight. Further, the contents of arabinose, rhamnose, and xylose are preferably 3-7% by mass of arabinose, 2-6% by mass of rhamnose, and 0-3% by mass of xylose, respectively, in the total neutral sugar.
At this time, the content of mannose and galactose is preferably 84 to 100% by mass in the total neutral sugar. The content of mannose is preferably 19 to 25% by mass in the total neutral sugar, and the content of galactose is preferably 65 to 75% by mass in the total neutral sugar.

The molecular weight of the polysaccharide of the present disclosure is preferably 3 to 9 × 10 ⁴ , more preferably 5 to 8 × 10 ⁴ in GPC measurement.
Moreover, since the polysaccharide of this indication is water-soluble, since it is easy to utilize for the use of various fields, it is suitable.

2. The polysaccharide production method of the present disclosure is not particularly limited as long as the polysaccharide production method of the present disclosure can produce the polysaccharide of the present disclosure. For example, microorganisms such as bacteria and algae and It is mentioned to use this modification. The modified body is a genetically modified or mutant microorganism having a function capable of producing the polysaccharide of the present disclosure, and a microorganism whose productivity and durability are improved as necessary It is.

As a preferable method for producing the polysaccharide of the present disclosure, the polysaccharide of the present disclosure is obtained by culturing Parachlorella unicellular green algae or a modified form by any culture method such as an independent culture method or a subordinate culture method. It is possible to manufacture with.
By using the green algae, the polysaccharide of the present disclosure can be produced and recovered outside the cells of the algae. By producing it extracellularly, it becomes easy to separate algal bodies and polysaccharides by physical means such as centrifugation. Thereby, it is easy to produce polysaccharides by continuous culture such as culture medium exchange. Therefore, using the Parachlorella unicellular green algae or the modified form of the present disclosure can greatly improve the productivity of the polysaccharide, is suitable for industrial production, and provides a stable supply of the polysaccharide of the present disclosure. Make it possible. Since stable supply is possible, the polysaccharide of the present disclosure can be used in a wide range of fields.

Of the Parachlorella unicellular green algae, Parachlorella kessreli is preferable, and Parachlorella kessleri-PNC1 strain (FERM BP-11493) is more preferable.
Panac Co., Ltd. New Business Development Department 2 obtained 14 strains of NIES-2152 to NIES-2162, 2177, 2178, and 2179 from the National Institute for Environmental Studies Microbial System Storage Facility (NIES Collection). While subculturing the obtained strain, a strain having a high extracellular polysaccharide productivity and suitable for subculture was searched for.
Specifically, the NIES-2152 strain was divided into a plurality of cells, and subculture was repeated under the subculture conditions shown in Examples below. Subculture was repeated in the same manner for other strains. While repeating subculture, a strain having high productivity of extracellular polysaccharide and suitable for subculture was found out of these, and this strain was designated as PNC1 strain.
Based on the morphological observation on the PNC1 strain and the molecular phylogenetic tree based on the rRNA gene sequence, it was judged to be a Parachlorella kessreli unicellular green algae and named Parachlorella kessleri-PNC1 strain. As a new algae, this strain was founded on July 19, 2012, 1-1-6 Higashi Tsukuba, Tsukuba City, Ibaraki Prefecture 305-8566, Tsukuba Center Chuo No. 6, National Institute for Product Evaluation Technology (NITE-) IPOD) was deposited as Parachlorella kessleri-PNC1 strain (FERM BP-11493).

As a medium used when culturing the single cell green algae of the genus Parachlorella of the present disclosure to produce a polysaccharide, a culture containing a carbon source containing a carbon source such as glucose in a basic medium capable of culturing general single cell green algae A medium is preferred. The basic medium in which unicellular green algae can be cultured is, for example, (Appl Microbiol Biotechnol. 2011 Jul; 91 (1): 31-46.Best practices in heterotrophic high-cell-density microalgal processes: achievements, potential and possible limitations. F, Cook S, Zachleder V, Hauser S, Kovar K].
Examples of inorganic salts in the basal medium include trace inorganic components such as KH ₂ PO ₄ and MgSO _{4, and} examples of nitrogen sources include ammonium sulfate and urea. Examples of the basal medium suitable for culturing the Parachlorella unicellular green algae of the present disclosure include the composition of the medium shown in Table 1, and the content of each component is preferably in the range of ± 10%. .
Examples of the carbon source include monosaccharides such as glucose and fructose; oligosaccharides such as sucrose. One or more of these may be selected 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 culturing for producing the polysaccharide of the present disclosure, culturing under an aerobic condition is preferable because the polysaccharide can be stably produced. Aeration culture is preferable from the viewpoint of improving the productivity of polysaccharides. Examples of the aeration means include stirring, shaking, aeration, and bubbling. These can be performed alone or in combination of two or more. Thereby, an appropriate gas comes to be mixed in the culture medium.
What is necessary is just to adjust dissolved oxygen (DO) in a culture medium so that it may be aerobic conditions, Preferably it is 3 ppm or more, More preferably, it is 5-13 ppm.
Although culture temperature is not specifically limited, What is necessary is just about the normal temperature of 5-40 degreeC.
Further, the pH (20 ° C.) in the culture medium is preferably 4-9, more preferably 5-8.
The culture period is not particularly limited, but it is preferable to produce polysaccharides by setting one cycle to 4 days to 2 weeks. Moreover, when performing preculture and main culture, the period of main culture should just be about 4 days-1 week. At this time, light irradiation may be performed, but a polysaccharide can be produced without light irradiation.
In addition, when increasing the number of algal bodies under autotrophic culture conditions, it is possible to use sunlight; light from artificial light sources such as lamps for plant cultivation and LEDs, and supply and agitate carbon dioxide. Is preferred.

  In addition, the polysaccharide production method of the present disclosure may be either a batch type or a continuous type, but the continuous type is preferable for improving productivity. Moreover, any of open system culture and closed system culture may be sufficient, for example, closed culture in a culture tank, open system open culture, etc. are mentioned. Closed culture is preferable from the viewpoint of culture condition management.

After the polysaccharide of the present disclosure is produced in the unicellular green algae or the modified body, the polysaccharide is separated from the alga body by physical means and chemical means such as aqueous solution washing, ultrasonic wave, centrifugation, filtration, etc. It is preferred to obtain a supernatant containing the disclosed polysaccharide. At this time, it is preferable to separate the polysaccharide from the surface of the cultured alga, since it is also possible to continuously produce the polysaccharide using the alga. For example, at the time of centrifugation, it may be 5000 to 9000 rpm for about 5 minutes. Further, it is preferable to perform filtration using a filter aid and a filtration filter alone or in combination because impurities can be further removed.
The obtained polysaccharide of the present disclosure may be appropriately adjusted to a diluted solution, a concentrated solution, a dried product, or the like. As the drying means, freeze-drying is preferable.

The polysaccharides of the present disclosure can remove inert impurities by a known separation / purification technique, such as liquid-liquid separation, solid-liquid separation, filtration membrane, activated carbon, adsorption resin, ion exchange resin, and the like. It may be purified.
It is preferable to dissolve the polysaccharide of the present disclosure in an aqueous solution, then remove the insoluble fraction and collect the water-soluble fraction. It is preferable to mix when dissolving in water, and the water temperature is preferably 5 to 40 ° C. After dissolution, it is preferably left at a low temperature (eg, 1 to 10 ° C.) (eg, 5 to 15 hours).
The insoluble fraction is preferably removed by physical means such as centrifugation and ultrafiltration membrane. Thereby, since a polysaccharide with higher water solubility can be obtained, it can be used for various applications. Further, the obtained water-soluble polysaccharide is preferably lyophilized.

3. Use of Polysaccharides of the Present Disclosure The polysaccharides of the present disclosure increase the amount of fibroblast growth factor-7 (FGF-7), which is one of the family of fibroblast growth factors, as shown in the Examples below. Is allowed to do. Therefore, the polysaccharide of the present disclosure has an FGF-7 production promoting action, a fibroblast proliferation activation action, a skin anti-aging prevention action, a wrinkle improvement action, a hair matrix cell proliferation action, a hair growth action and the like.

Here, it is known that the fibroblast growth factor has an angiogenesis action, an action for inhibiting the synthesis of collagen and fibronectin, and has a strong affinity for heparin.
In addition, fibroblasts are contained in the dermis, and by maintaining the proliferative function of fibroblasts, the moisture content, flexibility, elasticity, etc. of the skin are maintained in a good state, which is beautiful and healthy. The condition of the skin (skin) is maintained. Moreover, the production amount of the fiber component and the substrate of the skin decreases due to the decrease in the proliferation function of fibroblasts. This causes changes in the surface shape and physical properties of the skin, and causes skin roughness, rough skin, wrinkles, sagging, and the like. Therefore, it is considered that having a fibroblast proliferation activation action contributes to an anti-aging prevention action, wrinkle improvement action and the like of the skin.
FGF-7 is a factor secreted from hair papilla cells, and it is considered that this FGF-7 acts on hair matrix cells to promote hair matrix cell proliferation and promote hair growth. Therefore, it is considered that having FGF-7 production promoting action contributes to hair matrix cell growth action, hair growth action and the like.

Furthermore, as described above, the polysaccharide of the present disclosure has an FGF-7 production promoting action, a fibroblast proliferation activation action, and the like, thereby reducing FGF-7 production, reducing fibroblast proliferation ability, hair matrix. It is considered possible to prevent, ameliorate, or treat diseases and symptoms caused by reduced cell proliferative ability, fibroblast proliferative ability and the like.
Examples of various diseases and symptoms resulting from a decrease in FGF-7 production, a reduction in fibroblast proliferation ability, a reduction in hair matrix proliferation ability, and the like of the present disclosure include skin aging, wrinkle formation, and alopecia. . Examples of the alopecia include male pattern alopecia, female diffuse alopecia, alopecia areata and the like.

  Moreover, it is recognized that the polysaccharide of this indication increases the quantity of vascular endothelial growth factor (VEGF) as shown in the below-mentioned Example. Therefore, the polysaccharide of the present disclosure has a VEGF production promoting action, a vascular endothelial growth activating action, an angiogenesis promoting action, a hair matrix activation action, a hair growth promoting action and the like.

Here, vascular endothelial growth factor is known to act on vascular endothelial cells to promote cell proliferation and migration, promote angiogenesis, and enhance vascular permeability. Thus, having a vascular endothelial growth activating effect is thought to contribute to a vascular endothelial cell growth promoting action, an angiogenesis promoting action, and the like.
In addition, the polysaccharide of the present disclosure has vascular endothelial growth factor (VEGF), which is secreted from hair papilla cells, and this VEGF promotes angiogenesis, thereby activating hair matrix cells. It is thought that hair growth is promoted. Thus, having a VEGF production promoting action is considered to contribute to hair matrix activation, hair growth promoting action, and the like.
Furthermore, as described above, the polysaccharide of the present disclosure has a VEGF production promoting action and a vascular endothelial growth activating action, thereby causing various diseases caused by a decrease in VEGF production, vascular endothelial growth inhibition, hair matrix activity reduction, and the like. It is possible to prevent, improve or treat symptoms and symptoms.
Examples of various diseases and symptoms resulting from suppression of VEGF production, suppression of vascular endothelial growth, reduction of hair matrix cell activity, and the like of the present disclosure include vascular dysplasia, abnormal development of the vascular system, and alopecia.

The polysaccharides of the present disclosure have been found to activate hair papilla cells as shown in the Examples below. Therefore, since the polysaccharide of the present disclosure also has an FGF-7 production promoting action and a VEGF production promoting action as described above, it contributes very well to hair matrix cell growth activation action, hair growth action and the like. Conceivable.
In general, the search for a hair growth active substance is performed from a low molecular weight compound such as adenosine or an organic solvent extract derived from a plant, etc., but the polysaccharides of the present disclosure are different in molecular weight and properties. It is quite surprising that the polysaccharides of the present disclosure, which are molecular and polysaccharides, and which are novel, have an excellent hair-growth effect.

Therefore, the polysaccharide of the present disclosure has the above-described FGF-7 production promoting action, fibroblast proliferation activation action, cell activation action, hair papilla cell activation action, hair growth action, skin anti-aging prevention action, and wrinkle improvement. May be used for action, VEGF production promoting action, vascular endothelial growth stimulating action, etc., and FGF-7 production promoter, fibroblast proliferation activator, cell proliferation activator, hair papillary cell proliferation activator, It can be used in various preparations intended for use as described above, such as hair restorers, anti-aging agents for skin, wrinkle improving agents, VEGF production promoters, vascular endothelial growth activators, etc., and produce these various preparations Can be used for.
In addition, the “hair growth agent” in the present disclosure has effects such as growing hair, promoting hair growth, maintaining hair health, preventing hair loss, and reducing hair loss. It means to exert, and includes “hair restorer”.

Normally, melanin also plays a role in protecting the body from ultraviolet rays in the skin, but overproduction and uneven accumulation cause skin darkening and spots. In general, melanin is changed from tyrosine to dopa, from dopa to topaquinone, and then formed through an intermediate such as 5,6-dihydroxyindophenol by the action of an enzyme of tyrosinase produced in pigment cells. Therefore, in order to prevent or improve skin darkness (skin pigmentation), that is, for whitening, it is effective to inhibit the melanin production process or to lighten and bleach the already produced melanin. It has been.
As such a substance having an inhibitory action on tyroxynase activity, for example, JP-A-2002-370962 discloses that an ethanol extract from Fuji tea branch leaves has excellent tyrosinase activity.
Japanese Patent Application Laid-Open No. 2001-026530 discloses that a lipid called sphingolipid has an inhibitory action on melanin production of melanoma B16 cells.
As described above, since many whitening effects have been reported for low molecular weight systems such as plant extracts and sphingolipids, little knowledge has been found that white polysaccharides have a whitening action. .

The polysaccharide of the present disclosure has been observed to have a B16 melanoma cell growth inhibitory effect, as shown in Examples below. Therefore, the polysaccharide of the present disclosure has a melanin production inhibitory action and a whitening action.
Furthermore, the polysaccharide of the present disclosure has a melanoma cell growth inhibitory action and a melanin production inhibitory action, and thus is considered capable of preventing, improving, or treating diseases and symptoms caused by melanoma cell growth promotion and melanin production increase. It is done.
Examples of various diseases and symptoms resulting from the promotion of melanoma cell proliferation and increased melanin production of the present disclosure include skin cancer, skin darkening (skin pigmentation, malignant melanoma), and the like.
Therefore, the polysaccharide of the present disclosure may be used for the melanoma cell growth inhibitory action, the melanin production inhibitory action, the whitening action, etc. as described above, and the melanoma cell growth inhibitor, the melanin production inhibitor, It can be used in various preparations intended for use as described above, such as whitening agents, and can be used to produce these various preparations.

In addition, the polysaccharide of the present disclosure is ingested, administered or brought into contact with animals including humans, as described above, fibroblast proliferation activation action, FGF-7 production promotion action, skin anti-aging prevention action, wrinkle improvement It is a substance that can be used to achieve an action, a vascular endothelial growth activation action, a VEGF production promotion action, a hair growth action, a melanoma cell growth inhibition action, a melanin production inhibition action, a whitening action and the like.
Accordingly, the polysaccharides of the present disclosure have the above-described fibroblast proliferation activation, FGF-7 production promotion, skin anti-aging prevention, wrinkle improvement, vascular endothelial growth activation, VEGF production promotion, hair growth, and melanoma cell growth inhibition. For the purpose of inhibiting melanin production and whitening, it can be incorporated into external preparations for skin, cosmetics, pharmaceuticals, quasi drugs, foods and functional foods (for example, foods for specified health use), etc. Hair restorers, whitening agents and the like are useful as these external preparations for skin, cosmetics and the like.

The polysaccharide of the present disclosure is particularly preferably used for an external preparation for skin, cosmetics, and quasi drugs, and a preparation that is brought into contact with the skin for application or the like is suitable.
The content of the polysaccharide in the preparation such as the hair restorer or whitening agent is preferably 0.001 to 0.01% by mass, more preferably 0.01 to 0.1% by mass, and still more preferably 0.00. It is 1-1 mass%.

In addition, you may use for the said hair restorer, whitening agent, etc., combining the arbitrary component with the polysaccharide of this indication as needed. Other preferable components may be pharmaceutically or food-acceptable components. For example, cell activators, antioxidants, moisturizers, UV inhibitors, solvents (water, alcohols, etc.), oil agents , Surfactant, thickener, powder, chelating agent, pH adjuster, emulsifier, stabilizer, colorant, brightener, flavoring agent, flavoring agent, excipient, binder, disintegrant, lubricant , Diluents, osmotic pressure adjusting agents, fragrances and the like, and these may be blended according to the intended preparation.
The form of the hair-restoring agent and whitening agent is not particularly limited, and may be any form such as liquid, paste, gel, solid, and powder.

The present technology can employ the following configurations.
[1] A polysaccharide in which the sugar residue of the basic structure of the polysaccharide is composed of at least galactose and mannose, and the galactose has a furanose type.
[2] The polysaccharide according to [1], wherein the content of the galactose is 50 to 79% by mass in the total neutral sugar.
[3] The polysaccharide according to [1] or [2], wherein the polysaccharide has a molecular weight of 3 to 9 × 10 ⁴ in GPC measurement.
[4] The polysaccharide according to any one of [1] to [3], wherein a ratio of sugar residues of the galactose and mannose is 2.5 to 3.5: 1.
[5] The polysaccharide according to any one of [1] to [4], which is an extracellular polysaccharide derived from parachlorella. Preferred is a polysaccharide derived from Parachlorella kessreli. In addition, the polysaccharide is preferably water-soluble.

[7] FGF-7 production promoter, VEGF production promoter, fibroblast, wherein the sugar residue of the basic structure of the polysaccharide is composed of at least galactose and mannose, and contains a polysaccharide having a furanose type in the galactose Growth activator, vascular endothelial growth activator, cell proliferation activator, hair restorer, melanin production inhibitor, whitening agent, wrinkle improving agent, skin anti-aging agent, angiogenesis promoter.
[8] FGF-7 production promoter, VEGF production promoter, fibroblast proliferation activator, vascular endothelial proliferation activator containing the parachlorella extracellular polysaccharide according to any one of [2] to [5] Agent, cell proliferation activator, hair restorer, melanin production inhibitor, whitening agent, wrinkle improving agent, skin anti-aging agent, or angiogenesis promoter.
[9] The cell proliferation activator according to [7] or [8], wherein the cell proliferation activation is hair papilla cell proliferation activation.
[10] A skin external preparation containing a polysaccharide in which a sugar residue having a basic structure of a polysaccharide is composed of at least galactose and mannose, and the galactose has a furanose type.
[11] Preferably, the polysaccharide of the various preparations according to [7] to [13] is the polysaccharide according to any one of [2] to [5].

[12] FGF-7 production promoter, VEGF production promoter, fibroblast proliferation activator, vascular endothelial growth activator, cell proliferation activator, hair restorer, melanin production inhibitor, whitening agent, wrinkle improving agent, skin Use of a polysaccharide in which the sugar residue of the basic structure of a polysaccharide is composed of at least galactose and mannose, and the galactose has a furanose type, for the production of an anti-aging agent or angiogenesis-promoting agent.
The polysaccharide according to any one of [2] to [5] is preferable.
[13] FGF-7 production promoter, VEGF production promoter, fibroblast proliferation of polysaccharides in which the sugar residue of the basic structure of the polysaccharide is composed of at least galactose and mannose, and the galactose has a furanose type Use as an activator, vascular endothelial growth activator, cell proliferation activator, hair restorer, melanin production inhibitor, whitening agent, wrinkle improving agent, skin anti-aging agent or angiogenesis promoter.
The polysaccharide according to any one of [2] to [5] is preferable.
[14] Reduction of FGF-7 production, reduction of fibroblast proliferation ability, reduction of hair matrix proliferation ability, reduction of fibroblast proliferation ability, reduction of VEGF production, inhibition of vascular endothelial growth, reduction of hair matrix activity, (For prevention, improvement or treatment of diseases caused by melanoma cell proliferation promotion and melanin production increase, etc.) The sugar residue of the basic structure of the polysaccharide is composed of at least galactose and mannose, and the galactose has a furanose type Is a polysaccharide.
The polysaccharide according to any one of [2] to [5] is preferable.

Further, the present technology can employ the following configurations.
(1) A method for producing a polysaccharide, comprising subjecting Parachlorella unicellular algae or a modified body to subculture under aerobic conditions in a carbon source-containing culture medium and recovering the produced polysaccharide.
(2) The above-mentioned (1) to (1), wherein the polysaccharide is a polysaccharide in which the sugar residue of the basic structure of [1] polysaccharide is composed of at least galactose and mannose, and the galactose has a furanose type. 10. The method for producing a polysaccharide according to any one of 10). The polysaccharides [2] to [5] are preferred.
(3) The method for producing a polysaccharide according to (1) or (2) above, wherein the unicellular algae of Parachlorella is Parachlorella kessreli.
(4) The method for producing a polysaccharide according to any one of (1) to (3), wherein the concentration of the carbon source in the medium is 0.1 to 10% by mass.
(5) The method for producing a polysaccharide according to (1) to (4), wherein the carbon source is one or more selected from glucose, fructose and sucrose.
(6) The method for producing a polysaccharide according to any one of (1) to (5), wherein the culture is subordinate culture under aerated conditions.
(7) The method for producing a polysaccharide according to any one of (1) to (6), wherein the dissolved oxygen concentration in the culture is 3 to 13 ppm.
(8) The method for producing a polysaccharide according to any one of (1) to (7), wherein the culture temperature is 5 to 40 ° C.
(9) The method for producing a polysaccharide according to any one of (1) to (8), wherein the pH of the culture is 4 to 9.
(10) The method for producing a polysaccharide according to any one of (1) to (9), wherein the algal bodies are removed by centrifuging and filtration singly or in combination after the culturing.
(11) The method for producing a polysaccharide according to any one of (1) to (10), wherein the algal bodies are removed, freeze-dried, and further dissolved in an aqueous solution, and then the water-soluble fraction is collected.
(12) A polysaccharide obtained by the method for producing a polysaccharide according to any one of (1) to (11).
(13) The method for producing a polysaccharide according to any one of (1) to (12), which is an alga body named Parachlorella kessleri-PNC1 and deposited as FERM BP-11493.
(14) Algae named Parachlorella kessleri-PNC1 and deposited as FERM BP-11493.
[Examples of prior application]

Hereinafter, examples and comparative examples will be given to specifically describe the present invention (present technique), but the present invention (present technique) is not limited to the examples.

Panac Co., Ltd. New Business Development Department 2 obtained 14 strains of NIES-2152 to NIES-2162, 2177, 2178, and 2179 from the National Institute for Environmental Studies Microbial System Storage Facility (NIES Collection). Dividing each strain into a plurality of subcultures in the medium shown in Table 1, and from these, in the optical microscope, those that do not stain the ink body outside the cell body of the cell body very much The strain was searched for as a high productivity of extracellular polysaccharides.
Here, a large amount of polysaccharides are produced extracellularly; the separation of alga bodies and polysaccharides is easy and the polysaccharides can be easily recovered; the alga bodies can be reused for polysaccharide production. Being suitable for subculture, it is possible to cultivate in a large tank, and it is considered an algal body suitable for industrial production.
While repeating subculture, a strain having high productivity of extracellular polysaccharide and suitable for subculture was found out of these, and this strain was designated as PNC1 strain.

The algal PNC1 strain was subcultured in the medium shown in Table 1 and stained with ink using an optical microscope. A portion of the cell body that was not stained with ink was found outside the cell body. As a result of separating and analyzing the polysaccharides by the methods shown in the following Production Examples 1 to 3 for the portion not stained with the ink, it was found that the polysaccharides were produced outside the algal cells ( FIG. 1).
Further, by morphological observation and molecular phylogenetic tree based on the rRNA gene sequence (FIG. 2), the PNC1 strain was judged to be a Parachlorella kessreli unicellular green algae and named Parachlorella kessleri-PNC1 strain. Deposited as a Parachlorella kessleri-PNC1 strain (FERM BP-11493) at the National Institute for Microbiology (NITE-IPOD), National Institute of Technology and Evaluation.

Production Example 1: Production method of polysaccharide of the present disclosure
A slant medium (50 ml) containing Parachlorella kessleri-PNC1 strain (FERM BP-11493) is added to a 3 L baffled Erlenmeyer flask containing 250 ml of glucose-containing culture medium, and an illuminance of 8000 to 10,000 lux at 28 ° C. and 160 rpm. Pre-cultured for 4 days. In addition, you may increase the number of algal bodies preliminarily before preculture.
The glucose-containing culture medium contains 10 g of glucose per liter of the basal medium shown in Table 1.
240 ml of the preculture was transferred to a 30 L jar fermenter and main culture was carried out for 12 days, which was designated as the main culture. The culture conditions at this time were aerobic conditions of 30 ° C., 230 rpm, aeration 0.56 vvm, internal pressure 0.3 kgf / cm ² , pH 7.2, and no light irradiation was performed.
The main culture was centrifuged (7000 rpm (6500 G), 25 ° C.) and separated into a supernatant liquid containing the target polysaccharide and algal bodies. Further, the stock solution was concentrated to 2 L using a UF membrane (SIP1013) with a molecular weight cut off of 6000. 200 mL of an anion exchange resin was added to the concentrate (2 L), recovered to Brix 0.1, concentrated to 500 mL by UF membrane filtration, and freeze-dried to obtain a parachlorella-derived polysaccharide. The yield was 0.2 to 0.4 g per liter of the culture solution.

Production Example 2: Purification of polysaccharide 40 mL of distilled water was added to 1 g of a polysaccharide derived from Parachlorella and mixed and stirred to obtain a suspension. This suspension was allowed to stand at 4 ° C. overnight, and this suspension was centrifuged (7000 rpm, 6500 g, 5 minutes) to separate into a supernatant and a precipitate. The supernatant was freeze-dried to obtain a water-soluble parachlorella-derived polysaccharide (hereinafter referred to as “water-soluble parachlorella polysaccharide”) (0.4 to 0.5 g).

Example 1: Structural analysis of water-soluble parachlorella polysaccharide [molecular weight of polysaccharide]
As a result of measuring the molecular weight (Mw) of the water-soluble parachlorella polysaccharide by GPC analysis, it was 6.2 × 10 ⁴ , Mw (weight average molecular weight) / Mn (number average molecular weight) = 1.5 (FIG. 3). P1). Moreover, as a result of measuring the molecular weight (Mw) of the water-soluble parachlorella polysaccharide obtained by carrying out similarly to the above-mentioned manufacture examples 1 and 2 by GPC analysis, 6.7 * 10 < ⁴ >, Mw (weight average molecular weight) / Mn (number average molecular weight) = 1.5 (see P2 in FIG. 3).
<GPC analysis>
Measurement column: TSKgel GMPW _XL (7.6mmID x 300 mm: Tosoh Corporation) in series Measurement temperature: 40 ° C
Mobile phase: 0.1 M NaNO ₃
HPLC system: Shimadzu LC-20 system Detector: Differential refractometer LaboSystem RI-2000
Reference material: Pullulan (Showa Denko P-52)
Software used (GPC calculation): LC Solutions, GPC software (Shimadzu)

[Polysaccharide composition]
The water-soluble parachlorella polysaccharide was hydrolyzed with 2M-TFA (trifluoroacetic acid) at 120 ° C. for 2 hours. This hydrolyzate was reduced with NaBH ₄ 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 measurement was performed with a GL Science GC-353 gas chromatograph (column SP-2330 (Spelco); detector FID; constant temperature analysis at 220 ° C.).
They were arabinose (4.7%), rhamnose (4.0%), xylose (1.7%), mannose (22.5%) and galactose (67.1%).

Moreover, methylation analysis was performed about water-soluble parachlorella polysaccharide.
The polysaccharide sample was dissolved in DMSO, powdered NaOH was added and stirred, and then CH ₃ I was added to methylate the polysaccharide. After recovering the methylated polysaccharide, it was hydrolyzed at 120 ° C in 2M-TFA for 2 hours, reduced with NaBD ₄ and then acetylated with acetic anhydride and 1-methylimidazole. The obtained derivative was analyzed by GC-MS.
GC-MS was performed using Shimadzu QP-5000 GC-MS gas chromatograph mass spectrometer [column: SPB-50 (0.32 × 30 m)].
As the mannose residue of the polysaccharide, a non-reducing end, 1,2-linked, and 1,3-linked residues were observed. Further, most of the galactose residues of the polysaccharide are furanose, and as the galactose residues of the polysaccharide, non-reducing ends, 1,2-linked furanose, 1,5-linked furanose, 1,6-linked Furanose, 1,2,6-linked furanose, and 1,3-linked pyranose were observed.

In addition, by ¹ H-NMR analysis (Varian Unity plus 500, 1H: 500 MHz, 13 C: 125 MHz, [solvent: heavy water (D ₂ O), measurement temperature is 30 ° C., internal standard: DSS]), water-soluble parachlorella polysaccharide Many of the body's galactose residues were found to exist in the furanose form.
As a result of measuring the protein content (Raleigh method) and uronic acid content (carbazole-sulfuric acid method) of the water-soluble parachlorella polysaccharide, the presence of protein and uronic acid was recognized, although not in large quantities.

The DL of galactose and mannose present in the water-soluble parachlorella polysaccharide was examined.
A sample of the water-soluble parachlorella polysaccharide was hydrolyzed in 2M TFA at 120 ° C. for 2 hours, and then the acid was removed under a stream of nitrogen.
(S)-(+)-2-butanol (100 μL) and acetyl chloride (7.5 μL) were added to the hydrolyzate and heated at 60 ° C. overnight to form butyl glycoside. After removing the reagent under a nitrogen stream, the derivative obtained by trimethylsilylation was analyzed by GC. At this time, derivatives of known D-Gal, L-Gal, D-Man and L-Man were prepared in the same manner, and the absolute configuration of the constituent sugars was confirmed by comparison with the respective retention times. D-galactose and D-mannose were observed.
<GC conditions>
Column: SPB-1 (Spelco, 0.32 mm x 30 m)
GC: GL Science GC-353 gas chromatograph (detector FID)
Carrier gas: Nitrogen Oven temperature: 135-200 ° C (1 ° C / min)

  From this, it was estimated that the sugar residue of the basic structure of the polysaccharide of the present disclosure is mainly composed of at least galactose and mannose, and most of the galactose exists in the furanose type.

Example 2: Hair papilla cell activation test [human normal hair papilla cells]
For the preservation of cryopreserved human normal hair papilla cells (TOYOBO, Code No. CA60205a) during sleep, subculture, and growth to the required number of cells, a medium dedicated to hair papilla cells was used. The culture was performed in an incubator (5% CO ₂ , 37 ° C.).

In addition, it shows below about the reagent etc. which are used in Example 2 and Example 3 mentioned later.
<Reagent>
DMEM medium (Nacalai tesque, 14242-65)
FBS (Invitrogen, Cat. No. 10091-148)
Penicillin-streptmycin solution (Nacalai tesque, Cat.No.26253-84)
0.25% trypsin-EDTA solution (Nacalai tesque, 32777-44)
PBS (Nissui Pharmaceutical Co., Ltd., Code No. 05913)
Melanin (Sigma, Cat. No. M8631-100MG)
Phenyl-1-thiourea (PTU) (Wako, Cat. No. 166-13702)
Dimethyl sulfoxide (Nacalai tesque Cat. No. 13445-74)
Theophyline (Wako, Cat. No. 209-09932)
Hair papilla cell growth medium (TOYOBO, Code No.TMTPGM-250) additive-containing subculture set for hair papilla cells (TOYOBO, Code No. CA090K)
Viable cell count reagent SF (nacalai tesque, Cat. No. 07553)
Adenosine (Wako, Cat.No. 016-10493)
<Others>
Minisart 0.2 μm (Sartorius, Code No. 16534)
96 well culture plate (SUMILON, Cat. No. MS-8096F)
24 well cell culture cluster (TPP, Code No. 92424)
15 ml centrifuge tube (Corning, Code No. 430766)
Precision microplate reader (Molecular Devices)
Thermo Alumi Bath (IWAKI, ALB-121)
<Standard products>
[1] Theophylline was used to promote melanin production. Theophylline was dissolved in DMSO and sterilized by filtration to prepare a 36 mg / ml solution. At the time of use, it was diluted with a medium and adjusted to 90 μg / mL.
[2] Adenosine
Adenosine was used as a positive control for the dermal papilla cell activation test. Adenosine was dissolved in DMSO and sterilized by filtration to prepare a 100 mM solution. At the time of use, it was diluted with a medium and adjusted to 100 μM.

[Hair nipple toxicity test]
Papilla cells are seeded in a 96-well plate at a concentration of 1.4 × 10 ⁴ cells / 100 μL / well, and the following day (80% confluent), each concentration (0.01, 0.1, 1% by mass) of water-soluble parachlorella The test medium was replaced with a polysaccharide and cultured for 24 hours.
Cell proliferation was measured with a living cell number measuring reagent SF. A growth medium containing 10% of a viable cell count measurement reagent was added at 100 μL / well, and incubated at 37 ° C. and 5% CO ₂ . After 30 and 90 minutes, the absorbance of the reaction medium was measured using a microplate reader (measurement wavelength: 450 nm, control wavelength: 595 nm). The number of tests was three.
At any concentration of the water-soluble parachlorella polysaccharide of 0.01, 0.1 and 1% by mass, almost no cytotoxicity was observed on the hair papilla cells.

[Papilla cells / cell activation test]
Papilla cells are seeded in a 96-well plate at a concentration of 5 × 10 ³ cells / 100 μL / well and contain the test substance at each concentration (0.00001, 0.001, 0.1% by mass) the next day (50% confluent). The culture medium was changed to a test medium and cultured for 24 hours and 72 hours. Cell proliferation was measured using a living cell count reagent SF. A growth medium containing 10% of a viable cell count measurement reagent was added at 100 μL / well, and incubated at 37 ° C. and 5% CO ₂ . After 30 and 90 minutes, the absorbance of the reaction medium was measured using a microplate reader (measurement wavelength: 450 nm, control wavelength: 595 nm). The number of tests was 5.
As shown in Table 2, an excellent hair papillary cell proliferation activating effect was observed at a concentration of 0.00001% by mass or more of the water-soluble parachlorella polysaccharide.
<Significant difference test>
A significant difference test was performed in the comparative test section. The test was performed as Student T-test, and P <0.05 (the null hypothesis was less than 5%) was judged to be significant.

In general, the cell growth process can be broadly divided into an exponential growth phase in which division is active and a maintenance phase in which division is stopped and cells are stably maintained. In this test, in order to verify the effect of water-soluble parachlorella polysaccharide at each stage, water-soluble parachlorella polysaccharide was added at a cell density (confluent) of 40 to 60%, and the number of living cells after 24 hours of culture was measured. . Further, the effect on the cells in the maintenance phase was verified by further 48 hours of culture (total 3 days).
In the exponential growth phase (1 day of culture) and the maintenance phase (3 days of culture), the lowest concentration group of the water-soluble chlorella polysaccharide showed about 1.1 to 1.4 times the growth promotion as compared to the non-added group. This may be because the test substance has a cell division promoting effect and a maintenance promoting effect.
On the other hand, adenosine, which is a positive control, showed a growth promoting effect in the exponential growth phase, but in the maintenance phase, the number of living cells was the same as that in the non-added group. This may be because adenosine promotes cell division but does not act on cell maintenance after the cell density reaches 100%.
Therefore, it was confirmed that the polysaccharide of the present disclosure has an excellent cell proliferation activating effect and an excellent hair papillary cell proliferation activating effect.

Example 3: Whitening action (melanin production inhibition) test [cell culture]
B16 melanoma cells were seeded in a 24-well plate at 3 × 10 ⁴ cells / ml / well, and the following day, the test medium was replaced with 1 ml of a test medium containing water-soluble parachlorella polysaccharides of each concentration (0.01, 0.1% by mass). Cultured for 48 hours. Thereafter, the amount of intracellular melanin in all cells was measured. The number of tests was 5.
[Measurement of intracellular melanin]
After removing the culture supernatant, the cells adhering to the wells are washed with 1 ml of PBS. 100 μl of 0.25% trypsin-EDTA solution is added to the cells, and the cells are allowed to stand at 37 ° C. under 5% CO ₂ for 1 minute. Cells detached by trypsin treatment were collected with 1 ml of PBS and transferred to a 1.5 ml tube. Then, it centrifuged at 1,000 rpm and 4 degreeC for 5 minute (s), the supernatant was removed, and the cell pellet was wash | cleaned twice by PBS. After centrifuging the cell suspension at 3,000 rpm for 5 minutes, PBS was removed and the cells were suspended in 110 μl of 1N sodium hydroxide solution. In order to measure the total amount of melanin including detached cells, the culture medium supernatant and the washed PBS were collected and centrifuged at 3,000 rpm for 5 minutes, and the resulting precipitate and the cell suspension were mixed at 100 ° C. Cells were lysed by incubating for 30 minutes, 80 μl of this lysate was transferred to a 96-well plate, the absorbance at 405 nm was measured with a microplate reader, and the amount of intracellular melanin was calculated. Commercially available synthetic melanin was used as a standard product of melanin. A 2.5 to 160 μg / ml solution was prepared with a 1N sodium hydroxide solution, and a calibration curve was prepared from each absorbance. Based on this standard calibration curve, the amount of melanin in each specimen was calculated.
The results of the melanin production inhibition test are shown in Table 4. The water-soluble parachlorella polysaccharide showed significant inhibition of melanin production.
Therefore, it was recognized that the polysaccharide of this indication has the outstanding melanin production inhibitory effect and the outstanding whitening effect | action.

Example 4: Fibroblast proliferation activation test [culture supernatant]
The culture conditions of the dermal papilla cells and the test substance treatment conditions were the same as in Examples 1 and 2 above. The culture supernatant was stored frozen until measurement.
〔reagent〕
FGF7 Human ELISA kit (abcam, Code No. ablOO519)
〔Analysis equipment〕
Precision microplate reader (Molecular Devices)
[Preparation of reagents, etc.]
The required amount is 100 μL for FGF-7 measurement, and the required amount is 200 μL for VEGF measurement.
About 90 μL of the culture supernatant obtained in Example 2 was diluted about 4 times with 1 × PBS and used for FGF-7 measurement.
[Measurement of the amount of FGF-7 in the culture supernatant]
[1] FGF-7 was measured according to the protocol of the kit. Details are described below.
[2] Based on FGF-7 standard stock solution (50 ng / ml), 400, 133.3, 44.44, 14.81, 4.94, 1.65, 0.55 pg / Prepare ml FGF-7 standard solution.
[3] FGF-7 standard solution and 100 μl of specimen (culture supernatant) were added to each well of the FGF-7 solid-phased microplate and reacted at room temperature for 2 hours 30 minutes (primary reaction).
Remove the solution in the well and wash 4 times with the washing solution.
[4] 100 μl of biotin-labeled anti-FGF-7 antibody solution is added to each well and reacted at room temperature for 60 minutes (secondary reaction).
[5] The solution in the well is removed and the plate is washed 4 times with a washing solution.
[6] Add 100 μl of streptapidin solution to each well and react for 45 minutes at room temperature.
[7] Add 100 μl of the enzyme substrate solution to each well, and allow to stand at room temperature for 30 minutes in the dark (coloring reaction).
[8] Add 50 μl of the reaction stop solution to each well, mix with a plate mixer for 1 minute, and then measure the absorbance of each well with a plate reader (measurement wavelength: 450 nm).
[9] Calculate the FGF-7 concentration in the specimen from the standard curve.

The amount of FGF-7 produced in the dermal papilla cell culture supernatant collected in Example 2 was measured.
As shown in Tables 5 and 6, FGF-7 was not detected in the positive control, adenosine, in the dermal papilla cell culture for 1 day. However, the FGF-7 production promoting effect was observed in the water-soluble parachlorella polysaccharide. Further, in 3 days, a high FGF-7 production promoting effect was observed even at a concentration of 0.01 to 1% by mass of the water-soluble parachlorella polysaccharide compared with adenosine.
Therefore, it was confirmed that the polysaccharide of the present disclosure has a very excellent FGF-7 production promoting action and fibroblast proliferation activating action.

Example 5: Vascular endothelial growth activation test [culture supernatant]
The culture conditions of the dermal papilla cells and the test substance treatment conditions were the same as in Examples 1 and 2 above. The culture supernatant was stored frozen until measurement.
〔reagent〕
Human VEGF Quantikine ELISA (R & D Systems, Cat. No. DVEOO)
〔Analysis equipment〕
Precision microplate reader (Molecular Devices)
[Measurement of VEGF content in culture supernatant]
VEGF was measured according to the kit protocol. Details are described below.
[1] Based on VEGF standard stock solution (2000 pg / ml), prepare 1000, 500, 250, 125, 62.5, 31.2, 15.6 pg / ml VEGF standard solution using reaction buffer. .
[2] VEGF standard solution and 200 μl of specimen (culture supernatant) were added to a VEGF-immobilized microplate to which 50 μl of diluted solution was added to each well, and reacted at room temperature for 2 hours (primary reaction).
[3] The solution in the well is removed and washed with a washing solution three times.
[4] 200 μl of a horseradish oxidase-conjugated VEGF antibody solution is added to each well and reacted at room temperature for 2 hours (secondary reaction).
[5] The solution in the well is removed and washed with a washing solution three times.
[6] Add 200 μL of the substrate solution to each well and react at room temperature for 20 minutes.
[7] Add 50 μl of the reaction stop solution to each well, mix with a plate mixer for 1 minute, and then measure the absorbance of each well with a plate reader (measurement wavelength: 450 nm).
[8] Calculate the VEGF concentration in the specimen from the standard curve.
[Significant difference test]
A significant difference test was performed in the comparative test section. The test was performed as Student T-test, and a value of 0.05 (null hypothesis was less than 5%) was judged to be significant.

The amount of VEGF produced in the dermal papilla cell culture supernatant collected in Example 2 was measured.
As shown in Tables 7 and 8, the positive control, adenosine, had little VEGF production promoting effect. However, all the water-soluble parachlorella polysaccharides were found to promote VEGF production in the high concentration test section.
Therefore, it was confirmed that the polysaccharide of the present disclosure has a very excellent VEGF production promoting action and vascular endothelial growth activation action.

  In the actual hair tissue, dermal papilla cells are three-dimensionally proliferated. Therefore, it is considered that the production-promoted FGF-7 and VEGF function sufficiently, and further, the dermal papilla cell proliferation activation effect is also recognized. The polysaccharide of the present disclosure is considered to exhibit a very excellent hair-growth effect.

  Since the polysaccharide of the present disclosure has various effects and can be mass-produced, it can be applied in a wide range of fields. In addition, since the polysaccharide manufacturing method of the present disclosure can efficiently produce a high-value-added polysaccharide, the industrial applicability can be achieved in terms of manufacturing cost and stable supply. Very expensive.

(1) Parachlorella kessleri-PNC1 strain (FERM BP-11493) (Contract date: July 19, 2012)
Deposit place: 1-1-1 Higashi Tsukuba City, Ibaraki 305-8566, Tsukuba Center Chuo No. 6, National Institute for Product Evaluation Technology Patent Microbiology Deposit Center (NITE-IPOD).

[Claims of Prior Application]
[1]
A polysaccharide in which the sugar residue of the basic structure of the polysaccharide is composed of at least galactose and mannose, and the galactose has a furanose type.
[2]
The polysaccharide according to claim 1, wherein a content of the galactose is 50 to 79% by mass in all neutral sugars.
[3]
The polysaccharide according to claim 1 or 2, which is an extracellular polysaccharide derived from parachlorella.
[4]
A hair restorer containing a polysaccharide in which a sugar residue having a basic structure of a polysaccharide is composed of at least galactose and mannose, and the galactose has a furanose type.
[5]
A hair restorer containing the polysaccharide according to claim 2 or 3.
[6]
A hair matrix cell growth activator comprising the polysaccharide according to any one of claims 1 to 3.
[7]
The FGF-7 production promoter containing the polysaccharide of any one of Claims 1-3.
[8]
A VEGF production promoter containing the polysaccharide according to any one of claims 1 to 3.
[9]
The melanin production inhibitor containing the polysaccharide of any one of Claims 1-3.
[10]
The skin external preparation containing the polysaccharide of any one of Claims 1-3.
[11]
A method for producing a polysaccharide, comprising subjecting Parachlorella unicellular algae or a modified body to subculture under aerobic conditions in a carbon source-containing culture medium and recovering the produced polysaccharide.
[12]
The method for producing a polysaccharide according to claim 11, wherein the dissolved oxygen concentration in the culture is 3 to 13 ppm.
[13]
The polysaccharide production according to claim 12 or 13, wherein after the culturing, centrifugation and filtration are used alone or in combination to remove algal bodies and then freeze-dried and further dissolved in an aqueous solution, and then the water-soluble fraction is recovered. Method.
[14]
The method according to any one of claims 11 to 13, wherein the unicellular algae of the genus Parachlorella is Parachlorella kessreli.
[15]
The method for producing a polysaccharide according to any one of claims 11 to 14, wherein the unicellular algae of the genus Parachlorella is an algal body named Parachlorella kessleri-PNC1 and deposited as FERM BP-11493.
[16]
Algal body named Parachlorella kessleri-PNC1 and deposited as FERM BP-11493.

[Embodiments of the present invention]
Hereinafter, embodiments of the present invention using polysaccharides of prior applications will be described.

  In the present embodiment, the polysaccharide obtained by Production Example 1 of the prior application, particularly the polysaccharide obtained by centrifuging the algal culture and then purified by UF membrane filtration (hereinafter also referred to as purified polysaccharide) is used. Used as an immunostimulator. In the present embodiment, the immunostimulant refers to a drug having an action of enhancing the immune action in the administered subject. In addition, you may use the polysaccharide refine | purified by the manufacture example 2 of a prior application as an immunostimulator. Moreover, you may use the polysaccharide which is not refine | purified. The immunostimulant may contain components other than polysaccharides.

  In polysaccharides, the sugar residue of the basic structure is composed of at least galactose and mannose, and the galactose has a furanose type. Moreover, in a polysaccharide, it is preferable that content of galactose is 50-79 mass% in all the neutral sugars.

(Origin)
It is preferable to use a natural polysaccharide extracted from a living organism as the polysaccharide. As such an organism, it is preferable to use freshwater unicellular algae, particularly Parachlorella unicellular algae. The parachlorella unicellular algae is preferably an alga body named Parachlorella kessreli, particularly Parachlorella kessleri-PNC1, and deposited as FERM BP-11493. The deposit destination is as described above.

(Mechanism of action)
The immunostimulatory agent of the present embodiment is different from conventional therapeutic agents for viral infections such as acyclovir and oseltamivir phosphate (hereinafter referred to as conventional drugs), and produces neutralizing antibody amounts and secretory IgA antibody amounts of pathogens. Both are promoted. Secretory IgA antibodies play a central role in the mucosal immune defense system. The mucosal immune defense system is a local mucosal mechanism that protects the mucosal surface of an administration subject that is constantly exposed to antigens and microorganisms. As the mucosal immune defense system, for example, the digestive tract immune defense system, the respiratory tract immune defense system and the nasal cavity immune defense system are known.

  From the difference in the production pattern of the antibody amount, it is considered that the polysaccharide, which is the main component of the immunostimulant, has a different mechanism of action from oseltamivir phosphate. That is, it is speculated that polysaccharides not only act on pathogens but also act on the human immune system to promote antibody production. Specifically, for example, when a polysaccharide is orally administered, the polysaccharide is taken into the intestinal tract immune system due to the involvement of Peyer's patch. Thereafter, the polysaccharide is recognized by innate immune cells such as macrophages, dendritic cells, and NK cells, and the information is transmitted to T cells and B cells. Finally, it is presumed that when the acquired immune system is activated, the target pathogen-specific antibody producing ability is also improved. In this case, since the entire immune system is activated, it is expected that the immune system can function efficiently against this pathogen when another pathogen enters the body of the administration target next time. The

(Subject of administration)
The administration target of the immunostimulant of the present embodiment is not particularly limited. In addition to mammals such as humans, mice, pigs and cows, it can also be administered to birds such as chickens. Conventional drugs and vaccines greatly depend on the target animal species and pathogen type. On the other hand, it is considered that the immunostimulant of the present embodiment does not depend on the animal species or pathogen types to be administered.

(Method of administration)
The immunostimulant of the present embodiment is mainly composed of a polysaccharide that is produced by a harmless organism even if the administration subject eats it. Therefore, the administration period, dosage, and administration frequency are not particularly limited. For example, when administered to mice, 1 mg per dose may be administered twice a day, or 5 mg per dose once a day for 14 consecutive days. The immunostimulant of this embodiment is preferably administered continuously for 3 days or longer, more preferably 7 days or longer. Thereby, sufficient immunostimulatory function can be provided to the administration subject. The administration route is not particularly limited, but oral administration is preferred from the viewpoint of convenience and noninvasiveness.

  In addition, the immunostimulant of the present embodiment can be used alone as an inhibitor of influenza A virus, and functions of these drugs can be used in combination with anti-infectives such as conventional drugs and vaccines. It is thought that it can be reinforced.

(Pathogen)
The immunostimulant of this Embodiment can be used in order to raise the resistance with respect to the pathogen of administration object. As such pathogens, microorganisms such as bacteria, fungi, protozoa, and various viruses are mainly assumed. As bacteria, for example, Staphylococcus aureus, Mycobacterium tuberculosis, Vibrio cholerae, Diphtheria, Shigella, Bacillus anthracis, and protozoa such as Plasmodium are considered. As viruses, for example, RNA viruses such as influenza virus, AIDS virus and coronavirus, DNA viruses such as herpes virus (HSV-1, HSV-2, etc.) and adenovirus are envisaged. The immunostimulant of the present embodiment is considered to be particularly effective for general influenza viruses that grow in the respiratory tract. Conventional drugs and vaccines greatly depend on the type of target pathogen. On the other hand, the immunostimulant of the present embodiment has a growth inhibitory effect against a plurality of types of pathogens.

(Usefulness)
As described above, the immunostimulant of the present embodiment is mainly useful in the following points.
-Resistance to pathogens in administration subjects can be easily increased.
-The polysaccharide as the main component can be purified in large quantities using fresh water unicellular algae that can be easily cultured.
-The polysaccharide, which is the main component, is considered harmless to the administration subject. Therefore, the administration method can be appropriately selected.
・ There is no limit to the subject of administration. Therefore, it can be administered to a wide range of organisms.
・ Low specificity for pathogens. Therefore, it can be used to increase resistance against various pathogens.
・ It is considered that the mechanism of action is different from that of conventional drugs. Therefore, it is thought that it can be used together with a conventional drug for the purpose of reinforcing the conventional drug.
[Embodiments of the present invention]

Example 6: Comparison of chronological changes in symptoms in HSV-2 infected mice For HSV-2 infected mice, a purified polysaccharide (hereinafter referred to as “Parachlorella kessleri-PNC1: FERM BP-11493”) obtained periodically from Parachlorella kessleri-PNC1: The time course of the symptom was compared with and without administration of purified parachlorella polysaccharide. In this example, the following administration groups and methods were used.

(Administration group)
Control (saline) (single dose: 20 μl) (hereinafter, negative control group)
Parachlorella purified polysaccharide (PCP2) (single dose: 1 mg / 20 μl) (hereinafter, sample administration group)

(Method)
[1] BALB / c mice (female, 6 weeks old, n = 5) were subcutaneously injected with 17-medroxyprogesterone (Sigma) (3 mg / 0.1 ml / mouse) 6 days before and 1 day before virus inoculation. .
[2] HSV-2 (1 × 10 ⁴ PFU / 20 μl / mouse: lethal dose) was inoculated locally (genital).
[3] Administration was carried out by local administration twice a day (9:00 am and 6 pm) 1 hour before infection.
[4] The onset score was recorded from the day of infection (0 days later) to 14 days after infection. The score used is as follows. The score was calculated as an average score of 5 individuals.
0: asymptomatic, 1: mild redness locally, 2: severe redness / swelling locally, 3: appearance of exudate locally, 4: paralysis of hind limbs, 5: death

(result)
FIG. 4 is a graph showing changes over time in the pathology of mice infected with herpes simplex virus type 2 (HSV-2). FIG. 4 (A) is a graph showing changes over time in the onset score of mice infected with HSV-2. FIG. 4 (B) is a table showing the death status of mice infected with HSV-2.

  In the negative control group (Comparative Example 6), the disease developed from 4 days after infection (FIG. 4A), and all cases died 9 days after infection (FIG. 4B). On the other hand, in the sample administration group (Example 6), all cases died 11 days after the infection (FIG. 4B), but until then, the onset was significantly suppressed compared to the negative control group (FIG. 4). (A)).

Example 7: Comparison of the amount of HSV-2 proliferation in HSV-2 infected mice In Example 6, the amount of HSV-2 proliferation 3 days after infection was compared. Following the method [3] of Example 6, 3 days after infection, the local area was washed with phosphate buffered saline (100 μl), and the amount of virus in the washing solution was measured in Vero cells (African green monkey kidney) cultured in a 35 mm dish. (Derived cells) was measured by a plaque assay method. Student's t test was used for statistical processing of the obtained data.

  FIG. 5 is a graph showing the amount of growth of HSV-2 three days after infection in the mouse infected with HSV-2 in FIG.

  In the sample administration group (Example 7), although there was no significant difference due to large variation among individuals, the amount of virus 3 days after infection decreased compared to the negative control group (Comparative Example 7).

  From the above Examples 6 and 7, it was shown that the parachlorella purified polysaccharide can prolong the survival period of the mouse by suppressing the onset of genital herpes due to HSV-2.

Example 8: Comparison of changes over time in symptoms of influenza A virus-infected mice Changes in symptoms over time when the parachlorella purified polysaccharide was regularly administered to influenza A virus-infected mice. Compared. In this example, the following administration groups and methods were used.

(Administration group)
Control (sterile distilled water) (0.4 ml / day) (hereinafter, negative control group)
Parachlorella purified polysaccharide (PCP2) (5 mg / day) (hereinafter, sample administration group)

(Method)
[1] BALB / c mice (female, 6 weeks old, n = 10) were inoculated intranasally with influenza A virus (A / NWS / 33) (2 × 10 ⁴ PFU / 50 μl / mouse).
[2] Administration was carried out by oral administration twice a day (9:00 am and 6 pm) 7 days before infection.
[3] The change in body weight was recorded from the day of infection (0 days later) to 14 days after infection. The body weight was converted into a relative value when the body weight on the day of infection in each administration group was defined as 100 (%). Student's t test was used for statistical processing of the obtained data. *, **, and *** in FIGS. 7 to 11 indicate cases where p <0.05, p <0.01, and p <0.001, respectively, in comparison with the negative control group.

(result)
FIG. 6 is a graph showing changes in body weight over time in mice infected with influenza A virus.

  In the negative control group (Comparative Example 8) and the sample administration group (Example 8), no death was observed. In the sample administration group, the decrease in the average body weight of the mice was significantly suppressed from 1 day to 14 days after the infection as compared with the negative control group.

Example 9: Comparison of proliferation amount of influenza A virus in mice infected with influenza A virus In Example 8, the proliferation amount of influenza A virus 3 days after infection was compared. Following method [3] in Example 8, 3 days after infection, the viral load in lung and airway lavage fluid (BALF) was measured by plaque assay using MDCK cells (canine kidney-derived cells) cultured in 35 mm dishes. did.

(result)
FIG. 7 is a graph showing the proliferation amount of influenza A virus 3 days after infection in mice infected with the influenza A virus of FIG. 6 (5 mice in each group).

  In the sample administration group, the amount of influenza A virus was significant in both lung (Example 9-1) and airway lavage fluid (Example 9-2) compared to the negative control group (Comparative Examples 9-1 and 9-2). (P <0.01).

Example 10: Comparison of the amount of neutralizing antibody in mice infected with influenza A virus (5 mice per group) In Example 8, the amount of neutralizing antibody against influenza A virus 14 days after infection was compared. In this example, in addition to the sample administration group and the negative control group, an oseltamivir administration group administered with oseltamivir phosphate (Tamiflu (registered trademark), Roche) (0.2 mg / day) was used as a reference example. Following the method [4] of Example 8, 14 days after infection, the amount of neutralizing antibody in serum and airway lavage fluid (BALF) was measured by plaque assay using MDCK cells.

(result)
FIG. 8 is a graph showing the amount of neutralizing antibody 14 days after infection in mice infected with the influenza A virus of FIG. FIG. 8A is a graph showing the amount of neutralizing antibody in serum. FIG. 8B is a graph showing the amount of neutralizing antibody in the airway washing solution.

  In the oseltamivir-administered group, the amount of neutralizing antibody was significantly higher in both serum (Reference Example 10-1) and airway lavage fluid (Reference Example 10-2) than in the negative control group (Comparative Examples 10-1 and 10-2). Decreased (p <0.05 and p <0.001 respectively). On the other hand, in the sample administration group, the amount of neutralizing antibody was significantly increased in both serum (Example 10-1) and airway lavage fluid (Example 10-2) compared to the negative control group (p <0.001). .

Example 11: Comparison of the amount of virus-specific secretory immunoglobulin A (IgA) in mice infected with influenza A virus In Example 8, the amount of virus-specific secretory IgA antibody against influenza A virus 14 days after infection was compared. did. Similar to Example 10, in this example, in addition to the sample administration group and the negative control group, the oseltamivir administration group to which oseltamivir phosphate (Tamiflu (registered trademark), Roche) was administered (0.2 mg / day) Used as a reference example. Following the method [4] of Example 8, 14 days after infection, the amount of virus-specific secretory IgA antibody in airway lavage fluid (BALF) and excreta was measured by the following enzyme-linked immunosorbent assay (ELISA method). did.
1) Sample preparation: The airway washing solution is diluted 5-fold with physiological saline. The excreta is added with 10 times the amount of physiological saline, allowed to stand for 15 minutes, vortexed, centrifuged at 4 ° C. (3,000 rpm, 10 minutes), and the supernatant is collected.
2) An antigen (influenza virus purified by ultracentrifugation, 1 mg / ml) is added to each 96-well plate for ELISA at 50 ml / well and treated at 37 ° C. for 1 hour.
3) Wash 3 times with 0.05% Tween-20 phosphate buffered saline (PBS-T).
4) Add 5% skimmed milk at 100 ml / well and leave at 4 ° C. overnight to block.
5) Add 50 ml / well of the sample of 1) above and treat at 37 ° C. for 1 hour.
6) Wash 3 times with PBS-T.
7) Add 50 ml / well of secondary antibody (HRP-labeled anti-mouse IgA antibody) and treat at 37 ° C. for 1 hour.
8) Wash 5 times with PBS-T.
9) Add 50 ml / well of the substrate solution (0.4 mg / ml o-phenylenediamine + 10 ml / ml hydrogen peroxide) and let stand at room temperature for 20 minutes.
10) Add 4N H ₂ SO ₄ at 25 ml / well to stop the reaction.
11) Measure absorbance at 490 nm and determine the average of three measurements for each sample. The IgA amount of the sample is calculated from a separately prepared IgA calibration curve.
The amount of IgA antibody in the excrement serves as an index of the amount of IgA antibody secreted in the intestine.

(result)
FIG. 9 is a graph showing the amount of virus-specific secretory immunoglobulin A (IgA) 14 days after infection in mice infected with influenza A virus of FIG. FIG. 8 (A) is a graph showing the amount of IgA antibody in the airway washing solution. FIG. 8 (B) is a graph showing the amount of IgA antibody in excreta.

  In the oseltamivir-administered group, IgA antibody levels were significantly higher in both the airway lavage fluid (Reference Example 11-1) and the excreta (Reference Example 11-2) than in the negative control group (Comparative Examples 11-1 and 11-2). Decreased (p <0.001 and p <0.01, respectively). On the other hand, compared with the negative control group, the IgA antibody amount was significantly increased in the excrement (Example 11-2) in the sample administration group (p <0.001). Moreover, although a significant difference was not seen, the airway washing liquid (Example 11-1) also showed a higher value than the negative control group.

  From the results of Examples 8 to 11 described above, it was revealed that the purified parachlorella polysaccharide effectively suppresses the growth of influenza A virus. Also, unlike oseltamivir phosphate, both the neutralizing antibody amount and the IgA antibody amount were increased compared to the negative control group. This suggests that parachlorella purified polysaccharide has a different mechanism of action from oseltamivir phosphate. Specifically, it is speculated that the purified parachlorella polysaccharide has an effect of suppressing the growth of influenza A virus and stimulating the body's immune system to promote antibody production. Therefore, parachlorella purified polysaccharide can be used alone as an inhibitor of influenza A virus, and the function of these drugs can be enhanced by using it together with anti-infective drugs such as conventional drugs and vaccines. It is considered possible.

  The present invention is not limited to the above-described embodiments, and various modifications such as design changes can be added based on the knowledge of those skilled in the art. Embodiments to which such modifications are added Can also be included in the scope of the present invention.

Claims (7)

  An immunostimulant comprising a polysaccharide in which a sugar residue having a basic structure of a polysaccharide is composed of at least galactose and mannose, and the galactose has a furanose type.   The immunostimulant according to claim 1, wherein the content of the galactose is 50 to 79% by mass when the total amount of the neutral monosaccharide composition of the polysaccharide is 100% by mass.   The immunostimulant according to claim 1 or 2, wherein the polysaccharide is an extracellular polysaccharide derived from a unicellular alga of the genus Parachlorella.   The immunostimulant according to claim 3, wherein the polysaccharide is a polysaccharide obtained by separating and purifying a polysaccharide from the Parachlorella unicellular algae.   The immunostimulant according to claim 3 or 4, wherein the Parachlorella unicellular algae is Parachlorella kessreli.   The immunostimulant according to claim 5, wherein the parachlorella genus Kesseri is an algal body named Parachlorella kessleri-PNC1 and deposited as FERM BP-11493.   The immunostimulant according to any one of claims 1 to 6, which is used in combination with another anti-infective drug.