KR20230063343A - Combined Composition for preventing or treating hearing loss comprising Sarpogrelate and Vaccinium myrtillus extract - Google Patents

Abstract

The present invention relates to a composite composition for preventing or treating hearing loss containing sarpogrelate and a Vaccinium myrtillus extract as active components, wherein the composite composition of the present invention effectively inhibits hair cell death caused by reactive oxygen species generated by hydrogen peroxide in a small dose, and is confirmed to exhibit an excellent protective effect on zebrafish auditory hair cells, so that the composite composition can be usefully used as a pharmaceutical or health food for preventing or treating hearing loss.

Description

Complex composition for preventing or treating hearing loss containing sarpogrelate and bilberry extract as active ingredients {Combined Composition for preventing or treating hearing loss comprising Sarpogrelate and Vaccinium myrtillus extract}

The present invention relates to a composition for preventing or treating hearing loss in a combination preparation containing sarpogrelate and bilberry extract as active ingredients.

Hearing loss or hearing loss is a common disease that affects about 15-20% of the population, but it is a disease that greatly affects the quality of life. In recent years, the population with hearing loss is rapidly increasing due to the aging population and the spread of digital devices, and it is on the rise. Since hearing impairment is permanent and difficult to fundamentally treat, it is very important to prevent it before it occurs.

The ear is divided into the outer ear (eardrum to the eardrum), the middle ear (the eardrum to the cochlea), and the inner ear (inside the cochlea). Hearing loss caused by disorders of the outer and middle ear is cured when the disorder is treated, but hearing loss caused by disorders of the inner ear and auditory nervous system is not impaired. Even with treatment, recovery is very difficult.

Hearing loss can be classified into age-induced hearing loss, pediatric hearing loss, Meniere's disease, sudden hearing loss, noise-induced hearing loss, and ototoxic hearing loss, among which noise-induced hearing loss and ototoxic hearing loss caused by noise and drugs are the most common. Noise-induced hearing loss is rapidly increasing.

The human auditory organ is affected by noise above 75 dBA, and this level of noise is equivalent to living in noise harmful to the auditory organ in industrial society. In addition, there are many cases in which the auditory organs are exposed to loud sounds by using earphones, so the number of patients with noise-induced hearing loss is increasing in various age groups. Noise-induced hearing loss at a young age becomes more and more severe with age and affects people of various ages.

In addition, it is known that ototoxic hearing loss is caused by cell death caused by active oxygen generated by ototoxic drugs.

Antioxidants have been mainly used in studies to prevent and treat noise-induced hearing loss and ototoxic hearing loss, but there is no drug showing a clear preventive effect yet, and the safety of many drugs has not been proven.

Republic of Korea Patent Publication No. 10-2018-0089207 (2018.08.08. Publication)

The present invention is to provide a composition containing sarpogrelate and bilberry extract as active ingredients as a pharmaceutical composition and health food for preventing or treating hearing loss caused by reactive oxygen species generated by noise or drug exposure.

The present invention provides a pharmaceutical composition for preventing or treating hearing loss containing sarpogrelate and bilberry extract as active ingredients.

In addition, the present invention provides a health food for preventing or improving hearing loss containing sarpogrelate and bilberry extract as active ingredients.

According to the present invention, sapogrelate and bilberry extract are 1: (2 to 5) weight ratio (except for the case of 1: 3 weight ratio), that is, 1: (2 or more to less than 3) or 1: (more than 3 to 5 A composition containing as an active ingredient a complex composition mixed in a weight ratio selected from the following) effectively inhibits hair cell death caused by reactive oxygen species generated by hydrogen peroxide in a small dose, and has an excellent protective effect on zebrafish auditory hair cells As confirmed, the composition containing the sarpogrelate and bilberry extract as active ingredients can be provided as a composition for preventing or treating sensorineural hearing loss.

Figure 1 is the result of confirming the efficacy test on hair cells in the zebrafish drug efficacy model of the composite composition according to the composition ratio of the present invention.

Hereinafter, the present invention will be described in more detail.

Noise and ototoxic drugs generate active oxygen, and the active oxygen thus generated induces apoptosis of auditory cells, resulting in noise-induced hearing loss and ototoxic hearing loss. While researching a treatment for more effective treatment, it was confirmed that the complex composition containing sarpogrelate and bilberry extract as active ingredients in an optimal weight ratio exhibits excellent protective effect on auditory cells exposed to active oxygen even at a small dose. Accordingly, the present invention was completed.

The present invention contains sarpogrelate and bilberry extract as active ingredients, and the sapogrelate and bilberry extract are included in a weight ratio selected from 1: (2 or more to less than 3) or 1: (more than 3 to 5 or less) It is possible to provide a pharmaceutical composition for preventing or treating hearing loss.

More specifically, the hearing loss may be selected from the group consisting of noise-induced hearing loss, ototoxic hearing loss, age-related hearing loss and sudden hearing loss, and as an example, the hearing loss is noise-induced hearing loss induced by hydrogen peroxide or reactive oxygen species (ROS) Or it may be ototoxic hearing loss.

The pharmaceutical composition may be a composite composition consisting of sarpogrelate and bilberry extract in a weight ratio of 1: (1 to 8). Preferably, the weight ratio of sapogrelate and bilberry extract is a composite composition having a ratio of 1:2 to 5 (except for 1:3), more preferably, the weight ratio of sapogrelate and bilberry extract is 1:1 , It may be a composite composition having a weight ratio of 1:2, 1:4 or 1:5.

The composite composition may exhibit an increased hearing improvement effect rather than a simple additive increase effect than the effect of a substance consisting of a single component, sarpogrelate or bilberry extract. When the weight ratio of sarpogrelate and bilberry extract is less than 1:1, the content of bilberry that can be combined is low compared to the daily recommended amount of 300 mg of sarpogrelate, making it difficult to expect a synergistic effect as a complex composition, and sarpogrelate and bilberry extract Even when the weight ratio of is greater than 1:8, it is difficult to expect a synergistic effect as a composite composition because the content of sarpogrelate is low compared to the recommended daily amount of 510 mg of bilberry.

The bilberry extract used in the present invention is an extract prepared using water, C1 to C4 alcohol, or a mixed solvent of bilberry fruit/leaf parts as an extraction solvent. Using 5 to 30 times as much extraction solvent, it is extracted and concentrated by the cold acupuncture method, warm acupuncture method, percolation method, etc., and it is in the form of soft extract, or it is spray-dried or freeze-dried by mixing soft extract alone or mixed with excipients such as soft extract and dextran. It can be powdered.

Sarpogrelate, one of the constituents of the present invention, is (-)-4-[1-dimethylamino-3-[2-[2-(3-methoxyphenyl)ethyl]phenoxy]propan-2-yl]oxy-4- As oxobutanoic acid, its molecular structure is C ₂₄ H ₃₁ NO ₆ and it is a drug with the following structure, generally in the form of a hydrochloride salt. Sarpogrelate, which is a component of the present invention, is not limited to hydrochloride, and may include any salt including free base or hydrochloride.

[chemical formula]

The pharmaceutical composition may inhibit apoptosis of auditory cells and increase the number of hair cells.

According to an embodiment of the present invention, the complex extract inhibited apoptosis of auditory cell lines by up to 20% or more at the same concentration compared to a single administration of bilberry extract, and the number of hair cells in zebrafish was reduced with a single administration of sarpogrelate. It was confirmed that the effect was further increased by comparison.

In particular, in Example 3, in which sarpogrelate and bilberry extract were mixed at a weight ratio of 1:4, the effect was 20% higher than that of 50mpk and 100mpk doses of sapogrelate under the click tone condition in the 14-day auditory brainstem response test of mice. , it was confirmed that the threshold improvement effect was higher than that of the bilberry extract by 10% or more at the same dose.

In one embodiment of the present invention, the pharmaceutical composition for preventing or treating hearing loss containing the sarpogrelate and bilberry extract as active ingredients is an injection, granule, powder, tablet, pill, capsule, suppository, gel according to conventional methods. , Any one formulation selected from the group consisting of suspensions, emulsions, drops or liquids may be used.

In another embodiment of the present invention, the pharmaceutical composition is a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, lubricant, flavoring agent, antioxidant, buffer, bacteriostatic agent, diluent commonly used in the manufacture of pharmaceutical compositions , It may further include one or more additives selected from the group consisting of dispersants, surfactants, binders, and lubricants.

Specifically, carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil may be used, and solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules. These solid preparations may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for oral use, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base material of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

According to one embodiment of the present invention, the pharmaceutical composition is administered in a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered to the subject as

The preferred dosage of the composition containing the sarpogrelate and bilberry extract as active ingredients may vary depending on the condition and weight of the subject, the type and severity of the disease, the type of drug, the route and duration of administration, and may be appropriately selected by those skilled in the art. can According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or divided into several administrations, and the scope of the present invention is not limited thereby.

In the present invention, the 'subject' may be a mammal including a human, but is not limited to these examples.

In addition, the present invention contains sarpogrelate and bilberry extract as active ingredients, and the sapogrelate and bilberry extract are included in a weight ratio selected from 1: (more than 2 to less than 3) or 1: (more than 3 to less than 5) It is possible to provide a health food for preventing or improving hearing loss, characterized in that being.

The health food is used together with other foods or food additives in addition to the sarpogrelate and bilberry extract, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use thereof, for example, prevention, health or therapeutic treatment.

The effective dose of the compound contained in the health food may be used according to the effective dose of the therapeutic agent, but may be less than the above range in the case of long-term intake for the purpose of health and hygiene or health control. Since there is no problem in terms of safety, it is certain that the components can be used in an amount greater than the above range.

There is no particular limitation on the type of health food, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes; and the like.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Examples 1 to 4> Preparation of a composite composition of bilberry ethanol extract and sarpogrelate

The bilberry extract was first extracted by adding 100 g of bilberry to 1 L of 90 (v/v)% ethanol aqueous solution and stirring at 40 °C at 1600 rpm for 2 hours. The extract of the first extract was transferred, and 1L of 70 (v / v)% ethanol aqueous solution was added to the remaining residue, followed by agitation at 40 ° C. Thereafter, 1 L of 70 (v/v)% ethanol aqueous solution was added to the secondary residue, followed by third extraction in the same manner.

After mixing the first, second and third extracts, alcohol was evaporated while concentrating at a temperature of 50 °C or less. The concentrate obtained by the above process was diluted with 1.5±5% of Brix and centrifuged at 6900 rpm for 6 to 12 minutes.

The supernatant obtained through centrifugation was purified by compression filtration. Thereafter, 1200 L of water was added to the purified solution to perform first washing for 2 hours, and 5000 L of 70 (v/v)% ethanol was added to the first washed purified solution to perform second washing, and then the washed solution was collected and concentrated.

The concentrate obtained through the above process was spray-dried to prepare an ethanol extract of bilberry in powder form.

Sapogrelate hydrochloride (Manufacturer: Pharmacostech) was added to the powdered bilberry ethanol extract in the weight ratio shown in Table 1, and the bilberry extract and sapogrelate were added to a mixer and mixed at a speed of 50 rpm for 5 minutes to prepare a composite composition. did

Example 1 Example 2 Example 3 Example 4 Bilberry Ethanol Extract 10g 20g 40g 50g sarpogrelate 10g 10g 10g 30g Weight of composite composition 20g 30g 50g 80g

<Examples 5 to 8> Preparation of composite composition of bilberry ethanol extract and sarpogrelate

100 g of bilberry was added to 0.5L of 70 (v/v)% aqueous ethanol solution, and primary extraction was performed by stirring at 60° C. at 1600 rpm for 2 hours. The extract of the first extract was transferred, and 0.5 L of 70 (v / v)% ethanol aqueous solution was added to the remaining residue, followed by agitation at 60 ° C. . Thereafter, 0.5 L of 70 (v/v)% ethanol aqueous solution was added to the secondary residue, followed by third extraction in the same manner.

After mixing the first, second and third extracts, alcohol was evaporated while concentrating at a temperature of 50 ° C or less. The concentrate obtained by the above process was diluted with Brix 3±5% and centrifuged at 6900 rpm for 6 to 12 minutes.

The supernatant obtained through centrifugation was purified by pressure filtration. Thereafter, 1200 L of water was added to the purified solution to perform first washing for 2 hours, and 5000 L of 70 (v/v)% ethanol was added to the first washed purified solution to perform second washing, and then the washed solution was collected and concentrated. The obtained concentrate was freeze-dried to prepare an ethanol extract of bilberry in powder form.

Sapogrelate hydrochloride (manufacturer: Pharmacostech) was added to the powdered bilberry ethanol extract in the weight ratio shown in Table 2, and the bilberry extract and sapogrelate were added to a mixer and mixed at a speed of 50 rpm for 5 minutes to prepare a composite composition. did

Example 5 Example 6 Example 7 Example 8 Bilberry Ethanol Extract 10g 25g 35g 50g sarpogrelate 10g 10g 10g 20g Weight of composite composition 20g 35g 45g 70g

<Examples 9 to 10> Preparation of composite composition of bilberry methanol extract and sarpogrelate

Bilberry methanol extract was extracted at 28 to 30 ° C. by pulverizing frozen bilberry pulp and then immersing 100 g of the pulverized product in 0.5 to 1 L of 70 (v / v)% methanol aqueous solution. Thereafter, the supernatant was separated by centrifugation, citric acid was added to the supernatant, and the resulting precipitate was removed and purified. Then, the remaining extract was concentrated, diluted by adding ethanol, and dried at 60° C. for 36 hours to prepare a dry powder.

Sapogrelate hydrochloride (Manufacturer: Pharmacostec) was added to the bilberry methanol extract obtained through the above process in the weight ratio shown in Table 3, and the bilberry extract and sapogrelate were added to a mixer and mixed at a speed of 50 rpm for 5 minutes to obtain a composite composition was manufactured.

Example 9 Example 10 Bilberry Methanol Extract 10g 50g sarpogrelate 10g 10g Weight of composite composition 20g 60g

<Examples 11 to 12> Preparation of composite composition of bilberry methanol extract and sarpogrelate

100 g of bilberry was added to 1 L of a 70 (v/v)% methanol aqueous solution, followed by primary extraction by stirring at 30° C. for 2 hours at 1600 rpm. The extract of the first extract was transferred, and 1 L of 70 (v / v)% methanol aqueous solution was added to the remaining residue, followed by agitation at 30 ° C. Thereafter, 1 L of 70 (v/v)% methanol aqueous solution was added to the secondary residue, followed by third extraction in the same manner.

After mixing the first, second and third extracts, alcohol was evaporated while concentrating at a temperature of 50 ° C or less. The concentrate obtained by the above process was diluted with Brix 3±5% and centrifuged at 6900 rpm for 6 to 12 minutes.

The supernatant obtained through centrifugation was purified by pressure filtration. Thereafter, 1200 L of water was added to the purified solution for first washing for 2 hours, and 70 (v/v)% methanol (5000 L) was added to the first washed purified solution for second washing, and the washed solution was collected and concentrated. The obtained concentrate was spray-dried to prepare a powdered bilberry methanol extract.

Sapogrelate hydrochloride (manufacturer: Pharmacostech) was added to the bilberry methanol extract obtained through the above process in the weight ratio shown in Table 4, and the bilberry extract and sapogrelate were added to a mixer and mixed at a speed of 50 rpm for 5 minutes to obtain a composite composition was manufactured.

Example 11 Example 12 Bilberry Methanol Extract 25g 35g sarpogrelate 10g 10g Weight of composite composition 35g 45g

<Examples 13 to 16> Preparation of complex compositions of bilberry water extract and sarpogrelate

Bilberry water extract was prepared by putting 30 g of frozen bilberry pulp in 600 mL of water and extracting hot water at 110 ° C for 3 hours to obtain a hot water extract, which was concentrated with a vacuum concentrator until the solid content was 30%, and then freeze-dried to prepare a dry powder. .

Sapogrelate hydrochloride (Manufacturer: Pharmacostec) was added to the bilberry extract obtained through the above process in the weight ratio shown in Table 5, and the bilberry extract and sapogrelate were added to a mixer and mixed at a speed of 50 rpm for 5 minutes to prepare a composite composition. did

Example 13 Example 14 Example 15 Example 16 Bilberry Water Extract 10g 20g 40g 5g sarpogrelate 10g 10g 10g 30g Weight of composite composition 20g 30g 50g 80g

<Test Example 1> Confirmation of cell death inhibitory effect of auditory cell line by composite composition

House Ear Institute-Organ of Corti 1 (HEI-OC1), an auditory sensory hair cell derived from the organ of Corti, an auditory expression organ of mice, was cultured in 10% fetal bovine serum (FBS; WELGENE Inc., Gyeongsangbuk-do) without the addition of antibiotics. -do, Korea) and DMEM (high-glucose Dulbecco's Eagle's medium, Sigma-Aldrich Co., St. Louis, USA) culture medium containing 50 U/mL INF-γ (Peprotech Inc., Seoul, Korea) using, and cultured in an incubator at 33° C. and 10% CO ₂ .

A noise-induced hearing loss cell line was established by treating with hydrogen peroxide to generate oxygen species (ROS) by oxidative stress that occurs during noise-induced hearing loss.

The noise-induced hearing loss induced cell line was treated with the complex composition of Example 2 at concentrations of 0.001, 0.005, 0.01, 0.05, 0.1, and 0.5 μg/mL for 1 hour, and then treated with 100 μM hydrogen peroxide for 24 hours, followed by MTT analysis. Through this, the cytotoxic and apoptotic effects by hydrogen peroxide were quantitatively confirmed.

From the above results, it was confirmed that the composite compositions of Examples 1 to 4 can exhibit a superior apoptosis inhibitory effect than each single agent at a small dose.

<Test Example 2> Efficacy test on hair cells in zebrafish drug efficacy model

Zebrafish has an inner ear structure similar to that of humans, and is transparent for easy observation, and the lateral line system, an additional auditory organ, is exposed to the outside, so changes in auditory hair cells after noise exposure or drug administration can be seen as a live organism. Since it can be observed in the presence of oxygen, it is used as a model organism for reactive oxygen species-induced hearing loss.

To prepare zebrafish larvae, females and males were placed in a 1:1 ratio in a tank equipped with a net to separate eggs and adults, and zebrafish embryos were collected after 12 hours.

After collection, they were washed three times with 0.03% sea salt solution to remove foreign substances and reared in an incubator with a photoperiod of 28.5°C (14 persons/10 arms). Zebrafish fry (6 dpf) 6 days after fertilization were placed in a 24-well, treated with hydrogen peroxide at a concentration of 2.5 mM and exposed for 3 hours to prepare a hydrogen peroxide-induced hearing loss model. prepared.

The hydrogen peroxide-induced deafness zebrafish model was treated with concentrations of 0.1, 0.5, and 1 μg/mL from Example 1 to Example 5, respectively, and exposed for 12 hours.

The zebrafish fry for which the toxicity test was completed were anesthetized with 0.02% tricaine, and the auditory hair cells were stained with 0.1% YO-PRO for 30 minutes, and then the auditory hair cells were examined using a fluorescence microscope (Olympus 1x70, Olympus, Japan). It was counted after observation.

As a result, as shown in FIG. 1, all of the groups treated from Example 5 to Example 8 showed an effect of inhibiting cytotoxicity of zebrafish hair cells by hydrogen peroxide and increasing auditory hair cells related to hearing. 1A is Example 5, FIG. 1B is Example 6, FIG. 1C is Example 7, and FIG. 1D is Example 8 experimental results.

As above, specific parts of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

It contains sarpogrelate and bilberry extract as active ingredients, and the sapogrelate and bilberry extract are included in a weight ratio selected from 1: (2 or more to less than 3) or 1: (more than 3 to 5 or less) A pharmaceutical composition for preventing or treating hearing loss. The pharmaceutical composition for preventing or treating hearing loss according to claim 1, wherein the bilberry extract is obtained by extraction with purified water, C1-C4 alcohol, or a complex solvent thereof. The pharmaceutical composition for preventing or treating hearing loss according to claim 1, wherein the hearing loss is selected from the group consisting of noise-induced hearing loss, ototoxic hearing loss, age-related hearing loss, and sudden hearing loss. The pharmaceutical composition for preventing or treating hearing loss according to claim 1, wherein the pharmaceutical composition contains sarpogrelate and bilberry extract in a weight ratio of 1:1, 1:2.5, 1:3.5 or 1:5. The pharmaceutical composition for preventing or treating hearing loss according to claim 1, wherein the pharmaceutical composition inhibits apoptosis of auditory cells and increases the number of hair cells. It contains sarpogrelate and bilberry extract as active ingredients, and the sapogrelate and bilberry extract are included in a weight ratio selected from 1: (2 or more to less than 3) or 1: (more than 3 to 5 or less) Health food for preventing or improving hearing loss.

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