Vitality-improving agent, vitality-improving composition, and vitality-improving food composition

Abstract

The invention provides a vitality improving agent. The vitality improving agent takes pine needle extract as an effective component; a food composition for improving vigor, comprising the vigor-improving agent and a pharmaceutically acceptable carrier; and a food composition for improving vigor, which contains the vigor-improving agent.

Description

Technical Field

[0001] This invention relates to vitality enhancers, vitality enhancer compositions, and vitality enhancer food compositions. This application claims priority to Japanese Patent Application No. 2023-075024, filed April 28, 2023, the contents of which are incorporated herein by reference. Background Technology

[0002] As we age, various forms of vitality decline. For example, with age, we experience hair loss, a decrease in neuroregulation, and a decline in cognitive function. Additionally, depressive symptoms may develop with age.

[0003] Hair loss on the scalp is a symptom that occurs due to various reasons such as aging. While hair loss itself is not a dangerous condition, given its importance to appearance, it can significantly impact a person's quality of life. Therefore, various treatment methods for hair loss are being researched.

[0004] Currently available treatments for hair loss primarily involve the use of synthetic chemical ingredients. For example, the U.S. Food and Drug Administration (FDA) has approved minoxidil and finasteride as treatments for hair loss (see, for example, patent documents 1 and 2).

[0005] Furthermore, modern society is also known as a stressful society, where daily life is constantly filled with pressure. As a result, the number of people suffering from mental illnesses such as depression is increasing, a phenomenon that has become a significant social problem. The increased medical costs caused by these mental illnesses go without saying, but the economic losses and social losses resulting from suicides also have incalculable impacts on society.

[0006] Currently, various antidepressants such as bupropion, mirtazapine, and imipamine have been developed and used to treat patients with mental illnesses such as depression (see, for example, patent document 3).

[0007] Existing technical documents Patent documents Patent Document 1: US Patent No. 4,139,619 Patent Document 2: U.S. Patent Application Publication No. 2002 / 0042425 Patent Document 3: U.S. Patent Application Publication No. 2004 / 121010 Summary of the Invention The technical problem that the invention aims to solve However, the administration of existing hair loss treatments such as minoxidil can potentially cause many serious side effects (erectile dysfunction, dizziness, unintended hair growth, weakness, headache, rash, etc.). Therefore, there is a demand for hair growth agents derived from natural sources that have fewer side effects and are expected to be safe.

[0008] Furthermore, most existing antidepressants are synthetic low-molecular-weight compounds, leading to problematic side effects such as nausea, loss of appetite, drowsiness, and low blood pressure. Additionally, there is a risk of severe toxicity in cases of overdose. Therefore, there is a need for antidepressants derived from natural sources that have fewer side effects and are expected to be safer.

[0009] This creates a demand for technologies that safely improve age-related decline in vitality, such as hair loss and depression. Therefore, the present invention aims to provide a vitality enhancer derived from natural sources.

[0010] Solutions for solving technical problems The present invention includes the following methods.

[0011] [1] A vitality enhancer with pine needle extract as the active ingredient.

[0012] [2] According to the vitality enhancer described in [1], the pine needle extract is an extract in which water or a mixture of water and alcohol is used as the extraction solvent.

[0013] [3] According to the vitality enhancer described in [1] or [2], the pine needle extract contains 500 ppm to 9,500 ppm D-pineol and 30 ppm to 8,500 ppm shikimic acid.

[0014] [4] The vitality enhancer according to any one of [1] to [3], wherein the pine needle extract is substantially free of resin acids.

[0015] [5] A composition for enhancing vitality, comprising any one of [1] to [4] a vitality enhancer and a pharmaceutically acceptable carrier.

[0016] [6] A food composition for enhancing vitality, comprising any one of [1] to [4].

[0017] The present invention may also include the following methods.

[0018] [P1] A hair growth agent with pine needle extract as the active ingredient.

[0019] [P2] According to the hair growth agent described in [P1], the pine needle extract is an extract that uses alcohol or a mixture of water and alcohol as the extraction solvent.

[0020] [P3] A hair growth composition comprising the hair growth agent described in [P1] or [P2] and a pharmaceutically acceptable carrier.

[0021] [P4] An antidepressant with pine needle extract as its active ingredient.

[0022] [P5] According to the antidepressant described in [P4], the pine needle extract is an extract using alcohol or a mixture of water and alcohol as the extraction solvent.

[0023] [P6] A composition for treating depression, comprising the antidepressant described in [P4] or [P5] and a pharmaceutically acceptable carrier.

[0024] Invention Effects According to the present invention, it is possible to provide a vitality enhancer derived from natural substances. Attached Figure Description

[0025] Figure 1 This is a graph showing the results of the cell viability measurement in Experiment Example 4.

[0026] Figure 2 This is a graph showing the results of real-time PCR for the quantitative CTNNB1 gene in Experiment Example 6.

[0027] Figure 3 This is a graph showing the results of real-time PCR for the quantitative ALPL gene in Experiment Example 6.

[0028] Figure 4 This is a graph showing the results of real-time PCR for the quantitative FGF1 gene in Experiment Example 6.

[0029] Figure 5 This is a graph representing the results of evaluating cell proliferation in Experiment Example 8.

[0030] Figure 6 This is a graph showing the results of the tail suspension test in Experiment Example 9.

[0031] Figure 7 This is a graph representing the results of Experiment Example 10.

[0032] Figure 8 This is a graph showing the results of Gene Ontology (GO) analysis of the genomes that showed expression changes in the high-dose pine needle extract group in Experiment Example 11. Detailed Implementation

[0033] [Vitality Enhancer] In one embodiment, the present invention provides a vitality enhancer with pine needle extract as the active ingredient. As described later in the examples, by administering the vitality enhancer of this embodiment, age-related decline in vitality can be improved or treated in humans or non-human animals. Examples of age-related decline in vitality include hair loss and depression.

[0034] As non-human animals, examples include pet animals and livestock. As pets, examples include dogs, cats, rabbits, and hamsters. As livestock, examples include horses and cows.

[0035] [Vigor Enhancer (Hair Growth Agent)] The vitality enhancer of the present invention has a hair growth effect in humans or non-human animals. The same applies to non-human animals. Therefore, in one embodiment, the present invention can provide a hair growth agent with pine needle extract as the active ingredient. As described later in the examples, the inventors have demonstrated that pine needle extract has a hair growth promoting effect. The hair growth agent of this embodiment can be considered a treatment for alopecia, a hair growth agent, etc.

[0036] In the hair growth agent of this embodiment, "designated as an active ingredient" means that it contains an amount of pine needle extract sufficient to exert its effect as a hair growth agent. Alternatively, it means that it contains pine needle extract as the main active ingredient. The effect of the hair growth agent can be exemplified by promoting hair growth. Alternatively, examples can be given such that, compared to the absence of a hair growth agent, the expression level of hair growth-related genes in dermal papilla cells increases in the presence of the hair growth agent. Examples of hair growth-related genes include, for example, the CTNBB1 gene (β-linkin), the ALPL gene (alkaline phosphatase), and the FGF1 gene (Fiblobrast growth factor 1). As described later in the examples, the hair growth agent of this embodiment can significantly increase the expression level of hair growth-related genes in dermal papilla cells. Therefore, the hair growth agent of this embodiment can also be described as an expression promoter of the CTNBB1 gene, the ALPL gene, or the FGF1 gene.

[0037] The pine needles used as the material for pine needle extract can be any leaves of plants (pine trees) belonging to the genus Pinus of the family Pinaceae. There are no specific restrictions on the types of pine trees. Examples include: Red pine (Pinus densiflora), P. densata, P. fragilissima, Bosnian pine (P. heldreichii), P. henryi, P. hwangshanensis, Simao pine (P. kesiya), Ryukyu pine (P. luchuensis Mayr), Masson pine (P. massoniana), dwarf red pine (P. mugo), European black pine (P. nigra), resin pine (P. resinosa), Western red pine (P. sylvestris), Chinese pine (P. tabuliformis), black pine (P. thunbergii), tropical pine (P. tropicalis), P. yunnanensis, Italian rock pine (P. pinea), P. brutia, P. canariensis, Mediterranean pine (P. halepensis), P. latteri, Sumatran pine (P. merkusii), and coastal pine (P. pinaster). Ait.), P. roxburghii, P. leiophylla, P. lumholtzii, P. caribaea, P. cubensis, P. echinata, P. elliottii, P. glabra, P. occidentalis, P. palustris, P. pungens, P. rigida Mill.), P. serotina, P. taeda, P. banksiana, P. clausa, P. contorta, P. virginiana, P. attenuata, P. foisyi, P. greggii, P. herrerae, P. jaliscana, P. lawsonii, P. muricata, P. oocarpa, P. patula, P. praetermissa, P. pringlei, P. radiata D. Don, P. tecunumanii, P. teocote, P. apulcensis, P. arazonica, P. cooperi, P. coulteri, P. devoniana, P. durangensis, P. engelmannii, P. hartwegii, P. jeffreyi, P.johndayensis, P.maximinoi, P.montezumae, P.Ponderosa, P.pseudostrobus, P.sabiniana, P.torreyana, P.amamiana, P.armandii, P.ayacahuite, P.bhutanica, P.chiapensis, P.d abeshanensis, P.dalatensis, P.fenzeliana, P.flexilis, P.reflexa, P.lambertiana, P.monticola, P.parviflora, P.peuce, P.pumila, P.strobiformis, North American pine (P. Strombus, P. wallichiana, P. wangii, P. albicaulis, P. cembra, P. koraiensis, P. nelsonii, P. krempfii, P. bungeana, P. gerardiana, P. squamata, P. maximartinezii, P. pinceana, P. rzedowskii, P. cembroides, P. culminicola, P. discolor, P. edulis, P. johannis, P. monophylla, P. orizabensis, P. quadrifolia, P. remota, P. aristata, P. balfouriana, Pinus longaeva, etc.

[0038] Pine needles can be used for extraction directly in their raw state, or after drying. Alternatively, they can be crushed or pulverized before extraction.

[0039] Examples of extraction solvents include water, alcohols, or mixtures of water and alcohols. The alcohol is not particularly limited; examples include lower alcohols with 1 to 4 carbon atoms such as ethanol, methanol, propanol, butanol, and 1,3-butanediol. One alcohol may be used alone, or two or more may be used in combination.

[0040] Water can also be mixed with alcohol as an extraction solvent. In the hair growth agent of this embodiment, water or a mixture of water and alcohol is preferably used as the extraction solvent. The concentration of alcohol in the mixture of water and alcohol can be 10% to 98% by volume, for example, 20% to 98% by volume, for example, 40% to 98% by volume, for example, 60% to 98% by volume, for example, approximately 70% by volume.

[0041] Water is particularly preferred as the extraction solvent. In the examples, as described later, when water is used as the extraction solvent, the pine needle extract tends to have lower cytotoxicity.

[0042] As for the extraction method of pine needles, commonly used methods can be employed. For example, extraction can be carried out by immersing pine needles in the aforementioned extraction solvent. When the extraction solvent is a mixture of water and alcohol, the extraction temperature can be, for example, 1°C to 50°C, 10°C to 30°C, or room temperature of 15°C to 25°C. In this case, the extraction time can range from several hours to several weeks, for example, 1 day to 10 days, 3 days to 10 days, 5 days to 10 days, or approximately 7 days. Alternatively, the extraction solvent can be stirred during extraction.

[0043] When water is used as the extraction solvent, the extraction temperature can be above 60°C, 70°C, 80°C, 90°C, or 100°C. Alternatively, pressure can be applied during extraction. Under pressure, the extraction temperature can exceed 100°C. For example, the extraction temperature can be above 100°C, 105°C, or 110°C. In this case, the extraction time can be less than 24 hours, less than 12 hours, less than 6 hours, less than 3 hours, less than 1 hour, or approximately 30 minutes. Alternatively, the extraction solvent can be stirred during extraction. In this specification, "approximately" means including ±10%. Therefore, approximately 7 days can also be 6.3 to 7.7 days. Furthermore, approximately 30 minutes can also be 27 to 33 minutes.

[0044] When water is used as the extraction solvent, the extraction temperature is preferably 60°C or higher. Additionally, the extraction time is preferably 24 hours or less.

[0045] The obtained pine needle extract can be used directly after removing solids by filtration or centrifugation as needed, or it can be used as a soft extract by distillation to remove the solvent, or it can be used as a dried extract by vacuum drying, freeze drying, etc.

[0046] The pine needle extract preferably contains 500 ppm or more of D-pinyl alcohol, more preferably 700 ppm or more of D-pinyl alcohol, even more preferably 1,000 ppm or more of D-pinyl alcohol, particularly preferably 2,000 ppm or more of D-pinyl alcohol, preferably 9,500 ppm or less of D-pinyl alcohol, and more preferably 5,000 ppm or less of D-pinyl alcohol. Furthermore, the pine needle extract preferably contains 30 ppm or more of shikimic acid, more preferably 100 ppm or more of shikimic acid, even more preferably 1,000 ppm or more of shikimic acid, particularly preferably 2,000 ppm or more of shikimic acid, and preferably 8,500 ppm or less of shikimic acid. Furthermore, the pine needle extract preferably contains 500 ppm to 9,500 ppm of D-pineol and 30 ppm to 8,500 ppm of shikimic acid, more preferably 2,000 ppm to 9,500 ppm of D-pineol and 2,000 ppm to 8,500 ppm of shikimic acid. Pine needle extracts extracted under the conditions containing these components at the above-mentioned concentrations tend to have a higher expression enhancement effect on hair regrowth-related genes. In addition, the contents of D-pineol and shikimic acid in this embodiment, as described later in the examples, are converted values ​​when the non-volatile components of the pine needle extract are set to 3% by mass.

[0047] The pine needle extract preferably does not contain resin acids. Resin acids are the main components of rosin and refer to a mixture with various isomers such as abietic acid, dehydroabietic acid, longleaf abietic acid, piratic acid, and slash abietic acid as the main components. These resin acids also include resin acid derivatives such as hydrogenated resin acids. In the examples, as described later, pine needle extracts with a high content of resin acids tend to have higher cytotoxicity.

[0048] Here, "substantially free of resin acids" means that the pine needle extract is permitted to contain resin acids to a degree that does not pose a problem due to cytotoxicity. It can be said that it is substantially free of resin acids as long as the total amount of resin acids is approximately 200 ppm by mass or less.

[0049] The vitality enhancer of this embodiment is preferably formulated as a vitality enhancer composition comprising a pharmaceutically acceptable carrier. The vitality enhancer composition may also be a pharmaceutical composition. Considering the hair growth effect of the vitality enhancer, the vitality enhancer composition of this embodiment can be described as a hair growth composition or a hair development composition. In one embodiment, the hair growth composition includes pine needle extract and a pharmaceutically acceptable carrier.

[0050] There are no particular limitations on pharmaceutically acceptable carriers; examples include excipients, binders, disintegrants, lubricants, emulsifiers, thickeners, humectants, and solvents for injections. Furthermore, hair growth compositions may further contain additives.

[0051] As additives, there are no particular restrictions; examples include preservatives, pH adjusters, stabilizers, UV absorbers, antioxidants, colorants, and fragrances.

[0052] As a pharmaceutically acceptable carrier and additive, it can be made from common raw materials listed in the 18th edition of the Japanese Pharmacopoeia, for example.

[0053] Examples of dosage forms for hair growth compositions include oral dosage forms such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions, as well as non-oral dosage forms such as injections, suppositories, and topical skin preparations.

[0054] Examples of topical skin agents include creams, lotions, toners, emulsions, foundations, masks, foams, plasters, ointments, poultices, and aerosols.

[0055] Hair growth compositions can be used as treatments for alopecia, cosmetics, or food supplements.

[0056] The content of hair growth agent (pine needle extract) in the hair growth composition is calculated based on the solid components (dry weight) of the hair growth agent, for example, the ranges can be listed as 0.01% to 50% by mass, 0.01% to 30% by mass, 0.01% to 10% by mass, 0.01% to 5% by mass, and 0.01% to 1% by mass.

[0057] There are no particular restrictions on the method of administration for hair growth agents or hair growth compositions; it can be appropriately determined based on the symptoms, weight, age, and gender of the recipient. For example, tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions can be administered orally. In addition, injectable preparations can be administered intravenously, alone or mixed with common rehydration solutions such as glucose and amino acids, and further administered intra-arterially, intramuscularly, intradermally, subcutaneously, or intraperitoneally as needed. Suppositories can be administered rectally. Topical preparations can be applied, patched, or sprayed onto the affected area.

[0058] The dosage of hair growth agents or hair growth compositions varies depending on the recipient's symptoms, weight, age, and gender, and cannot be generalized. For oral administration, a daily dose of 0.01 mg / kg to 5,000 mg / kg of the active ingredient (pine needle extract) is generally sufficient. For injectable formulations, a daily dose of 0.01 mg to 500 mg of the active ingredient is generally sufficient. For suppositories, a daily dose of 0.01 mg to 1,000 mg of the active ingredient is generally sufficient. For topical applications, a daily dose of 0.01 mg to 500 mg of the active ingredient is generally sufficient.

[0059] [Food composition for enhancing vitality (food composition for hair growth)] In one embodiment, the present invention provides a food composition for hair growth containing pine needle extract as an active ingredient. The food composition of this embodiment includes the aforementioned hair growth agent. By consuming the food composition of this embodiment, hair growth can be promoted in humans or non-human animals. Alternatively, by consuming the food composition of this embodiment, the expression level of hair growth-related genes in dermal papilla cells can be increased in humans or non-human animals. The same applies to non-human animals. The food composition of this embodiment can also be described as a food composition for promoting hair growth, a food composition for hair development, etc.

[0060] The preferred intake of the food composition of this embodiment is the same as that of the hair growth agent or hair growth composition described above, for example, the amount of active ingredient (pine needle extract) taken daily from 0.01 mg / kg to 5,000 mg / kg body weight can be listed. The food composition can be taken once or about 2 to 4 times a day.

[0061] The food composition of this embodiment can be, for example, a supplement, a beverage, a solid, semi-solid, or gel-like food, or any cooked food.

[0062] Examples of supplements that can be taken in forms such as powders, granules, tablets, and capsules. Examples of beverages that can be taken in forms such as coffee, tea, black tea, sports drinks, fruit and vegetable drinks, and carbonated drinks.

[0063] The food composition of this embodiment can also be a functional display food. Functional display food refers to food whose functionality is displayed on its packaging based on scientific evidence, and refers to food delivered to consumer goods stores.

[0064] [Vitality enhancers (antidepressants)] The vitality enhancer of the present invention has an antidepressant effect in humans or non-human animals. The same applies to non-human animals. Therefore, in one embodiment, the present invention can provide an antidepressant with pine needle extract as the active ingredient. As described later in the embodiments, the inventors have demonstrated that pine needle extract has neuroprotective effects. The antidepressant of this embodiment is derived from a natural product and is therefore considered to have fewer side effects and is safe.

[0065] In the antidepressant of this embodiment, "as an active ingredient" means that it contains an amount of pine needle extract sufficient to exert its effect as an antidepressant. Alternatively, it means that it contains pine needle extract as the main active ingredient. Effects as an antidepressant may include, for example, neuroprotective effects.

[0066] Neuroprotective effects can be categorized as antidepressant effects, anti-stress effects, etc. In other words, the antidepressant in this embodiment can be considered a treatment for depression, a neuroprotective agent, an anti-stress agent, etc.

[0067] In the antidepressant of this embodiment, the pine needle extract is the same as described above. The pine needle extract is preferably an extract using ethanol or a mixture of water and ethanol as the extraction solvent, and more preferably an extract using water as the extraction solvent.

[0068] The vitality enhancer of this embodiment is preferably formulated as a vitality enhancer composition comprising a pharmaceutically acceptable carrier. The vitality enhancer composition may also be a pharmaceutical composition. Considering the antidepressant effect of the vitality enhancer, the vitality enhancer composition of this embodiment can be considered a composition for treating depression. In one embodiment, the composition for treating depression comprises pine needle extract and a pharmaceutically acceptable carrier.

[0069] As pharmaceutically acceptable carriers, the same carriers as those described above for hair growth compositions can be used. Furthermore, compositions for treating depression may further contain additives, which are also the same as those described above. As pharmaceutically acceptable carriers and additives, for example, commonly used raw materials listed in the 18th edition of the Japanese Pharmacopoeia, etc., can be used.

[0070] Examples of dosage forms for compositions used to treat depression include oral dosage forms such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions, as well as non-oral dosage forms such as injections, suppositories, and topical preparations.

[0071] Examples of topical skin agents include creams, lotions, toners, emulsions, foundations, masks, foams, plasters, ointments, poultices, and aerosols.

[0072] Compositions for treating depression can be medications for treating depression, cosmetics, or food supplements.

[0073] The content of the antidepressant (pine needle extract) in the composition for treating depression is calculated in terms of the solid component (dry weight) of the antidepressant, and for example, ranges of 0.01% to 50% by mass, 0.01% to 30% by mass, 0.01% to 10% by mass, 0.01% to 5% by mass, and 0.01% to 1% by mass can be listed.

[0074] There are no particular restrictions on the method of administration for antidepressants or compositions for the treatment of depression; it can be appropriately determined based on the symptoms, weight, age, and gender of the recipient. For example, tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions can be administered orally. In addition, injectable preparations can be administered intravenously, alone or mixed with common rehydration solutions such as glucose and amino acids, and further administered intra-arterially, intramuscularly, intradermally, subcutaneously, or intraperitoneally as needed. Suppositories can be administered rectally. Topical preparations can be applied, patched, or sprayed onto the affected area.

[0075] The dosage of antidepressants or compositions for treating depression varies depending on the patient's symptoms, weight, age, and sex, and cannot be generalized. For oral administration, a daily dose of 0.01 mg / kg to 5,000 mg / kg of the active ingredient (pine needle extract) is generally sufficient. For injectable formulations, a daily dose of 0.01 mg to 500 mg of the active ingredient is generally sufficient. For suppositories, a daily dose of 0.01 mg to 1,000 mg of the active ingredient is generally sufficient. For topical applications, a daily dose of 0.01 mg to 500 mg of the active ingredient is generally sufficient.

[0076] [Energy-boosting food composition (Depression-relieving food composition)] In one embodiment, the present invention provides a food composition for improving depression containing pine needle extract as an active ingredient. The food composition of this embodiment contains the aforementioned antidepressant. By consuming the food composition of this embodiment, a neuroprotective effect can be exerted in humans or non-human animals. The neuroprotective effect can be described as an antidepressant effect, an anti-stress effect, etc. That is, the food composition of this embodiment can be described as a food composition for improving depression, a neuroprotective food composition, an anti-stress food composition, etc.

[0077] The preferred intake of the food composition of this embodiment is the same as that of the hair growth agent or hair growth composition described above, for example, the amount of active ingredient (pine needle extract) taken daily from 0.01 mg / kg to 5,000 mg / kg body weight can be listed. The food composition can be taken once or about 2 to 4 times a day.

[0078] The food composition of this embodiment can be, for example, a supplement, a beverage, a solid, semi-solid, or gel-like food, or any cooked food.

[0079] Examples of supplements that can be taken in forms such as powders, granules, tablets, and capsules. Examples of beverages that can be taken in forms such as coffee, tea, black tea, sports drinks, fruit and vegetable drinks, and carbonated drinks.

[0080] The food composition of this embodiment can be a functional display food. Functional display food refers to food whose functionality is displayed on its packaging based on scientific evidence, and refers to food delivered to consumer goods stores.

[0081] [Other Implementation Methods] In one embodiment, the present invention provides a treatment for age-related decline in vitality, comprising administering an effective amount of pine needle extract to a patient requiring treatment.

[0082] In one embodiment, the present invention provides a pine needle extract for treating age-related decline in vitality.

[0083] In one embodiment, the present invention provides the use of a pine needle extract for the manufacture of a therapeutic agent for age-related decline in vitality.

[0084] In one embodiment, the present invention provides a method for treating alopecia, comprising administering an effective amount of pine needle extract to a patient requiring treatment.

[0085] In one embodiment, the present invention provides a pine needle extract for treating alopecia.

[0086] In one embodiment, the present invention provides the use of a pine needle extract for the manufacture of a treatment for hair loss.

[0087] In one embodiment, the present invention provides a treatment for depression, comprising administering an effective amount of pine needle extract to a patient in need of treatment.

[0088] In one embodiment, the present invention provides a pine needle extract for the treatment of depression.

[0089] In one embodiment, the present invention provides the use of a pine needle extract for the manufacture of a therapeutic agent for depression.

[0090] In these embodiments, the pine needle extract, administration method, dosage, etc., are the same as described above. The pine needle extract is preferably formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is the same as described above.

[0091] [Example] The present invention will now be illustrated in more detail with examples, but the invention is not limited to the following examples.

[0092] [Experimental Example 1] (Preparation of pine needle extract from manufacturing example 1) Dried pine needles derived from red pine seedlings were pulverized using a mixer. Then, 100 mg of the pulverized pine needles were mixed with 1 mL of 70% (v / v) ethanol aqueous solution and extracted for one week in the dark at room temperature to obtain the pine needle extract of Preparation Example 1. During extraction, the sample was vortexed daily. The supernatant was then collected by centrifugation at 1,000 × g for 15 minutes. The supernatant was then sterilized by passing it through a 0.22 μm filter and stored at -20°C. The pine needle extract was used directly after dilution in the cell culture medium used in each experiment.

[0093] [Experimental Example 2] (Preparation of pine needle extracts from manufacturing examples 2-7) The pine needles shown in Table 1 were extracted using the extraction methods described in Table 1 below. The extracts were then centrifuged at 1,000 × g for 15 minutes, and the supernatant was collected. The supernatant was then sterilized by passing it through a 0.22 μm filter to obtain the pine needle extracts of Examples 2-7. Each pine needle extract was stored at -20°C.

[0094] [Table 1]

[0095] [Experiment Example 3] (Component analysis of pine needle extracts from manufacturing examples 1-7) The D-pinol, shikimic acid, and resin acids contained in the pine needle extracts of Manufacturing Examples 1-7 were analyzed by gas chromatography-mass analysis (GC / MS) and high-performance liquid chromatography (HPLC). Specifically, D-pinol and shikimic acid in the pine needle extracts were analyzed by HPLC, and resin acids in the pine needle extracts were analyzed by GC / MS. In the GC / MS analysis, the sample was methylated by reacting with diazomethane. Furthermore, a standard curve was prepared using catechol to determine the contents of D-pinol, shikimic acid, and resin acids, and the conversion values ​​were calculated using catechol.

[0096] The GC / MS apparatus and measurement conditions are described below.

[0097] GC: Agilent 7890A (Made by Agilent Technologies) MS: Agilent 5977A (made by Agilent Technologies) Automated sampler: MPS robotic pro (manufactured by GERSTEL) Column: VF-5ms (0.25mm×0.25μm×30m) Carrier gas: He The apparatus and conditions for HPLC determination are described below.

[0098] Column: Inert Sustain Amide (3.0×250mm i.d., 3mm, manufactured by GL Science) Column oven temperature: 40℃ Sample dilution solvent: methanol Injection volume: 1μL Sample concentration: 50% by mass Flow rate: 0.5 mL / min UV detector: 210nm Mobile phase A: Acetonitrile Mobile phase B: 100 mM ammonium formate aqueous solution Mobile phase composition condition A:B = 80:20 Measurement time: 20 min Tables 2 and 3 below show the amounts of D-pinol, shikimic acid, and resin acids in each pine needle extract. In Tables 2 and 3, the units for the amounts of each component are ppm (mass). Furthermore, the contents of D-pinol and shikimic acid are converted values ​​assuming the non-volatile components of the pine needle extract are 3% by mass. The method for determining the non-volatile components of the pine needle extract is described below.

[0099] Bg of pine needle extract was weighed into a 20mL threaded tube (Ag), dried in a co-current dryer at 80°C for 4 hours, and then cooled in a desiccator. The mass (Cg) of the cooled threaded tube was measured, and the non-volatile components of the pine needle extract were calculated using the following formula.

[0100] Non-volatile components (mass%) of pine needle extract = (CA) / B × 100 For example, if the non-volatile component of the pine needle extract is calculated to be 1.5% by mass using the above determination method, and the pine needle extract contains 1,000 ppm D-pineol and 1,000 ppm shikimic acid, the conversion value when the non-volatile component is set to 3% by mass is calculated to be 2,000 ppm D-pineol and 2,000 ppm shikimic acid.

[0101] [Table 2]

[0102] [Table 3]

[0103] As a result, the pine needle extracts of Manufacturing Examples 1-7 contained 500 ppm to 9,500 ppm of D-pineol and 30 ppm to 8,500 ppm of shikimic acid when the non-volatile components were set at 3% by mass. Furthermore, the pine needle extracts of Manufacturing Examples 6 and 7 showed a high content of resin acids.

[0104] [Experiment Example 4] (Cytotoxicity study of pine needle extract from manufacturing example 1) The cytotoxicity of the pine needle extract from Manufacturing Example 1 was studied. Mesenchymal cells isolated from the papillae of hair follicles in normal human scalp, namely human primary dermal papilla cells (HFDPC, purchased from Promocel), were used as the cells. The cells were cultured in 96-well plates coated with collagen at a concentration of 5 × 10⁻⁶. 4 FFDPCs were seeded per well and incubated at 37°C in a humidified environment with 5% CO2. A dermal papilla cell growth medium supplemented with growth factors (fetal bovine serum, insulin, transferrin, triiodothyronine, bovine pituitary extract, and cycloacetone solution) was used as the culture medium.

[0105] After 24 hours, the cell culture medium was replaced with a medium containing 0 (control), 1 / 6,000, 1 / 5,000, 1 / 4,000, 1 / 3,000, 1 / 2,000, 1 / 1,000, 1 / 500, 1 / 250, and 1 / 100 volumes of pine needle extract, and the cells were incubated for 48 hours.

[0106] Next, cell viability was determined using 3-(4,5-dimethyl-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT). Specifically, MTT reagent (5 mg / mL) was added to the cells, and after incubation for 8 hours, 10% sodium dodecyl sulfate (SDS) was added, and incubation continued overnight. The absorbance at 570 nm was then measured using a microplate reader, and cell viability was quantified as the percentage of absorbance relative to that of untreated cells.

[0107] Figure 1 It is a graph representing the results of cell viability measurements. Figure 1 In this context, "ns" indicates no significant difference. "" indicates a significant difference at P < 0.0001. The results show that adding 1 / 6,000 to 1 / 250 volume of pine needle extract to the culture medium did not exhibit cytotoxicity. When 1 / 100 volume of pine needle extract was added to the culture medium, cell viability decreased to approximately 30%.

[0108] [Experiment Example 5] (Cytotoxicity study of pine needle extracts from manufacturing examples 3, 4, 6, and 7) The cytotoxicity of the pine needle extracts from Manufacturing Examples 3, 4, 6, and 7 was studied using the same method as in Experimental Example 4. The pine needle extract from Manufacturing Example 6, like that from Manufacturing Example 1, exhibited slightly higher cytotoxicity. Furthermore, the pine needle extract from Manufacturing Example 7 showed high cytotoxicity, making it impossible to study its impact on the germinal growth-related genes described later.

[0109] The results are presented in Table 4 below. In Table 4, "cytotoxicity" indicates the evaluation results of cytotoxicity. The evaluation criteria for cytotoxicity are described in Table 4 below.

[0110] (Cytotoxicity evaluation criteria) -: No cytotoxicity was confirmed.

[0111] ±: Reduces cell viability by more than 40% in 1 / 100 volume.

[0112] +: Reduces cell viability by more than 10% in 1 / 10,000 volume.

[0113] ++: Reduces cell viability by more than 40% in 1 / 10,000 volume.

[0114] [Table 4]

[0115] [Experiment Example 6] (Study on the effect of pine needle extract from manufacturing example 1 on hair growth-related genes) Pine needle extract from Preparation Example 1 was added to the HFDPC culture medium, and the expression of hair regrowth markers was determined by quantitative real-time PCR. The CTNBB1 gene (β-linkin), ALPL gene (alkaline phosphatase), and FGF1 gene (Fiblobrast growth factor 1) were investigated as hair regrowth markers. β-linkin is known to induce the anagen phase of the hair cycle and promote keratinocyte proliferation. ALPL is a representative hair regrowth marker in dermal papilla cells. FGF1 is a marker for follicular formation.

[0116] HFDPC at 5×10 4 Cells were seeded per well in 6-well plates and incubated at 37°C for 24 hours. The medium was then removed and replaced with medium containing 1 / 4,000 or 1 / 2,000 volume of pine needle extract. Additionally, for comparison, cells were prepared with minoxidil (0.1 μM), an existing treatment for hair loss, added to the medium.

[0117] Next, after 48 hours, the cells were washed with cold PBS, and total RNA was extracted using the ISOGEN kit (NIPPONGEN). Then, the total RNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) for quantitative real-time PCR analysis.

[0118] First, cDNA was synthesized from the extracted total RNA using the SuperScript III reverse transcription kit (Thermo Fisher Scientific) through a cycle of 95°C for 10 minutes, 95°C for 15 seconds for 40 cycles, and 60°C for 1 minute.

[0119] Next, real-time PCR was performed on a 7500 Fast Real-Time PCR system (Software 1.3.1, Thermo Fisher Scientific) and using specific TaqMan probes for CTNBB1, ALPL, and FGF1. GAPDH was used as an endogenous control, and 2... -ΔΔ The Ct method was used to compare the relative mRNA levels with those of GAPDH.

[0120] Figure 2 This is a graph representing the results of real-time PCR for the CTNBB1 gene. Figure 3 This is a graph representing the results of real-time PCR for the ALPL gene. Figure 4 This is a graph showing the results of real-time PCR for the FGF1 gene. Figures 2-4In the chart, the values ​​represent the mean ± standard deviation (n=3). Additionally, "ns" indicates no significant difference. "This indicates a significant difference when P < 0.05" "This indicates a significant difference when P < 0.01." "" indicates that there is a significant difference when P < 0.0001.

[0121] The results showed that the expression levels of CTNBB1 and FGF1 genes in HFDPCs were significantly increased by using culture media containing 1 / 4,000 and 1 / 2,000 volumes of pine needle extract. Furthermore, the expression level of the ALPL gene in HFDPCs was significantly increased by using culture media containing 1 / 2,000 volumes of pine needle extract. Moreover, the increase in FGF1 gene expression exceeded the effect of treatment with 0.1 μM minoxidil (a hair growth drug) used as a positive control. These results indicate that pine needle extract has a hair growth promoting effect.

[0122] [Experiment Example 7] (Study on the effects of pine needle extracts from manufacturing examples 3-6 on hair growth-related genes) The effects of the pine needle extracts from Preparations 3-6 on hair regrowth-related genes were investigated using the same method as in Experimental Example 6. Similarly, D-pinenein and shikimic acid were also studied. The pine needle extracts from Preparations 3-6 were added to the cell culture medium at 1 / 6,000 volume or 1 / 10,000 volume. Furthermore, the pine needle extracts from Preparations 1 and 6 were added to the cell culture medium at concentrations where no cytotoxicity was observed. D-pinenein was added to the cell culture medium at 1 μM and 10 μM. Shikimic acid was added to the cell culture medium at 1 μM and 10 μM.

[0123] The results are presented in Table 5 below. In Table 5, “CTNBB1,” “ALPL,” and “FGF1” represent the evaluation results of the enhanced expression of each germinal growth-related gene. The evaluation criteria for the impact on germinal growth-related genes are described in Table 5 below.

[0124] (Evaluation criteria for the impact on genes related to hair growth) -: Suppressed expression.

[0125] ±: No significant difference was identified in expression enhancement at at least some concentrations.

[0126] +: Significant differences were confirmed at all concentrations in terms of expression enhancement, p < 0.05 or higher.

[0127] ++: Significant differences were confirmed at all concentrations in terms of expression enhancement, p < 0.01 or higher.

[0128] [Table 5]

[0129] The results showed that while D-pineol and shikimic acid enhanced the expression of CTNBB1 and FGF1, they did not enhance the expression of ALPL. In contrast, the pine needle extracts from Examples 3-6 showed enhanced ALPL expression. Furthermore, the pine needle extracts from Examples 4-6 showed enhanced expression of all three gene types: CTNBB1, ALPL, and FGF1.

[0130] [Experiment Example 8] (Study on the neuroprotective effect of pine needle extract from manufacturing example 1) Using an in vitro depression model evaluation system, we investigated the neuroprotective effects (antidepressant and anti-stress effects) of pine needle extract. More specifically, we investigated whether treatment of SH-SY5Y cells (a human neuroblastoma cell line) with dexamethasone (a neurotoxin) in the presence of pine needle extract could inhibit dexamethasone-induced neuronal death.

[0131] SH-SY5Y cells were loaded at 2.0 × 10⁻⁶. 4 Cells / well were inoculated into 96-well plates and cultured at 37°C in the presence of 5% CO2 for 24 hours. As the culture medium, a mixture of DMEM and Ham's F-12 nutrient mixture in a 1:1 (v:v) ratio was used, supplemented with 15% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 1% non-essential amino acids.

[0132] After 24 hours, the culture medium was replaced with medium supplemented with 0 (control), 1 / 35,000, 1 / 30,000, 1 / 25,000, 1 / 20,000, 1 / 15,000, 1 / 10,000, and 1 / 5000 volumes of pine needle extract, and incubated for 1 hour. Additionally, for comparison, cells were prepared with rosmarinic acid (10 μM), known for its neuroprotective effects, added to the culture medium. After 1 hour, 0.5 mM dexamethasone was added to each well, and incubation continued for another 48 hours.

[0133] Next, MTT reagent (5 mg / mL) was added to the cells, and after incubation for 24 hours, 10% sodium dodecyl sulfate (SDS) was added, and incubation was continued for another 24 hours. Then, the absorbance at 570 nm was measured using a microplate reader, and cell proliferation was quantified as the ratio (%) of absorbance to that of untreated cells.

[0134] Figure 5 This is a graph representing the results of evaluating cell proliferation. In Figure 5 In the charts, the values ​​represent the mean ± standard deviation (n=6). Additionally, "Ctrl" indicates the control group (no dexamethasone or pine needle extract added), "Dex" indicates the results with dexamethasone only, "RA" indicates the results with rosmarinic acid added, and "Pine" indicates the results with pine needle extract added. Furthermore, compared to cells with only dexamethasone added, "This indicates a significant difference at P < 0.05 compared to cells treated with only dexamethasone." "" indicates that there is a significant difference when P < 0.01.

[0135] The results showed that SH-SY5Y cells supplemented with pine needle extract exhibited higher cell proliferation than SH-SY5Y cells supplemented with dexamethasone alone. This indicates that pine needle extract has a significant neuroprotective effect against dexamethasone. These results also suggest that pine needle extract may be useful as an antidepressant and anti-stress agent.

[0136] [Experiment Example 9] (A study on the effects of pine needle extract on depressive behavior) As a pine needle extract, the pine needle extract from Manufacturing Example 2 was used to investigate its effects on depressive behavior in vivo using a tail suspension test in mice. The tail suspension test (TST) measures the time a mouse struggles while suspended in the opposite direction, and is used as an indicator of depressive-like behavior. While the suspended mouse moves back and forth to escape, the time it remains still (immobility time) gradually increases. It is known that mice administered antidepressants show a reduced immobility time in the tail suspension test. A longer immobility time is interpreted as indicating an increase in depressive-like behavior, while a shorter immobility time indicates a decrease in depressive-like behavior.

[0137] Mice were prepared according to the groups shown in Table 6 below. In Table 6, "LPS" represents lipopolysaccharide. Bupropion, an antidepressant, was administered to the positive control group. Mice in each group were fed and acclimatized for 7 days, and then orally administered pine needle extract daily for 7 days. Then, LPS was administered intraperitoneally once to induce neuroinflammation in the brain, leading to depression. A tail suspension test was performed the day after LPS administration. A SMART 3.0 video image behavioral analysis device (Panlab) was used in the tail suspension test. The brain was harvested the day after the tail suspension test, and the hippocampus was recovered for subsequent experiments.

[0138] [Table 6]

[0139] Table 7 below shows the changes in body weight and total food intake of mice in each group during oral administration of pine needle extract. The results showed that 7 days of oral administration of pine needle extract had no effect on body weight or total food intake. Based on these results, pine needle extract is considered not to exhibit serious toxicity.

[0140] [Table 7]

[0141] Figure 6 It is a graph representing the results of the tail suspension test. Figure 6 In this context, "Saline" represents physiological saline, "Bup" represents bupropion, "LowPN" represents the results of the low-dose pine needle extract group, and "HighPN" represents the results of the high-dose pine needle extract group. Additionally, " "" indicates that there is a significant difference when p < 0.05.

[0142] The results showed that oral administration of a high dose of pine needle extract (50 mg / kg) significantly inhibited the prolongation of immobility time caused by LPS-induced neuroinflammation. On the other hand, no significant effect was observed with oral administration of a low dose of pine needle extract (5 mg / kg). These results suggest that pine needle extract has an inhibitory effect on depressive behavior.

[0143] [Experiment Example 10] (Protein expression analysis in the hippocampus of mouse brain) The hippocampus of each group of mice recovered in Experiment 9 was provided to ELISA to quantify mature BDNF, dopamine, norepinephrine, serotonin, and acetylcholine.

[0144] Figure 7 It is a chart representing quantitative results. Figure 7 In this context, "Saline" represents physiological saline, "Bup" represents bupropion, "LowPN" represents the results of the low-dose pine needle extract group, and "HighPN" represents the results of the high-dose pine needle extract group. Additionally, " "Indicates a significant difference when p < 0.05" "" indicates that there is a significant difference when p < 0.01.

[0145] The results showed that oral administration of pine needle extract significantly inhibited the decrease in mature BDNF expression induced by LPS-induced neuroinflammation. Furthermore, in the high-dose pine needle extract group, the decrease in dopamine and norepinephrine expression induced by LPS-induced neuroinflammation was significantly suppressed. Additionally, in the low-dose pine needle extract group, the decrease in serotonin expression induced by LPS-induced neuroinflammation was significantly suppressed. On the other hand, oral administration of pine needle extract did not affect acetylcholine expression.

[0146] [Experiment Example 11] (Comprehensive gene expression analysis in the hippocampus of mouse brain) The hippocampal DNA microarray analysis of mice recovered from each group in Experiment 9 was provided to comprehensively analyze gene expression in the high-dose pine needle extract group and the LPS group.

[0147] Among the genes with a Fold Change exceeding 1.5 between the high-dose pine needle extract group and the LPS group, 330 genes showed increased expression and 196 genes showed decreased expression. The expression ratio was calculated using the following formula.

[0148] Expression ratio = Expression level in the high-dose pine needle extract group / Expression level between LPS groups Figure 8 This is a figure showing the results of a GeneOntology (GO) analysis of the genomes that showed expression changes in the high-dose pine needle extract administration group. Figure 8 In this context, "BP" represents biological processes, "CC" represents cellular components, and "KEGG" represents the Kyoto Encyclopedia of Genes and Genomes (KEGG), a database of gene and compound associations related to enzyme reactions and signal transduction centered on metabolic pathways. The results showed increased expression of genes associated with metabolism and nerves, and decreased expression of genes associated with inflammation. These results suggest that administration of pine needle extract may potentially induce neurogenesis, inhibit neuroinflammation, and enhance neurometabolism.

[0149] Industrial utilization potential According to the present invention, a vitality enhancer derived from natural substances can be provided.

Claims

1. A vitality enhancer, wherein, The vitality enhancer uses pine needle extract as its active ingredient.

2. The vitality enhancer according to claim 1, wherein, The pine needle extract is an extract that uses water or a mixture of water and alcohol as the extraction solvent.

3. The vitality enhancer according to claim 1 or 2, wherein, The pine needle extract contains 500 ppm to 9,500 ppm of D-pineol and 30 ppm to 8,500 ppm of shikimic acid.

4. The vitality enhancer according to claim 1 or 2, wherein, The pine needle extract does not actually contain resin acids.

5. A composition for enhancing vitality, wherein, The vitality-enhancing composition comprises the vitality-enhancing agent as described in claim 1 or 2 and a pharmaceutically acceptable carrier.

6. A food composition for enhancing vitality, wherein, The vitality-enhancing food composition comprises the vitality enhancer as described in claim 1 or 2.

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