KR20180089207A - Pharmaceutical composition or health functional food for preventing or treating of sensorineural hearing loss comprising eisenia bicyclis extract as an active ingredient - Google Patents

Abstract

The present invention provides a pharmaceutical composition or health functional food for preventing or treating sensorineural hearing loss comprising eisenia bicyclis extract as an active ingredient. The composition according to the present invention has been found to restore reduced hair cells due to ototoxicity substance, to restore hair cells damaged by diabetic acute neuropathy, and to effectively suppress the rise of a hearing threshold due to noise. Therefore, it is effective in preventing or treating hearing loss, in particular acoustic trauma, and temporary or permanent noise-induced hearing loss.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition or a health functional food for preventing or treating sensory neuronal deafness, which contains seaweed extract of rhubarb as an active ingredient. [0002]

The present invention relates to a method for the treatment of seaweed rhizopenia ( Eisenia The present invention relates to a composition for preventing or treating sensorineural hearing loss containing an extract of bicyclis as an active ingredient.

Rhubarb ( Eisenia bicyclis ) is a group of brown algae belonging to the Lessoniaceae, also called sea oak, arame. The length varies depending on the depth of the habitat, but the larger one grows more than 1.5 m in diameter and is 2 to 3 cm in diameter. They are distributed mainly in Korea (Ulleungdo or Dokdo) and Japan, and they live in the lower rocky area of the intertidal zone within 10m depth. Rhubarb is widely used for food in Korea and Japan. In Ulleungdo, it has been cooked for a long time in the form of wrapped, salted or spiced fish. In Japan, it has been used for a short time before being eaten. It is also used as a substitute for sea tangle or as a raw material for alginic acid, together with moxa or ganoderma, and is known as a Japanese name arame, and is used as an edible seaweed.

Rhubarb is rich in minerals such as calcium, iodine, iron, and magnesium, and also contains a large amount of vitamin A, which can be used for antiinflammation, anticancer, antioxidant, hypoglycemic, Have been reported.

The ear classifies the ear and external auditory meatus into an external ear while the ear and auditory ossicles classify the middle ear and the cochlea and auditory nerve as internal ear do. Sound is acoustical energy transmitted through the auricle and external auditory canal to vibrate the eardrum. The vibration of the eardrum is transferred to the ossicles consisting of three small bones attached to the eardrum with mechanical energy. The last bone of the ossicle, the spine, connects to the cochlea, which transfers energy to the lymph fluid in the cochlea. The delivered energy causes the lymphatic fluid to oscillate, which causes the hair cells in the cochlea to be stimulated. As the hair cells move, the neurons are transferred to the auditory nerves attached to the hair cells, and the sound energy is transferred from the auditory nerve to the brain in the form of electrical energy.

<Figure 1. Ear structure>

As an organ that delivers sounds to the outside, it can be recovered by treatment or surgery, and the resulting hearing loss can also be improved after treatment. This hearing loss is called conductive hearing loss. On the other hand, the hearing loss caused by the brain responsible for the hearing, which plays a comprehensive role in the discrimination and understanding of the sound, is caused by the sensorineural hearing loss ). The body's sensory organ The auditory organ is one of the most basic senses in communication, one of the most important senses to acquire language, acquire knowledge, live a social life and live a human life. Most of the hearing loss is related to sensory neural deafness, which has no treatment method other than prevention until now. Once hearing loss occurs, hearing aids are assisted by using auxiliary means such as hearing aids or by implanting mechanical devices into the body.

Sensory neural deafness can be classified according to the cause and timing of the onset. Depending on the timing, it is classified into congenital hearing loss and acquired hearing loss.

Congenital hearing loss has been damaged before birth and is caused by problems in heredity, in the uterus, or in the birth process. Most congenital hearing impairments are very severe and even learning a language is impossible without training and education. In congenital hearing loss, most hearing aids and cochlear implants (implants of the device by stimulating the auditory nerves by electrical stimulation and listening to them while wearing separate external devices) are implanted to hear the sound. However, hearing aids and cochlear implants, such as hearing impairment degree is less effective and normal listening ability is different from the daily life is still a lot of inconvenience.

In the case of acquired hearing loss, there are noise, drugs, aging, trauma, and viruses that are caused by illness, noise, drugs, or accidents after birth. Of these, the most notable increase in recent years is hearing loss due to noise and aging. The development of science and technology has prolonged the life span of human beings, and the elderly population is rapidly increasing worldwide. Because senile hearing loss is also the most common sensory neural deafness, there is no drug or treatment for treatment or improvement other than prevention and management.

As the society has become industrialized, the number of hearing impairments due to noise has also increased rapidly in recent years. In addition to occupational noise-induced hearing loss such as workers and soldiers working in a noisy environment, noise-induced hearing loss due to cultural and leisure activities is also increasing. According to the industrial material solution in Korea, when exposed to environmental noise of 90 dBA or more, it can damage the hearing. Occupational Safety and Health Administration (OSHA) has conducted noise management on noise levels above 85 dBA. Research has shown that human hearing organs are affected by noise above 75 dBA. Since the noise of 75 dBA is the noise level of the road on which the car is moving, it can be seen that in the industrial society, everyone lives in a degree of harmful noise to the auditory organ. In addition to the environmental noise that is inevitably heard, many youths are exposed to loud sounds in leisure activities including MP3 use. Recently, noise-induced hearing loss has appeared in various ages. As a result, hearing impairment due to the use of MP3s in his youth is harmful to the hearing so that conversation is possible only if hearing aid is used from his 40s. When physical aging is accompanied, the degree of hearing becomes more and more severe. Hearing aids and other ancillary tools are less effective as the degree of hearing loss becomes worse, resulting in serious hearing damage that eventually causes serious communication problems. That is, when the young generation experiencing the current noise-induced hearing loss becomes an elderly person, the degree of hearing loss becomes worse, and hearing loss has an important influence on the quality of life of various generations, from the elderly to the young.

<Figure 2. Comparison of hair cells in patients with sensory nerve damage and normal people>

Most of the sensory hearing impairments seen in noise - induced hearing loss, poisoning hearing loss, and senile hearing loss are caused by functional problems of inner ear, especially damage of cochlear hair cells. In addition to congenital loss of genetic factors, hair cells may be damaged by various factors such as noise exposure in daily life, antibiotics, drug use such as anticancer drugs, and aging. Therefore, it is possible to treat many sensory neural deafness including noise-induced hearing loss if it can promote regeneration of wah hair cells.

Recently, preclinical studies on antioxidants, N-methyl-D-aspartate (NMDA) antagonists, apoptosis inhibitors, growth factors and the like have been reported as research for finding effective substances for prevention and treatment of hearing loss (Prasher D., Lancet, 352, pp. 1240-1242, 1998).

In the early stage of the study, the use of antioxidants to neutralize reactive oxygen species (ROS) and reactive nitrogen species (RNS) inhibited the cell damage of cochlea, It was not developed as a.

N-acetylcysteine (NAC) and methionine (MET) have been reported to be useful for the prevention of hearing loss, and NAC enhances production of GSH as a prodrug of GSH (Meister A., Pharmacol Ther., 51, pp155-194, 1991). High-dose NAC is a mucopolysaccharide hydrolysing enzyme for respiratory-related diseases, which is effective in preventing noise-induced hearing loss. MET is converted to cysteine as a pure amino acid.

Other researchers have also been interested in inhibiting apoptosis and have reported that the function of the GPx mimic (GPx mimic) in relation to antioxidants plays a protective role in the outer hair cell damage due to noise. Among these studies, Mg, NAC and Ebselen have been shown to be effective in the clinical stage. It has been reported that 300 mg of Mg granules per day of 4 mg of granules per day reduced the severity of permanent hearing impairment after a week of exposure compared to the placebo effect control group (Attias J. et al., Am J. Otolaryngol., 15, pp26-32, 1994). Administration of Mg to low-intensity noise-exposed soldiers reduced transient hearing impairment, and 600 US Navy personnel showed a reduction in permanent hearing impairment in the NAC-administered group for two weeks of arms training. In animal models, high doses of NAC have been shown to have preventive effects on noise-induced hearing loss, but there are limitations to permanent hearing damage (Kopke RD et al., Hear. Res., 149, pp138-146, 2000; Kramer S. et al., American Academy of Audiology Annual Convention and Expo, Washington, DC, USA, Poster Presentation, pp502, 2005). It has been reported that epicelenium is administered to 60 US soldiers during a two-week weapon training to prevent temporary hearing loss and permanent hearing loss (Yamasoba T. et al., Neurosci. Lett., 380 , pp234-238, 2005)

To date, no approved drugs have been approved for the prevention and treatment of noise-induced hearing loss, and studies reported at the preclinical stage of the academia have only reported Mg, NAC and ephedrine drugs. On the other hand, in the field of oriental medicine, it has been reported that Sukjipphi has been used for inner ear disease such as tinnitus and hearing loss, and that ethanol extract of Sukjipjang inhibits lipid peroxidation and eliminates the activity of free radicals, (Hyeon-Hee Yu et al., Journal of Ethnopharmacology, Volume 107, Issue 3, pp383-388, 2006).

As a result of efforts to find various active substances that can prevent or treat hearing loss in natural substances having excellent safety, the present inventors have found that the extract of seaweed rhubarb is useful for ototoxicity deafness due to kanamycin or salicylic acid, The present inventors confirmed that the effect of protecting the hair cell from the hearing loss and the increase of the hearing threshold due to the noise are effectively suppressed.

It is an object of the present invention to provide a process for the preparation of algae rhubarb The present invention also provides a composition for preventing or treating sensorineural hearing loss, which contains bicyclic extract as an active ingredient.

In order to achieve the above object, the present invention rhubarb (Eisenia The present invention provides a pharmaceutical composition for the prevention or treatment of sensorineural hearing loss containing an extract of bicyclis as an active ingredient.

In addition, the present invention relates to the use of rhubarb ( Eisenia The present invention provides a health functional food for improving sensorineural hearing loss containing an extract of bicyclis as an active ingredient.

The present invention provides a pharmaceutical composition for prevention or treatment of hearing loss containing a seaweed extract of rhubarb as an active ingredient or a health functional food for improving hearing loss. It has been confirmed that the composition according to the present invention restores hair cells reduced by this toxic substance, restores hair cells damaged by diabetic ear neuropathy, and effectively suppresses the increase of hearing threshold due to noise, Acoustic trauma, and temporary or permanent noise-induced hearing loss.

Fig. 1 shows the effect of a seaweed extract of rhizomes on injured hair cells of zebrafish which caused toxicity to kanamycin:
Normal group: normal hair cells;
Control group: hair cells treated with kanamycin;
DHE 1: Experimental group treated with 1 ㎍ / ml of rhubarb extract after treatment with kanamycin; And
DHE 5: Experimental group treated with 5 ㎍ / ml of rhubarb extract after treatment with kanamycin.
FIG. 2 shows the effect of the seaweed extract of rhubarb on the damaged hair cells of the zebrafish causing hearing damage by salicylic acid:
Normal group: normal hair cells;
Control group: hair cells treated with salicylic acid; And
DHE 1: Experimental group treated with 1 ㎍ / ml of rhubarb extract after treatment with salicylic acid.
FIG. 3 shows the effect of seaweed extract of rhubarb on the damaged hair cells of zebrafish which caused diabetic neuropathy due to hyperinsulinemia:
Normal group: normal hair cells;
Control group: hair cells treated with insulin; And
DHE 1: Experimental group treated with 1 ㎍ / ml of rhubarb extract after treatment with insulin.
FIG. 4 shows the effect of the seaweed extract of the seaweed on the hearing thresholds after the noise exposure, using the click sound of the auditory brainstem response (ABR) test:
4A: hearing threshold shift value before and after noise exposure;
4b: hearing threshold according to change of stimulus sound after noise exposure;
NIHL: untreated control after noise exposure;
DHE 100 mg / kg: treated with 100 mg / kg of seaweed extract of rhododendron after noise exposure; And
DHE 300 mg / kg: Experimental group treated with 300 mg / kg of seaweed extract of rhizome after noise exposure.
FIG. 5 is a graph showing the effect of the rhizome extract of seaweed on the hearing threshold after the noise exposure, with the stimulus sound of 8 kHz pure tone in the auditory evoked potential test:
5A: hearing threshold shift value before and after noise exposure;
FIG. 5b: Hearing threshold value according to change of stimulus sound after noise exposure;
NIHL: untreated control after noise exposure;
DHE 100 mg / kg: treated with 100 mg / kg of seaweed extract of rhododendron after noise exposure; And
DHE 300 mg / kg: Experimental group treated with 300 mg / kg of seaweed extract of rhizome after noise exposure.
FIG. 6 shows the effect of the extract of seaweed rhubarb extracts on the hearing threshold after noise exposure, using a 16 kHz pure tone for stimulus sounding potential test:
6A: hearing threshold shift value before and after noise exposure;
FIG. 6B: Hearing threshold value according to change of stimulus sound after noise exposure;
NIHL: untreated control after noise exposure;
DHE 100 mg / kg: treated with 100 mg / kg of seaweed extract of rhododendron after noise exposure; And
DHE 300 mg / kg: Experimental group treated with 300 mg / kg of seaweed extract of rhizome after noise exposure.
FIG. 7 shows the effect of the seaweed extract of the seaweed on the hearing threshold after the noise exposure, with the stimulus sound of 24 kHz pure tone in the auditory evoked potential test:
7A: hearing threshold shift value before and after noise exposure;
Fig. 7B: hearing threshold according to change of stimulus sound after noise exposure;
NIHL: untreated control after noise exposure;
DHE 100 mg / kg: treated with 100 mg / kg of seaweed extract of rhododendron after noise exposure; And
DHE 300 mg / kg: Experimental group treated with 300 mg / kg of seaweed extract of rhizome after noise exposure.
FIG. 8 shows the effect of the seaweed extract of the seaweed on the hearing threshold after the noise exposure, in which the stimulus sound was 32 kHz pure tone during the auditory evoked potential test:
8A: hearing threshold shift value before and after noise exposure;
8b: hearing threshold value according to change of stimulus sound after noise exposure;
NIHL: untreated control after noise exposure;
DHE 100 mg / kg: treated with 100 mg / kg of seaweed extract of rhododendron after noise exposure; And
DHE 300 mg / kg: Experimental group treated with 300 mg / kg of seaweed extract of rhizome after noise exposure.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating sensorineural hearing loss containing Eisenia bicyclis extract as an active ingredient.

The rhubarb extract may be prepared by a process comprising the steps of:

1) preparing an extract by adding an extracting solvent to rhubarb;

2) filtering the extract of step 1); And

3) Concentrating the filtered filtrate of step 2) under reduced pressure and drying.

In the above production process, the extraction solvent may be water, alcohol or a mixture thereof. The alcohol may be a C ₁ to C ₂ lower alcohol, and specifically, the alcohol may be ethanol or methanol. In one embodiment of the present invention, the extraction solvent may be ethanol. The extraction solvent may be added in an amount of 1 to 1,000 ml per 1 g of dry weight of the rhubarb which is used for extraction.

The extraction method may be shaking extraction, Soxhlet extraction or reflux extraction. At this time, the extraction time may be 10 to 48 hours and 15 to 30 hours. The extraction may be repeated three to five times, specifically three times.

On the other hand, the vacuum concentration in the step 3) may be performed using a vacuum decompression concentrator or a vacuum rotary evaporator. The drying may be vacuum drying, vacuum drying, boiling drying, spray drying or freeze drying, and may be freeze-drying.

The sensory neural deafness may be caused by damage of inner ear's hair cells and surrounding tissue, and the sensory neural deafness may be caused by noise-induced hearing loss, presbycusis, diabetic ear neuropathy Or hearing loss due to ototoxicity medication.

The diabetic ear neuropathy may be due to a high concentration of insulin, but is not limited thereto. In the present invention, insulin resistance is modeled by exposing high-concentration insulin to zebrafish. Diabetic peripheral neuropathy, which is one of the diabetic complications, induces auditory neuropathy, which leads to improvement of rhabdomyolysis in zebrafish, Efficacy was confirmed.

Wherein the toxic drug is selected from the group consisting of streptomycin, kanamycin, gentamicin, neomycin, amikacin, tobramycin, neilmicin, dibekacin, sisomycin, livodomycin, cisplatin, carboplatin, erythromycin, furosemide, ethacrin, But are not limited to, ethacryn acid, bumex, vincristine, quinine, mercury, lead, aspirin, salicylic acid, Viagra, levitra, cialis, toluene, styrene, xylene, vancomycin but is not limited to, one or more selected from the group consisting of vancomycin, bleomycin, acetazolamide, chloroquine, carbon monoxide and carbon disulfide. . Salicylic acid has been used for a long time since it has been widely used as an antiinflammatory, antipyretic, and analgesic agent. Salicylic acid at high concentration induces various side effects, such as reversible hearing loss and tinnitus. Or tinnitus in animals.

In a specific embodiment of the present invention, the inventors have made rhubarb extracts and found that the rhubarb extract significantly increases hair cells reduced to kanamycin or salicylic acid (see Figures 1 and 2) (Fig. 3). In the case of the hair cells, In addition, the inventors have confirmed that the rhubarb extract significantly reduces the increased hearing threshold in mice exposed to noise (see FIGS. 4-8).

Therefore, the rhubarb extract of the present invention can be used for preventing or treating sensory neuron deafness such as hearing damage caused by toxic substances and temporary or permanent noise (acoustic traumatic) hearing loss.

The pharmaceutical composition according to the present invention may contain 10 to 95% by weight of an effective ingredient, a rhubarb extract, based on the total weight of the composition. In addition, the pharmaceutical composition of the present invention may further contain at least one active ingredient which exhibits the same or similar functions in addition to the above-mentioned effective ingredients.

The pharmaceutical compositions of the present invention may include carriers, diluents, excipients or mixtures thereof conventionally used in biological preparations. Pharmaceutically acceptable carriers can be used as long as they are suitable for delivering the composition in vivo. Specifically, the carrier is selected from Merck Index, 13th ed., Merck & Sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture thereof. If necessary, conventional additives such as an antioxidant, a buffer, a bacteriostatic agent and the like may be added.

When the composition is formulated, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient may be added.

The compositions of the present invention may be formulated into oral or parenteral formulations. Oral preparations may include solid preparations and liquid preparations. The solid preparation may be a tablet, a pill, a powder, a granule, a capsule, or a troche. The solid preparation may be prepared by adding at least one excipient to the composition. The excipient may be starch, calcium carbonate, sucrose, lactose, gelatin or a mixture thereof. In addition, the solid preparation may comprise a lubricant, examples of which include magnesium stearate, talc, and the like. On the other hand, the liquid preparation may be a suspension, a solution, an emulsion or a syrup. At this time, the liquid preparation may contain an excipient such as a wetting agent, a sweetener, a fragrance, a preservative, and the like.

The parenteral preparation may contain injections, suppositories, respiratory inhalation powders, aerosol formulations for spraying, powders and creams. The injection may include a sterilized aqueous solution, a non-aqueous solvent, a suspending agent, an emulsion, and the like. As the non-aqueous solvent or suspension, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and injectable esters such as ethyl oleate may be used.

The composition of the present invention can be administered orally or parenterally according to the desired method. Parenteral administration may include intraperitoneal, rectal, subcutaneous, intravenous, intramuscular or intrathecal injection.

The composition may be administered in a pharmaceutically effective amount. This may vary depending on the type of disease, severity, activity of the drug, sensitivity of the patient to the drug, administration time, route of administration, duration of treatment, However, for a desired effect, the amount of the active ingredient contained in the pharmaceutical composition according to the present invention may be 0.0001 to 100 mg / kg, specifically 0.001 to 10 mg / kg. The administration may be several times per day.

The composition of the present invention may be administered alone or in combination with other therapeutic agents. When administered concomitantly, the administration can be sequential or simultaneous.

In addition, the present invention relates to the use of rhubarb ( Eisenia The present invention provides a health functional food for improving sensorineural hearing loss containing an extract of bicyclis as an active ingredient.

The rhubarb extract may be prepared by a process comprising the steps of:

1) preparing an extract by adding an extracting solvent to rhubarb;

2) filtering the extract of step 1); And

3) Concentrating the filtered filtrate of step 2) under reduced pressure and drying.

In the above production process, the extraction solvent may be water, alcohol or a mixture thereof. The alcohol may be a C ₁ to C ₂ lower alcohol, and specifically, the alcohol may be ethanol or methanol. In one embodiment of the present invention, the extraction solvent may be ethanol. The extraction solvent may be added in an amount of 1 to 1,000 ml per 1 g of dry weight of the rhubarb which is used for extraction.

The extraction method may be shaking extraction, Soxhlet extraction or reflux extraction. At this time, the extraction time may be 10 to 48 hours and 15 to 30 hours. The extraction may be repeated three to five times, specifically three times.

On the other hand, the vacuum concentration in the step 3) may be performed using a vacuum decompression concentrator or a vacuum rotary evaporator. The drying may be vacuum drying, vacuum drying, boiling drying, spray drying or freeze drying, and may be freeze-drying.

The sensory neural deafness may be caused by damage of inner ear's hair cells and surrounding tissue, and the sensory neural deafness may be caused by noise-induced hearing loss, presbycusis, diabetic ear neuropathy Or hearing loss due to ototoxicity medication.

The diabetic ear neuropathy may be due to a high concentration of insulin, but is not limited thereto. In the present invention, insulin resistance is modeled by exposing high-concentration insulin to zebrafish. Diabetic peripheral neuropathy, which is one of the diabetic complications, induces auditory neuropathy, which leads to improvement of rhabdomyolysis in zebrafish, Efficacy was confirmed.

Wherein the toxic drug is selected from the group consisting of streptomycin, kanamycin, gentamicin, neomycin, amikacin, tobramycin, neilmicin, dibekacin, sisomycin, livodomycin, cisplatin, carboplatin, erythromycin, furosemide, ethacrin, But are not limited to, ethacryn acid, bumex, vincristine, quinine, mercury, lead, aspirin, salicylic acid, Viagra, levitra, cialis, toluene, styrene, xylene, vancomycin but is not limited to, one or more selected from the group consisting of vancomycin, bleomycin, acetazolamide, chloroquine, carbon monoxide and carbon disulfide. . Salicylic acid has been used for a long time since it has been widely used as an antiinflammatory, antipyretic, and analgesic agent. Salicylic acid at high concentration induces various side effects, such as reversible hearing loss and tinnitus. Or tinnitus in animals.

In a specific embodiment of the present invention, the inventors have made rhubarb extracts and found that the rhubarb extract significantly increases hair cells reduced to kanamycin or salicylic acid (see Figures 1 and 2) (Fig. 3). In the case of the hair cells, In addition, the inventors have confirmed that the rhubarb extract significantly reduces the increased hearing threshold in mice exposed to noise (see FIGS. 4-8).

Therefore, the rhubarb extract of the present invention can be usefully used for the improvement of sensory neural deafness such as hearing damage caused by toxic substances and temporary or permanent noise (acoustic traumatic) hearing loss.

The composition of the present invention can be added directly to food or used together with other food or food ingredients. At this time, the content of the active ingredient to be added may be determined depending on the purpose, and may generally be 0.01 to 90 parts by weight of the total food weight.

The form and kind of the health functional food are not particularly limited. Specifically, the health functional food may be in the form of a tablet, a capsule, a powder, a granule, a liquid and a ring. The health functional food may contain various flavors, sweeteners or natural carbohydrates as an additional ingredient. The sweeteners may be natural or synthetic sweeteners, examples of natural sweeteners include tau martin and stevia extract. On the other hand, examples of synthetic sweeteners include saccharin and aspartame. The natural carbohydrates may be monosaccharides, disaccharides, polysaccharides, oligosaccharides, sugar alcohols, and the like.

The health functional food of the present invention may contain, in addition to the above-described additional ingredients, a nutrient, a vitamin, an electrolyte, a flavoring agent, a colorant, a pectin and a salt thereof, an alginic acid and its salt, an organic acid, a protective colloid thickening agent, , Glycerin, alcohol, and the like. These components may be used independently or in combination. The proportion of the additive may be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are intended to be illustrative of the present invention, but the present invention is not limited thereto.

Seaweed rhubarb ( Eisenia bicyclis ) Preparation of extract

500 g of seaweeds from Ulleungdo was firstly extracted with 9 L of 70% ethanol at 90 ℃ for 120 min. 4.5 liters of 70% ethanol was further added and the mixture was extracted twice at the same temperature for 60 minutes. The extract was filtered and concentrated at 70-80 rpm at 40 ° C in a water bath. The concentrate was frozen at -50 ° C and lyophilized for 7 days. After freeze-drying, it was stored in a -50 ° C freezer. A total of 500 g of rhubarb was extracted to obtain 79.9 g of seaweed rhubarb extract. The yield was 16.0%.

< Experimental Example  1>

Seaweed rhubarb  By extract With Kanameishin  Identification of improvement effects of induced sensory neuron auditory damage

The effect of the seaweed extract prepared in Example 1 on kanamycin-reduced hair cells was confirmed by the following method.

Specifically, 6 days post fertilization zebrafish larvae were placed in 24 wells and exposed to 20 μM kanamycin for 30 minutes. Then, it was exposed for 12 hours by replacing with seaweed extract (DHE) prepared in Example 1, and 0.03% sea salt solution was treated as a control. Fluorescence microscopy (Olympus 1 × 70, Olympus, Japan) was used for direct hair cell observation and stained with 0.1% YO-PRO for 30 min for fluorescence microscopy. The data were analyzed by counting the number of hair cells obtained through fluorescence microscopy.

As a result, as shown in Fig. 1, it was confirmed that the number of hair cells was significantly increased in the group treated with 1 or 5 μg / mL of seaweed extract (DHE) compared to the control group (Fig. 1).

< Experimental Example  2>

Seaweed rhubarb  Identification of the effect of salicylic acid-induced sensory-nerve aural damage by extracts

Salicylic acid has been used for a long time as a drug effective for inflammation, fever and pain. However, high concentrations of salicylic acid can cause many side effects. The most common symptoms are reversible hearing loss or ototoxic symptoms such as tinnitus, which induces tinnitus in humans or animals. The effect of the seaweed extract prepared in Example 1 on salicylic acid-reduced hair cells was confirmed by the following method.

Specifically, five days post-fertilization zebrafish larvae were placed in 24 wells and exposed to 5 mM salicylic acid for 48 hours. Then, it was exposed for 12 hours by replacing with seaweed extract (DHE) prepared in Example 1, and 0.03% sea salt solution was treated as a control. Fluorescence microscopy (Olympus 1 × 70, Olympus, Japan) was used for direct hair cell observation and stained with 0.1% YO-PRO for 30 min. The data were analyzed by counting the number of hair cells obtained through fluorescence microscopy.

As a result, as shown in FIG. 2, the number of hair cells was significantly increased in the group treated with 1 μg / mL of seaweed extract (DHE) than the control group (FIG. 2).

< Experimental Example  3>

Seaweed rhubarb  The effect of extracts on hyperinsulinemic sensory neurons

In order to create a model of diabetic hearing loss, insulin resistance was modeled by exposing high insulin to zebrafish, which leads to auditory neuropathy due to diabetic peripheral neuropathy, one of the diabetic complications. In order to confirm whether the rhubarb extracts showed the improvement effect on the damaged hair cells of the diabetic zebrafish, the following method was used.

Specifically, three days post-fertilization zebrafish larvae were placed in 6 wells and exposed to 10 μM insulin for 48 hours. Then, it was exposed for 48 hours by replacing with seaweed extract (DHE) prepared in Example 1, and 0.03% sea salt solution was treated as a control. Fluorescence microscopy (Olympus 1 × 70, Olympus, Japan) was used for direct hair cell observation and stained with 0.1% YO-PRO for 30 min. The data were analyzed by counting the number of hair cells obtained through fluorescence microscopy.

As a result, as shown in FIG. 3, the number of hair cells was significantly increased in the group treated with 1 μg / mL of seaweed extract (DHE) than the control group (FIG. 3).

< Experimental Example  4>

Seaweed rhubarb  Improvement of hearing loss after noise exposure by extract

<4-1> Broadband Stimulant Click sound  Used Hearing threshold  Confirm

To investigate the effects of seahorse rhubarb extract on hearing threshold after noise exposure, hearing threshold measurements were performed using the sensory evoked potential. The auditory brainstem response (ABR) measurement is a method of evaluating the response to sound by measuring the electrical energy when a sound stimulus is transmitted to an electrical signal in the auditory nerve. When the sound reaches the auditory nerves through the outer ear, middle ear, and cochlea, it reflects all of the states of the ear, middle ear, and cochlea, reflecting the actual sound energy to which the sound energy reaches the brain. Hearing threshold refers to the minimum sensory point of sound that can be heard. In normal mice, the response is observed even on the average of 20 dB. A click stimulus is a measure of the ability to hear, telling the total audio frequency.

Specifically, mice to be administered with 100 mg / kg or 300 mg / kg of seaweed extract (DHE) prepared in Example 1 and an untreated control mouse were each divided into three groups of eight mice each. The noise was exposed for 1 hour with a 110 dB compound tone. After 24 hours of exposure to the noise, the seaweed extract of Example 1 was orally administered at the same time every day. Hearing thresholds were assessed before, during and 1 day after exposure.

Ketamine (4.57 mg / kg) and silazane (0.43 mg / kg) were anesthetized with an intramuscular injection and evaluated at a temperature of 37 ± 0.5 ° C. During the auditory evoked potential test, the stimulus sound was evaluated by lowering the sound gradually from 80 dB to 5 dB at the click of a broadband stimulus sound, and the lowest sound of the response was the threshold.

As a result, as shown in Fig. 4, in the group administered with 300 mg / kg of seaweed extract (DHE), the threshold value decreased at 14 days (Fig. 4b) 26 dB and 23 dB in the group administered with 100 mg / kg of seaweed extract (DHE) and 18 dB in the group administered with 300 mg / kg, respectively (Fig. 4A). It was confirmed that the hearing threshold improvement was suppressed in the group administered with DHE 300 mg / kg compared to the control group, indicating significant hearing improvement effect.

<4-2> 8 kHz  TB Stimulation sounds  Used Hearing threshold  Confirm

During the auditory evoked potential test, the stimulus sound was evaluated as 8 kHz pure tone, gradually decreasing the sound from 80 dB to 5 dB. The frequencies used in Experimental Examples <4-3>, <4-4>, and <4-5> were evaluated by frequency subdivided in the audible range.

As a result, as shown in FIG. 5, the threshold value of the untreated control group (NIHL) after the noise exposure was 42 , and 37 dB for the test group administered with 100 mg / kg of seaweed extract (DHE) and 20 dB for the test group administered with 300 mg / kg of DHE (FIG. 5A). It was confirmed that the hearing threshold improvement was suppressed in the group administered with DHE 300 mg / kg compared to the control group, indicating significant hearing improvement effect.

<4-3> 16 kHz  TB Stimulation sounds  Used Hearing threshold  Confirm

The auditory evoked potential was measured at 16 kHz pure tone and gradually decreased by 5 dB from 80 dB.

As a result, as shown in FIG. 6, in the group administered with 300 mg / kg of seaweed extract (DHE), the threshold value decreased on day 14 (FIG. 6b) dB, 44 dB for the test group administered with 100 mg / kg of seaweed extract (DHE) and 32 dB for the test group administered with 300 mg / kg of DHE (FIG. 6A). It was confirmed that the hearing threshold improvement was suppressed in the DHE 100 or 300 mg / kg group compared to the control group, indicating significant hearing improvement.

<4-4> 24 kHz  TB Stimulation sounds  Used Hearing threshold  Confirm

During the auditory evoked potential test, the stimulus sound was evaluated as a 24 kHz pure tone with the sound gradually decreasing from 80 dB to 5 dB.

As a result, as shown in FIG. 7, in the group administered with 300 mg / kg of seaweed extract (DHE), the threshold value decreased on the 14th day (FIG. 7b) and the threshold value of the untreated control group (NIHL) , 39 dB in the experimental group administered with 100 mg / kg of seaweed extract (DHE) and 23 dB in the experimental group administered with DHE 300 mg / kg (FIG. 7A). It was confirmed that the hearing threshold improvement of the group administered with DHE 300 mg / kg was suppressed compared to the control group, indicating that there was a significant hearing improvement effect.

<4-5> 32 kHz  TB Stimulation sounds  Used Hearing threshold  Confirm

The auditory evoked potential was 32 kHz pure tone, and the auditory evoked potential was gradually decreased from 80 dB to 5 dB.

As a result, as shown in Fig. 8, in the group administered with 300 mg / kg of seaweed extract (DHE), the threshold value decreased at 14 days (Fig. 8b) dB, and 25 dB for the test group administered with 100 mg / kg of seaweed rhubarb extract (DHE) and 10 dB for the test group administered with DHE 300 mg / kg (FIG. 8A). This suggests that hearing threshold improvement was suppressed in the experimental group administered with DHE 300 mg / kg, as compared to the control group.

Claims (10)

Eisenia The present invention relates to a pharmaceutical composition for preventing or treating sensorineural hearing loss, which comprises an extract of bicyclis as an active ingredient.
The pharmaceutical composition according to claim 1, wherein the extract is extracted with water, a C ₁ to C ₂ lower alcohol or a mixture thereof.
The pharmaceutical composition according to claim 2, wherein the lower alcohol is ethanol or methanol.
The pharmaceutical composition according to claim 1, wherein the sensory neuron deafness is caused by damage to hair cells.
2. The method of claim 1, wherein the sensory neural deafness is selected from the group consisting of noise-induced hearing loss, presbycusis, hearing loss due to diabetic aural neuropathy or hearing loss due to ototoxicity drug, A pharmaceutical composition for preventing or treating hearing loss.
6. The method of claim 5 wherein said toxic drug is selected from the group consisting of streptomycin, kanamycin, gentamicin, neomycin, amikacin, tobramycin, neilmicin, dibekacin, sisomycin, livodomycin, cisplatin, carboplatin, erythromycin, furosemide, eta But are not limited to, ethacryn acid, bumex, vincristine, quinine, mercury, lead, aspirin, salicylic acid, Viagra, levitra, cialis, toluene, styrene, xylene, Prevention of sensory nerve impairment, at least one selected from the group consisting of vancomycin, bleomycin, acetazolamide, chloroquine, carbon monoxide and carbon disulfide. The pharmaceutical composition for the treatment.
Eisenia bicyclis ) extract as an active ingredient for the improvement of sensorineural hearing loss.
8. The health functional food according to claim 7, wherein the sensory neuron deafness is caused by damage of hair cells.
8. The method of claim 7, wherein the sensory neural deafness is selected from the group consisting of noise-induced hearing loss, presbycusis, hearing loss due to diabetic aural neuropathy or hearing loss due to ototoxicity drugs, Health functional food for hearing loss improvement.
10. The method of claim 9, wherein the toxic drug is selected from the group consisting of streptomycin, kanamycin, gentamicin, neomycin, amikacin, tobramycin, neilmicin, dibekacin, sisomycin, livodomycin, cisplatin, carboplatin, erythromycin, furosemide, eta But are not limited to, ethacryn acid, bumex, vincristine, quinine, mercury, lead, aspirin, salicylic acid, Viagra, levitra, cialis, toluene, styrene, xylene, The present invention relates to a method for improving sensory nerve tone deafness, which is selected from the group consisting of vancomycin, bleomycin, acetazolamide, chloroquine, carbon monoxide and carbon disulfide River supplements.

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