KR20150122470A - Composition for prevention and treatment of inner ear damage comprisiong subfraction of Piper longum L. extract - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a food composition for preventing and treating inner ear damage, containing a subfraction of a Piper longum L. extract as an active ingredient. More specifically, the subfraction of the Piper longum L. extract is prepared by a method comprising the following steps: preparing a primary Piper longum L. extract by immersing Piper longum L. powder, obtained by washing, drying, and grinding Piper longum L., in an extractant; and preparing the subfraction of the Piper longum L. extract by immersing the primary Piper longum L. extract in a mixed extractant of hexane and ethanol. The subfraction of the ethanol extract of Piper longum L. in accordance with the present invention has excellent anti-apoptotic activity and activity as a radical scavenger, and thus has an excellent effect in preventing and treating inner ear damage induced by aminoglycoside antibiotics.

Description

TECHNICAL FIELD The present invention relates to a composition for prevention and treatment of damage to inner ear comprising an active ingredient of a sub-

The present invention relates to a pharmaceutical composition and a food composition for preventing and treating damage to inner ear which contains a sub-fraction of the extract of Pepper as an active ingredient.

The loss of hearing personally causes a great deal of disruption to daily life, and also causes a huge socioeconomic loss. In particular, sensory nerve impairment is caused by hair cell damage of the inner ear which converts the vibration of the sound wave into an electric signal. The case of ototoxicity caused by various drugs is called hearing loss. The damage to the inner ear caused by these toxic agents occurs through various routes, including systemic administration such as oral administration, intramuscular injection, intravenous injection, or topical administration such as spotting, as well as inhalation of contaminated air have. Symptoms may occur immediately after administration, may occur spontaneously after weeks or months after discontinuation of the drug, may also be temporary, but may result in permanent damage and may occur in one or both sides.

A typical drug causing this toxicity is aminoglycoside antibiotics such as gentamicin (GM), and when it is taken in long term / high dose, sensorineural hearing loss and dizziness are induced. This is because inner ear hair cells, which convert sound signals into electrical stimuli, are vulnerable to damage caused by aminoglycoside antibiotics. Among them, apoptosis is a major aspect of inner ear hair cell damage, oxygen species, ROS) plays an important role in the progression of cell necrosis.

Several drugs have been reported to have a protective effect against this toxicity by blocking the production of these reactive oxygen species or scavenging reactive oxygen species. These include vitamin E, alpha-lipoic acid, Ebselen, and melatonin. In addition, various herbal extracts have also been studied as protective agents against aminoglycoside-induced toxicity, including Ginkgo biloba extract biloba , Gu Sui Bu, and Tanshinone, a phenolic acid derivative of danshen.

Piper longum L. was originated from the southern plant, Pibul, which was called Phil Bali in the ancient kingdom of Magadan, and it was called the middle point of the northern branch of Afghanistan. (Purum: 林 国) was called as Ariata (阿 梨 阿 陀). The petals have an unusual direction, the taste is spicy, the properties are hot, the appearance is the columnar shape, and the fruit of the small grain is attached to the periphery of the 果 axis. In general, it is used for abdominal pain, vomiting, loss of appetite, diarrhea, dysentery, toothache and the like, which is caused by side effects. The pharmacological effect is antibiotic action, anticonvulsive action, skin vasodilating action, antioxidant action, And hypolipidemic action have been reported. Korean Patent Laid-Open Publication No. 10-2013-0004949, which is a prior art by the present inventors, discloses an example of using a petal extract as an agent for prevention and treatment of inner ear damage.

In the above patent documents, it has been shown that the ethanol extract of petroleum jelly has a protective effect against damaging inner ear hair cells caused by gentamicin. The extract has a complex mixture form, and sub-fractions from the crude extract can exhibit a variety of biological activities. Therefore, the biological activity of these sub-fractions is likely to exhibit different biological properties than whole ethanol extracts of the petals, and may even exhibit prophylactic and therapeutic effects on better inner ear damage.

The present inventors have further studied the biological activity of the subfractions prepared using various solvents for the ethanol extract of petroleum liquor which has been reported previously, And to provide a pharmaceutical composition and a food composition for preventing and treating inner ear damage induced by antibiotics and containing a petal extract as an effective ingredient without affecting antibiotic action.

Therefore, in order to solve the first problem,

A step of washing the powdered hair, drying and pulverizing the powdered powder, and immersing the obtained powdered powder in an extraction solvent to prepare a primary powdered extract; And

Immersing the primary wil extract in a mixed extraction solvent of hexane and ethanol to prepare a subfraction of the wilted extract, thereby preventing inner ear damage containing the subfraction of the wilted extract as an active ingredient And a pharmaceutical composition for therapeutic use.

Further, in order to solve the second problem,

The present invention provides a food composition for prevention and improvement of damage to inner ear which contains as an active ingredient a sub-fraction of the extract.

The subfraction of the ethanol extract according to the present invention has excellent anticancer necrosis activity and radical scavenger activity and thus has excellent preventive and therapeutic effects against the inner damage induced by the aminoglycoside antibiotics do.

FIG. 1 is a graph showing phalloidin-labeled outer ear hair cells and inner ear hair cells. (AC) represent the number of hair cells per 100 μm length of the basal, middle and distal portions of the cochlea, respectively. Bundles labeled with Paloidein were counted under fluorescence microscopy and expressed as mean ± SD. The number of hair cells in a single sample was the average of five fields, and 5-11 samples were analyzed for each condition. The results were significant (* P <0.01, ** P <0.005) compared to the gentamicin alone group. IHC, inner ear hair cells; OHC, foreign hair cells; GM, gentamicin; PL, Lilac.
Fig. 2 is a microscopic image of Paloinden-labeled inner ear hair cells and outer ear hair cells. All samples were obtained from corresponding basal, mid and end regions of the explants. (AC) are images of the basal, middle, and distal regions from untreated grouped explants. (DF) are images of the basal, mid and end regions of the group treated with gentamicin (GM; 0.3 mM). (GI) are images of the basal, mid- and distal regions of 1 μg sub-fraction of SPL extract and GM-treated group. (JL) are images of the basal, mid- and distal regions of the 5 μg sub-fraction of SPL extract and the GM-treated group. (MO) are images of the basal, mid- and distal regions of the 10 μg sub-fraction of SPL extract and the GM-treated group. GM, gentamicin; PL, Lilac; SPL, subfraction of the petals.
Figure 3 is a microscopic image of anti-myosin-7a (red) and TUNEL (green) double-labeled P4 organs of Corti slices. All samples were obtained from corresponding basal, mid and end regions of the explants. (AC) is an image for control specimens. (DF) is an image for gentamicin (0.3 mM) treated sections. (GI) are images of 1 μg sub-fraction and GM-treated sections of SPL extract. (JL) are images of 5 μg sub-fraction and GM-treated sections of SPL extract. (MO) are images of 10 μg sub-fraction and GM-treated fragments of SPL extract. GM, gentamicin; PL, Lilac; SPL, subfraction of the petals.
FIG. 4 is a graph showing counts of TUNEL-labeled outer ear hair cells (OHC) and inner ear hair cells (IHC). The bars indicate the number of exocrine hair cells and inner ear hair cells per 100 μm length of the cochlea, respectively. TUNNEL labeled bundles were counted under fluorescence microscopy and expressed as the mean ± SD of the outer ear hair cells and inner ear hair cells per 100 μm length of the cochlea. The number of hair cells in a single sample was the average of five fields, and 5-11 samples were analyzed for each condition. The results were significant compared to the control group (* P < 0.05). GM, gentamicin; PL, Lilac.
Figure 5 shows the radical scavenging activity of the wilted extract sub-fraction (SPL). Percent increase in scavenging activity was plotted against the concentration of SPL extract. The results were significant at 1 μg / mL (* P <0.0001). The error bars represent the SD of quadruple samples.

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

The present invention provides a pharmaceutical composition for preventing and treating inner ear damage, which comprises a petroleum extract as an active ingredient. More specifically,

A step of washing the powdered hair, drying and pulverizing the powdered powder, and immersing the obtained powdered powder in an extraction solvent to prepare a primary powdered extract; And

Immersing the primary wil extract in a mixed extraction solvent of hexane and ethanol to prepare a subfraction of the wilted extract, thereby preventing inner ear damage containing the subfraction of the wilted extract as an active ingredient And a pharmaceutical composition for therapeutic use.

As described above, the inventors of the present invention have developed a wilt extract for preventing and treating inner ear damage, and the first step of the present invention, that is, the step of preparing the primary wilt extract, It is also disclosed in Laid-Open Publication No. 10-2013-0004949, which is incorporated herein by reference in its entirety.

Specifically, the primary wil extract may be produced by the following method. Piper longum L., PL is washed with water to remove foreign matter, dried in the shade and crushed. The petals may be used without restrictions, such as cultivated or commercially available products. A suitable amount of solvent is added to the ground pulp so that it is completely immersed. The extraction solvent is preferably a solvent selected from water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof, more preferably ethanol. Next, the above-obtained Lilium extract is filtered and concentrated under reduced pressure to obtain a final extract.

The sub-fraction of the extract of Liliaceae is prepared by the additional extraction process of the first lineal extract obtained by the above process, which is carried out using a mixed extraction solvent of hexane and ethanol. Specifically, the primary wil extract may be extracted using a mixed solvent of hexane and ethanol in various volume ratios. The mixing ratio may be 1: 9 to 9: 1 by volume ratio of hexane: ethanol, 2: 8. The final product can be obtained by removing the solvent through processes such as filtration, depressurization and concentration.

As can be seen from the following examples, the sub-fraction of the present invention prepared by the above-mentioned process has a protective effect against the inner damage caused by the antibiotic of the aminoglycoside even at a very low concentration , And further exhibits effects such as anti-cell necrosis activity and radical-scavenging activity.

The pharmaceutical composition for preventing and treating inner ear damage comprising the sub-fraction of the present invention as an active ingredient comprises 0.1 to 90% by weight, more preferably 0.1 to 50% by weight, of the sub-fraction based on the total weight of the composition do. However, the composition as described above is not necessarily limited to this, but may vary depending on the condition of the patient, the type of disease, and the degree of progression.

The inner ear damage disease as defined in the present invention is characterized by being caused by the death of auditory hair cells and includes tinnitus, hearing loss (hearing loss), dizziness, dizziness and the like. In addition, this toxicity (ototoxicity) refers to damage to the ear by the toxin, particularly the cochlea, the auditory nerve, and the vestibular organs, which is usually caused by medicines. This toxicity can cause sensory nerve damage or hearing loss, or both. Both can be recoverable and temporary, but they can be irreversible and permanent.

Examples of drugs which are toxic to the inner ear nerve include aminoglycoside antibiotics such as amikacin, arbekacin, gentamycin, kanamycin, neomycin, nethylamycin, paromomycin, streptomycin and tobramycin, Diuretics such as cisplatin, cisplatin, or cisplatin; and chemotherapeutic agents based on platinum such as cisplatin or carboplatin. It also includes, but is not limited to, nonsteroidal anti-inflammatory drugs (NSAIDS) such as meloxicam, heavy metals such as quinine or tin, mercury and lead, aspirin, Viagra, levitra, cialis and the like.

The composition of the present invention can be used not only as a sub-fraction of the extract of the present invention as an active ingredient, but also in combination with a conventionally known preventive or therapeutic agent for inner ear damage. The pharmaceutical composition of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, Or a flavoring agent. In addition, the pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration.

The pharmaceutical form of the pharmaceutical composition may be a granule, a powder, a tablet, a coated tablet, a capsule, a suppository, a liquid, a syrup, a juice, a suspension, an emulsion, a drip agent or an injectable liquid agent. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using methods disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, as appropriate in the field.

An effective amount of a pharmaceutical composition according to the present invention means an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, an animal or a human being considered by a researcher, veterinarian, physician or other clinician, &Lt; / RTI &gt; inducing a reduction of the symptoms of the disease or disorder. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like. In the treatment method of the present invention, in the case of an adult, it is preferable to administer the composition of the present invention at a dose of 1 / kg to 250 / kg once to several times a day. The compositions of the present invention may be administered in a conventional manner via oral, rectal, intravenous, intra-arterial, intraperitoneal, intramuscular, intrasternal, percutaneous, topical, intra-ocular or intradermal routes.

Meanwhile, the present invention provides a food composition for prevention and improvement of inner ear damage containing the sub-fraction of the petroleum extract as an active ingredient.

The food composition according to the present invention can be formulated in the same manner as the above-mentioned pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meats, chocolates, foods, confectionery, pizza, ramen noodles, gums, ice cream, alcoholic beverages, vitamin complexes, . The amount of the food composition according to the present invention added to the food or beverage may be 0.01 to 15% by weight of the total food, and the health functional beverage composition may be added in a proportion of 0.01 to 10 g, preferably 0.3 to 1 g, .

The food composition according to the present invention may contain various flavors or natural carbohydrates as an additional ingredient as well as a conventional food composition, in addition to containing a sub-fraction of the extract of the present invention as an effective ingredient. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. The above-described flavors can be advantageously used as natural flavorings (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.).

In addition, the food composition may contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid, Salts of alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like. In addition, the food composition of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks.

Since the subfraction of the extract of Liliaceae, which is an active ingredient of the present invention, is a natural substance, it has little toxicity and side effects, so that it can be safely used for long-term use for hearing protection purposes.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following Examples are intended to assist the understanding of the present invention and are not intended to limit the scope of the present invention.

Petal  Ethanol extract

Jinpyeol was purchased from the Pharmaceutical Department of Ilsan Hospital, Dongguk University. The dried fruit powder was extracted twice at 85 ° C-90 ° C for 3 hours with 10 times volume of 30% ethanol and filtered using a 40 μm filter. Ethanol was removed in a vacuum at 60 ° C and the yield was 4.7%. The extract was resuspended in phosphate buffered saline (PBS, pH 7.2).

Petal  Sub-fractionation of ethanol extracts

The subfraction of PL (SPL) extract for the ethanol extract of the petal was extracted with various combinations of hexane and ethanol for 3 hours at 85 ° C-90 ° C and filtered as described above. The solvent was removed in vacuo at 60 &lt; 0 &gt; C. The subfraction obtained using a combination of hexane: ethanol in a volume ratio of 2: 8 was analyzed for protective effects on antioxidant activity and GM-induced hair cell loss in neonatal cochlea cultures.

Cochlear organ type Culture  ( Cochlea organotypic culture )

The cochlear organ type culture was prepared as previously reported (Du XF, Song JJ, Hong S, Kim J. Ethanol extract of Piper longum L. attenuates gentamicin-induced hair cell loss in neonatal cochlea cul-tures. Pharmazie. Jun; 67 (6): 559-63). All protocols were in line with the guidelines of the Animal Protection Ethics Committee at Dongguk University Ilsan Hospital. ICR mouse (Koatech, Pyeongtaek, Korea) Koatch, Pyeongtaek, Korea) The offspring were beheaded on the fourth day of life. The cochlea was carefully dissected in a flat preparation of the Corti organ. (Sigma-Aldrich, St. Louis, Mo., USA) and 2% sodium &lt; RTI ID = 0.0 &gt; (Nunc, Rochester, NY, USA) was added to the mixture, and the mixture was gelled. Thereafter, 1 mL of serum-free Dulbecco's modified Eagle's medium (Welgene, South Dakota) supplemented with 1% N-1 supplement (Sigma-Aldrich) and 50 U / mL penicillin G (Sigma-Aldrich) was added. The Corti organ was gently pressed onto the collagen surface using forceps and fixed by the surface tension of the culture medium. The cochlear culture was incubated overnight at 37 ° C in a humidified atmosphere of 5% CO ₂ and exposed to media containing GM (0.3 mM) for 24 hours. To assess the protective effect, the explants were treated with SPL extract for 1 h (0, 1, 5, and 10 μg / mL) prior to treatment with GM for 24 h.

Hair cell count

After culturing for 24 hours, the culture was fixed, permeabilized (permeabilized) and stained with Alexa Fluor 350 phalloidin (Invitrogen, Carlsbad, CA, USA). The slices were mounted on slides containing Gel Mount Biomedia (Fisher Scientific, Waltham, MA, USA), covered and covered with a DMIL microscope (Leica, Jena, Germany). Each labeled ciliated cilia was counted in three outer hair cells (OHC) columns and one inner hair cell (IHC) column. Cells were considered to be lost if gaps were present in the normal geometric arrangement and stereocilia or cuticular plates were not apparent. The number of cells was counted over a distance of 100 [mu] m from five randomly selected fields for the base, middle, and distal ends of each section. The average from the five fields was considered as a single sample and 5-11 samples were evaluated for each condition.

TUNEL  And Myosin -7a double marker

The anti-cell necrosis effect was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). DNA fragments were analyzed in situ by using a TUNEL staining kit (Trevigen, Gaithersburg, MD, USA) with slight modifications to the manufacturer's instructions. Fixed cochlear organ type cultures were placed in 0.01 M PBS for 10 minutes and then treated with proteolytic enzyme K solution for at least 15 minutes at room temperature. Washed twice with deionized water for 2 minutes, and then each culture was immersed in 1 x TdT label buffer for 5 minutes. The culture, at 37 ℃ of the humidification chamber for 60 minutes and cultured in a 50 × TdT dNTP, 50 × cations ^{(Mg 2 +), 50 ×} TdT enzyme and 1 × TdT TUNEL mixture comprising labeling buffer. The reaction was terminated by washing in 1 x TdT stop buffer for 5 minutes. Cultures were washed in 0.01 M PBS-Tween (PBS-T), treated with Strep-Fluor solution for 30 min at room temperature in the dark and then mounted on glass with cover slip using fluorescence mounting media. Samples were observed under a fluorescence microscope using a 495 nm filter. In the case of myosin-7a labeling, the cultures were fixed, permeabilized, and myosin-7a rabbit polyclonal antibody (1: 300 dilution; Proteus BioSciences, Ramona, CA, USA) followed by Alexa Fluor 594 donkey anti- 500 dilution; Invitrogen). The slices were mounted on a slide with Gel Mount Biomedia (Fisher Scientific), then cover slips were covered and examined with a model DP70 fluorescence microscope (Olympus, Tokyo, Japan). Five wells in a 96-well plate were used for each concentration. Results were obtained from three replicate experiments.

2,2- Diphenyl -One- Picrylhydrazyl  ( DPPH ) analysis

Free radical scavenging activity evaluation was performed. SPL extract stocks were diluted in 99% ethanol to produce concentrations of 1, 5, and 10 μg / mL, respectively. DPPH (100 mM; Sigma Aldrich) solution was prepared in 95% ethanol. SPL extract was diluted twice in 50% ethanol and DPPH was diluted 100-fold in 95% ethanol (DPPH solution). A 0.5 mL volume of the sample was mixed with 3.0 mL (1 mM) DPPH and then left at room temperature for 30 minutes under light protection. Absorbance at 540 nm was measured using a spectrometer (Molecular Devices, Sunnyvale, Calif., USA). The absorbance difference between the test and control groups was calculated and expressed as a percentage (%). The ability to scavenge DPPH radicals was calculated using the following equation:

Scavenging activity (%) = [1- (sample value-margin / control group)] × 100

Statistical analysis

Results were expressed as mean ± SD. Student t-test was used to determine statistically significant differences between groups. P <0.05 was considered significant.

result

GM - for induced hair cell damage SPL  Protective effect of extract

After birth, cochlear culture from mice was cultured in medium containing 1, 5, and 10 μg / mL of SPL extract for 1 hour and then treated with GM (0.3 mM). Viable phalloidin-labeled hair cells were counted after 24 hours. In GM-treated groups, OHC and IHC numbers were significantly reduced. On the other hand, no reduction in OHC and IHC was observed in the SPL extract-treated sections. SPL extracts at concentrations of 1 [mu] g / mL and 5 [mu] g / mL protected OHC and IHC significantly (P < 0.01), respectively, in a dose-dependent manner in the basal region of the cochlea (FIG. 1A). Similarly, SPL extracts at concentrations of 1 [mu] g / mL and 5 [mu] g / mL showed a significant (P < 0.01) protective effect against GM-induced OHC and IHC damage in the middle region of the cochlea (Fig. The SPL extract at a concentration of 5 μg / mL showed a significant (P <0.01) protective effect on the OHC and IHC at the distal end of the cochlea (FIG. 1C).

Compared to untreated controls (Fig. 2A-C), GM severely impaired the anatomical structure of the Corti organ in GM-treated groups. In this group, hair cells showed deformation and disturbances and gaps were seen (Fig. 2D-F). SPL extract did not show toxicity to the cochlea and decreased GM-induced toxicity (Fig. 2G-O); In the SPL extract treated group, the alignment of the cells in the Corti organ was somewhat irregular, while the hair cells maintained their shape and floating cilia (Fig. 2G-O). These results support the fact that SPL extracts have a significant protective effect against GM-induced hair cell damage and that this protective effect is more pronounced for OHC than IHC. Among the basal, mid and end regions of the cochlea, the protective effect of the SPL extract was more pronounced in the basal and middle hairy cells than in the terminal hairy cells.

SPL  The anti-cytotoxic activity of the extract

The cell death was assessed by TUNEL assay, which stained DNA fragments in necrotic cells and detected hair cell damage by myosin-7a labeling. In the untreated group, necrotic cells were absent and no reduction in OHC and IHC was observed by TUNEL and myosin-7a double labeling (Fig. 3A-C). TUNEL-positive (necrotic) cells were observed in cochlear cultures after 24 hours of 0.3 mM GM treatment (Figure 3D-F). Significant OHC and IHC damage were detected in the basal, middle and end regions of the cochlea by myosin-7a labeling. The number of TUNEL-positive cells (green fluorescence) was significantly reduced and the number of hair cells (red fluorescence) was increased when the fragment was pretreated with SPL extract (1 μg / mL) (FIG. 3G-I) . SPL extracts at 1-10 μg / mL significantly inhibited GM-induced apoptotic cell death in both IHC and OHC. In the sections exposed to 1-10 μg / mL of SPL extract, TUNEL-positive cells were significantly reduced (FIG. 3G-O). The number of TUNEL-labeled OHC and IHC was significantly increased in the 0.3 mM GM treated specimens. The number of OHC and IHC decreased when the intervertebral bodies were treated with SPL extract prior to GM treatment. The anti-cytotoxic effect of SPL extracts was dose dependent (Figure 4). At concentrations greater than 5 μg / mL, SPL extracts significantly (P <0.05) protected OHC and IHC against GM-induced cell necrosis and hair cell damage. These results indicate that the SPL extract gives a protective effect to OHC and IHC in GM-induced hair cell damage and shows that the anti-cell necrotic activity of the extract is highly concentration dependent.

SPL  Extract Radical - Scavenging  activation

The radical scavenging activity of the SPL extract was measured by DPPH analysis. It was clear that the scavenging activity of the SPL extract increased in a concentration-dependent manner. Percent scavenging activity of SPL extracts was significantly increased (P < 0.05) at a concentration ≥1 μg / mL when compared to the control (FIG. 5). The scavenging activities of SPL extracts 1, 5, and 10 μg / mL were 25.65%, 48.59%, and 60%, respectively.

Review

The results of this example show that the sub-fraction of the volatile ethanol extract has a protective effect against GM-induced hair loss. The sub-fraction of the volatile ethanol extract showed an antioxidative effect in a dose-dependent manner and exhibited a protective effect against GM-induced hair cell damage in cochlea due to anti-cell necrotic activity. Further, it suppresses hair cell death by (1.25 to 10 mg / ml) and even at a very low concentration (1 μg / ml) than the prior art of Korean Patent Laid-Open Publication No. 10-2013-0004949 by the present inventors. Thus, it can be seen that the sub-fraction of the ethanol extract according to the present invention has more potent antioxidative and anti-cell necrotic activity than the ethanol extract of the prior art of the prior art.

GM is widely used as an aminoglycoside antibiotic. However, the clinical utility of aminoglycosides such as GM is known to be limited due to the risk of this toxicity and nephrotoxicity (Forge A, Schacht J. Aminoglycoside antibiotics. Audiol Neurotol. 2000 Jan-Feb; 5 (1): 3-22). GM is able to induce hearing loss and balance impairment by destroying inner ear hair cells (Theopold HM. Comparative surface studies of ototoxic effects of various aminoglycoside antibiotics on the organ of Corti in the guinea pig: a scanning electron microscopic study. Acta Otolaryngol 1977 Jul-Aug; 84 (1-2): 57-64). Cell death is known to be a major mode of hair cell damage in this toxicity, and ROS is known to play an important role as an initiator of cell necrosis (Forge A, Li L. Apoptotic death of hair cells in mammalian vestibular sensory epithelia. Hear Res 2000 Jan; 139 (1-2): 97-115). Aminoglycosides such as GM can form complexes with iron, which react with unsaturated fatty acids to form superoxide (O2) radicals and lipid peroxides (Sha SH, Schacht J. Salicylate attenuates gentamicin-induced ototoxicity. Lab Invest., 1999 Jul; 79 (7): 807-13). Typically, O2 is converted to hydrogen peroxide by superoxide dismutase and deoxidized by water and oxygen by catalysis. However, under very high oxidative environments, the endogenous antioxidant pathway may be overwhelmed and the free oxygen radicals become enriched (Schacht J. Antioxidant therapy attenuates aminoglycoside-induced hearing loss. Ann NY Acad Sci. 1999 Nov; 884: 125-30 Sha SH, Taylor R, Forge A, Schacht J. Differential vulnerability of basal and apical hair cells based on intrinsic susceptibility to free radicals Hear Res 2001 May; 155 (1-2): 1-8). Aminoglycosides such as GM can activate inductive nitric acid synthase in inner ear tissues and cause nitrate increase (Takumida M, Popa R, Anniko M. Free radicals in the guinea pig inner ear following gentamicin exposure ORL J Otorhinolaryngol Relat Spec. 1999 Mar-Apr; 61 (2): 63-70). Under these circumstances, O2 free radicals can react with available nitric acid to form destructive peroxynitrite radicals or directly initiate hair cell death.

Hair cell protection is an important goal to prevent GM-induced toxicity. The common signaling pathway involved in GM-induced toxicity is ROS production and cell death. Therefore, many drugs have been evaluated in terms of protecting the hair cells from GM-induced toxicity through prevention of ROS overproduction and reduction of cell necrosis (Choung YH, Kim SW, Tian C, Min JY, Lee HK, Park, SN, et al. Korean red ginseng prevents gentamicin-induced hearing loss in rats. Laryngoscope 2011 Jun; 121 (6): 1294-302 .; Jeong HJ, Choi Y, Kim MH, Kang IC, Lee JH, Park C , et al .: Rosmarinic acid, an active component of Dansam-Eum attenuates ototoxicity of cochlear hair cells through blockage of caspase-1 activity PLoS One 2011 Apr 15; 6 (4): e18815 .; Chang J, Jung HH, Yang JY , Choi J, Im GJ, Chae SW, Park JM, Lee BD, Chae K. Department of Cardiovascular Surgery, SW, Chi J, Kwon SK, Song JJ. Protective effect of NecroX, a novel necroptosis inhibitor, on gentamicin-induced ototoxicity. Such compounds include antioxidants such as lipoic acid, a-tocopherol, ebselen, D-methionine, salicylic acid, as well as iron chelators such as desferoxamine and dihydroxybenzoate, Also included are conventional drugs (Xie J, Talaska AE, Schacht J. New developments in aminoglycoside therapy and ototoxicity. Hear Res. 2011 Nov; 281 (1-2): 28-37).

On the other hand, it is a well-known traditional medicinal substance in the Asian and Pacific Island regions and has been used for a variety of biological activities including anti-amoeba, anti-giardial, immunostimulatory, anti-ulcerative, anti- (Kumar S, Kamboj J, Suman, Sharma S. Overview for various aspects of the health benefits of Piper longum linn: fruit J Acupunct Merid-ian Stud. 2011 Jun; 4 (2): 134-40). The main chemical components of the volatile oils are volatile oils, resins and alkaloids, namely piperine, piperlongumine and piperlonguminine (Shankaracharya NB, Rao LJ, Naik JP, Nagalakshmi S. Characterization of chemical constituents of Indian Long Pepper (Piper-longum L). J Food Sci Tech. 1997; 34 (1): 73-5).

The present inventors have disclosed a related art Korean Patent Publication No. 10-2013-0004949 and related prior art (Du XF, Song JJ, Hong S, Kim J. Ethanol extract of Piper longum L. attenuates gentamicin-induced hair cell loss in neonatal cochlea cultures. Pharmazie. 2012 Jun; 67 (6): 559-63) discloses that the ethanol extract of the petals has a protective effect against GM-induced inner ear hair cell damage 1.25 to 10 mg / ml in the case of publication No. 10-2013-0004949, and 5 mg / mL in the case of the prior art). In contrast, the present invention showed that the sub-fraction of the ethanol extract of peanut showed a significant protective effect against GM-induced toxicity even at a low concentration of 1 μg / mL. Furthermore, the antioxidative activity of the extract of Liliaceae was more remarkable than that of the aforementioned prior patents and prior arts.

Thus, the main cause for the extract of the present invention to have better efficacy compared to the related prior arts is that the petals contain polyphenols and these polyphenols are quantitatively and qualitatively found in the subfractions extracted with hexane and ethanol As well. Furthermore, it is believed that the wilted extract extracted with various solvents will exhibit various biological activities, which means that by optimizing the extraction process, the biological activity of the natural compound can be enhanced.

In conclusion, the sub-fraction of the ethanol extract of the present invention has excellent anti-cell necrosis activity and radical scavenger activity, and thus provides excellent prevention and treatment for the inner damage induced by the aminoglycoside antibiotics Effect.

Claims (9)

A step of washing the powdered hair, drying and pulverizing the powdered powder, and immersing the obtained powdered powder in an extraction solvent to prepare a primary powdered extract; And
Immersing the primary wil extract in a mixed extraction solvent of hexane and ethanol to prepare a subfraction of the wilted extract, thereby preventing inner ear damage containing the subfraction of the wilted extract as an active ingredient &Lt; / RTI &gt; The pharmaceutical composition according to claim 1, wherein the extraction solvent used in the step of preparing the primary extract is water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The pharmaceutical composition according to claim 1, wherein the mixed extraction solvent used in the step of preparing the sub-fraction has a mixing volume ratio of hexane: ethanol of 1: 9 to 9: 1. The pharmaceutical composition according to claim 1, wherein the mixed extraction solvent has a mixing volume ratio of hexane: ethanol of 2: 8. The pharmaceutical composition according to claim 1, wherein the inner ear lesion is caused by the death of the auditory hair cell. 2. The pharmaceutical composition according to claim 1, wherein the inner damage is caused by an aminoglycoside antibiotic. 7. The method of claim 6, wherein the aminoglycoside antibiotic is any one selected from the group consisting of amikacin, arbekacin, gentamicin, kanamycin, neomycin, nethilmine, paromomycin, streptomycin, tobramycin &Lt; / RTI &gt; The pharmaceutical composition according to claim 1, wherein the inner ear damage is tinnitus, deafness or dizziness. A food composition for prevention and improvement of damage to inner ear which comprises the subfraction of the extract of Pemphorus according to claim 1 as an active ingredient.

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