KR102481709B1 - A composition for treating dry eye syndrome comprising a extract of Vaccinium Uliginosum as an active ingredient - Google Patents

Abstract

The present invention provides a composition for preventing, improving, and treating dry eye syndrome comprising a blueberry extract, a blueberry fraction extract, or both.
The blueberry extract and the blueberry fraction extract of the present invention exhibit excellent therapeutic effects in inhibiting corneal inflammation and corneal damage, and thus can be used as pharmaceutical compositions and health functional foods useful for preventing or treating dry eye syndrome.

Description

A composition for treating dry eye syndrome comprising a blueberry extract as an active ingredient {A composition for treating dry eye syndrome comprising a extract of Vaccinium Uliginosum as an active ingredient}

The present invention relates to a composition for preventing, treating, or improving dry eye syndrome comprising a blueberry extract and/or a blueberry fraction extract.

Dry eye syndrome is a general term for diseases in which abnormalities in the tear film occur due to damage to one or all of the structures on the ocular surface responsible for tear film production (cornea, conjunctiva, goblet cells, lacrimal gland, etc.) . This is a disorder of the tear film due to lack of tear production or excessive evaporation, which causes damage to the ocular surface and causes discomfort. Recently, it has been defined as a disease of tears and ocular surface caused by various causes, accompanied by damage to the ocular surface, causing discomfort, visual impairment, destabilization of the tear film, and the like, and also accompanied by increased osmotic pressure of the tear film and inflammation of the ocular surface.

Such dry eye syndrome can be said to be caused by lacrimal gland dysfunction such as lacrimal duct obstruction, changes in tear film stability and composition, and inflammation and cell death of the ocular surface. Systemic factors include age, gender, hormonal changes, autoimmune diseases, and contact lens wear, and environmental factors include dry living conditions, use of computers, videos, and mobile devices. Dry eye syndrome is a disease in which the prevalence increases with age, and the incidence rate is higher in women than in men. It is characterized by the high prevalence of people in their 20s recently, and it can be seen that it is not irrelevant to the increase in smartphone use.

Restasis eye drops (cyclosporine 0.05%), an immunomodulator, is the only specialty treatment for dry eye syndrome currently on the market, but since it exhibits drug efficacy through anti-inflammatory action, it must be used repeatedly for several months to show satisfactory efficacy to patients, and it may cause burning sensation, etc. It has the disadvantage of side effects of (Ophthalmology, 107:631-9, 2000: Thomson Pharma, www.thomson-pharma.com). In addition, artificial tears preparations that temporarily supplement insufficient tears and treatments for corneal and conjunctival epithelial disorders accompanying dry eye syndrome are commonly used, but these are only temporary therapies and cannot be said to be practical treatments for dry eye syndrome. That is, the development of a therapeutic agent for dry eye syndrome that can more fundamentally treat dry eye syndrome while being safe and free from side effects without simply symptomatic treatment has not yet been developed.

Dry eye syndrome is a multifactorial disease caused by various causes, and various approaches to treatment have been studied. Among them, studies on inflammation and apoptosis of the ocular surface are being actively conducted. In particular, it has been reported that dry stress activates NF-κb and AP-1 and increases IL-1b, causing corneal barrier damage along with inflammation of the ocular surface (Ophthalmology, 154(1):63-71, 2012 , Mol Vis, 17:533-42, 2011). Under this background, it is necessary to develop a treatment method through suppression of inflammation of the ocular surface and restoration of the resulting corneal barrier damage.

On the other hand, blueberry (Bog bilberry, vaccinium) uliginosum L) is a deciduous broad-leaved small shrub of the Ericaceae family ( Vaccinium uliginosum ), the stem is about 1 meter high and dark brown. The fruit is a round berry, ripens black in autumn, and is edible as blueberry. It is common in cold regions of high mountains and plateaus, and is distributed in Korea, Manchuria, Mongolia, Japan, Europe, and North America. Blueberries contain flavonoids and anthocyanins.

Republic of Korea Patent Registration No. 2011-0102246 discloses the therapeutic effect of blueberry extract or fraction extract on macular degeneration. However, the fractionation method of the above patent is a solid phase extraction (SPE) fractionation method and uses organic solvents such as ethyl acetate, so it is not suitable as a health functional food material, and C18 Sep-Pak cartridges etc. are used in the manufacturing process It uses a fractionation method that is not suitable for mass production process. In addition, macular degeneration is a disease that occurs in the nerve tissue located in the center of the inner retina of the eye, and the treatment of the disease is completely different from that of dry eye syndrome.

Under this background, there is a need to develop a therapeutic agent capable of fundamentally preventing, improving, and treating dry eye syndrome.

Republic of Korea Patent Publication No. 2011-0102246

The present invention provides a pharmaceutical composition for preventing or treating dry eye syndrome comprising a blueberry extract and/or a blueberry fraction extract as an active ingredient.

The present invention also provides a health food for preventing or improving dry eye syndrome, comprising a blueberry extract and/or a blueberry fraction extract as an active ingredient.

While carrying out research and development on the use of blueberry extract and blueberry fraction extract that can be commercially mass-produced suitable for the human body, the present inventors found that the blueberry extract and blueberry fraction extract expressed NF-κb and activating protein-1 (AP-1). It was confirmed that the effect of inhibiting inflammation and ocular surface damage was excellent through the action effects such as inhibiting, and accordingly, it was confirmed that the blueberry extract and the blueberry fraction extract could be used as a treatment for dry eye syndrome, and the present invention has been completed.

The present invention provides a pharmaceutical composition for preventing or treating dry eye syndrome comprising a blueberry extract, a blueberry fraction extract, or both of them as an active ingredient.

The pharmaceutical composition for preventing or treating dry eye syndrome according to the present invention exhibits excellent effects in preventing or treating dry eye syndrome through inhibition of inflammation through inhibition of expression of NF-κb and AP-1, recovery of ocular surface damage, and the like. In addition, it shows excellent action and effect even through oral administration, and the medication compliance is very high.

In the present invention, the term "fractionated extract" refers to a product obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various components.

The blueberry extract and the blueberry fraction extract according to the present invention are not limited thereto, but may preferably have impurities such as sugar removed and a high total polyphenol content as a marker component.

The blueberry extract and the blueberry fraction extract according to the present invention may contain 1% to 60% by weight of polyphenols based on the total weight. In particular, the blueberry fraction extract may contain polyphenols in a high content of preferably 2 to 60% by weight, more preferably 3 to 15% by weight, and still more preferably 4 to 12% by weight, based on the total weight.

In the present invention, the term "dry eye syndrome (DES)" refers to dry eyes caused by dry eyes such as keratitis sicca, xerophthalmia, keratoconjunctivitis sicca (KCS) or cornea sicca. including all diseases.

In the present invention, blueberry ( vaccinium uliginosum L) may be extracted from flowers, fruits, leaves or bark of blueberry trees, and preferably from fruits of blueberry trees. In addition, collected, cultivated, or commercially available products may be used without limitation.

The blueberry extract can be prepared by extracting the blueberry extract in the presence of water, anhydrous lower alcohol having 1 to 4 carbon atoms or a hydrous lower alcohol having 1 to 4 carbon atoms.

Anhydrous lower alcohols having 1 to 4 carbon atoms are lower alcohols that do not contain water and include, but are not limited to, methanol, ethanol, propanol, butanol, and isobutanol. In addition, the hydrous lower alcohol having 1 to 4 carbon atoms is a lower alcohol mixed with water, such as methanol, ethanol, propanol, butanol, or a mixed solvent of isobutanol and water, but is not limited thereto. Preferably, the solvent may be water.

As an extraction method, it is preferable to use shaking extraction, Soxhelt extraction or reflux extraction, but is not limited thereto.

Preferably, the blueberry extract according to the present invention may be prepared by treating blueberries with an enzyme in water, anhydrous lower alcohol having 1 to 4 carbon atoms, or anhydrous lower alcohol having 1 to 4 carbon atoms, followed by heating and extracting.

The enzyme may be treated with blueberries to enhance the polyphenol content of blueberries. Preferably, the enzyme is amylase, glugoamylase, protease, oxidase, polymethylgalacturonase, polygalacturonase, glucosidase, cellulase ) or pectinase, and more preferably, pectinase may be used. After the enzyme treatment, heating may be performed for 1 hour to 10 hours, preferably 1 hour to 4 hours, and the reaction may be performed at a reaction temperature of 40 to 65 °C, preferably 45 to 60 °C.

Preparation of the blueberry extract of the present invention may further include filtering, concentrating, and drying the extract after extracting the blueberry extract. The filtration may be performed by filtering the solution according to a conventional method in the art or obtaining a filtrate or supernatant through centrifugation. The concentration is preferably concentrated under reduced pressure, and the concentration under reduced pressure preferably uses a vacuum vacuum concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, drying is preferably performed under reduced pressure, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.

The blueberry fraction extract can be prepared by extracting the blueberry extract in water, anhydrous lower alcohol having 1 to 4 carbon atoms or anhydrous lower alcohol having 1 to 4 carbon atoms, and passing it through a resin. In preparing the blueberry fraction extract, the preparation steps of the blueberry extract are the same as those of the above.

Preferably, the blueberry fraction extract according to the present invention is obtained by: (a) extracting by treating blueberries with an enzyme in water, anhydrous lower alcohol having 1 to 4 carbon atoms or hydrated lower alcohol having 1 to 4 carbon atoms, and heating; (b) passing the extract of step (a) through a resin.

In step (a), the enzyme may be treated with blueberries to increase the polyphenol content of blueberries. Preferably, the enzyme is amylase, glugoamylase, protease, oxidase, polymethylgalacturonase, polygalacturonase, glucosidase, cellulase ) or pectinase, and more preferably, pectinase may be used. After the enzyme treatment, heating may be performed for 1 hour to 10 hours, preferably 1 hour to 4 hours, and the reaction may be performed at a reaction temperature of 40 to 65 °C, preferably 45 to 60 °C.

The step of passing the blueberry extract of step (b) through a resin is preferably a resin filtration method using an adsorption resin or a resin column method. XRD resin, XDA resin, Amberlite XAD resin, Dowex resin, Superlite, DAX resin, Diaion HP-10, HP-20, HP-30, HP-40 and HP-50 resin are available. and, preferably, XDA resin, XRD resin, HP-20 resin, or a mixed resin thereof may be used.

Preparation of the blueberry fraction extract of the present invention may further include filtering, concentrating, and drying the extract after extracting the blueberry extract. The filtration may be performed by filtering the solution according to a conventional method in the art or obtaining a filtrate or supernatant through centrifugation. The concentration is preferably concentrated under reduced pressure, and the concentration under reduced pressure preferably uses a vacuum vacuum concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, drying is preferably performed under reduced pressure, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.

The blueberry fraction extract filtered or eluted with the anhydrous lower alcohol having 1 to 4 carbon atoms or the hydrous lower alcohol having 1 to 4 carbon atoms may be concentrated and dried. Preferably, it may be concentrated under reduced pressure, and it is preferable to use a vacuum vacuum concentrator or a vacuum rotary evaporator for concentration under reduced pressure, but is not limited thereto. In addition, drying is preferably performed under reduced pressure, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.

By passing the extracted blueberry extract through a resin, a blueberry fraction in which sugar components of the blueberry extract are removed and the total polyphenol content is greatly increased can be prepared. This is preferable in that it does not use an organic solvent harmful to the human body and can show excellent operational effects in human application.

According to the method for preparing a blueberry extract according to an embodiment of the present invention, a blueberry extract may be prepared by treating the blueberry extract with pectinase and heating it.

According to the resin filtration method of the manufacturing method of the blueberry fraction extract according to an embodiment of the present invention, the extract of step (a) is loaded on the resin, and anhydrous lower alcohol having 1 to 4 carbon atoms or lower hydrous alcohol having 1 to 4 carbon atoms is loaded. A blueberry fraction extract can be prepared by filtering with alcohol, preferably 20 to 99% methanol or 20 to 99% ethanol, 1 to 3 times. The anhydrous lower alcohol having 1 to 4 carbon atoms or hydrous lower alcohol having 1 to 4 carbon atoms may further include 0.01% to 1% acetic acid. In addition, various impurities including sugar may be removed by filtering with water before filtration with the lower alcohol having 1 to 4 carbon atoms.

According to the method of using a resin column in the method for preparing a blueberry fraction extract according to an embodiment of the present invention, the extract of step (a) is loaded on a resin in a column tube, and anhydrous lower alcohol having 1 to 4 carbon atoms or carbon atoms 1 to 4 is added. A blueberry fraction extract can be prepared by eluting with a hydrous lower alcohol, preferably 20 to 99% methanol or 20 to 99% ethanol, 1 to 3 times. The anhydrous lower alcohol having 1 to 4 carbon atoms or hydrous lower alcohol having 1 to 4 carbon atoms may further include 0.01% to 1% acetic acid. In addition, impurities may be removed by filtering with water before filtration with the lower alcohol having 1 to 4 carbon atoms.

The blueberry extract, the blueberry fraction extract, or all of them prepared according to the above preferred method is free from impurities, such as sugar, and has a high total polyphenol content.

The blueberry extract and the blueberry fraction extract according to the present invention may contain 1% to 60% by weight of total polyphenols based on the total weight. In the case of blueberry fraction extract, prevention and improvement of dry eye syndrome by having a polyphenol content of preferably 2 to 60% by weight, more preferably 3 to 15% by weight, and most preferably 4 to 12% by weight relative to the total weight And shows a remarkable effect in the treatment.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the blueberry extract, blueberry fraction extract, or both. The pharmaceutically acceptable carrier includes those commonly used in the art, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. In addition, the pharmaceutical composition of the present invention may include fillers, extenders, binders, wetting agents, disintegrants, diluents or excipients such as surfactants, and other pharmaceutically acceptable additives.

The composition of the present invention may be administered orally or parenterally (eg, by application or intravenous, subcutaneous, or intraperitoneal injection), but oral administration is preferred. Solid preparations for oral administration include powders, granules, tablets, capsules, soft capsules, pills, and the like. Liquid preparations for oral use include suspensions, solutions for oral use, emulsions, syrups, aerosols, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. can Formulations for parenteral administration are sterilized aqueous solutions, solutions, non-aqueous solutions, suspensions, emulsions, eye drops, eye ointments, syrups, suppositories, aerosols, etc., and sterilized injections, respectively, according to conventional methods. It can be used, preferably by preparing a pharmaceutical composition of cream, gel, patch, spray, ointment, warning agent, lotion, liniment agent, eye ointment, eye drop, pasta agent or cataplasma agent. , but is not limited thereto. Compositions for topical administration may be in anhydrous or aqueous form depending on the clinical prescription. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 99.9 parts by weight based on the composition.

A preferred dosage of the composition of the present invention varies depending on the degree of absorption of the active ingredient in the body, the age, sex and degree of obesity of the patient, but can be appropriately selected by those skilled in the art. However, for a desirable effect, in the case of oral administration, the composition of the present invention per 1 kg of body weight per day is generally administered to adults at 0.0001 to 100 mg / kg per day, preferably 0.001 to 100 mg / kg. It is good. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way.

The composition for preventing or treating dry eye syndrome of the present invention may contain at least one active ingredient exhibiting the same or similar function in addition to the blueberry extract, the blueberry fraction extract, or both.

The present invention provides a health functional food for preventing or improving dry eye syndrome, comprising a blueberry extract, a blueberry fraction extract, or both of them as an active ingredient.

The health functional food for preventing or improving dry eye syndrome according to the present invention shows excellent effects in preventing or treating dry eye syndrome through suppression of inflammation through inhibition of expression of NF-κb and AP-1, recovery of ocular surface damage, and the like. In addition, it shows excellent action and effect even through oral administration, and the medication compliance is very high.

When the blueberry extract, blueberry fraction extract, or both of them of the present invention are used as a health functional food or health functional beverage additive, the blueberry extract, blueberry fraction extract, or both are added as is or used together with other foods or food ingredients, It can be used appropriately depending on the method. The amount of the blueberry extract, the blueberry fraction extract, or both of them may be appropriately determined depending on the purpose of use (prevention, health or improvement, or therapeutic treatment). In the case of long-term intake for the purpose of health and hygiene or health control, since the blueberry extract and blueberry fraction extract have no problem in terms of safety, long-term intake is possible.

There is no particular limitation on the type of health functional food. Examples of foods to which the blueberry extract and/or the blueberry fraction extract can be added include meat, sausages, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, and various These include soups, sodas, teas, soft drinks, alcoholic beverages, and vitamin complexes. When formulated into a beverage, the liquid component added in addition to the blueberry extract, the blueberry fraction extract, or all of them is not limited thereto, but, like a conventional beverage, various flavoring agents or natural carbohydrates may be included as additional components. Examples of the aforementioned natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.) and polysaccharides (eg, common sugars such as dextrins, cyclodextrins, etc.), and xylitol. , sorbitol, erythritol, etc. are sugar alcohols. In addition, natural sweeteners [eg, taumarin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)] and synthetic sweeteners (eg, saccharin, aspartame, etc.) may be used as the sweetener.

As another embodiment, the health functional food of the present invention is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof , organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain fruit flesh for preparing fruit and vegetable beverages. These components may be used alone or in combination, and the proportion of these additives is generally selected from the range of 0.001 to 50 parts by weight per total weight of the composition.

As another embodiment, the blueberry extract, blueberry fraction extract, or both of them of the present invention may be powder, granule, tablet, capsule, syrup or beverage.

The blueberry extract, blueberry fraction extract, or all of them of the present invention exhibits excellent therapeutic effects in inhibiting corneal inflammation and corneal damage, and thus can be used as a pharmaceutical composition and health functional food useful for preventing or treating dry eye syndrome.

1 shows a graph in which the degree of corneal damage treatment of a blueberry fraction extract against scopolamine induced dessicating stress is scored. (*p<0.05, **p<0.01 vs control)
Figure 2 shows a picture taken with a slit-ramp microscope to confirm the degree of corneal damage treatment of the blueberry fraction extract against scopolamine induced dessicating stress.

Hereinafter, the present invention will be described in more detail through the following examples and experimental examples. However, the following Examples and Experimental Examples are intended to illustrate the present invention, and the scope of the present invention is not limited by these Examples and Experimental Examples.

< Example 1> Blueberry Preparation of extract

600 kg of frozen blueberry raw material (fruit, origin: Heilongjiang, China) was thawed at room temperature. After adding 1,200 L of water and adding 0.6 kg of pectinase (Nanning Longbo (China, No.50126)), the mixture was heated and stirred at 50-55 °C for 2 hours. Then, it was filtered and the filtrate was concentrated to 65 brix to obtain 45 kg of blueberry extract.

< Example 2> Resin column used Blueberry Preparation of Fractional Extracts

10 kg of resin (HP-20 resin) was washed with 10 L of ethanol and placed in a column tube. The resin was washed again with 10 L of water, and 1 kg of the blueberry extract obtained in Example 1 was loaded. After eluting with 10 L of water and eluting with 25 L of 50% ethanol (0.1% Acetic acid solution). 10 L of ethanol was eluted, and the blueberry fraction extract solution corresponding to the 50% ethanol (0.1% acetic acid solution) fraction was concentrated under reduced pressure at 45 to 60 ° C. Then, maltodextrin was added and spray-dried to obtain 180 g of blueberry fraction extract. got it

< Example 3> using resin filtration Blueberry Preparation of Fractional Extracts

After putting 10 kg of resin (HP-20 resin) in a filter, washing with water, and loading 4 kg of blueberry extract obtained in Example 1. Filtered with 35 L of water and discarded. After adding 50 L of 50% ethanol (0.1% acetic acid solution), filtering, and concentrating the filtrate under reduced pressure at 45-60 ° C, maltodextrin was added and spray-dried to obtain 790 g of blueberry fraction extract.

< Example 4> Blueberry Mass production of fractional extracts

600 kg of frozen blueberry raw material (origin: Heilongjiang, China) was thawed at room temperature. After adding 1,200 L of water and 0.6 kg of pectinase, heat and stir at 50 to 55 ° C. for 2 hours. The stirred solution was filtered, and the supernatant was separated by centrifugation at 3,300-3,600 r/min. Then, 600 kg of resin (HP-20 resin) was put into the column tube, and the separated supernatant was added thereto, followed by flowing down at a flow rate of 10 to 12 L/min. Then, 600 L of water was added and flowed down at a flow rate of 10 to 12 L/min. Finally, 1,800 L of 50-55% ethanol (0.1% acetic acid solution) was added and flowed down at a flow rate of 5-6 L/min to obtain the fractions. In addition, 90 to 95% ethanol was added and flowed down at a flow rate of 5 to 6 L/min to obtain a 95% ethanol fraction. After concentrating, maltodextrin was added and spray-dried to obtain 30 kg of blueberry fraction extract.

< Example 5> blueberry extract total polyphenols (Total polyphenol ) concentration assay

In order to analyze the total polyphenol concentration of the blueberry extract and the blueberry fraction extract prepared in <Example 1> to <Example 4>, UV/visible spectrophotometer analysis was performed, and standard substances were analyzed. Analysis was performed according to the Association of Official Analytical Chemists (AOAC) method using tannic acid as a furnace.

For the AOAC analysis, a tannic acid standard solution was prepared as a standard material (solution). For the standard solution, approximately 100 mg of tannic acid was precisely measured and placed in a 1,000 ml volumetric flask, distilled water was added, and after mixing well, the solution was used regularly. Then, the standard solution was diluted to an appropriate concentration to prepare a standard solution of 0.1 to 0.5 mg/ml.

In addition, for the AOAC analysis, about 30 mg of blueberry extract and blueberry fraction extract samples were precisely measured as test solutions and placed in a 10 ml volumetric flask, and then ultrasonically shaken for 20 minutes with distilled water. The solution was used as a test solution by adjusting the mark with distilled water and shaking well.

For analysis, 75 ml of distilled water was put into a volumetric flask, and 1 ml each of the standard solution and the test solution prepared above was added thereto. 5 ml of Folin-denis reagent and 10 ml of 35% sodium carbonate were added to the test tube, mixed, and diluted with distilled water. Then, after standing at room temperature for 30 minutes, absorbance at 760 nm was measured using SPWCTRAmax PLUS (Molecular Devices).

To calculate the content concentration, a calibration curve of the standard material was prepared using the difference between the absorbance of each concentration of the standard substance and the absorbance of each blank test, and the difference between the absorbance of the sample and the blank test absorbance of the sample was applied to the calibration curve to determine the total polyphenol content of the test solution. The concentration of phenol (total polyphenol) was determined.

The results are shown in Table 1.

[Table 1] blueberry extract and of the fractional extract of total polyphenols content

As confirmed in Table 1, it was confirmed that the total polyphenol content was greatly increased in the blueberry extract and the blueberry fraction extract prepared according to the preparation methods of Examples 1 to 4. That is, high total polyphenol content was confirmed in the prepared blueberry extract and blueberry fraction extract. In particular, a high total polyphenol content of 5% or more was confirmed in the prepared blueberry fraction extract.

< Example 6> Blueberry Effects of fractional extracts on scopolamine-induced mouse model (High fat diet-induced obese mouse model)

Using the blueberry fraction extract obtained in <Example 3>, the inhibitory effect on dry eye in a mouse dry eye model induced by scopolamine was evaluated. For the preparation of the scopolamine-induced dry eye mouse model, 6-week-old male C57/BL6 male mice (Daehan Biolink, Korea) were fed standard diet and water at 22-24 °C and 60-80% humidity for 1 week. After domestication, 15 mg/kg of scopolamine was administered three times a day (at 10:00, 14:00, and 16:00), and the humidity in the breeding room was maintained at 34%, and dry eyes were induced for 2 weeks. After measuring the weight of each individual, the top 70% was selected as the experimental animal, and the bottom 30% was eliminated. The selected top 70% of mice were divided into 8 mice in each group based on body weight, and each drug was orally administered at 10:00 am every day for 2 weeks. As a positive control group, cyclosporine 0.05% was instilled twice a day.

Corneal fluorescence staining was performed on the mice after drug administration for 2 weeks. After staining with Fluorscein sodium (F6377, sigma Aldrich) for 1 minute, wsahing with PBS was performed, and pictures were taken through a fluorescence filter of a slit-lamp microscope. The staining score was scored using a 15-point grading system established by the National Eye Institute (NEI).

The results are shown in Figure 1 and Table 2.

[graph 2] Corneal staining score (N=8)

As shown in Figure 1 and Table 2, compared to the negative control group, the blueberry fraction extract-administered group had lower corneal staining scores in a dose-dependent manner. In particular, in the 100 mg/kg administration group, the corneal staining score was 82.64% lower than that of the negative control group, and the treatment efficacy was similar to that of cyclosporin, a drug administered by eye drop.

In addition, as confirmed from the staining results shown in FIG. 2, it was confirmed that corneal recovery was remarkably achieved in a concentration-dependent manner in the blueberry fraction extract-treated group.

That is, it was confirmed that oral administration of the blueberry fraction extract can exhibit efficacy higher than that of eye drops.

< Example 7> Blueberry Inflammation inhibitory mechanism confirmation test of fraction extract

After culturing the A549 cell line in a 6-well plate, pNF-κb-luc or pAP1-luc reporter plasmid was introduced into the cells. For transfection, each plasmid (4 ug) was mixed in liquid phase with lipofector Q (APTABIO) and OptiMem (Invitrogen) and left at room temperature for 20 minutes to form a DNA complex for transfection. It was added to cells in culture and kept at 37 ℃ for 6 hours. It was completed by culturing in In order to activate each NF-κb and AP-1 pathway, PMA (phorbol myristate acetate) was treated at a concentration of 10 ng/ml to the transfected cells for 6 hours. The activity of luciferase induced by PMA treatment was measured in the presence of luciferase substrate using a luminometer (Victor X3. PerkinElmer). To measure the activity of the blueberry fraction extract or component, the cell line transformed by transfection was treated with the blueberry fraction extract or component at a predetermined concentration 1 hour before adding PMA. The expression suppression rate of the reporter gene was calculated as the decrease rate of luciferase activity compared to DMSO treatment, and the experimental results are shown in Table 3.

[Table 3] Blueberry fraction according to administration NF - κb and expression inhibition rate of AP-1 (N=8)

As confirmed in Table 3, it was confirmed that the expression of NF-κb and AP-1 was suppressed in a concentration-dependent manner according to the treatment of the blueberry fraction extract. This shows a significant therapeutic effect of the blueberry fraction extract, which can suppress the action that causes corneal barrier damage along with inflammation of the ocular surface as IL-1b is increased by activation of NF-κb and AP-1.

< manufacturing example 1> Preparation of injection preparation

Blueberry extract or blueberry fraction extract of the present invention 100 mg

Sodium phosphate monobasic 6.0 mg

Benzyl Alcohol 0.002 mL

Sodium Chloride 15 mg

Appropriate amount of sterile distilled water for injection

After mixing the above components to make 2 ml in a conventional manner, fill in an ampoule with a capacity of 2 ml and sterilize to prepare an injection.

< manufacturing example 2> Manufacture of tablets

Blueberry extract or blueberry fraction extract of the present invention 200 mg

Lactose monohydrate 100 mg

Microcrystalline Cellulose 50 mg

Crospovidone 15 mg

magnesium stearate Appropriate amount

Tablets were prepared by mixing the above ingredients and tableting according to a conventional tablet manufacturing method.

< manufacturing example 3> Manufacture of capsules

100 mg of blueberry extract or blueberry fraction extract of the present invention

Lactose 50mg

Substituted Hydroxypropyl Cellulose 20mg

Silicon Dioxide 5mg

Talc 2mg

Appropriate amount of magnesium stearate

Capsules were prepared by mixing the above ingredients and filling them into gelatin capsules according to a conventional capsule preparation method.

< formulation example 4> Preparation of liquid formulation

1000 mg of blueberry extract or blueberry fraction extract of the present invention

20g per bag

Anhydrous Citric Acid 3g

Fructose 20g

Cherry Flavor

Add purified water to make a total of 100 ml

A liquid formulation was prepared by mixing the above components according to a conventional liquid formulation preparation method, filling a 100 ml brown bottle, and sterilizing the mixture.

Claims (22)

Blueberry ( Vaccinium uliginosum L. ) A pharmaceutical composition for preventing or treating dry eye syndrome comprising a blueberry extract, a blueberry fraction extract, or both of them as an active ingredient. The pharmaceutical composition for preventing or treating dry eye syndrome according to claim 1, wherein the blueberry extract and the blueberry fraction extract contain polyphenols in an amount of 1 to 60% by weight based on the total weight of the extract. The pharmaceutical composition for the prevention or treatment of dry eye syndrome according to claim 1, wherein the prevention or treatment of dry eye syndrome is by inhibiting corneal inflammation. [Claim 4] The pharmaceutical composition for preventing or treating dry eye syndrome according to claim 3, wherein the inhibition of inflammation is by inhibiting the expression of AP-1 and NF-κb. The pharmaceutical agent for preventing or treating dry eye syndrome according to claim 1, wherein the blueberry extract is extracted by treating blueberry with an enzyme under water, anhydrous lower alcohol having 1 to 4 carbon atoms, or anhydrous lower alcohol having 1 to 4 carbon atoms and then heating it. academic composition. The method of claim 1, wherein the blueberry fraction extract is obtained by: (a) extracting by treating blueberries with an enzyme in water, anhydrous lower alcohol having 1 to 4 carbon atoms, or anhydrous lower alcohol having 1 to 4 carbon atoms and heating; (B) A pharmaceutical composition for preventing or treating dry eye syndrome, which is a blueberry fraction extract prepared by passing the extract of step (a) through a resin. The pharmaceutical composition for preventing or treating dry eye syndrome according to claim 6, wherein the solvent in step (a) is water, methanol, ethanol, propanol, butanol, isobutanol, or a mixed solvent thereof. The method of claim 6, wherein the enzyme in step (a) is selected from amylase, glugoamylase, protease, oxidase, polymethylgalacturonase, polygalacturonase, glucosidase, cellulase or pectinase. A pharmaceutical composition for preventing or treating dry eye syndrome, which is any one of them. The pharmaceutical composition for preventing or treating dry eye syndrome according to claim 6, wherein the extraction in step (a) is carried out at 40 to 65 °C for 1 hour to 10 hours. The method of claim 6, wherein the resin in step (b) is XRD resin, XDA resin, Amberlite XAD resin, Dowex resin, Superlite, DAX resin, Diaion HP-10, HP-20, HP-30, HP A pharmaceutical composition for preventing or treating dry eye syndrome, which is any one selected from -40 or HP-50 resin. The drug for preventing or treating dry eye syndrome according to claim 6, wherein the step of passing the resin through the resin in step (b) is eluting or filtering with anhydrous lower alcohol having 1 to 4 carbon atoms or hydrated lower alcohol having 1 to 4 carbon atoms. academic composition. Blueberry ( Vaccinium uliginosum L. ) A health functional food for preventing or improving dry eye syndrome, comprising a blueberry extract, a blueberry fraction extract, or both of them as active ingredients. The health functional food for preventing or improving dry eye syndrome according to claim 12, wherein the blueberry extract and the blueberry fraction extract contain polyphenols in an amount of 1 to 60% by weight based on the total weight of the extract. The functional food for preventing or improving dry eye syndrome according to claim 12, wherein the prevention or improvement of dry eye syndrome is caused by inhibition of corneal inflammation. [Claim 15] The functional food for preventing or improving dry eye syndrome according to claim 14, wherein the suppression of inflammation is by inhibiting the expression of AP-1 and NF-κb. 13. The method of claim 12, wherein the blueberry extract is extracted by treating blueberry with an enzyme under water, anhydrous lower alcohol having 1 to 4 carbon atoms, or anhydrous lower alcohol having 1 to 4 carbon atoms, and then heating the extract. functional food. The method of claim 12, wherein the blueberry fraction extract is obtained by: (a) extracting by treating blueberries with an enzyme in water, anhydrous lower alcohol having 1 to 4 carbon atoms, or hydrated lower alcohol having 1 to 4 carbon atoms and heating; (b) A health functional food for preventing or improving dry eye syndrome, which is a blueberry fraction extract prepared by passing the extract of step (a) through a resin. The health functional food for preventing or improving dry eye syndrome according to claim 17, wherein the solvent in step (a) is water, methanol, ethanol, propanol, butanol, isobutanol, or a mixture thereof. The method of claim 17, wherein the enzyme in step (a) is selected from amylase, glugoamylase, protease, oxidase, polymethylgalacturonase, polygalacturonase, glucosidase, cellulase or pectinase Health functional food for preventing or improving dry eye syndrome, which is any one. The health functional food for preventing or improving dry eye syndrome according to claim 17, wherein the extraction in step (a) is carried out at 40 to 65 ° C for 1 hour to 10 hours. The method of claim 17, wherein the resin in step (b) is XRD resin, XDA resin, Amberlite XAD resin, Dowex resin, Superlite, DAX resin, Diaion HP-10, HP-20, HP-30, HP A health functional food for preventing or improving dry eye syndrome, which is any one selected from -40 or HP-50 resin. The method of claim 17, wherein the step of passing through the resin in step (b) is eluting or filtering with anhydrous lower alcohol having 1 to 4 carbon atoms or hydrated lower alcohol having 1 to 4 carbon atoms. functional food.

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