Agents for treating, improving, and / or preventing age-related hearing loss

Abstract

To provide novel agents capable of treating, ameliorating and / or preventing age-related hearing loss.SOLUTION: As a result of intensive studies to solve the above problems, the present inventors found that the progression of hearing loss is significantly suppressed by administering Ninjinyoeito or Ryokeijutsukanto to age-related deafness model mice compared with the untreated group. The present invention provides an agent for treating, ameliorating and / or preventing age-related hearing loss, containing as an active ingredient Atractylodis Rhizoma, Wolfiporia cocos Ryvarden et Gilbertson, Cinnamomum cassia Blume and Glycyrrhiza uralensis Fischer, or extracts thereof.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an agent and a pharmaceutical composition for treating, improving and / or preventing presbycusis.

Background Art

[0002] Knowledge regarding the treatment and prevention of presbycusis (presenile deafness) is extremely limited. According to the estimation of the World Health Organization, the number of people with hearing impairment in the world is 466 million (6.1% of the world population), and about one-third of them are elderly people aged 65 years or older.

[0003] In Japan, the number of presbycusis patients aged 65 years or older exceeds 15 million. Since deafness is not recognized as a critical disease directly related to death, research on the treatment and prevention of presbycusis has not been actively pursued so far. However, it has been reported that deafness not only leads to a decline in communication ability but also causes isolation, depression, dementia, and other coexisting diseases associated with presbycusis.

[0004] Regarding hearing impairment, established countermeasures such as hearing aids and cochlear implants exist. However, some hearing-impaired people cannot purchase hearing aids due to cost issues. In addition, even if they have a hearing aid, there are some people who do not use it due to reasons such as discomfort, a sense of unnecessaryness, and shyness. Furthermore, when the damage to the inner ear and nerve cells progresses, the effects of hearing aids and cochlear implants may become poor. Therefore, in addition to countermeasures such as the use of hearing aids and the wearing of cochlear implants, it is important to develop a method for controlling the progression of presbycusis.

[0005] Presbycusis often progresses gradually and can be one of the causes leading to a decline in the quality of life of the elderly. Therefore, if useful components for preventing presbycusis can be found, it may be helpful for improving the overall quality of life of the elderly population.

[0006] Age-related hearing loss has been reported pathologically to be caused by decreased blood flow to the cochlea, leading to oxygen and nutrient deficiencies, which in turn generate reactive oxygen species derived from mitochondria, causing cell death in cells related to hearing (Non-Patent Literature 1). From the perspective of traditional East Asian medicine, the decreased blood flow to the cochlea seen in age-related hearing loss is attributed to decreased kidney function, and it is believed that restoring kidney function can restore blood flow, leading to improvement in hearing loss and tinnitus.

[0007] Regarding the prevention of age-related hearing loss, several components have been reported to be useful (Non-Patent Documents 2 and 3). However, no pharmaceuticals effective in suppressing the progression of age-related hearing loss have yet been reported.

[0008] Patent Document 1 discloses an agent containing a substance that increases the expression of endothelin receptor B, as a preventive or therapeutic agent for age-related hearing loss, noise-induced hearing loss, or tinnitus.

[0009] Furthermore, as a drug for treating tinnitus patients accompanied by age-related hearing loss, Patent Document 2 discloses a drug containing neramexane or a pharmaceutically acceptable salt thereof.

[0010] However, the drugs disclosed in Patent Documents 1 and 2 have not been clinically established as treatments or preventive agents for age-related hearing loss. Currently, there are no drugs that are effective in fundamentally treating or preventing age-related hearing loss. Non-Patent Document 6 states that "(for age-related hearing loss) vitamins B, E, ATP, etc. are used, but their effectiveness is not very promising." [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] Japanese Patent Publication No. 2011-37738 [Patent Document 2] Japanese Patent Publication No. 2016-138145 [Non-patent literature]

[0012] [Non-Patent Document 1] Lyu AR et al., “Mitochondrial damage and necroptosis in aging cochlea.”, Int J Mol Sci., 2020, Vol.21, p.2505. doi: 10.3390 / ijms21072505 [Non-Patent Document 2] Darrat I et al., “Auditory research involving antioxidants.”, Curr Opin Otolaryngol Head Neck Surg., 2007, Vol.15, p.358-63. [Non-Patent Document 3] Seidman MD., “Effects of dietary restriction and antioxidants on presbyacusis.” Laryngoscope., 2000, Vol.110, p.727-38. [Non-Patent Document 4] Honkura Y et al., “NRF2 Is a key target for prevention of noise-induced hearing loss by reducing oxidative damage of cochlea.”, Sci Rep., 2016, Vol.6, p.19329. [Non-Patent Document 5] Kane KL et al., “Genetic background effects on age-related hearing loss associated with Cdh23 variants in mice.”, Hear Res., 2012, Vol.283, p.80-8. [Non-Patent Document 6] Author: Ichiro Kaitai, “New Otorhinolaryngology”, Revised 11th Edition, Nansando, February 14, 2013, p.199 [Summary of the Invention] [Problems to be Solved by the Invention]

[0013] As mentioned above, knowledge regarding the treatment and prevention of age-related hearing loss is very limited, and effective methods for slowing its progression have yet to be established.

[0014] Therefore, the present invention aims to provide a novel agent capable of treating, improving, and / or preventing age-related hearing loss. [Means for solving the problem]

[0015] The inventors diligently investigated the above problems and found that administering Ninjin-Yōeitō or Ryōkeijutsukantō to age-related hearing loss model mice significantly suppressed the progression of hearing loss compared to an untreated group. Furthermore, the inventors found that Ninjin-Yōeitō and Ryōkeijutsukantō significantly suppressed the decrease in the density of viable auditory nerve cells in age-related hearing loss model mice. These results suggest that Ninjin-Yōeitō and Ryōkeijutsukantō have the effect of suppressing age-related atrophy, degeneration, and death of auditory cells. Additionally, analysis of auditory nerve cells located in the basal rotation of the cochlea, which are involved in high-frequency hearing, revealed a significant difference in high-frequency hearing (16 and 32 kHz) and the density of viable auditory nerve cells (cells / mm²). 2 A strong positive correlation was observed between the two. Therefore, it was strongly suggested that Ninjin'yoei-to and Ryokeijutsukan-to significantly suppress the high-frequency hearing loss characteristic of age-related hearing loss. Based on these findings, the inventors completed the present invention.

[0016] The present invention provides an agent for treating, improving, and / or preventing age-related hearing loss, comprising a formula containing all of Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Licorice root, or containing extracts thereof as active ingredients.

[0017] The present invention also provides an agent for treating, improving and / or preventing age-related hearing loss, wherein the active ingredient is Ninjin'yoei-to or Ryokeijutsukan-to.

[0018] The present invention also provides an agent for suppressing the progression of presbycusis, which contains a prescription including all of Atractylodes macrocephala Koidzumi, Poria cocos Wolf, Cinnamomum cassia Blume, and Glycyrrhiza glabra L., or an extract thereof as an active ingredient.

[0019] The present invention also provides an agent for suppressing the progression of presbycusis, wherein the above active ingredient is Hitoshi-eki-to or Reikei-shukangan-to.

[0020] The present invention also provides an agent for suppressing age-related hearing loss, which contains a prescription including all of Atractylodes macrocephala Koidzumi, Poria cocos Wolf, Cinnamomum cassia Blume, and Glycyrrhiza glabra L., or an extract thereof as an active ingredient.

[0021] The present invention also provides an agent for suppressing age-related hearing loss, wherein the above active ingredient is Hitoshi-eki-to or Reikei-shukangan-to.

[0022] The present invention also provides an agent for suppressing atrophy, degeneration, and / or death of the auditory nerve associated with aging, which contains a prescription including all of Atractylodes macrocephala Koidzumi, Poria cocos Wolf, Cinnamomum cassia Blume, and Glycyrrhiza glabra L., or an extract thereof as an active ingredient.

[0023] The present invention also provides an agent for suppressing atrophy, degeneration, and / or death of the auditory nerve associated with aging, wherein the above active ingredient is Hitoshi-eki-to or Reikei-shukangan-to.

[0024] The present invention also provides a food composition for improving and / or preventing presbycusis, which contains a prescription including all of Atractylodes macrocephala Koidzumi, Poria cocos Wolf, Cinnamomum cassia Blume, and Glycyrrhiza glabra L., or an extract thereof as an active ingredient.

[0025] The present invention also provides a food composition, wherein the above active ingredient is Hitoshi-eki-to or Reikei-shukangan-to.

Effects of the Invention

[0026] Renjin'yoei-to and Ryokeijutsukan-to, which contain Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Glycyrrhiza uralensis, have the effect of suppressing the progression of age-related hearing loss. Furthermore, Renjin'yoei-to and Ryokeijutsukan-to have the effect of suppressing age-related atrophy, degeneration, and death of auditory nerves. Moreover, Renjin'yoei-to and Ryokeijutsukan-to can significantly suppress the high-frequency hearing loss characteristic of age-related hearing loss. Therefore, the present invention can provide novel agents, pharmaceuticals, and food compositions that can treat, improve, and / or prevent age-related hearing loss. [Brief explanation of the drawing]

[0027] [Figure 1] This figure shows the ABR hearing evaluation results for the baseline group at 5 months of age, the untreated group at 7 months of age, and the group treated with Ninjin'yoeito (a traditional Chinese medicine). [Figure 2] This figure shows the results of histological examination (hematoxylin and eosin staining) of specimens from the baseline group at 5 months of age, the untreated group at 7 months of age, and the group treated with Ninjin'yoeito (a traditional Chinese medicine). [Figure 3] This figure shows the neuronal cell survival rate (cell count / mm2) in the baseline group at 5 months of age, the untreated group at 7 months of age, and the group treated with Ninjin'yoeito (a traditional Chinese medicine). [Figure 4] This figure shows the correlation between hearing at a frequency of 4 kHz and the density of viable auditory nerve cells in a baseline group at 5 months of age, an untreated group at 7 months of age, and a group treated with Ninjin'yoeito (a traditional Chinese medicine). [Figure 5] This figure shows the correlation between hearing at a frequency of 8 kHz and the density of viable auditory nerve cells in a baseline group at 5 months of age, an untreated group at 7 months of age, and a group treated with Ninjin'yoeito (a traditional Chinese medicine formula). [Figure 6] This figure shows the correlation between hearing at a frequency of 12 kHz and the density of viable auditory nerve cells in a baseline group at 5 months of age, an untreated group at 7 months of age, and a group treated with Ninjin'yoeito (a traditional Chinese medicine formula). [Figure 7] This figure shows the correlation between hearing at a frequency of 16 kHz and the density of viable auditory nerve cells in a baseline group at 5 months of age, an untreated group at 7 months of age, and a group treated with Ninjin'yoeito (a traditional Chinese medicine). [Figure 8]This figure shows the correlation between hearing at a frequency of 32 kHz and the density of viable auditory nerve cells in a baseline group at 5 months of age, an untreated group at 7 months of age, and a group treated with Ninjin'yoeito (a traditional Chinese medicine). [Figure 9] This figure shows the ABR hearing evaluation results for the baseline group at 5 months of age, the untreated group at 7 months of age, and the group treated with Linggui Zhu Gan Tang. [Figure 10] This figure shows the correlation between hearing at a frequency of 4 kHz and the density of viable auditory nerve cells in a baseline group at 5 months of age, an untreated group at 7 months of age, and a group treated with Linggui Zhu Gan Tang. [Figure 11] This figure shows the correlation between hearing at a frequency of 8 kHz and the density of viable auditory nerve cells in a baseline group at 5 months of age, an untreated group at 7 months of age, and a group treated with Linggui Zhu Gan Tang. [Figure 12] This figure shows the correlation between hearing at a frequency of 12 kHz and the density of viable auditory nerve cells in a baseline group at 5 months of age, an untreated group at 7 months of age, and a group treated with Linggui Zhu Gan Tang. [Figure 13] This figure shows the correlation between hearing at a frequency of 16 kHz and the density of viable auditory nerve cells in a baseline group at 5 months of age, an untreated group at 7 months of age, and a group treated with Linggui Zhu Gan Tang. [Figure 14] This figure shows the correlation between hearing at a frequency of 32 kHz and the density of viable auditory nerve cells in a baseline group at 5 months of age, an untreated group at 7 months of age, and a group treated with Linggui Zhu Gan Tang. [Modes for carrying out the invention]

[0028] The present invention provides an agent for treating, improving, and / or preventing age-related hearing loss, comprising a formulation containing all of Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Licorice root, or containing extracts thereof as active ingredients. The present invention also provides an agent for inhibiting the progression of age-related hearing loss, comprising a formulation containing all of Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Licorice root, or containing extracts thereof as active ingredients. The present invention also provides an agent for inhibiting age-related hearing loss, comprising a formulation containing all of Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Licorice root, or containing extracts thereof as active ingredients. The present invention also provides an agent for inhibiting age-related atrophy, degeneration, and / or death of auditory nerves, comprising a formulation containing all of Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Licorice root, or containing extracts thereof as active ingredients.

[0029] In this specification, "age-related hearing loss" refers to sensorineural hearing loss that progresses with age. Hearing loss includes conductive hearing loss, which occurs when the conduction of sound to the inner ear is obstructed due to damage to the outer or middle ear, and sensorineural hearing loss, which is mainly caused by abnormalities in the inner ear and auditory nerve. Furthermore, hearing loss includes acute hearing loss, such as sudden hearing loss, and chronic hearing loss, such as age-related hearing loss.

[0030] In age-related hearing loss (presbycusis), the hearing threshold gradually increases with age, peaking in the 20s, and becoming more pronounced in the high-frequency range after the age of 50. Generally, physiological hearing loss associated with age is called presbycusis in a broad sense, while hearing loss significantly exceeding the age average is called presbycusis in a narrow sense. Age-related degeneration of the auditory organs appears in the middle ear, inner ear, and central nervous system, and is mostly characterized by atrophic degeneration of the spiral organ epithelium in the high-frequency portion of the inner ear, and atrophy and / or reduction of spiral ganglion cells. In addition, if the stria vascularis atrophies, bilateral horizontal hearing loss is observed. Age-related hearing loss can also be caused by decreased motility due to lipid deposition, calcification, and hyalinization of the basilar plate (Non-Patent Literature 6, p. 198).

[0031] On the other hand, sudden sensorineural hearing loss can occur as a sudden, unexplained, severe hearing loss in healthy individuals with no prior history of ear disease. Sudden sensorineural hearing loss is usually unilateral, with bilateral cases accounting for only about 7% of all cases (Non-Patent Literature 6, page 193). While age-related hearing loss is particularly pronounced in the high-frequency range, sudden sensorineural hearing loss can affect hearing in all frequencies, not just the high-frequency range. Another difference is that age-related hearing loss is often bilateral, while sudden sensorineural hearing loss is usually unilateral. Although progress has been made in developing treatments and medications for acute hearing loss such as sudden sensorineural hearing loss, effective treatments for chronic hearing loss have not yet been established.

[0032] In this specification, the term “treatment” includes improving or eliminating age-related hearing loss or its symptoms, achieving remission of the disease covered by the present invention, and achieving an improved prognosis.

[0033] In this specification, the term “improvement” includes alleviating or reducing the disease or symptoms in question, and inhibiting, suppressing, delaying or eliminating the progression of the symptoms.

[0034] In this specification, the term “prevention” includes inhibiting, suppressing, or delaying the onset (onset) of the disease or symptom in question, and inhibiting, suppressing, or delaying the recurrence of the disease or symptom in question.

[0035] In this specification, "suppressing the progression of age-related hearing loss" means inhibiting, suppressing, or delaying the worsening of symptoms or conditions of age-related hearing loss, inhibiting, suppressing, or delaying the onset of symptoms or conditions of age-related hearing loss, and inhibiting, suppressing, or delaying the recurrence of symptoms or conditions of age-related hearing loss. Symptoms or conditions of age-related hearing loss include various symptoms and conditions such as age-related hearing loss, age-related hearing loss in the high-frequency range, and tinnitus and dizziness associated with age-related hearing loss. The agent of the present invention can contribute to the treatment, improvement, and / or prevention of age-related hearing loss by suppressing its progression.

[0036] In this specification, "suppression of age-related hearing loss" includes significantly inhibiting, suppressing, delaying, or eliminating the progression of age-related hearing loss compared to an untreated group. The agent of the present invention can effectively suppress hearing loss in the high-frequency range, which is particularly characteristic of age-related hearing loss. The high-frequency range refers to frequencies such as 16-32 kHz in mice and 4-8 kHz in humans in audiometry.

[0037] In this specification, “suppression of age-related atrophy, degeneration, and / or death of auditory nerves” includes significantly inhibiting, suppressing, delaying, or eliminating the progression of age-related decline in the density or number of viable auditory nerve cells compared to an untreated group.

[0038] The active ingredient in the agent of the present invention may be Ninjin'yoei-to or Ryokeijutsukan-to.

[0039] Ninjin'yoeito is a traditional Chinese medicine that combines ginseng, astragalus, angelica, rehmannia, atractylodes, poria, peony, cinnamon, dried tangerine peel, polygala, schisandra, and licorice. Ninjin'yoeito in this invention may be a commercially available traditional Chinese medicine or may be manufactured by conventionally known methods. Furthermore, Ninjin'yoeito in this invention may be a chopped preparation of crude drugs prepared for extraction.

[0040] The preferred proportions of each herbal ingredient in Ninjin'yoei-to are 2-4 parts by weight of ginseng, 1.5-3 parts by weight of astragalus, 3-4 parts by weight of angelica, 2-4 parts by weight of rehmannia, 3-4 parts by weight of Atractylodes macrocephala, 2-4 parts by weight of Poria cocos, 2-4.5 parts by weight of peony root, 2-3 parts by weight of cinnamon bark, 2-3 parts by weight of dried tangerine peel, 1-2 parts by weight of Polygala tenuifolia, 1-2 parts by weight of Schisandra chinensis, and 1-3 parts by weight of licorice. Furthermore, as described in "Kampo Kosei Yoho Koshu" (by Michiaki Yakazu), 0.5-2 parts by weight of ginger and / or 0.5-2 parts by weight of jujube may also be added.

[0041] Linggui Zhu Gan Tang is a traditional Chinese medicine combining Atractylodes macrocephala (Byakujutsu), Poria cocos (Bukuryo), Cinnamon bark (Keihi), and Licorice root (Kanzo). Atractylodes lancea (Sojutsu) may be used instead of Atractylodes macrocephala (Byakujutsu). The Linggui Zhu Gan Tang used in this invention may be a commercially available traditional Chinese medicine or may be manufactured by conventionally known methods. Furthermore, the Linggui Zhu Gan Tang used in this invention may be a pre-cut preparation of crude drugs for extraction.

[0042] The preferred proportions of each herbal ingredient in Linggui Zhu Gan Tang are 2-4 parts by weight of Atractylodes macrocephala (or Atractylodes lancea), 4-6 parts by weight of Poria cocos, 3-4 parts by weight of Cinnamon bark, and 2-3 parts by weight of Licorice root.

[0043] Ginseng is a crude drug made from medicinal ginseng. Medicinal ginseng includes varieties such as Korean ginseng, Panax ginseng, Panax ginseng, and Panax notoginseng. Ginseng can be, for example, the root of Panax ginseng (Panax ginseng CAMeyer (Panax schinseng Nees)) or other related plants (Araliaceae), with the fine roots removed, or the root lightly blanched.

[0044] Astragalus is a crude drug made from the roots of Astragalus membranaceus Bunge, Astragalus mongholicus Bunge, or other related plants (Leguminosae).

[0045] Angelica root is a crude drug made from the roots of Angelica acutiloba Kitagawa, Angelica acutiloba Kitagawa var. sugiyamae Hikino, or other related plants (Umbelliferae). For example, these roots may be blanched before use.

[0046] Rehmannia glutinosa Liboschitz var. purpurea Makino and Rehmannia glutinosa Liboschitz, among others, is a crude drug made from the roots of plants of the same genus (Scrophulariaceae), and may be steamed, for example.

[0047] Byakujutsu is a crude drug made from the rhizome of Atractylodes japonica Koidzumi ex Kitamura (wabyakujutsu), Atractylodes macrocephala Koidzumi (karabyakujutsu), or other plants of the same genus.

[0048] Poria cocos is a crude drug made from the sclerotia of Wolfiporia cocos Ryvarden et Gilbertson (Poria cocos Wolf) or other related plants (Polyporaceae), and may be used with most of the outer layer removed.

[0049] Peony is a crude drug made from the roots of the peony plant (Paeonia lactiflora Pallas) or other related plants (Paeoniaceae).

[0050] Cinnamon bark is a crude drug made from the bark or part of the periderm of Chinese cinnamon (Cinnamomum cassia Blume) or other related plants (Lauraceae).

[0051] Chenpi is a crude drug made from the mature fruit peel of Satsuma mandarins (such as Citrus unshiu Marcowicz and Citrus reticulata Blanco) or other related plants (Rutaceae).

[0052] Onshi is a crude drug made from the roots of Polygala tenuifolia Willdenow or other related plants (Polygalaceae).

[0053] Gomi-shi is a crude drug made from the fruit of Schisandra chinensis Baill, a plant in the Schisandraceae family, and dried fruit may also be used.

[0054] Licorice is a crude drug made from the roots and stolons of Ural licorice (Glycyrrhiza uralensis Fischer), Spanish licorice (Glycyrrhiza glabra Linne), or other related plants (Leguminosae), and may also be used with the periderm removed (peeled licorice).

[0055] Each herbal ingredient in Ninjin'yoei-to and Ryokeijutsukan-to may be the plant itself, or it may be a powder obtained by crushing and drying the plant, or an extract from the plant. The extract can be obtained using conventionally known extraction methods, for example, by adding the plant or its pulverized material to a solvent such as water, heating and extracting, filtering, and then drying.

[0056] In the preparation of the present invention, Ninjin-Yōeitō and Ryōkeijutsukantō may be contained in the form of extracts. Extracts of Ninjin-Yōeitō and Ryōkeijutsukantō can be obtained using conventionally known extraction methods, for example, by adding Ninjin-Yōeitō or Ryōkeijutsukantō to a solvent such as water, heating and extracting, filtering, and then drying. Alternatively, the preparation of the present invention may be a preparation of chopped crude drugs prepared for extraction. The preparation may be, for example, a mixture of chopped crude drugs. Alternatively, the preparation of the present invention may be in the form of essential oils obtained from Ninjin-Yōeitō and Ryōkeijutsukantō. Essential oils can be isolated, for example, during the extraction process of Ninjin-Yōeitō and Ryōkeijutsukantō.

[0057] The agent of the present invention can be any form of formulation. As an orally administered formulation, the agent of the present invention can be, for example, tablets such as sugar-coated tablets, buccal tablets, coated tablets and chewable tablets; lozenges; pills; powders; capsules including hard capsules and soft capsules; granules; and liquid formulations such as suspensions, emulsions, syrups and elixirs.

[0058] Furthermore, the agent of the present invention may be a parenteral administration preparation such as intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, transdermal administration, transnasal administration, transpulmonary administration, enteral administration, oral administration, and transmucosal administration. The agent of the present invention may be, for example, an injectable preparation, a transdermal absorption tape, an aerosol preparation, and a suppository. The agent of the present invention may also be in the form of an essential oil, which may be used by applying it to the skin or by inhaling the aromatic components of the essential oil through the nose.

[0059] Furthermore, the agent of the present invention can be in any form, such as solid, liquid, granular, granular, powder, jelly, oil, capsule, cream, and paste.

[0060] The agent of the present invention may further contain any components commonly used in pharmaceuticals, quasi-drugs, or foods. For example, the agent of the present invention may further contain pharmaceutically acceptable bases, carriers, excipients, binders, disintegrants, lubricants, and colorants.

[0061] Examples of carriers and excipients used in the agents of the present invention include lactose, glucose, sucrose, mannitol, dextrin, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, and crystalline cellulose.

[0062] Examples of binders include starch, gelatin, syrup, tragacanth gum, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, hydroxypropyl cellulose, methylcellulose, ethylcellulose, and carboxymethylcellulose.

[0063] Examples of disintegrants include starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, sodium alginate, sodium carboxymethylcellulose, and calcium carboxymethylcellulose.

[0064] Examples of lubricants include magnesium stearate, hydrogenated vegetable oils, talc, and macrogol. For colorants, any colorant permitted for use in pharmaceuticals, quasi-drugs, and food products may be used.

[0065] Furthermore, the agent of the present invention may be coated with one or more layers of sucrose, gelatin, purified shellac, gelatin, glycerin, sorbitol, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, cellulose phthalate acetate, hydroxypropylmethylcellulose phthalate, methyl methacrylate, and methacrylic acid polymers, if necessary.

[0066] Furthermore, the agent of the present invention may optionally contain pH adjusters, buffers, stabilizers, preservatives, antiseptics, diluents, coating agents, sweeteners, flavorings, and solubilizers.

[0067] The amounts of Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Glycyrrhiza uralensis in the preparation of the present invention should be such that they exert their effects sufficiently, and can be appropriately set according to the target, purpose, and method of administration (method of intake). For example, when administering Ninjin'yoei-to orally to a human as an active ingredient, it is preferable to include Ninjin'yoei-to as a crude drug in an amount of 0.3 to 300 g per day, more preferably 1 to 100 g, and even more preferably 10 to 50 g. Alternatively, it is preferable to include an extract of Ninjin'yoei-to in an amount of 0.05 to 100 g per day, more preferably 0.5 to 50 g, and even more preferably 3 to 15 g. Furthermore, when administering Ryokeijutsukanto orally to a human as an active ingredient, it is preferable to include Ryokeijutsukanto as a crude drug in an amount of 0.3 to 300 g per day, more preferably 1 to 100 g, and even more preferably 10 to 50 g. For example, Linggui Zhu Gan Tang can be included in such a way that the daily intake amount is 31g, which is the full amount of crude drug. Preferably, the extract of Linggui Zhu Gan Tang can be included in such a way that the daily intake amount is 0.05 to 100g, more preferably 0.5 to 50g, and even more preferably 3 to 15g. For example, Linggui Zhu Gan Tang can be included in such a way that the daily intake amount is 15g, which is the full amount of extract.

[0068] The present invention also provides a pharmaceutical composition for treating, improving, and / or preventing age-related hearing loss, comprising Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Licorice root or extracts thereof as active ingredients. The pharmaceutical composition of the present invention may further contain any ingredients commonly used in pharmaceuticals, for example, pharmaceutically acceptable bases, carriers, excipients, binders, disintegrants, lubricants, and colorants. The pharmaceutical composition of the present invention may be constructed in the same manner as the agents described above.

[0069] The pharmaceutical composition of the present invention may be used in combination with vitamins, steroids, and / or drugs effective against tinnitus. These drugs may be administered in combination with the pharmaceutical composition of the present invention or incorporated into the pharmaceutical composition of the present invention. Examples of vitamins include vitamin C and vitamin B12. Examples of steroids include dexamethasone, vecramsazone, corticosteroids, prednisone, prednisolone, and methylprednisolone. Examples of drugs effective against tinnitus include adenosine triphosphate disodium (ATP), vitamin B12 preparations, isosorbide, diphenidol hydrochloride, nicotinamide, papaverine hydrochloride, loflazepate ethyl, alprazolam, clotiazepam, etizolam, kallidinogenase, and tofisopam.

[0070] The present invention also provides a method for treating, improving, and / or preventing age-related hearing loss, comprising administering Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Glycyrrhiza uralensis, or extracts thereof, to a subject. The present invention also provides a method for suppressing the progression of age-related hearing loss, comprising administering Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Glycyrrhiza uralensis, or extracts thereof, to a subject. The present invention also provides a method for suppressing atrophy, degeneration, and / or death of auditory nerves, comprising administering Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Glycyrrhiza uralensis, or extracts thereof, to a subject. In the methods of the present invention, Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Glycyrrhiza uralensis, or extracts thereof, may be administered in the form of the above-described agents or compositions.

[0071] In the method of the present invention, the dosage of Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Licorice root, or extracts thereof, may be set appropriately according to the target, purpose, and method of administration (ingestion) to which it is applied. For example, when administered orally to a human, preferably, the daily intake of Ninjin-yoei-to as a crude drug is 0.3 to 300 g, more preferably 1 to 100 g, and even more preferably 10 to 50 g. Alternatively, preferably, the daily intake of an extract of Ninjin-yoei-to is 0.05 to 100 g, more preferably 0.5 to 50 g, and even more preferably 3 to 15 g. Alternatively, preferably, the daily intake of Ryokeijutsukan-to as a crude drug is 0.3 to 300 g, more preferably 1 to 100 g, and even more preferably 10 to 50 g. Furthermore, it is preferable that the extract of Linggui Zhu Gan Tang be administered in a daily intake amount of 0.05 to 100 g, more preferably 0.5 to 50 g, and even more preferably 3 to 15 g.

[0072] The methods of the present invention are applicable to mammals such as humans, mice, rats, rabbits, cats, dogs, cattle, horses, and monkeys. In the methods of the present invention, the subjects to be administered the treatment are not particularly limited, as long as they are subjects that need to be treated, improved, and / or prevented for age-related hearing loss.

[0073] The present invention also provides Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Licorice root, or extracts thereof, for use in the treatment, improvement, and / or prevention of age-related hearing loss. The present invention also provides the use of Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Licorice root, or extracts thereof, for the treatment, improvement, and / or prevention of age-related hearing loss. The present invention also provides the use of Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Licorice root, or extracts thereof, for the manufacture of agents, compositions, pharmaceutical compositions, or food compositions for the treatment, improvement, and / or prevention of age-related hearing loss.

[0074] The present invention also provides a food composition for improving and / or preventing age-related hearing loss, comprising a formula containing all of Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Glycyrrhiza uralensis, or containing extracts thereof as active ingredients. The food composition of the present invention may be constructed in the same manner as the agents and compositions described above.

[0075] In this specification, "food composition" includes not only general food and beverages, but also foods for the sick, health foods, functional foods, foods for specified health uses, nutritional supplements, and other supplements. General food and beverages include, for example, various beverages, various foods, processed foods, liquid foods (soups, etc.), seasonings, nutritional drinks, and confectionery. In this specification, "processed food" refers to products made by processing and / or cooking natural ingredients (animals and plants, etc.), and includes, for example, processed meat products, processed vegetable products, processed fruit products, frozen foods, retort foods, canned foods, bottled foods, and instant foods. The food composition of the present invention may be a food product that bears a label indicating that it treats, improves and / or prevents age-related hearing loss, inhibits the progression of age-related hearing loss, or inhibits age-related hearing loss. The food composition of the present invention may also be provided in a form sealed in bags and containers. The bags and containers used in the present invention can be any bags and containers that are normally used for food. [Examples]

[0076] (mouse) The 5-month-old C57BL / 6J female mice used in the examples were purchased from Oriental Yeast Co., Ltd. The mice were acclimated for one week at 23±3°C in an environment with free access to food and water under a 12-hour light-dark cycle (lights on from 08:00 to 20:00).

[0077] (Reagents and feed) Ketamine was purchased from Daiichi Sankyo Co., Ltd., and xylazine was purchased from Fujifilm Wako Pure Chemical Industries, Ltd. Standard mouse feed (MF) was purchased from Clear Japan.

[0078] (Test method) Five-month-old C57BL / 6J female mice were divided into three groups (baseline group, untreated group, and test sample-treated group) (n=10). Ten mice in the test sample-treated group were fed a 4% (w / w) test sample diet for two months from five to seven months of age. As a control, ten mice in the untreated group were fed MF (normal diet) for two months. To establish a baseline for the hearing of the mouse strain used, auditory evaluation was performed on ten five-month-old mice in the baseline group. Auditory evaluation was performed under anesthesia two months after the start of treatment in the test sample-treated and untreated groups (mice at seven months of age). After evaluating auditory brainstem response (ABR), the mice were sacrificed, and cochlear tissue was collected and analyzed.

[0079] (ABR hearing assessment method) Based on Non-Patent Document 4, an ABR hearing evaluation was conducted.

[0080] Mice were anesthetized by intraperitoneal administration of ketamine (100 mg / kg) and xylazine (20 mg / kg). ABR was evaluated in a soundproof room. Three subcutaneous electrodes (base electrode, reference electrode, and active electrode) were placed 2-3 mm under the skin to assess the auditory threshold. The active electrode was inserted subcutaneously into the forehead. The reference electrode and ground electrode were inserted subauricular and dorsal to the right ear, and ABR recordings were acquired using the TDT System 3 auditory evoked potential workstation and analyzed using BioSigRP software (Tucker-Davis Technologies, Alachua, FL, USA).

[0081] ABR was used to induce responses using pure tone bursts at frequencies of 4, 8, 12, 16, and 32 kHz. Evoked responses were averaged over 1,000 sweeps. ABR was evaluated starting at 100 dB and gradually decreasing in 5 dB steps down to 10 dB. Threshold shift was defined as the lowest acoustic intensity that could elicit at least one peak in the average ABR. In this experiment, the threshold was evaluated using the V-wave. Under blinded conditions, the ABR threshold was determined by clearly identifying the V-wave from the raw data. This was double-checked by the assigned caregiver and a blinded caregiver.

[0082] (Cochlear tissue analysis method) After hearing evaluation using ABR, cochlear tissue was collected from mice. Following sample collection, the cochlea was fixed by replacing the perilymph by injecting 4% paraformaldehyde through the oval or round window using a syringe. Subsequently, decalcification was performed using 10% EDTA, followed by stepwise dehydration with 70-100% ethanol. Paraffin blocks were then prepared, and thin tissue sections were obtained. Deparaffinization was performed using xylene, and the paraffin was removed with ethanol.

[0083] The slides were washed with water. The cells were stained with Meyer's hematoxylin for 5 minutes and washed with water for 30 seconds. The cells were immersed in phosphate-buffered saline for 1 minute and washed with water for 2 minutes. Eosin staining was performed for 1 minute, and then the cells were washed with water. The density of intact auditory nerve cells and the area of ​​the stria vascularis in the basal turn (the region responsible for high frequencies) of the cochlea were measured.

[0084] (statistical analysis) All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), the graphical user interface for R-2.3-0 (R Foundation for Statistics Computing, Vienna, Austria). All data are presented as mean ± standard error of the mean. Statistical comparisons were performed using one-way ANOVA followed by Tukey's test. For data with a Bartlett test p-value <0.01 or a Kolmogorov-Smirnov test (Shapiro-Wilk normality test) p-value <0.05, the Steel-Dwass test was used.

[0085] A difference of p<0.05 indicates statistical significance.

[0086] (Correlation between hearing ability and the density of living auditory nerve cells) The correlation between hearing ability and the density of viable auditory nerve cells was evaluated based on the correlation coefficient R, using the following criteria. |R|=0.7~1 There is a fairly strong correlation. |R|=0.4~0.7 Slight correlation |R|=0.2~0.4 Weak correlation present. |R|=0~0.2 Almost no correlation

[0087] [Example 1: Ginseng Nourishing Decoction] (Ginseng Nourishing Decoction) Ninjin-Yōeitō (NYT) was prepared by Kracie Pharmaceutical Co., Ltd. (Toyama, Japan) as a powder containing the following crude drugs: Rehmannia glutinosa (4g), Angelica sinensis (4g), Atractylodes macrocephala (4g), Poria cocos (4g), Ginseng (3g), Cinnamon bark (2.5g), Polygala tenuifolia (2g), Peony root (2g), Citrus reticulata (2g), Astragalus membranaceus (1.5g), Glycyrrhiza uralensis (1g), and Schisandra chinensis (1g). These crude drugs were identified based on their external form and certified based on marker compounds in accordance with the Japanese Pharmacopoeia and our company's standards. The Ninjin-Yōeitō extract powder (lot number: E1712111A0) was mixed with a normal diet at a 4% (w / w) concentration.

[0088] (HPLC analysis) 0.5 g of Ninjin-Yōeitō extract was mixed with 50% methanol (50 mL), shaken, and extracted by sonication for 30 minutes. The supernatant was filtered using a membrane filter (0.22 μm), and three-dimensional high-performance liquid chromatography (HPLC) fingerprint analysis was performed. The HPLC system consisted of an LC-30AD pump, an SPD-M30A diode array detector (Shimadzu Corporation, Kyoto, Japan), and a YMC-Triart C18 column (φ3.0 × 150 mm, YMC Co., Ltd., Kyoto, Japan). The mobile phase was either 0.2% phosphate-containing ultrapure water / 0.2% phosphate-containing acetonitrile (6:4) or 0.2% phosphate-containing ultrapure water / 0.2% phosphate-containing acetonitrile (8:2). During the HPLC analysis, the flow rate was controlled at 0.5 mL / min by the LC-30AD. The eluent from the column was monitored, and the 3D data was processed using an SPD-M30A diode array detector.

[0089] (HPLC of Ninjin-Yōeitō extract) The composition of the Ninjin-Yōeitō extract was identified using HPLC. Identification was performed by chromatographic profiling based on retention time and ultraviolet spectrum, using corresponding standards. As a result, (E)-cinnamic acid, glycyrrhizic acid, (E)-cinnamic acid aldehyde, (E)-2-methoxycinnamaldehyde, nobiletin, schizandrine, atractylenolide III, and gomisin A were identified using 0.2% phosphate-containing ultrapure water / 0.2% phosphate-containing acetonitrile (8:2) as the mobile phase. Furthermore, paeoniflorin, naringin, hesperidin, (E)-cinnamic acid, ginsenoside Rg1, and (E)-cinnamic acid aldehyde were identified using 0.2% phosphate-containing ultrapure water / 0.2% phosphate-containing acetonitrile (8:2) as the mobile phase.

[0090] (ABR hearing evaluation results) ABR hearing was evaluated in mice two months after treatment with Ninjin-Yōeitō. Table 1 and Figure 1 show the results of the ABR hearing evaluation. Data are expressed as mean ± standard error of the mean (n=10) (Steel-Dwass test). As shown in Figure 1, the untreated group (7 months; 7M) had significantly lower hearing compared to the baseline group at 5 months of age (5 months; 5M). On the other hand, the hearing of the Ninjin-Yōeitō treated group (7 months + Ninjin-Yōeitō; NYT), which was administered for two months starting at 5 months of age, was almost the same as that of the baseline group at 5 months of age. In other words, the hearing loss seen in the untreated group did not progress in the Ninjin-Yōeitō treated group, and their hearing was significantly higher than that of the untreated group. Furthermore, the hearing of the Ninjin-Yōeitō treated mice was significantly higher than that of the untreated group at all frequencies: 4, 8, 12, 16, and 32 kHz.

[0091] [Table 1]

[0092] (Cochlear tissue analysis results) Next, the collected cochlear tissue was analyzed. Figure 2 shows the histological examination (hematoxylin and eosin staining) results of specimens from the baseline group at 5 months of age, the untreated group at 7 months of age, and the Ninjin-Yōeitō-treated group. Table 2 and Figure 3 show the viable cell density (cell count / mm³) of neurons in the baseline group at 5 months of age (5M), the untreated group at 7 months of age (7M), and the Ninjin-Yōeitō-treated group (NYT). 2 ) indicates.

[0093] As shown in Figure 3, the density of viable auditory nerve cells in the untreated group at 7 months of age was significantly lower compared to the baseline group at 5 months of age. On the other hand, the density of viable auditory nerve cells in the Ninjin-Yōeitō-treated group at 7 months of age was significantly higher than in the untreated group. These results suggest that Ninjin-Yōeitō has the effect of suppressing the atrophy, degeneration, and death of auditory cells, including auditory nerve cells, that are associated with aging.

[0094] [Table 2]

[0095] Next, we analyzed auditory nerve cells located in the basal rotator cleft of the cochlea, which are involved in high-frequency hearing. Figures 4-8 show the correlation between hearing and the density of viable auditory nerve cells at frequencies of 4, 8, 12, 16, and 32 kHz. In Figures 4-8, squares represent the baseline group at 5 months of age, triangles represent the untreated group at 7 months of age, and circles represent the group treated with Ninjin'yoeito at 7 months of age.

[0096] As a result, the correlation coefficients between hearing ability and the density of viable auditory nerve cells were R=0.426 at 4kHz, R=0.453 at 8kHz, R=0.462 at 12kHz, R=0.471 at 16kHz, and R=0.514 at 32kHz. A correlation was observed at all frequencies, particularly between hearing ability and the density of viable auditory nerve cells (cells / mm²) in the high-frequency range (16 and 32kHz). 2 A strong positive correlation was observed between (r=0.5).

[0097] Furthermore, no atrophy of the stria vascularis region was observed in this model mouse between 5 and 7 months of age (not shown).

[0098] These results suggest that Ninjin-Yōeitō has an effect of suppressing age-related hearing loss and protecting auditory neuroprotection. ABR results at each frequency revealed that hearing in mice treated with Ninjin-Yōeitō was significantly higher than that of untreated mice in the high-frequency range. This suggests that Ninjin-Yōeitō is effective against the high-frequency hearing loss characteristic of age-related hearing loss (Figure 1). The suppression of age-related hearing loss progression in mice treated with Ninjin-Yōeitō may be due to its protective effect on auditory nerve cells.

[0099] The C57BL / 6J female mice used in this example have been reported to develop age-related hearing loss at 6 months of age (Non-Patent Literature 5), and are widely used as an evaluation model for age-related hearing loss. Based on the results of the hearing evaluation and the correlation evaluation of living cells in this example, it was suggested that Ninjin'yoei-to suppresses the progression of age-related hearing loss and maintains hearing by protecting the auditory nerves that degenerate or die with age.

[0100] [Example 2] (Ling Guiju Sweet Tang) Linggui Zhu Gan Tang (RKZ) was prepared by Kracie Pharmaceutical Co., Ltd. (Toyama, Japan) as a powder containing the herbal medicines Atractylodes macrocephala (1.5g), Poria cocos (3.0g), Cinnamon bark (2.0g), and Licorice root (1.0g). These herbal medicines were identified based on their external form and certified based on marker compounds in accordance with the Japanese Pharmacopoeia and our company's standards. The Linggui Zhu Gan Tang extract powder (lot number: 16110425) was mixed with a normal diet at a concentration of 1.0% (w / w).

[0101] (ABR hearing evaluation results) ABR hearing was evaluated in mice two months after treatment with Linggui Zhu Gan Tang. Table 3 and Figure 9 show the results of the ABR hearing evaluation. Data are expressed as mean ± standard error of the mean (n=10) (Steel-Dwass test). As shown in Figure 9, the untreated group (7 months; 7M) had significantly lower hearing compared to the baseline group at 5 months of age (5 months; 5M). On the other hand, the hearing of the Linggui Zhu Gan Tang-treated group (7 months + Linggui Zhu Gan Tang; RKZ), which was administered for two months starting at 5 months of age, was almost the same as that of the baseline group at 5 months of age. In other words, the hearing loss seen in the untreated group did not progress in the Linggui Zhu Gan Tang-treated mice, and their hearing was significantly higher than that of the untreated group. Furthermore, mice treated with Linggui Zhu Gan Tang had significantly higher hearing at frequencies of 12 and 32 kHz compared to the untreated group.

[0102] [Table 3]

[0103] Next, we analyzed auditory nerve cells located in the basal rotator cleft of the cochlea, which are involved in high-frequency hearing. Figures 10-14 show the correlation between hearing and the density of viable auditory nerve cells at frequencies of 4, 8, 12, 16, and 32 kHz. In Figures 10-14, squares represent the baseline group at 5 months of age, triangles represent the untreated group at 7 months of age, and circles represent the group treated with Linggui Zhu Gan Tang at 7 months of age.

[0104] As a result, the correlation coefficients between hearing ability and the density of viable auditory nerve cells were R=0.285 at 4kHz, R=0.301 at 8kHz, R=0.297 at 12kHz, R=0.439 at 16kHz, and R=0.272 at 32kHz. A positive correlation was observed at all frequencies, and a slightly positive correlation was particularly observed at 16kHz, which is in the high-frequency range.

[0105] Furthermore, no atrophy of the stria vascularis region was observed in this model mouse between 5 and 7 months of age (not shown).

[0106] These results suggest that Reikeijutsukanto has an effect of suppressing age-related hearing loss and protecting auditory neuroprotection. ABR results at various frequencies revealed that hearing in mice treated with Reikeijutsukanto was significantly higher than in untreated mice in the high-frequency range. This suggests that Reikeijutsukanto is effective against the high-frequency hearing loss characteristic of age-related hearing loss. The suppression of age-related hearing loss progression in mice treated with Reikeijutsukanto may be due to its protective effect on auditory nerve cells.

[0107] The C57BL / 6J female mice used in this example have been reported to develop age-related hearing loss at 6 months of age (Non-Patent Literature 5), and are widely used as an evaluation model for age-related hearing loss. Based on the results of the hearing evaluation and the correlation evaluation of living cells in this example, it was suggested that Reikeijutsukanto suppresses the progression of age-related hearing loss and maintains hearing by protecting the auditory nerves that degenerate or die with age. [Industrial applicability]

[0108] The present invention is suitably applicable to agents, pharmaceuticals, and food compositions for treating, improving, and / or preventing age-related hearing loss.

Claims

1. A formula containing all of Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Licorice root, or containing only extracts thereof as active ingredients, for the suppression of age-related atrophy, degeneration, and / or death of auditory nerves in subjects before the onset of age-related hearing loss.

2. The inhibitor of age-related atrophy, degeneration, and / or death of auditory nerves according to claim 1, wherein the active ingredient is Ninjin'yoei-to or Ryokeijutsukan-to.

3. A food composition for suppressing age-related atrophy, degeneration, and / or death of auditory nerves in subjects before the onset of age-related hearing loss, comprising a formula containing all of Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Licorice root, or containing only extracts thereof as active ingredients.

4. The food composition according to claim 3, wherein the active ingredient is Ninjin'yoei-to or Ryokeijutsukan-to.

5. A formula containing all of the following ingredients: Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Licorice root, or containing only extracts of these ingredients as active ingredients, for the purpose of preventing the onset of age-related hearing loss in subjects who have not yet developed age-related hearing loss.

6. A formula containing all of the following: Atractylodes macrocephala, Poria cocos, Cinnamon bark, and Licorice root, or containing only extracts of these as active ingredients, as a protective agent for auditory nerve cells in subjects before the onset of age-related hearing loss.

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