KR101320792B1 - Composition for treatment or prevention of ototoxic drug-induced hearing loss comprising Red Ginseng extracts - Google Patents

Abstract

The present invention relates to a composition for treating or preventing a toxic drug-induced auditory injury comprising red ginseng extract. The red ginseng extract of the present invention can reduce the hearing damage induced by a toxic drug such as an aminoglycoside antibiotic, and thus, prolonged administration of an aminoglycoside antibiotic having a toxic effect such as tuberculosis and deep infections. Even if this is unavoidable, antibiotics can be reliably administered to treat infectious diseases. It can also reduce the side effects of anticancer drugs, known as toxic drugs, and treat and prevent hearing damage that is not related to toxic drugs such as sudden hearing loss.

Description

Composition for treatment or prevention of ototoxic drug-induced hearing loss comprising Red Ginseng extracts}

The present invention relates to a composition for treating or preventing a toxic drug-induced auditory injury comprising red ginseng extract.

The ear is classified as the outer ear, the ear canal and the ear canal, and the eardrum and the osseous bone are classified as the middle ear, and the cochlea and auditory nerve as the inner ear. Sound is acoustic energy that is transmitted through the ear canal and ear canal to vibrate the eardrum. The vibrations of the tympanic membrane are mechanical energy that is transmitted to the osseous bone, which consists of three small bones attached to the eardrum. The spine, the last bone of the iso bone, is connected to the cochlea, which transfers energy to lymphatic fluid in the cochlea. The energy delivered causes waves in the lymph, which stimulate the hair cells in the cochlea. As ionic changes occur through the movement of hair cells, neurotransmitters are delivered to the auditory nerves attached to the hair cells, and sound energy is transmitted from the auditory nerve to the brain in the form of electrical energy to recognize sound.

This hearing may be damaged by auditory blast or nerve damage, where the damage may be caused by genetic disorders, noise, toxicity, or any other specific stressor. In particular, the drug or chemicals that damage the hearing function or balance function of the body, the hearing can be damaged mainly by ototoxicity drugs that damage the inner ear (inner ear).

Among the toxic drugs, especially aminoglycoside antibiotics are mainly used for Gram-negative bacterial infections, tuberculosis, and deep infections that do not respond well to general antibiotics, but they show side effects of toxic and renal toxicity that cause hearing and equilibrium dysfunction in the inner ear. Have a problem. These side effects can occur not only in the overdose of antibiotics, but also in long-term or moderate doses at appropriate therapeutic doses.

Ginseng is a perennial ginseng belonging to the ginseng of Panax ginseng as a plant taxonomy and about 11 species are known on the earth. Red ginseng is steamed and dried ginseng, which has sedation and excitement effects on the central nervous system and prevents hypertension or arteriosclerosis by acting on the circulatory system. It is known to have a back. The main ingredients are saponin (ginsenosides), panaxynol, beta-elemene and maltol. Aqueous extract of red ginseng is used to treat anemia, diabetes, insomnia, gastritis, blood pressure abnormalities, dyspepsia, overstrain, and fatigue It has been studied to contain a variety of triterpene glycosides called saponin (ginsenosides) (Baranov AI, J. Ethnopharmacol., 6, 339-53, 1982; Chong SK, et al., Postgrad Med. J., 64, 841-6, 1988).

In the case of tuberculosis, deep infection, etc., it is inevitable to administer aminoglycoside antibiotics having long-term toxicity, and thus no effective substance has been reported that can suppress the side effects of the toxic drugs such as antibiotics. Accordingly, the present inventors have made a thorough study, and confirmed that the red ginseng extract can reduce the hearing damage side effects induced by a toxic drug such as aminoglycoside antibiotics, and completed the present invention.

The present invention is to provide a pharmaceutical composition for treating or preventing hearing damage comprising red ginseng extract as an active ingredient.

In addition, the present invention is to provide a supplement for inhibiting antibiotic side effects comprising red ginseng extract as an active ingredient.

In addition, the present invention is to provide a composition for treating an infectious disease comprising the adjuvant and antibiotic.

In addition, the present invention is to provide a food composition for preventing or improving hearing damage comprising red ginseng extract as an active ingredient.

In one embodiment, the present invention provides a pharmaceutical composition for treating or preventing hearing damage, comprising red ginseng extract as an active ingredient.

The red ginseng extract can be prepared using red ginseng or commercially available red ginseng directly prepared using ginseng. The red ginseng extract of the present invention can be extracted using an organic solvent such as water or alcohol as an extraction solvent. Specifically, extraction solvents such as water, an aqueous lower alcohol having 1 to 4 carbon atoms or anhydrous lower alcohol, a mixed solvent of the lower alcohol and water, or acetone, ethyl acetate, chloroform, 1,3-butylene glycol, and butyl acetate are used. Can be obtained. Preferably, the red ginseng extract may be prepared using a hydrous lower alcohol, most preferably ethanol. The extract of the present invention includes red ginseng extracts having substantially the same effect as prepared using the aforementioned extraction solvent as well as other extraction solvents.

In addition, the red ginseng extract of the present invention includes not only the extract by the above-described extraction solvent, but also an extract that has undergone a conventional purification process. Activity obtained through various purification methods additionally performed, for example, separation using ultrafiltration membranes having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity). The fraction is also included in the red ginseng extract of the present invention. In addition, the red ginseng extract of the present invention may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze drying or spray drying.

For example, the red ginseng extract according to an embodiment of the present invention may be prepared as follows: Red ginseng prepared by the conventional red ginseng manufacturing method by steaming ginseng is washed with water and cooled for 1 to 48 hours, preferably 10 hours. The red ginseng chilled for a while is put in a polar solvent such as 1 to 15 times water, ethanol or methanol, preferably 5 to 10 times water, heated at 40 to 100 ° C., preferably 70 to 90 ° C., and then heated. The extract was repeatedly extracted 1 to 10 times, filtered under reduced pressure, and the filtered extract was mixed and concentrated under reduced pressure at 20 to 100 ° C., preferably at 50 to 70 ° C., using a rotary vacuum concentrator to remove the crude extract. Can be.

The red ginseng is preferably Korean Red Ginseng. Korean red ginseng is red ginseng manufactured using Korean ginseng. Korean ginseng is classified as Panax ginseng C.A. It has a plant name Meyer.

The red ginseng extract of the present invention may include one or more components selected from the group consisting of ginsenoside Rb1 and ginsenoside Rb2. Ginseng includes a ginseng-derived glycoside saponin component called ginsenoside, and may include ginsenoside Rb1 or ginsenoside Rb2 belonging to a saponin based on Protopanaxadiol (PD).

"Hearing impairment" of the present invention means impairment of the sense of perception of sound and thereby includes a deficiency of the ability to perceive sound. The hearing damage may be due to auditory blast or neuronal damage and includes damage to cells present in the Corti organs or cochlea. The damage here can be caused by genetic disorders, noise, toxicity, or any other specific stressor. Defects in the ability to recognize sounds due to hearing impairment are sensorineural hearing loss, conductive hearing loss, combined hearing loss, mild (25-40 dB), moderate (41-55 dB), moderately severe (56-70). dB), severe (71 to 90 dB), and very severe (greater than 90 dB) hearing loss, hearing loss before and after speech, unilateral (affects one ear) and bilateral (affects both ears) ) Hearing loss, or any combination thereof, ie sensorineural / severe / after language / bilateral hearing loss. In one embodiment of the present invention, hearing damage occurs in both ears in the form of a sudden high hearing loss mainly at high frequencies of 4000 Hz or more, and mainly includes damage to the basal turn of the cochlea. do.

In the "prophylaxis or treatment" of the present invention, "prevention" refers to any action that inhibits or delays the onset of hearing damage by administering a pharmaceutical composition comprising the red ginseng extract, "treatment" refers to the administration of the pharmaceutical composition Any action that improves or benefits the hearing loss.

In the present invention, the hearing damage may be induced by a toxic drug. Ototoxicity drugs are drugs or chemicals that damage the hearing function or the body's balance function, and mainly cause damage to the inner ear.

Such toxic drugs include antibiotics, anticancer agents (e.g. cisplatin, bleomycin, vincristine), diuretics (e.g. acetazolamide, furosemide, bumetanide, etaclinic) and the like, in particular aminoglycosides Contains antibiotics. The aminoglycoside antibiotic may be at least one selected from the group consisting of streptomycin, kanamycin, gentamycin, neobaicin, amikacin, tobramycin, netylmycin, dibecacin and sisomycin.

In addition, the composition of the present invention may inhibit apoptosis. The auditory hair cell death may be induced by a toxic drug. In a specific embodiment of the present invention, especially when treated with ginsenoside Rb1 and ginsenoside Rb2 of the red ginseng extract, the effect of inhibiting auditory hair cell death was excellent.

As another aspect, the present invention provides an adjuvant for inhibiting antibiotic side effects comprising red ginseng extract as an active ingredient. In particular, the antibiotic may be an aminoglycoside antibiotic. Aminoglycoside antibiotics are mainly used for Gram-negative bacterial infections, tuberculosis, and deep infections that do not respond well to general antibiotics. The toxicity of glycoside antibiotics is manifested in the form of hearing damage in about 5-33% of users. Antibiotic side effects in the present invention means that the drug administered at the time of antibiotic administration damages the hearing due to toxicity. These side effects can occur not only in the overdose of antibiotics, but also in long-term or moderate doses at appropriate therapeutic doses. In particular, in the case of tuberculosis, deep infection, etc., it is inevitable to administer aminoglycoside antibiotics for a long time, so it is preferable to use an adjuvant that can suppress antibiotic side effects.

The present invention provides a composition for treating an infectious disease comprising the adjuvant and an antibiotic together. The kind of antibiotic that can be used in the composition of the present invention preferably includes aminoglycoside antibiotics. The aminoglycoside antibiotic may be one or more selected from the group consisting of streptomycin, kanamycin, gentamycin, neobaicin, amikacin, tobramycin, netylmycin, dibecacin and sisomycin, but are not limited thereto.

In an embodiment of the present invention, when the red ginseng extract was administered with the aminoglycoside antibiotic gentamycin, hearing loss was prevented compared to the group administered with only gentamycin. Specifically, the hearing thresholds of 16KHz and 32KHz were measured by ABR (brain hepatic trigger test), and the hearing thresholds were better in the group treated with gentamicin and red ginseng extract than the group treated with gentamicin only. In other words, the red ginseng extract was confirmed to prevent hearing loss by gentamicin. In addition, as a result of analysis of auditory cell morphology, when the red ginseng extract was administered with the aminoglycoside antibiotic gentamycin, compared with the group which received only gentamycin, the basal turn area and the middle turn The extra auditory hair cell damage at) was significantly lower. In other words, the red ginseng extract was found to prevent hearing cell damage caused by gentamicin. In addition, considering that the basal rotation site (32KHz) was more susceptible to the toxic drug than the intermediate rotation site, the red ginseng extract of the present invention showed an excellent preventive effect on hearing damage of the basal rotation site to high frequency. Can be.

The composition of the present invention may include a pharmaceutically acceptable carrier, and the term "pharmaceutically acceptable carrier" in the present invention means a carrier that does not significantly stimulate the organism and does not inhibit the biological activity and properties of the administered compound or Refers to a diluent. The composition may be formulated with a carrier. In the case of oral dosage forms, for example, they may be formulated as troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. It is not limited to this. To prepare into formulations such as tablets and capsules, binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin and excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch and magnesium stearate Lubricants, such as calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of capsule formulation, in addition to the above-mentioned substances, it may include a liquid carrier such as fatty oil.

To formulate into parenteral dosage forms, the red ginseng extract may be formulated for injection, such as subcutaneous, intravenous or intramuscular injection, for suppository injection, or for aerosols that allow inhalation through the respiratory system. . To formulate into injectable formulations, the red ginseng extract is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is formulated for unit administration of ampoules or vials. To inject into suppositories, it may be formulated into a rectal composition such as suppositories or medicated enema containing conventional suppository bases such as cocoa butter or other glycerides. When formulated for sprays such as aerosols, propellants or the like may be combined with the additives to disperse the dispersed dispersion or wet powder.

In another aspect, the present invention provides a method of inhibiting antibiotic side effects by administering red ginseng extract. The present invention also provides a method for preventing or treating hearing damage by administering red ginseng extract.

The term "administering" as used herein means introducing the composition of the present invention to a patient in any suitable manner, and the administration route of the composition of the present invention may be administered through any conventional route so long as it can reach the target tissue have. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, pulmonary administration, rectal administration, intranasal administration, intraperitoneal administration or intradural administration, but is not limited thereto. . The composition according to the present invention may be administered separately with the antibiotic or at a certain time before or after the administration of the antibiotic, but is preferably administered after the antibiotic is administered. The composition of the present invention may be administered once, or may be administered twice or three times at regular time intervals.

As another aspect, the present invention provides a food composition for preventing or improving hearing damage, which comprises red ginseng extract as an active ingredient. The food composition includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof, organic acids , Protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition it may contain extracts for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. In addition, the food composition is any one of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soup, beverages, tea, functional water, drinks, alcoholic beverages and vitamin complexes Can be.

The red ginseng extract of the present invention can reduce the hearing damage induced by a toxic drug such as an aminoglycoside antibiotic, and thus, prolonged administration of an aminoglycoside antibiotic having a toxic effect such as tuberculosis and deep infections. Even if this is unavoidable, antibiotics can be reliably administered to treat infectious diseases. It can also reduce the side effects of anticancer drugs, known as toxic drugs, and treat and prevent hearing damage that is not related to toxic drugs such as sudden hearing loss.

Figure 1 shows the result of hearing function measurement to confirm the effect of KRG to prevent hearing loss by GM. At both 16 kHz and 32 kHz, hearing threshold changes were significantly less after GM administration in the KRG + GM group than in the GM group ( P <0.05).
FIG. 2 is a result of evaluating hair cell damage at the basal turn (recognition of 32 kHz) and middle turn (recognition of 16 kHz) of the cochlea using a scanning electron microscope (SEM). Exogenous hair cells (OHC) showing intact microvilli (stereocilia) were much more observed in the KRG + GM group than in the GM group ( P <0.05). Exogenous hair cell damage was more severe at basal rotation than at intermediate rotation.
Figure 3 shows the results of observing the changes in the auditory hair cells of the corti organs through paloidine staining. Basal and mid-rotational auditory hair cell damage in the GM group was statistically more severe than in the KRG + GM group. Exogenous hair cell damage was more severe at basal rotation (arrow) compared to intermediate rotation (*).
Figure 4 is an MTT assay result for confirming the effect of the red ginseng extract components on auditory cell line killing by gentamicin.
Figure 5 shows the results of the annexinV / PI FACS assay for confirming the effect of the red ginseng extract components on auditory cell line killing by gentamicin.
Figure 6 is a Western blotting result for confirming the effect of the red ginseng extract components on auditory cell line killing by gentamicin.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Example  One. Gentamicin ( GM ) Effect of Red Ginseng Extract on Induction Hearing Loss (In Vivo Experiment)

Animal preparation

Sprague-Dawley female rats (200-250 g) at 8 weeks of age were treated in four groups. Gentamicin (Sigma) was administered intraperitoneally for 5 days at 160 mg / kg / day dose, and Korean red ginseng powder (Korea Ginseng Society) was administered orally every 7 days before GM administration at 500 mg / kg / day dose.

a. GM group (15): 5 days of intraperitoneal injection of gentamicin

b. KRG + GM group (12 animals): 5 days of intraperitoneal injection of gentamicin + 12 days of oral administration of Korean red ginseng

c. KRG group (4 animals): 12 days of oral administration of Korean red ginseng

d. Saline group (control, 4): saline intraperitoneal injection 5 days

Hearing function measurement

The tone burst stimulus of 16 kHz and 32 kHz was heard using a Biosig 32 ABR (Terebral-Davis Technologies, Gainesville, FL, USA . ) From 75 dB to 10 dB. As a result, the minimum stimulus loudness (dB) from which the wave V waveform appears was determined as the hearing threshold. Hearing was measured 1 day before drug administration and 1 day after administration.

<Result>

The average hearing threshold in Group 4 before treatment was 18.5 ± 1.1dB at 16 kHz and 21.4 ± 1.8dB at 32 kHz. After GM administration, the hearing thresholds of GM, KRG + GM, KRG and saline groups were 27.7 ± 7.2 dB, 23.1 ± 4.1 dB, 16.9 ± 2.6 dB, 21.3 ± 3.5 dB, and 30.5 at 32 kHz, respectively, at 16 kHz. ± 6.6 dB, 25.2 ± 4.3 dB, 22.5 ± 2.7 dB, 22.5 ± 3.8 dB (Fig. 1).

Hearing thresholds after GM administration worsened in both GM and KRG + GM groups (mean difference at 16 kHz = 7.23 dB, 95% Confidence interval (CI) = 5.65 to 8.81, P = 0.000, mean difference at 32 kHz = 7.94 dB, 95% CI = 6.61-9.27, P = 0.000). However, at 16 kHz and 32 kHz, hearing threshold changes were significantly less after GM administration in the KRG + GM group than in the GM group. That is, it was confirmed that KRG prevented hearing loss by GM.

Hair Cell Morphology Analysis

(1) Scanning electron microscopy (SEM) evaluated hair cell damage at basal turn (recognition of 32kHz) and middle turn (middle turn (recognition of 16kHz)).

(2) 1 hour staining with Texas Red ^��- X ^paloidine (Invitrogen, Eugene, Oregon, USA), followed by confocal microscopy (LSM710, Carl Zeiss, Jena, Germany) for auditory hair cells of corti organs. Morphological changes were observed.

<Result>

(1) The number of cells showing intact microvilli at the mid-rotation (16 kHz) site was 4.0 ± 6.9 cells per 30 exogenous hair cells in the GM group, 28.7 ± 1.5 in the KRG + GM group and the KRG group. Was 29.7 ± 0.6 in the saline group and 29.7 ± 0.6 in the saline group. The number of intact cells per 30 exogenous hair cells at the basal rotation (32 kHz) site was 0.5 ± 1.5 cells in the GM group, 19.3 ± 5.1 in the KRG + GM group, 29.0 ± 1.0 in the KRG group and 29.3 ± in the saline group. 0.6 cells. Hair cells showing intact microvilli (stereocilia) were much more observed in the KRG + GM group than in the GM group ( P <0.05) (FIG. 2).

(2) Paloidine staining results were similar to SEM results. Basal and mid-rotational auditory hair cell damage in the GM group was statistically more severe than in the KRG + GM group. In other words, the red ginseng extract was confirmed to prevent auditory cell damage by GM. On the other hand, the rats treated with the red ginseng extract were normal as in the saline group. Therefore, it was confirmed that there are no side effects of red ginseng extract administration (FIG. 3).

Example  2. Red Ginseng Extract Ingredients Auditory cell line  Inhibitory effect on in vitro Assay

Cell culture

HEI-OC1 cells in mouse auditory cells were passaged in a 5% CO ₂ incubator in DMEM medium (Dulbecco's minimum essential medium) with 10% FBS, 30 U / ml penicillin.

Drug treatment

The cell lines were divided into three groups as follows and treated for each ginsenoid component of the red ginseng extract (Rb1, Rb2, Rg, Re).

Group 1 (GM)-48m exposure to gentamicin 1 mM.

Group 2 (GM + KRG)-Each ginsenoid component (Rb1, Rb2, Rg1, Re) of red ginseng extract was pretreated with 50, 200, 800, 1000 pg / ml for 12 hours and then exposed to 1 mM of gentamicin for 48 hours. Let's do it.

Control Group-Add DMSO.

Cell survival rate measurement- MTT Assay

The cell lines of each group were incubated for 48 hours by dispensing 100ul per well into 5 × 10 ³ cells / 100ul in 96 well cell culture tubes, and then dissolving MTT (3- [4,5-dimethlythiazol-2-yl] -2,5 in PBS. 40ul / well of -diphenyltetra zolium bromide, Sigma) solution (5mg / ml) was added and incubated in a CO ₂ incubator for 4 hours, and the solution was removed. Formazon formed by viable cells was dissolved in 100ul / well of DMSO solution for 30 minutes, and then absorbance was measured at 540 nm using an ELISA reader.

<Result>

As can be seen in Figure 4, Rb1, Rb2, Rg of the red ginseng extract components showed a cytoprotective effect on GM in a dose-dependent manner.

Cell death apoptosis ) Inhibition measurement- Annexin  V / PI  ( Annexin  V / Propidium  Iodide) Assay

After incubating the cells of each group in 6 wells, the supernatant of the wells was removed, and 0.25% Trappine-EDTA (GIBCO) was added in 500 ul each to separate the cells, followed by centrifugation at 1500 rpm for 10 minutes. The supernatant was removed, and 5 μl of Annexin V-FITC, PI solution was added and reacted for 15 minutes. 400 μl of 1 × binding buffer was analyzed by flow cytometry (FACScanto ^™ II (BD, San Diego, USA)).

<Result>

As can be seen in Figure 5, it was confirmed that Rb1, Rb2 in the red ginseng extract component has an effect of preventing GM-induced cell death, respectively.

Cell death apoptosis ) Inhibition measurement- Western Blotting

After the cells of each group were loaded by a general western blotting method, protein expression was compared using antibodies against poly-ADP-ribose polymerase (PARP), caspase-3, and β-actin.

<Result>

As can be seen in Figure 6, as the concentration of Rb1, Rb2 in the red ginseng extract component was confirmed that the expression of the cleaved-PARP decreases. It was confirmed that Rb1 and Rb2 among the red ginseng extract components have an effect of preventing GM-induced cell death.

Claims (18)

A pharmaceutical composition for treating or preventing hearing damage induced by an aminoglycoside antibiotic comprising at least one component selected from the group consisting of ginsenoside Rb1 and ginsenoside Rb2 as an active ingredient. delete delete delete delete delete The composition of claim 1, wherein the antibiotic is at least one selected from the group consisting of streptomycin, kanamycin, gentamycin, neobaicin, amikacin, tobramycin, netylmycin, dibecacin, and sisomycin. The composition of claim 1, wherein the composition inhibits auditory hair cell death. An adjuvant for inhibiting hearing damage induced by an aminoglycoside antibiotic comprising at least one component selected from the group consisting of ginsenoside Rb1 and ginsenoside Rb2 as an active ingredient. delete 10. The method of claim 9, wherein the aminoglycoside antibiotic is at least one selected from the group consisting of streptomycin, kanamycin, gentamicin, neobaicin, amikacin, tobramycin, netylmycin, dibecacin and sisomycin. Supplements. delete A composition for the treatment of an infectious disease comprising an adjuvant and an antibiotic according to claim 9. The composition of claim 13, wherein the infectious disease is tuberculosis or deep infection. Food composition for preventing or ameliorating hearing damage induced by an aminoglycoside antibiotic comprising at least one component selected from the group consisting of ginsenoside Rb1 and ginsenoside Rb2 as an active ingredient. delete delete delete

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