JP6152205B1 - Cosmetics, pharmaceutical compositions, and methods for producing them - Google Patents

Abstract

The present invention provides cosmetics and various pharmaceutical compositions that can be produced safely and stably even when applied to humans, and methods for producing them. A cosmetic and various pharmaceutical compositions containing a culture supernatant obtained by culturing mesenchymal stem cells derived from horses. These production methods include a collection step of collecting cells from a racehorse or a racehorse; a selection step of selecting mesenchymal stem cells from the collected cells; none containing ammonium metavanadate, phenol red, and mercaptoethanol. A culture step of culturing the mesenchymal stem cells using a serum medium; and a recovery step of separating and recovering the culture supernatant produced in the culture step from the serum-free medium and the mesenchymal stem cells. [Selection] Figure 8

Description

  The present invention relates to cosmetics, various pharmaceutical compositions, and methods for producing them.

  Various cosmetics effective for relieving or preventing skin wrinkles or improving skin gloss and smoothness have been proposed so far. The active ingredients of these cosmetics can be broadly classified into those derived from plants and those derived from animals. Known active ingredients derived from animals include extracts that are directly collected from animals such as placenta and collagen, and those that use animal cell culture supernatants.

  Patent Document 1 describes that a cosmetic product is obtained by freeze-drying a culture supernatant of stem cells collected from the pulp, porcine bone marrow, or porcine adipose tissue of a porcine fallen deciduous tooth. This cosmetic product is said to have effects such as wrinkle removal, whitening, hair growth, and hair growth. Patent Document 2 describes a composition for the treatment of an injured part using a culture supernatant of dental pulp stem cells of human deciduous deciduous teeth as a medicinal ingredient.

International Publication No. 2013/118877 Pamphlet International Publication No. 2011/118795 Pamphlet

  The culture supernatant is produced as a supernatant by culturing cells in a medium. In particular, since various growth factors and cytokines are contained in the culture supernatant produced when culturing mesenchymal stem cells, various cosmetic effects and medicinal effects are expected.

  In obtaining cosmetics and various pharmaceutical compositions intended to be applied to humans, two aspects are required: safety and stem cell culture rate. In terms of safety, both the viewpoint of not containing ingredients that should not be administered to humans such as deleterious substances and poisons, and the viewpoint of no risk of infectious diseases and epidemics are conceivable. In addition, since the number of stem cells to be cultured and the amount of the culture supernatant are approximately proportional, the culture rate of the stem cells is an important viewpoint for producing the culture supernatant stably in a commercial manner. In order to increase the culture rate of stem cells, it is possible to collect a large number of stem cells (primary stem cells) from the living organism of the donor animal, and the cell division at each generation is fast when the collected stem cells are subjected to primary culture and subculture. It becomes important.

  However, when a pig is a donor animal as in Patent Document 1, it is difficult to eliminate infectious diseases and epidemics due to problems in the breeding environment. In addition, regarding the pulp and bone marrow of porcine fallen deciduous teeth, it is difficult to collect a large amount of primary stem cells because there are few stem cells. Similarly, when using dental pulp stem cells of human deciduous deciduous teeth as in Patent Document 2, it is difficult to collect sufficient primary stem cells. Further, according to the study of the present inventor, since stem cells derived from dental pulp or bone marrow are difficult to proliferate even if they are cultured, a large amount of stem cells cannot be obtained even if they are subcultured. For this reason, it is difficult for the methods of Patent Document 1 and Patent Document 2 to stably produce a safe culture supernatant.

  The present invention has been made in view of the above problems, and provides cosmetics and various pharmaceutical compositions that can be produced safely and stably even when applied to humans, and methods for producing the same. To do.

ADVANTAGE OF THE INVENTION According to this invention, the cosmetics containing the culture supernatant obtained by culturing the mesenchymal stem cell derived from the fat of a racehorse are provided. The mesenchymal stem cell is a fat-derived mesenchymal stem cell collected from the racehorse or a passage stem cell obtained by subculturing the mesenchymal stem cell, and the cosmetic of the present invention is ammonium metavanadate. It is characterized by not containing any of phenol red, mercaptoethanol and antibiotics.
Further, according to the present invention, the composition for anti-allergy treatment, composition for hair growth or hair growth, composition for anti-inflammation treatment comprising a culture supernatant obtained by culturing mesenchymal stem cells derived from fat of a racehorse And a composition for promoting skin metabolism are provided.

Further, according to the present invention, a collecting step of collecting adipocytes from a racehorse ; a selecting step of selecting mesenchymal stem cells from the collected adipocytes ; any of ammonium metavanadate, phenol red , mercaptoethanol, and antibiotics A culture step of culturing the mesenchymal stem cells using a serum-free medium not containing; and the culture supernatant produced in the culture step is separated and recovered from the serum-free medium and the cultured mesenchymal stem cells A method for producing a cosmetic or pharmaceutical composition comprising a recovery step.

  According to the cosmetics, various pharmaceutical compositions and methods for producing them of the present invention, cosmetics and various pharmaceutical compositions that can be produced safely and stably even when applied to humans are obtained.

(A) And (b) is an affected part photograph which shows the state before and behind applying an Example composition to the skin of a dog infected with bacteria (Example 1). (A) And (b) is an affected part photograph which shows the state before and behind applying an Example composition to hair (Example 2). It is a graph which shows a time-dependent transition of the number of hair loss (Example 3). (A)-(d) is a photograph which shows the state before and behind applying an Example composition to eyelashes and eye periphery (Example 4). (A) And (b) is an affected part photograph which shows the state before and behind applying an Example composition to a postoperative mark (Example 5). (A)-(c) is a photograph which shows the state before and behind applying an Example composition to a wolf (Example 6). (A)-(d) is a photograph which shows the state before and behind applying an Example composition to atopic dermatitis (Example 7). It is a graph which shows the improvement effect of atopic dermatitis. (A)-(c) is a photograph which shows the state before and behind applying an Example composition to seborrheic eczema (Example 8). It is a graph which shows the improvement effect of hay fever (Example 9).

  Hereinafter, embodiments of the present invention will be described.

  The cosmetic and pharmaceutical composition of the present invention contains a culture supernatant obtained by culturing mesenchymal stem cells derived from horses. Examples of the pharmaceutical composition include an antiallergic treatment composition, a hair growth or hair growth composition, an antiinflammatory treatment composition, and a skin metabolism promoting composition. The medicinal effects of these compositions will be described in detail using examples described later.

  The culture supernatant used in the present embodiment is liquid, and is a culture solution after culturing mesenchymal stem cells. The culture supernatant contains a number of growth factors, and exhibits an antioxidant effect, wound healing effect, wrinkle improvement or prevention effect, hair regeneration effect and the like.

  The cosmetic and pharmaceutical composition of the present embodiment can be applied to humans, but is not limited to humans, and is widely applied to non-human mammals such as pets such as dogs and cats and domestic animals such as cows and horses. May be. As an example of application to humans, it can be used for various uses such as skin care, improvement of hair, suppression of various inflammations, and healing of trauma. In addition, even when applied to non-human mammals, it can be used for various purposes such as healing of body surface trauma and eczema, regeneration of body hair, treatment of tumors such as oral maxillomatosis of pet animals and domestic animals. .

  The mesenchymal stem cells for obtaining the culture supernatant of this embodiment are stem cells collected from horses or passage stem cells obtained by subculturing the stem cells. Stem cells have self-renewal ability and pluripotency, and from their differentiation ability, mesenchymal stem cells, hematopoietic stem cells, neural stem cells, embryonic stem cells and the like are known. Of these, mesenchymal stem cells, which are somatic stem cells derived from mesodermal tissue (mesenchyma), are used in the present embodiment. Mesenchymal stem cells are involved in the remodeling of many tissues in the body such as skin, bone, blood vessels, muscles or tendons. There is no particular limitation as to which mesenchymal stem cells are collected from which tissue of the horse. For example, stem cells derived from umbilical cord, umbilical cord blood or placenta (hereinafter also referred to as umbilical cord or the like) (umbilical cord stem cells) in addition to stem cells derived from fat (adipose stem cells). These stem cells are stem cells that can be collected while keeping the horse alive. In addition, adipose-derived stem cells can collect a large amount of stem cells relatively easily compared to bone marrow-derived stem cells, are excellent in mass culture, have excellent multipotency to differentiate into various cells, and differentiate into organ stem cells It is preferable in terms of a high ratio.

  The primary stem cells of mesenchymal stem cells for obtaining the culture supernatant used in this embodiment are collected from horses. Horses are a general term for equine animals, and include horses, zebras and donkeys in a narrow sense. Of these, racehorses and racehorses are particularly preferable.

  The racehorse includes not only a horse registered as a racehorse but also a breeding horse that can be registered as a racehorse. Race horses are obliged to register their horse names. In Japan, for example, names that have passed the horse name examination by a specified organization (Japan Studbook International as of 2016) are registered as official horse names. . A competition horse refers to a horse that can participate in a competition to conduct a competition. Examples of competitions include horse riding competitions, equestrian competitions, cattle chasing, rodeos, polos, and halters. The racehorse includes a racehorse having a pedigree and registered with pedigree information and a racehorse registered without pedigree information.

  In particular, the mesenchymal stem cells for obtaining the culture supernatant of the present embodiment include stem cells collected from racehorses or racehorses having pedigree information, or passage stem cells obtained by subculturing such stem cells. preferable. By using a racehorse or racehorse with pedigree information as an animal that provides mesenchymal stem cells, physical management is always performed, such as continuous vaccination, and drug administration histories and injury histories are generated for multiple generations ( In the case of a racehorse, it is possible to grasp over at least 5 generations), and thus the safety of the culture supernatant can be ensured. Furthermore, it is preferable to use an active racehorse or racehorse having pedigree information as a providing animal. By being an active racehorse or racehorse, the medication administration history and injury history are more reliably guaranteed.

The method for producing a cosmetic or pharmaceutical composition of the present embodiment (hereinafter sometimes abbreviated as the present method) includes at least a collection step, a selection step, a culture step, and a recovery step.
In the collection step, cells are collected from the donor animal. Particularly in this method, cells are preferably collected from an active racehorse or racehorse.
In the selection step, mesenchymal stem cells are selected from the collected cells.
In the culturing step, the mesenchymal stem cells are cultured using a serum-free medium. The serum-free medium used in this method is characterized by not containing any of ammonium metavanadate, phenol red and mercaptoethanol.
In the recovery step, the culture supernatant produced in the above culture step is separated and recovered from the serum-free medium and mesenchymal stem cells.

  Hereinafter, this method will be described in more detail.

(Collection process)
In the collection step of this method, cells are collected from a horse that is a donor animal. The method of collecting stem cells from horses is not particularly limited, and includes a method of collecting only stem cells from any tissue in animals, or a method of collecting stem cells together with any other biological component other than stem cells. It can be adopted as appropriate. As an example, in order to collect mesenchymal stem cells without killing the donor animal, it is preferable to collect subcutaneous fat in the buttocks. Racehorses, in particular, have low body fat, but a few grams of fat cells can be collected per head by excising the skin of the buttocks and collecting the subcutaneous fat. Thereafter, the skin can be transplanted to the affected area where fat cells are collected, whereby the collecting step of the present method can be performed while the donor animal is living. In addition, when the donor animal is a mare, mesenchymal stem cells may be collected from the umbilical cord or the like at the time of delivery. As a result, mesenchymal stem cells can be obtained without special surgical action at the time of delivery.

  According to the inventor's study, it was confirmed that the mesenchymal stem cells fractionated from the racehorse fat showed a good growth rate in the subsequent subculture. Therefore, in this method, it is desirable to culture a fat-derived mesenchymal stem cell collected from a racehorse to obtain a culture supernatant.

(Selection process)
In the selection step, sample cells such as fat cells and umbilical cords collected in the above-described collection step are finely cut. On the other hand, an enzyme solution containing collagenase is prepared in advance. A sample cell is treated with an enzyme solution in a thermostatic chamber, and then centrifuged to obtain a stromal vascular fraction containing mesenchymal stem cells (Stromal Vascular Fraction). Thereby, mesenchymal stem cells are selected from the cells collected from the donor animal. Hereinafter, the mesenchymal stem cells may be abbreviated as “stem cells”.

  The selected stem cells may be subsequently subjected to the following culture process or may be cryopreserved.

(Culture process)
In the culturing step, the stem cell sampled in the selection step, or the stem cell that has been cryopreserved and thawed after being cryopreserved, is subjected to primary culture in a medium and, if necessary, subculture. Cryopreservation and thawing steps may be performed between generations of subculture.

  The culture step of this method is performed by seeding stem cells in a medium accommodated in a culture container such as a dish or a flask. It is preferable to use a serum-free medium that does not contain animal-derived components. By using a serum-free medium, it is possible to obtain a medium that does not contain unknown components and contains only known components. More specifically, the serum-free medium used in the present method preferably contains at least human serum albumin, insulin, linoleic acid and transferrin and does not contain a poisonous or deleterious substance. Poisonous deleterious substances include ammonium metavanadate, phenol red and mercaptoethanol. Since the culture medium does not contain any of these poisonous and deleterious substances, the culture supernatant of this embodiment, and cosmetics and pharmaceutical compositions prepared using this, contain ammonium metavanadate, phenol red, and mercaptoethanol. None of them will be contained. For this reason, even when the cosmetic or pharmaceutical composition obtained by this method is applied to humans, the occurrence of side effects can be suppressed and can be safely applied. Moreover, it is preferable that the serum-free culture medium used for a culture | cultivation process does not contain an antibiotic. Note that cosmetics and pharmaceutical compositions do not contain the above components means that the concentration of these components is below the lower limit of detection in a general commercially available high performance liquid chromatography (HPLC) apparatus. Say.

  By using a serum-free medium containing only known ingredients in the medium used for the culturing process and containing no poisonous deleterious substances, the providing animal is a racehorse or racehorse having pedigree information as in the present method. The culture supernatant obtained by the above can be compliant with the “Act on the Ensuring Safety of Regenerative Medicine, etc.” prescribed by the Ministry of Health, Labor and Welfare.

  In the culturing step, a culture supernatant is produced as the supernatant of the medium. The culture supernatant contains biological components such as cytokines secreted from the stem cells. Cytokines are physiologically active proteins produced from cells and are generally known to be low molecular weight molecules having a molecular weight of 80,000 or less, more often 30,000 or less. The physiological activity means a property that a chemical substance acts on a specific physiological regulatory function in a living body. Examples of cytokines include, but are not limited to, chemokines, cell growth factors, interleukins, or interferons, or derivatives thereof. The chemokine is a low molecular weight protein of about 8 kDa to about 14 kDa, which is roughly classified into CC chemokine, CXC chemokine, C chemokine or CX3C chemokine. Chemokines act through chemokine receptors. Preferable examples of the cytokine contained in the culture supernatant include CXCL12 or a derivative thereof. The stem cell-containing solution containing CXCL12 or a derivative thereof preferably contains CXCR4 or a derivative thereof which is a receptor for CXCL12.

  In the culturing step, the medium may be changed, for example, every 3 to 5 days until the majority of the culture vessel becomes sub-confluent covered with stem cells and the rate of increase in the number of cells is reduced.

  In the case of subculture, when the cells become sub-confluent or confluent, the cultured stem cells are detached from the medium, the medium is replaced with a new one having the same composition, and the stem cells are seeded. In the case of stem cells obtained from adipocytes of racehorses, for example, it is possible to obtain 4 billion stem cells and 40 liters of culture supernatant from 2 grams of primary stem cells by 4 generations of subculture. . According to the study of the present inventor, this is more than 1000 times the number of stem cells (about 1 million) collected when subcultured stem cells collected from bone marrow.

(Recovery process)
In the recovery step, the culture supernatant produced in the culture step is separated and recovered from the serum-free medium and mesenchymal stem cells. Since the culture supernatant is produced as a supernatant of the culture medium, the culture supernatant can be aspirated and collected using a commercially available suction device. The timing of collecting the culture supernatant is not particularly limited, but may be performed, for example, at the timing of exchanging the medium.

  In addition, in this method, various post processes may be optionally applied to the collected culture supernatant. Examples of such post-process include a filtration process for filtering the collected culture supernatant, a dilution process for diluting the culture supernatant, and an addition process for adding other compounding ingredients as a cosmetic or pharmaceutical composition. For example, in obtaining cosmetics, examples of the ingredients added to the culture supernatant include alcohol, humectant, emulsifier, gelling agent, cream, fragrance, and preservative.

As described above, cosmetics and various pharmaceutical compositions containing the culture supernatant are prepared by this method.
The cosmetic product obtained by this method gives various cosmetic effects (skin care effect, hair care effect) to the skin and hair. Examples of hair include head hair, body hair, eyelashes, and eyebrows.
Specific skin benefits include moisturizing and keeping moisture, preventing dry skin, preventing rough skin, giving skin firmness, reducing the appearance of pores, giving skin gloss, These include the effects of adjusting the texture of the skin, suppressing bears and slack under the eyes, suppressing dullness of the skin, suppressing itching and scalp (dandruff) of the scalp. With regard to the hair, there are effects such as imparting firmness and stiffness to the hair, imparting moisture to the hair, making the hair supple, improving combing, and imparting gloss to the hair. In addition, the cosmetics referred to in the present invention are not particularly limited as long as they are applied or dispersed on human skin or hair and used for beauty, such as skin lotions, creams, milky lotions, cosmetics, facial cleansers, etc. Basic cosmetics; makeup cosmetics such as makeup base and teak; hair care products such as shampoos, conditioners and hairdressing products; oral care products such as mouthwashes; and other cosmetics such as perfumes, whitening agents and hair removal agents Can do. In addition, the cosmetics referred to in the present invention include medicinal cosmetics that exhibit medicinal effects as exemplified below.

  In addition, the cosmetic of the present invention may be provided as a fluid such as a liquid or cream in a container such as a bottle or a spray container, or may be provided by impregnating a sheet such as a nonwoven fabric or gauze. Good.

In addition, as a medicinal effect of the pharmaceutical composition obtained by this method, at least antiallergic action, hair growth and hair growth action, antiinflammatory action, and skin metabolism promoting action have been confirmed.
More specifically, the antiallergic action has been confirmed to improve allergic rhinitis (hay fever), allergic conjunctivitis, suppress bronchial asthma symptoms, and maintain asthma asymptomatic conditions for a long time.
As hair growth and hair growth action, it has been confirmed that hair is born, hair is reduced, hair loss is reduced, eyelash is elongated, and eyelashes are raised.
Anti-inflammatory effects include improvement and healing of endogenous eczema such as atopic dermatitis, healing of dermatitis (seborrheic eczema), healing of itching and swelling of contact dermatitis (rash) such as insect bites, dryness Healing of dermatosis (sebum-deficient eczema) and other common eczema has been confirmed.
Skin metabolism means metabolism of the existing keratin of the skin and does not include dermal regeneration such as hemostasis or healing of trauma (wound). Specific skin metabolism promoting effects include skin beauty effects such as improving skin gloss and smoothness, reducing localized keratin proliferation, and reducing abnormal growth of skin pigment cells. Or metabolic reduction of fibrous tissue overproduced in trauma scars and postoperative scars. The pharmaceutical composition obtained by the present method promotes skin metabolism by reducing and eliminating localized stratum corneum such as metatarsal stamatoma (wort and octopus), reducing wrinkles associated with abnormal proliferation of pigment cells, and Disappearance, reduction of trauma and postoperative scars, healing of burns and bruises have been confirmed.

The pharmaceutical composition of the present invention is a composition formulated as the main medicinal component of an antiallergic therapeutic agent, a hair growth or hair growth agent, an anti-inflammatory therapeutic agent, and a skin metabolism promoter. The reason why the culture supernatant of the present invention exhibits various medicinal effects as described above is not necessarily clear, but it is considered that the growth factor contained in the culture supernatant exhibits the following effects (i) to (v). It is done.
(i) Basic fibroblast growth factor: The culture supernatant of the present invention promotes angiogenesis of capillaries in the skin, and promotes the proliferation of fibroblasts secreting collagen, thereby exerting an effect of promoting skin metabolism. In addition, it has anti-allergic and anti-inflammatory effects due to its tissue repair and neuroprotective actions.
(ii) Epidermal growth factor: The culture supernatant of the present invention prevents and eliminates wrinkles by creating new skin cells, smoothes the skin, and regenerates new skin cells, thereby enhancing skin metabolism. It is thought that it demonstrates.
(iii) Transforming effect: The culture supernatant of the present invention increases the elasticity of the skin by strengthening the structure of collagen and elastin, promotes skin growth and wound healing, and produces new cells. It is thought to prevent and improve wrinkles.
(iv) Platelet-derived growth factor: It is considered that the culture supernatant of the present invention promotes skin cell regeneration and collagen synthesis, thereby preventing and improving wrinkles and promoting hair growth.
(v) Insulin-like growth factor: The culture supernatant of the present invention prevents and eliminates wrinkles by creating new skin cells, improves collagen feel and stimulates hair roots by increasing collagen, elastin and hyaluronic acid This is thought to promote hair growth.
(vi) Vascular Endothelial Cell Growth Factor: The culture supernatant of the present invention promotes hair growth by transporting nutrients to the hair root, prevents and improves wrinkles by producing new cells, and energy to the skin It is thought to improve skin gloss by giving.

  Examples of the present invention will be described below, but the present invention is not limited thereto.

(Collecting stem cells)
An active racehorse was used as a donor animal, and its hip was cut open, and about 5 g of fat cells were collected to obtain a sample for collecting stem cells. The sample was cut to about 1 mm square and then treated with a collagenase solution at 37 ° C. for 1 hour. Next, the treated sample is centrifuged for about 10 minutes with a centrifugal force of 400 × g, and undigested adipose tissue fragments are removed with a cell strainer equipped with a 100 μm mesh, and then adipose-derived mesenchymal stem cells A stromal blood vessel fraction containing was obtained.

(Stem cell culture)
An original serum-free medium containing human serum albumin, insulin, linoleic acid and transferrin, and not containing any of ammonium metavanadate, phenol red and mercaptoethanol was prepared and placed in a flask. The above-obtained fraction was seeded in the flask, and liquid stationary culture was performed under conditions of 37 ° C./5% CO ₂ . Every 2 days, the entire supernatant (culture supernatant) of the medium was collected and replaced with a fresh medium. The culture is terminated when it is confirmed that the cells adhere to an area of 80% or more and 90% or less with respect to the total area of the bottom surface of the flask with a phase contrast microscope, and the culture supernatant generated so far is removed. Collected.

  The collected culture supernatant was filtered with a mesh having an opening of 20 nm, and further diluted 50-fold with purified water to obtain Example compositions.

(Comparative Example 1)
The donor animal was changed from a racehorse to a dog, and subcutaneous fat was collected. Other than that, stem cells were cultured in the same manner as in the above Example.

(Comparative Example 2)
The donor animal was changed from a racehorse to a human, and subcutaneous fat was collected. Other than that, stem cells were cultured in the same manner as in the above Example.

  In the case of Comparative Example 1 and Comparative Example 2, the stem cell culture was stopped halfway, and the stem cells did not grow to an area of 80% or more with respect to the total area of the bottom surface of the flask. From this, it was confirmed that by using the donor animal as a horse, the collected stem cells proliferate well and a large amount of culture supernatant can be obtained.

  The Example composition obtained above was applied to various animals and humans as test animals, and the drug efficacy was verified.

Example 1
The subject animal was a dog whose skin was infected with bacteria, and the skin was inflamed as shown in FIG. 1 (a). When the example composition was sprayed on the inflammatory affected area of this subject animal at intervals of about 1 day, the swelling and redness of the inflammatory affected area disappeared as shown in FIG. 1 (b) one week after the start of spraying.
In addition, when 15 dogs of the same breed were sampled and the composition of the example was sprayed on the body surface and oral cavity at intervals of about 1 day for 2 weeks, the following was confirmed.
・ Natural hair growth ・ Cloud fat (dandruff) no longer appears ・ Moist hair is moist ・ Foul breath is suppressed ・ Severe oral edema that has developed in the upper jaw and penetrated to the nose has improved ・ Eczema As described above, according to this example, the hair growth effect and anti-inflammatory treatment effect of the present invention were confirmed.

(Example 2)
The subject animal (subject) is a middle-aged man (human) with thinned hair. As shown in FIG. 2 (a), when the hair at the top of the head was thin, the example composition was applied to the head at intervals of about 1 day, and after about 8 months from the start of application, FIG. 2 (b) As shown in FIG. 4, remarkable hair growth was observed, and the density of the hair on the parietal region increased dramatically.
According to the present Example, the hair growth effect and the hair growth effect of this invention were confirmed.

(Example 3)
The subject animal (subject) is an adult female (human) who suffers from hair loss. The Example composition was applied to the head every day to check the number of hair loss. FIG. 3 is a graph showing the time course of the total number of hair loss during shampooing, drying, and the total of these. On February 3, the third day from the start of application, the number of hair loss was 75 at the time of shampooing and 50 at the time of the dryer. However, the number of hair loss was shampooed on February 5, the fifth day from the start of application. The number of hair loss decreased to 60 at the time of shampooing and 38 at the time of drying. Furthermore, on February 21, the 21st day from the start of application, the number of hair loss decreased markedly at 32 during shampooing and 30 during drying.
According to this example, the hair growth effect (reduction of hair loss) of the present invention was confirmed.

Example 4
The subject animal (subject) is an adult woman (human) who suffers from short eyelashes. Example compositions were applied daily around the eyes. FIG. 4A shows the state at the start of application (January 29, 2016). FIG. 4B shows the state on the fourth application day (February 2, 2016), and FIG. 4C shows the state on the seventh application day (February 5, 2016). ) Is the state on the 11th day of application (February 8, 2016). Thus, it was confirmed that some eyelashes extended on the 4th day from the start of application (FIG. 4B), and on the 7th day, it was confirmed that the eyelashes extended as a whole.
Further, not only the eyelashes became longer, but also the number density was improved, and the eyelashes increased in elasticity and stiffness from the root to the tip, so that the eyelashes face upward as shown in FIG. 4 (c) and FIG. 4 (d). It was confirmed that it began to grow like a stand up.
Furthermore, it was confirmed that not only the hair growth effect of eyelashes but also the pupils were clearer and the double eyelashes were clearer than before and after application of the example compositions.
According to the present Example, the hair-growth effect (elongation of eyelashes) of the present invention and the elasticity increase effect of eyelashes (skin) were confirmed.

(Example 5)
The subject animal (subject) is a man in 30s (human). As shown in FIG. 5 (a), when the composition of the example was sprayed daily on the postoperative marks that were conspicuously generated on the head, as shown in FIG. In addition, the postoperative scars became assimilated with the surrounding skin, and the scars themselves became smaller.
According to this example, postoperative scar reduction, that is, skin metabolism promoting effect was confirmed.

(Example 6)
The subject animal (subject) is a woman (human) in their 40s. As shown in FIG. 6 (a), when the Example composition was sprayed daily on the women that had developed on the little toes of the foot, the pain disappeared and disappeared on the 6th day from the start of spraying ( (Refer FIG.6 (b)). After that, even after 8 months had passed, wort did not recur and remained in a normal state (see FIG. 6 (c)).
According to the present Example, the reduction | decrease of the localized keratin, ie, the skin metabolism promotion effect, was confirmed.

(Example 7)
The subject animal (subject) is a male child (human) who has developed atopic dermatitis. As shown in FIG. 7 (a), on January 14, 2016 before spraying the example compositions, a wide range of inflamed areas accompanied by a sore was confirmed under the armpits. When the Example composition was sprayed every day, the sores began to subside on January 25 of the same day on the 10th day (see FIG. 7B), and inflammation markedly on March 20 of the 65th day of the same year. It decreased (see FIG. 7 (c)), and there was almost no inflammation on the 94th day of April 18 of the same year, and significant healing was observed.

  Moreover, the graph which shows the result of having sprayed an Example composition every day over 14 days as a test animal (subject) as 20 adults (human) including man and woman who also develop atopic dermatitis. It is. Before the start of spraying and the last day of spraying, No. From the viewpoint of 1 to 14, the symptoms of atopic dermatitis were self-evaluated in 6 stages from 0 to 5. The questionnaire results are also shown in Table 1. Point 0 is "I don't care at all", Point 1 is "I don't care", Point 2 is "Neither", Point 3 is "Somewhat worried", Point 4 is "I'm worried", Point 5 is Corresponding to “Very anxious”, the smaller the symptom, the smaller the point. FIG. 8 shows the last spray point in Table 1 on the left side and the pre-spray point on the right side in comparison with each monitor name. In Table 1, the improvement point is a difference between the pre-spray point and the last spray point, and the improvement rate is a value obtained by dividing the improvement point by the pre-spray point.

According to the results shown in FIG. 8 and Table 1, the symptoms of atopic dermatitis are greatly improved. As a secondary effect, the psychological influence of the symptoms on one's own mental aspect is also improved. I found out.
As described above, according to this example, the anti-inflammatory action of the present invention was confirmed.

(Example 8)
The subject animal (subject) is a zero-year-old child (human). As shown in FIG. 9 (a), seborrheic eczema was observed over a wide area of the breast before the application of the present invention. When the subject composition was sprayed once in the morning and evening every day on such a subject, the redness of eczema was reduced as shown in FIG. 9 (b) on the first day of spraying, and surprisingly On the second day of spraying, it disappeared as shown in FIG.
According to this example, the anti-inflammatory action of the present invention was confirmed.

Example 9
The test animals (subjects) were 20 adults (humans) who developed hay fever and had a sex ratio of 6: 4, and the Example compositions were sprayed under the following conditions. The average age of the subjects was 45.9 years and the average hay fever history was 7.8 years. That is, subjects who have been suffering from hay fever for many years were targeted. During the monitoring period, other drugs were not taken (concomitant use).

(1) The subject sprayed at least twice a day (morning and evening) after washing the face.
(2) The subject sprayed once or twice around the right eye and the left nose (in no particular order).
(3) After spraying, the subjects lightly massaged each part and the surrounding area with their fingers to familiarize them.
(4) The number of days to spray was 3 days or more and 23 days or less, and it was arbitrary for each subject according to the symptom improvement state.

  Before the start of spraying, 48 hours after the start of spraying, and on the last day of spraying, a questionnaire covering 20 items was conducted, and symptom of hay fever was self-evaluated in 5 stages from 1 to 5. In Table 2 below, the top 12 questionnaire items with the highest degree of improvement are shown as “monitor names”, and the questionnaire results are also shown in Table 2. Point 1 corresponds to “no symptom”, point 2 corresponds to “mild”, point 3 corresponds to “moderate”, point 4 corresponds to “severe”, and point 5 corresponds to “highest severity”. FIG. 10 shows the points on the last day of spraying in Table 2 on the left side, the point after 48 hours from the spraying in the center, and the point before spraying on the right side in comparison with each monitor name. In Table 2, the improvement point is the difference between the point before spraying and the point on the last day of spraying, and the improvement rate is a value obtained by dividing the improvement point by the point before spraying.

  According to the results shown in FIG. 10 and Table 2, most of the symptoms of hay fever were improved within a short period of 48 hours from the start of spraying, and the symptoms were further improved by continuing spraying thereafter. From this, according to the present Example, the antiallergic action of this invention was confirmed, and it turned out that an antiallergic action is exhibited very rapidly.

  From the above embodiments and examples, it was demonstrated that the culture supernatant obtained by the present invention exhibits at least an antiallergic action, hair growth and hair growth action, antiinflammatory action, and skin metabolism promoting action.

The above embodiments and examples include the following technical ideas.
(1) A cosmetic containing a culture supernatant obtained by culturing mesenchymal stem cells derived from horses.
(2) The cosmetic according to (1), wherein the mesenchymal stem cell is a stem cell collected from a racehorse or a racehorse or a passaged stem cell obtained by subculturing the stem cell.
(3) Cosmetics as described in said (1) or (2) characterized by not containing any ammonium metavanadate, phenol red, and mercaptoethanol.
(4) An antiallergic composition containing a culture supernatant obtained by culturing mesenchymal stem cells derived from horses.
(5) A composition for hair growth or hair growth containing a culture supernatant obtained by culturing mesenchymal stem cells derived from horses.
(6) An anti-inflammatory therapeutic composition comprising a culture supernatant obtained by culturing mesenchymal stem cells derived from horses.
(7) A composition for promoting skin metabolism comprising a culture supernatant obtained by culturing mesenchymal stem cells derived from horses.
(8) Collection step of collecting cells from racehorses or horses; Selection step of selecting mesenchymal stem cells from the collected cells; Serum-free medium containing none of ammonium metavanadate, phenol red and mercaptoethanol A culturing step for culturing the mesenchymal stem cell using; and a collecting step for separating and recovering the culture supernatant produced in the culturing step from the serum-free medium and the mesenchymal stem cell; A method for producing a composition.

Claims (8)

A cosmetic containing a culture supernatant obtained by culturing mesenchymal stem cells derived from a racehorse fat ,
The mesenchymal stem cell is a fat-derived mesenchymal stem cell collected from the racehorse or a passage stem cell obtained by subculturing the mesenchymal stem cell,
Cosmetics characterized by not containing any of ammonium metavanadate, phenol red, mercaptoethanol and antibiotics .   The cosmetic according to claim 1, comprising insulin, linoleic acid and transferrin. An antiallergic treatment composition comprising a culture supernatant obtained by culturing mesenchymal stem cells derived from a racehorse fat ,
The mesenchymal stem cell is a fat-derived mesenchymal stem cell collected from the racehorse or a passage stem cell obtained by subculturing the mesenchymal stem cell,
An antiallergic treatment composition characterized by not containing any of ammonium metavanadate, phenol red, mercaptoethanol and antibiotics . A composition for hair growth or hair growth containing a culture supernatant obtained by culturing mesenchymal stem cells derived from fat of a racehorse ,
The mesenchymal stem cell is a fat-derived mesenchymal stem cell collected from the racehorse or a passage stem cell obtained by subculturing the mesenchymal stem cell,
A composition for hair growth or hair growth, which does not contain any of ammonium metavanadate, phenol red, mercaptoethanol and antibiotics . A composition for anti-inflammatory treatment comprising a culture supernatant obtained by culturing a mesenchymal stem cell derived from a racehorse fat ,
The mesenchymal stem cell is a fat-derived mesenchymal stem cell collected from the racehorse or a passage stem cell obtained by subculturing the mesenchymal stem cell,
A composition for anti-inflammatory treatment, which does not contain any of ammonium metavanadate, phenol red, mercaptoethanol and antibiotics . A composition for promoting skin metabolism comprising a culture supernatant obtained by culturing a mesenchymal stem cell derived from a fat of a racehorse ,
The mesenchymal stem cell is a fat-derived mesenchymal stem cell collected from the racehorse or a passage stem cell obtained by subculturing the mesenchymal stem cell,
A composition for promoting skin metabolism, which does not contain any of ammonium metavanadate, phenol red, mercaptoethanol and antibiotics . A collecting step of collecting fat cells from the racehorse ;
A selection step of selecting mesenchymal stem cells from the collected adipocytes ;
A culturing step of culturing the mesenchymal stem cells using a serum-free medium containing none of ammonium metavanadate, phenol red , mercaptoethanol and antibiotics ; and the culture supernatant produced in the culturing step as the serum-free medium And a recovery step of separating and recovering from the cultured mesenchymal stem cells;
A method for producing a cosmetic or pharmaceutical composition comprising:   The method for producing a cosmetic or pharmaceutical composition according to claim 7, wherein the serum-free medium used in the culturing step contains insulin, linoleic acid and transferrin.