WO2010062034A2 - Liquid eye drop or gel type pharmaceutical composition containing vitis vinifera seed extract - Google Patents

Abstract

The present invention provides a liquid eye drop or gel type pharmaceutical composition which contains vitis vinifera seed extract; and an antioxidant selected from a group consisting of ascorbic acid or a salt thereof, sodium hydrogen sulfite, potassium pyrosulfite, and L-threonine or a salt thereof. The pharmaceutical composition according to the present invention maintains a physical and chemical stability over a long period of time. Therefore, the pharmaceutical composition of the present invention can be stored over a long period of time, and valuably used in the field of drug manufacture.

Description

Pharmaceutical compositions in the form of eye drops or gels containing seed extracts of European grapes

The present invention relates to pharmaceutical compositions in the form of eye drops or gels containing a seed extract of the European species Vitis vinifera .

Vitis is a vine that belongs to Rhamnales and Vitaceae, and grows or grows all over the earth except near the equator and above 50 ° latitude. There are 700 genera of 11 genera, Vitis vinifera , Vitis labrusca , Vitis riparia , Vitis rupestris , Vitis rupestris , and Vitis berladieri , and Vitis. coignetiae ) and Vitis amurensis are mainly grown for fruit use.

Among these, extracts obtained from seeds of the European species Vitis vinifera include (-) epicatechin, proanthocyanidins B1 and B2, (+) catechins, and mixtures of polymerized derivatives thereof, which are procyano Known as stone or flavonol oligomers (GB-A-1541469 and FR-A-2092743).

The extract mainly acts on glycosaminoglycans such as connective tissue, blood vessels, lymph and joints selectively to promote synthesis and inhibit degradation of fibers related to the binding ability of collagen, elastin, fibronectin, etc. It has been reported to inhibit the capillary permeability and quickly restore the elasticity of veins. [Arteres et veines Vol. 5 (5), 397-401 (1986); Sem-dex Hopitaux 48/47, 2009-2013 (1981)]

In addition, grape seed extract has been reported to have a therapeutic effect on capillary vulnerability, hypertension-related retinopathy, and residual retinal edema after retinal detachment in diabetic patients [Gazette Mde France Vol. 88, No. 14, 2035-2038]. (1981), which protects the various structures of the retina, speeds up the regeneration of visual pigment, and has been reported to be effective in restoring the retina after exposure to scintillation in clinical trials [Bull. Soc. Opth. France Vol 88 (2), 173-4, 177-9 (1988).

In addition, oral tablets containing seed extracts of the European species Vitis vinifera are commercially available as adjuvants for disorders related to retina and choroid circulation. However, the direct pharmacological effect can be expected when the seed extract of the European grape varieties ( Vitis vinifera ) is directly administered to the eye. However, topical ophthalmic eye drops or gel-based external preparations have been reported. none. Particularly, since eye drops or gels are faster than tablets in solid form due to the characteristics of the formulation, securing stability, particularly long-term stability for about three years, is a very important factor in formulation design.

The present inventors have conducted various studies to develop an ophthalmic preparation comprising the seed extract of the European grape ( Vitis vinifera ) as an active ingredient, that is, a formulation having excellent physical and chemical stability as an eye drop or gel. As a result, it has been found that, when formulated in the form of a liquid or gel by containing a specific antioxidant, physical and chemical stability can be maintained for a long time, and therefore, it can be usefully applied to the pharmaceutical field since it can be stored for a long time. .

Accordingly, the present invention provides pharmaceutical compositions in the form of eye drops or gels containing seed extracts of Vitis vinifera having excellent stability.

According to one aspect of the invention, seed extract of the European species grape ( Vitis vinifera ); And an antioxidant selected from the group consisting of ascorbic acid or salts thereof, sodium bisulfite, potassium pyrosulfite, and L-threonine or salts thereof, there is provided a pharmaceutical composition in the form of eye drops or gels.

The content of the antioxidant may be 0.01 to 15% by weight based on the total weight of the composition, the content of the seed extract of the European species grape ( Vitis vinifera ) as an active ingredient may be 0.1 to 30% by weight relative to the total weight of the composition. . In addition, the seed extract of the grape varieties ( Vitis vinifera ) is a procyanidolic value (PCV) of 80 to 130; Content of (+) catechin and (−) epicatechin of up to 30%; And a proanthocididine content of 95 to 105%.

The seed extract of the grape varieties Vitis vinifera is (a) extract the seeds of the pulverized grape varieties ( Vitis vinifera ) at room temperature with a mixed solvent of acetone and water having a volume ratio of acetone and water of 1: 1 to 2, and filtered step; (b) distilling the filtrate obtained in step (a) to remove acetone, then saturating sodium chloride and filtering; (c) extracting the filtrate obtained in step (b) with ethyl acetate and concentrating; And (d) adding chloroform to the concentrate obtained in step (c) and filtering to obtain a precipitate.

Also, optionally, the seed extract of the grape varieties Vitis vinifera may be (1) (i) a mixed solvent of acetone and water having a volume ratio of acetone and water of 3-5 to 1: 1 in the ground seed of the grape varieties Vitis vinifera . Extracting with filtration and filtering; (ii) concentrating the filtrate obtained in step (i) to remove acetone and filtering; (iii) extracting the filtrate obtained in step (ii) with ethyl acetate; (iv) drying the extract obtained in step (iii) to obtain a first extract; (2) (p) extracting the seeds of crushed Vitis vinifera with water and filtering; (q) extracting the filtrate obtained in step (p) with ethanol and filtering; (r) drying the filtrate obtained in step (q) to obtain a second extract; And (3) it can be obtained by a manufacturing method comprising the step of mixing the first extract and the second extract.

The pharmaceutical composition in the form of eye drops or gels according to the present invention comprises a seed extract of grape varieties Vitis vinifera as an active ingredient, ascorbic acid or salts thereof, sodium hydrogen sulfite, potassium pyrosulfite, and L-threonine or By including an antioxidant selected from the group consisting of salts thereof, physical and chemical stability can be maintained for a long time. Therefore, the pharmaceutical composition in the form of eye drops or gels according to the present invention can be usefully applied to the pharmaceutical field because it can be stored for a long time.

The present invention is a seed extract of European species grape ( Vitis vinifera ); And an antioxidant selected from the group consisting of ascorbic acid or a salt thereof, sodium bisulfite, potassium pyrosulfite, and L-threonine or a salt thereof.

The pharmaceutical compositions of the present invention enhance the physical and chemical stability of eye drops or gels obtained by the inclusion of certain antioxidants, namely ascorbic acid or salts thereof, sodium bisulfite, potassium pyrosulfite, and / or L-threonine or salts thereof. Let's do it. The amount of the antioxidant is different depending on the type of antioxidant used, but may be 0.01 to 15% by weight, preferably 0.0125 to 1% by weight based on the total weight of the composition. Specifically, about 0.1% by weight of ascorbic acid or a salt thereof, about 0.3% by weight of sodium hydrogen sulfite, 0.0125 to 0.025% by weight of potassium pyrosulfite, 0.3% by weight of L-threonine or a salt thereof It may contain.

In the pharmaceutical composition of the present invention, the content of the seed extract of the grape varieties Vitis vinifera contained as an active ingredient may be in a range sufficient to provide a therapeutically effective amount. For example, the content of the seed extract of the grape varieties Vitis vinifera may range from 0.1 to 30% by weight, preferably from 0.5 to 5.0% by weight and more preferably from about 1.0% by weight relative to the total weight of the composition. .

Seed extracts of the European grape varieties ( Vitis vinifera ) include a procyanidolic value (PCV) of 80-130; Content of (+) catechin and (−) epicatechin of up to 30%; And those having a proanthocididine content of 95 to 105% are particularly preferably used.

As used herein, the term "procyanidolic value" (hereinafter referred to as "PCV") means a value calculated by the following method and formula.

① Preparation of Standard Solution

100 mg of the standard extract of the seed extract of the European grape ( Vitis vinifera ) is precisely weighed, and isopropanol is added to make 50 ml. Take 10 ml of this solution, add 10 ml of 3M hydrochloric acid, add isopropanol to make 50 ml.

② Preparation of Test Solution

100 mg of the sample is precisely weighed and operated in the same manner as the standard solution to prepare a sample solution.

③ operation

10 ml of the sample solution and 10 ml of standard solution are placed in 5 test tubes, and capped, and then heated in a 100 ° C. water bath for 45 minutes. After heating, the test tubes were cooled in cold water, 2 ml of each test tube was taken, and 20 ml of isopropanol was added thereto. Take the reacted test solution and the standard solution, and measure the absorbance at 550 nm using isopropanol as a reference solution.

④ Formula

PCV = 105 X [A (t) X Mt X (100-Et)] / [A (s) X M X (100-E)]

 A (t): average absorbance of reacted sample solution

 A (s): average absorbance of the reacted standard solution

 Mt: Standard dose (mg)

 M: amount of sample taken (mg)

 Et: Moisture Content (%) of the Standard

 E: Moisture content of the sample (%)

The content of the (+) catechin and (-) epicatechin means a value quantified as follows.

① Preparation of Standard Solution

50 mg of each of the (+) catechin standard and (-) epicatechin standard is precisely weighed and dissolved in acetonitrile and diluted phosphate mixture (5:95) to make 100 ml.

② Preparation of Test Solution

50 mg of the sample is precisely weighed, dissolved in an acetonitrile and dilute phosphoric acid mixed solution (5:95), and 10 ml is used as the sample solution.

③ Analysis condition

Column: column filled with octadecylsilylated silica gel (0.46 X 25 cm, 5 um)

Mobile phase (Table 1)

Table 1 Minutes Mobile phase A Mobile phase B 0 95 5 50 85 15 60 20 80 70 95 5

Mobile phase A: Dilute phosphoric acid (dilute phosphoric acid: 0.3%, V / V aqueous solution)

 Mobile Phase B: Acetonitrile

Flow rate: 0.7 ml / min

Detector: ultraviolet absorbance measurement (wavelength: 278 nm)

Injection volume: 10 ul

④ Formula

% Of catechin as epicatechin

 = [A1 X Me X 100 X 100] / [Ae X M1 X (100-E) X 10]

Epicatechin content (%)

 = [A2 X Me X 100 X 100] / [Ae X M1 X (100-E) X 10]

 A1: peak area of catechin in the sample solution

 A2: peak area of epicatechin in the sample solution

 Ae: Peak area of epicatechin in standard solution

 Me: amount of epicatechin in standard solution (mg)

 M1: amount of extract in sample (mg)

 E: Water content in extract (%)

In addition, the "proanthocididine content" means a value calculated by the following method and formula.

① Preparation of internal standard solution

Accurately weigh 30.0 mg of 2,6-di-tert-butyl-4-methylphenol (BHT) into a 100 ml volumetric flask and mark it with a mobile phase. It is done.

② Preparation of Test Solution 1

Accurately weigh 10 mg of the sample into a 10 ml volumetric flask and dissolve it in a 5 ml internal standard solution.

③ Preparation of Test Solution 2

Accurately weigh 10 mg of the sample into a 10 ml volumetric flask and dissolve it in a 5 ml internal standard solution.

④ Preparation of calibration curve of standard solution

-Standard solution 1: 8 mg of proanthocyanidin standard is precisely weighed into a 10 ml volumetric flask and dissolved in 5 ml of internal standard solution.

-Standard solution 2: Accurately weigh 10 mg of proanthocyanidin standard into a 10 ml volumetric flask and dissolve in 5 ml of internal standard solution.

-Standard solution 3: 12 mg of proanthocyanidin standard is precisely weighed into a 10 ml volumetric flask, and dissolved in 5 ml of internal standard solution.

⑤ Operation conditions

Column: PL Gel Column (7.6 X 300 mm, 5 um) or similar column

Detector: ultraviolet absorbance photometer (wavelength: 280 nm)

Mobile phase: mixed solvent of tetrahydrofuran and aqueous lithium bromide solution (95: 5)

 Lithium bromide aqueous solution: 1.04 g of lithium bromide is precisely taken and placed in a 1000 ml volumetric flask.

Flow rate: 1.0 ml / min

Injection volume: 10 uL

Measurement time: 15 minutes

⑥ How to operate

Standard solutions 1, 2, 3 and sample solutions 1, 2 are analyzed twice with the following liquid chromatography method. However, the calibration curve of the standard solution is prepared using the concentration of the standard solution and the corresponding main peak to IS peak area ratio (A _{proanthocyanidin} / A _BHT ) and calculated according to the following formula.

⑦ Formula

Ti% = {[(A _{proanthocyanidin} / A _BHT ) _test -a] / b} x (1 / C _test ) x 100

A _{proanthocyanidin} = peak area of _{proanthocyanidins in the} sample solution

A _BHT = peak area of BHT in the sample solution

 a = Y intercept of the standard calibration curve

 b = slope of the standard calibration curve

C _test = concentration of the sample solution (mg / ml)

Proanthocyanidin content (%) = Ti% x [(100-KF _std ) / (100-KF _test )]

KF _std = standard product moisture compensation

KF _test = sample moisture compensation

In addition, the seed extract of the grape varieties Vitis vinifera may be preferably used by the improved manufacturing method developed by the present inventors (ie, WO2009 / 031826).

That is, the seed extract of the grape varieties ( Vitis vinifera ) is (a) the seeds of the pulverized grape varieties ( Vitis vinifera ) are extracted at room temperature with a mixed solvent of acetone and water having a volume ratio of acetone and water of 1: 1 to 2, Filtration; (b) distilling the filtrate obtained in step (a) to remove acetone, then saturating sodium chloride and filtering; (c) extracting the filtrate obtained in step (b) with ethyl acetate and concentrating; And (d) adding chloroform to the concentrate obtained in step (c) and filtering to obtain a precipitate.

Seeds of crushed European grapes ( Vitis vinifera ) are washed with skin, seeds and branches obtained by pressing European grapes ( Vitis vinifera ) in water, dried using an oven, etc., and then the seeds are separated, and the conventional method is used. It can be obtained by grinding.

The manufacturing method is compared to the conventional extraction method (for example, GB-A-1541469) A water-acetone mixed solvent having a low acetone content is used as the primary extraction solvent, and the primary extraction process is performed at room temperature (about 25 ° C.) without additional heating. As a water-acetone mixed solvent used in the preparation method, a mixed solvent having a volume ratio of acetone and water of 1: 1 to 2, preferably about 1: 1.5 may be used. The first extraction may be performed once or repeatedly, more preferably 2 to 3 times. The filtration of step (a) may be carried out in a conventional manner, and the filtrate is recovered for carrying out the next step.

In the step of distilling the filtrate obtained in step (a) to remove acetone, and then saturating sodium chloride and filtering [step (b)], the acetone having a relatively low boiling point is removed by the distillation and is dissolved in acetone. The impurities are precipitated. The distillation may be performed according to a conventional distillation method, for example, may be carried out by distillation under reduced pressure. Preferably it is carried out under reduced pressure conditions of about 50 ℃ or less. The extract obtained through the distillation process is subjected to saturation of sodium chloride and filtration immediately, without a separate organic solvent extraction process. When the extract (that is, the extract obtained by distilling acetone) is saturated with sodium chloride, impurities such as a tannin component are precipitated, which is removed through a filtration process. The impurity precipitation by saturation of sodium chloride and filtration is preferably filtered after saturating sodium chloride and then leaving it for 2 to 3 hours. The filtration can be carried out in a conventional manner, and the filtrate is recovered for carrying out the next step.

In the step of extracting the filtrate obtained in step (b) with ethyl acetate and concentrating [step (c)], the extraction with ethyl acetate (secondary extraction) may be performed once or repeatedly, preferably Preference is given to repeating 2-3 times. In addition, the concentration is preferably carried out to be concentrated to 0.4 to 0.7 times the total volume of the extract (ie filtrate) obtained in step (b).

In the step [c] of adding the chloroform to the concentrate obtained in step (c), and filtering to obtain a precipitate, when the chloroform is added, active ingredients including oligomers which are not dissolved in chloroform are precipitated and formed. The precipitates can be isolated by simply filtering the seed extract of Vitis vinifera . The precipitate obtained by filtration can be obtained in the form of a dry powder by drying according to a conventional method, and the drying can be carried out by drying under reduced pressure, for example, a reduced pressure of 50 ℃ or less.

Optionally, the seed extract of the grape varieties Vitis vinifera contained as an active ingredient in the pharmaceutical composition of the present invention (1) (i) the seed of the ground grape varieties ( Vitis vinifera ) is a volume ratio of acetone and water of 3 ~ 5: extraction with a mixed solvent of acetone and water of 1 and filtration; (ii) concentrating the filtrate obtained in step (i) to remove acetone and filtering; (iii) extracting the filtrate obtained in step (ii) with ethyl acetate; (iv) drying the extract obtained in step (iii) to obtain a first extract; (2) (p) extracting the seeds of crushed Vitis vinifera with water and filtering; (q) extracting the filtrate obtained in step (p) with ethanol and filtering; (r) drying the filtrate obtained in step (q) to obtain a second extract; And (3) it can be obtained by the method of producing a seed extract of the European species grape ( Vitis vinifera ) comprising the step of mixing the first extract and the second extract.

The preparation method is performed by separately preparing the first extract and the second extract, and mixing them to obtain an extract. Therefore, the manufacturing method not only reduces the amount of acetone used as a whole, but also lacks a sodium chloride saturation process and a chloroform extraction process, thereby simplifying extraction and minimizing the problem of environmental pollution due to the use of an organic solvent. In addition, the yield of the resulting extract can be greatly increased by about 10 times.

In the preparation of the first extract, step (i) extracts the seeds of pulverized European grapes ( Vitis vinifera ) with a mixed solvent of acetone and water having a volume ratio of acetone and water of 3-5 to 1, more preferably 4: 1, , By filtration. The first extraction may be performed once or repeatedly, more preferably 2 to 3 times. The filtration of step (i) can be carried out in a conventional manner, and the filtrate is recovered for the performance of the next step.

In preparing the first extract, step (ii) is carried out by concentrating the filtrate obtained in step (i) to remove acetone and filtering. The acetone having a relatively low boiling point is removed by the concentration, and impurities dissolved in acetone are precipitated. The concentration may be carried out by conventional vacuum concentration (or distillation under reduced pressure), for example, may be carried out by distillation under reduced pressure conditions. After concentration, the precipitate is removed by filtration and the filtrate is recovered.

In preparing the first extract, step (iii) is carried out by extracting the filtrate obtained in step (ii) with ethyl acetate. Extraction using the ethyl acetate (secondary extraction) may be performed once or repeatedly, preferably 2 to 3 times. In addition, step (iii) may further include a dehydration process using anhydrous sodium sulfate after performing the extraction process using the ethyl acetate.

In preparing the first extract, step (iv) is carried out by drying the extract obtained in step (iii). The drying may be carried out by conventional methods, for example drying under reduced pressure of up to 50 ° C. More preferably, the extract obtained in step (iii) may be concentrated to remove ethyl acetate, and the resulting concentrate is dissolved in water and then spray dried.

In the preparation of the second extract, step (p) can be carried out by extracting the seeds of pulverized European grape ( Vitis vinifera ) with water and filtering; Step (q) can be carried out by extracting the filtrate obtained in step (p) with ethanol and filtering. The extraction of step (q) may be performed once or repeatedly, preferably two to three times.

In addition, in the preparation of the second extract, step (r) may be carried out by drying the filtrate obtained in step (q). The drying of step (r) is carried out by spray drying the filtrate obtained in step (q); It is preferably carried out by concentrating the filtrate obtained in step (q) and then spray drying the obtained concentrate. The concentration may be concentrated to 0.4 to 0.7 times the total volume of the filtrate obtained in step (q), but is not limited thereto.

Mixing of the first extract and the second extract obtained as described above may be carried out by simply mixing the extract, the mixing ratio of the first extract and the second extract may be a weight ratio of 1: 0.5 to 1.5.

The pharmaceutical composition of the present invention may contain additives commonly used in the pharmaceutical field for the preparation of eye drops or gels, such as stabilizers, thickeners, buffers, isotonic agents, preservatives, pH adjusting agents and the like.

The stabilizer comprises D-sorbitol, glycerin, sodium edetate and the like; The thickener includes sodium carboxymethyl cellulose; The buffer includes phosphoric acid or a salt thereof, boric acid, borax, citric acid or a salt thereof, and the like; The tonicity agent includes sodium chloride and the like; The preservative includes benzalkonium chloride, methyl paraoxybenzoate, propyl paraoxybenzoate, chlorobutanol and the like; The pH adjusting agent (pH 5-8) includes hydrochloric acid, sodium hydroxide and the like.

The amount of the additive may be in the range conventionally used in the pharmaceutical field, for example, 0.01-15 wt% of the buffer and 0.01-50 wt% of the stabilizer (specifically, 0.01 g of the concentrated glycerin) based on the total weight of the composition. ~ 15.0%, D-sorbitol solution (70%) is 0.01 to 50% by weight), the thickener may be in the range of 0.01 to 30.0% by weight, the content of the additive may be appropriately selected according to the purpose of use.

Hereinafter, the present invention will be described in more detail through Examples and Test Examples. However, these Examples and Test Examples are for illustrating the present invention, and the scope of the present invention is not limited to these Examples and Test Examples.

Extract Preparation Example 1. European Grape ( Vitis vinifera Preparation of Seed Extract

European grapes ( Vitis vinifera ) were pressed, the skins, seeds and branches were collected, washed in water and dried in a rotary oven, and the seeds were separated. 1 kg of the obtained seed was pulverized, and then 500 ml of an acetone aqueous solution consisting of 300 ml of purified water and 200 ml of acetone were added thereto, followed by extraction at room temperature. The extraction process was repeated three times, and all of the obtained extracts were combined and filtered. The filtrate obtained was distilled under reduced pressure of 50 ° C. or lower to remove acetone, and then saturated with sodium chloride, and then allowed to stand at room temperature for about 3 hours and then filtered. The filtrate obtained was extracted three times with 250 ml of ethyl acetate and then dehydrated with anhydrous sodium sulfate. The resulting extract was concentrated under reduced pressure until the volume was reduced to about 125 ml. About 600 ml of chloroform was added to the obtained concentrate, and a precipitate was produced and filtered. The precipitate obtained by filtration was dried in a vacuum oven at 50 ° C. or lower to obtain about 3.5 g of the seed extract of Vitis vinifera as a brown powder. The obtained extract was hydrolyzed by heating in a dilute acid solution, and then the procyanidolic value (PCV) was measured by quantifying the content of procyanidolic oligomer, and showed a high value of about 105. In addition, the proanthocyanidin content was measured as described above and found to be 103%. Therefore, the extract contains a large amount of oligomers in which two or more monomers of (+) catechin and (-) epicatechin are polymerized.

Extract Preparation Example 2 European Grape ( Vitis vinifera Preparation of Seed Extract

European grapes ( Vitis vinifera ) were pressed, the skins, seeds and branches were collected, washed in water and dried in a rotary oven, and the seeds were separated. 1 kg of the obtained seed was pulverized, and then 500 ml of acetone aqueous solution (acetone / water = 8/2, v / v) was added and extracted. The extraction process was repeated three times, and all of the obtained extracts were combined and filtered. The filtrate was concentrated under reduced pressure to remove acetone and then filtered. The filtrate obtained was extracted three times with 250 ml of ethyl acetate and then dehydrated with anhydrous sodium sulfate. The obtained extract was concentrated under reduced pressure to remove ethyl acetate, and the obtained concentrate was dissolved in 500 ml of water, followed by spray drying to obtain about 20 g of a powdery extract (first extract).

European grapes ( Vitis vinifera ) were pressed, the skins, seeds and branches were collected, washed in water and dried in a rotary oven, and the seeds were separated. 1 kg of the obtained seed was pulverized, and 500 ml of purified water was added thereto, followed by extraction. The extraction process was repeated three times, and all of the obtained extracts were combined and filtered. The filtrate obtained was extracted three times with 250 ml of ethanol and then filtered. The resulting filtrate was concentrated under reduced pressure, and then the obtained concentrate was spray dried to obtain about 15 g of a powdery extract (second extract).

The first extract and the second extract were mixed to obtain about 35 g of the seed extract of the grape varieties Vitis vinifera . The obtained extract was hydrolyzed by heating in a dilute acid solution, and then the procyanidolic value (PCV) was measured by quantifying the content of procyanidolic oligomer, showing a high value of about 98. In addition, the proanthocyanidin content was measured as described above and found to be 98.5%. Therefore, the extract contains a large amount of oligomers in which two or more monomers of (+) catechin and (-) epicatechin are polymerized.

Example 1 Eye Drop Solution

In 800 ml of sterile purified water, 10 g of seed extract of the European type grape ( Vitis vinifera ), 8 g of boric acid, 9 g of borax, 2 g of concentrated glycerin, 15 g of D-sorbitol solution (70%), 3 g of sodium bisulfite, edetic acid 0.5 g of sodium was added thereto to dissolve, and then, sterile purified water was added thereto, and the final volume was adjusted to 1,000 ml to prepare an eye drop solution. (pH 6.0)

Example 2 Eye Drop Solution

In 800 ml of sterile purified water, 10 g of seed extract of the European type grape ( Vitis vinifera ), 8 g of boric acid, 8 g of borax, 2 g of concentrated glycerin, 15 g of D-sorbitol solution (70%), 0.125 g of potassium pyrosulfite, edetic acid 0.5 g of sodium was added thereto to dissolve, and then, sterile purified water was added thereto, and the final volume was adjusted to 1,000 ml to prepare an eye drop solution. (pH 6.0)

Example 3 Eye Drops

In 800 ml of sterile purified water, 10 g of seed extract of Vitis vinifera , boric acid 8 g, borax 9 g, concentrated glycerin 2 g, D-sorbitol solution (70%) 15 g, ascorbic acid 1 g, sodium edate 0.5 g was added to dissolve, and then sterile purified water was added to the final volume to prepare a solution for eye drops. (pH 6.0)

Example 4 Eye Drops

In 800 ml of sterile purified water, 10 g of seed extract of Vitis vinifera , boric acid 8 g, borax 9 g, concentrated glycerin 2 g, D-sorbitol solution (70%) 15 g, L-threonine 3 g, edetic acid 0.5 g of sodium was added thereto to dissolve, and then, sterile purified water was added thereto, and the final volume was adjusted to 1,000 ml to prepare an eye drop solution. (pH 6.0)

Comparative Example 1. Eye Drop

In 800 ml of sterile purified water, 10 g of seed extract of Vitis vinifera , 6.8 g of potassium dihydrogen phosphate, and 5 g of sodium chloride were added to dissolve, and adjusted to pH 6.0 by addition of 1N sodium hydroxide solution. To the final volume was adjusted to 1,000 ml to prepare an eye drop solution. (pH 6.0)

Comparative Example 2. Eye Drop

To 800 ml of sterile purified water, 10 g of seed extract of the European grape ( Vitis vinifera ), 1 g of citric acid, 19 g of sodium citrate, 0.5 g of sodium edetate, and 5 g of sodium chloride were added to dissolve, followed by addition of 1N sodium hydroxide solution. An eye drop solution was prepared by adjusting the pH to 6.0 and adding sterile purified water to a final volume of 1,000 ml. (pH 6.0)

Comparative Example 3. Eye Drop Solution

In 800 ml of sterile purified water, 10 g of seed extract of the European grape ( Vitis vinifera ), 8 g of boric acid, 8 g of borax, 2 g of concentrated glycerin, 15 g of D-sorbitol (70%), and 0.5 g of sodium edate were dissolved. Next, sterile purified water was added to adjust the final volume to 1,000 ml to prepare an eye drop solution. (pH 6.0)

Example 5 Eye Drop Gel

In 800 mL of sterile purified water, 10 g of seed extract of the European grape ( Vitis vinifera ), 16.5 g of sodium carboxymethylcellulose, 50 g of D-sorbitol solution (70%), 3 g of sodium bisulfite, and 1 g of sodium edite Dissolved. 0.1N-sodium hydroxide solution was added to this solution to adjust the pH to 6.0, and then sterile purified water was added to the final weight to 1,000 g to prepare an eye drop gel. (pH 6.0)

Example 6 Eye Drop Gel

In 800 mL of sterile purified water, 10 g of seed extract of European grape ( Vitis vinifera ), 16.5 g of sodium carboxymethylcellulose, 50 g of D-sorbitol solution (70%), 0.25 g of potassium pyrosulfite, and 1 g of sodium edite Dissolved. 0.1N-sodium hydroxide solution was added to this solution to adjust the pH to 6.0, and then sterile purified water was added to the final weight to 1,000 g to prepare an eye drop gel. (pH 6.0)

Example 7 Eye Drop Gel

Homogeneously dissolve 10 g of seed extract of Vitis vinifera , 16.5 g of sodium carboxymethylcellulose, 50 g of D-sorbitol solution (70%), 1 g of ascorbic acid and 1 g of sodium edate in 800 mL of sterile purified water. I was. 0.1N-sodium hydroxide solution was added to this solution to adjust the pH to 6.0, and then sterile purified water was added to the final weight to 1,000 g to prepare an eye drop gel. (pH 6.0)

Example 8 Eye Drop Gel

In 800 mL of sterile purified water, 10 g of the seed extract of Vitis vinifera , 16.5 g of sodium carboxymethylcellulose, 50 g of D-sorbitol solution (70%), 3 g of L-threonine, and 1 g of sodium edate Dissolved. 0.1N-sodium hydroxide solution was added to this solution to adjust the pH to 6.0, and then sterile purified water was added to the final weight to 1,000 g to prepare an eye drop gel. (pH 6.0)

Comparative Example 4. Eye Drop Gel

In 800 mL of sterile purified water, 10 g of the seed extract of European grape ( Vitis vinifera ), 16.5 g of sodium carboxymethylcellulose, 50 g of D-sorbitol solution (70%), and 1 g of sodium edate were dissolved homogeneously. 0.1N-sodium hydroxide solution was added to this solution to adjust the pH to 6.0, and then sterile purified water was added to the final weight to 1,000 g to prepare an eye drop gel. (pH 6.0)

Test Example 1 Physical and Chemical Stability Test

The eye drops and gel preparations prepared in Examples 1 to 8 and Comparative Examples 1 to 4 were filled in a container, and 12 months under room temperature conditions (25 ± 2 ° C./60±5% relative humidity) and accelerated conditions (40 ± 2 ° C.) Physical and chemical stability were tested for 6 months at 75 ± 5% relative humidity). (Container Material: Eye Drop-PET, Ophthalmic Gel-Aluminum)

(1) physical stability

The change in appearance (color and sediment formation) and pH change were measured every 3 months. Color change and sediment formation were observed visually. The test results for the change in properties are shown in Table 2 and Table 3, and the test results for the change in pH are shown in Table 4.

TABLE 2 Change of color Storage condition Early 3 months 6 months 9 months 12 months Example 1 25 ± 2 ℃ / Relative Humidity 60 ± 5% Light yellow Light yellow Light yellow Light yellow Light yellow 40 ± 2 ℃ / relative humidity 75 ± 5% Light yellow Light yellow Light yellow - - Example 2 25 ± 2 ℃ / Relative Humidity 60 ± 5% Light brown Light brown Light brown Light brown Light brown 40 ± 2 ℃ / relative humidity 75 ± 5% Light brown Light brown Brown - - Example 3 25 ± 2 ℃ / Relative Humidity 60 ± 5% Light brown Light brown Light brown Light brown Light brown 40 ± 2 ℃ / relative humidity 75 ± 5% Light brown Light brown Light brown - - Example 4 25 ± 2 ℃ / Relative Humidity 60 ± 5% Light brown Light brown Light brown Light brown Light brown 40 ± 2 ℃ / relative humidity 75 ± 5% Light brown Light brown Brown - - Comparative Example 1 25 ± 2 ℃ / Relative Humidity 60 ± 5% Brown Dark brown Earthy brown Earthy brown Earthy brown 40 ± 2 ℃ / relative humidity 75 ± 5% Brown Earthy brown Earthy brown - - Comparative Example 2 25 ± 2 ℃ / Relative Humidity 60 ± 5% Light brown Brown Earthy brown Earthy brown Earthy brown 40 ± 2 ℃ / relative humidity 75 ± 5% Light brown Earthy brown Earthy brown - - Comparative Example 3 25 ± 2 ℃ / Relative Humidity 60 ± 5% Brown Dark brown Earthy brown Earthy brown Earthy brown 40 ± 2 ℃ / relative humidity 75 ± 5% Brown Earthy brown Earthy brown - - Example 5 25 ± 2 ℃ / Relative Humidity 60 ± 5% Light yellow Light yellow Light yellow Light yellow Light yellow 40 ± 2 ℃ / relative humidity 75 ± 5% Light yellow Light yellow Light yellow - - Example 6 25 ± 2 ℃ / Relative Humidity 60 ± 5% Light brown Light brown Light brown Light brown Light brown 40 ± 2 ℃ / relative humidity 75 ± 5% Light brown Light brown Brown - - Example 7 25 ± 2 ℃ / Relative Humidity 60 ± 5% Light brown Light brown Light brown Light brown Light brown 40 ± 2 ℃ / relative humidity 75 ± 5% Light brown Light brown Light brown - - Example 8 25 ± 2 ℃ / Relative Humidity 60 ± 5% Light brown Light brown Light brown Light brown Light brown 40 ± 2 ℃ / relative humidity 75 ± 5% Light brown Light brown Brown - - Comparative Example 4 25 ± 2 ℃ / Relative Humidity 60 ± 5% Brown Dark brown Earthy brown Earthy brown Earthy brown 40 ± 2 ℃ / relative humidity 75 ± 5% Brown Earthy brown Earthy brown - -

TABLE 3 Sediment Formation Storage condition Early 3 months 6 months 9 months 12 months Example 1 25 ± 2 ℃ / Relative Humidity 60 ± 5% ND ND ND ND ND 40 ± 2 ℃ / relative humidity 75 ± 5% ND ND ND - - Example 2 25 ± 2 ℃ / Relative Humidity 60 ± 5% ND ND ND ND ND 40 ± 2 ℃ / relative humidity 75 ± 5% ND ND ND - - Example 3 25 ± 2 ℃ / Relative Humidity 60 ± 5% ND ND ND ND ND 40 ± 2 ℃ / relative humidity 75 ± 5% ND ND ND - - Example 4 25 ± 2 ℃ / Relative Humidity 60 ± 5% ND ND ND ND ND 40 ± 2 ℃ / relative humidity 75 ± 5% ND ND ND - - Comparative Example 1 25 ± 2 ℃ / Relative Humidity 60 ± 5% ND ND + ++ +++ 40 ± 2 ℃ / relative humidity 75 ± 5% ND ++ ++++ - - Comparative Example 2 25 ± 2 ℃ / Relative Humidity 60 ± 5% ND + ++ +++ ++++ 40 ± 2 ℃ / relative humidity 75 ± 5% ND +++ ++++ - - Comparative Example 3 25 ± 2 ℃ / Relative Humidity 60 ± 5% ND ND ND ND ND 40 ± 2 ℃ / relative humidity 75 ± 5% ND ND ND - - Example 5 25 ± 2 ℃ / Relative Humidity 60 ± 5% ND ND ND ND ND 40 ± 2 ℃ / relative humidity 75 ± 5% ND ND ND - - Example 6 25 ± 2 ℃ / Relative Humidity 60 ± 5% ND ND ND ND ND 40 ± 2 ℃ / relative humidity 75 ± 5% ND ND ND - - Example 7 25 ± 2 ℃ / Relative Humidity 60 ± 5% ND ND ND ND ND 40 ± 2 ℃ / relative humidity 75 ± 5% ND ND ND - - Example 8 25 ± 2 ℃ / Relative Humidity 60 ± 5% ND ND ND ND ND 40 ± 2 ℃ / relative humidity 75 ± 5% ND ND ND - - Comparative Example 4 25 ± 2 ℃ / Relative Humidity 60 ± 5% ND ND ND ND ND 40 ± 2 ℃ / relative humidity 75 ± 5% ND ND ND - -

※ N.D. : Not detected

Table 4 pH change Storage condition Early 3 months 6 months 9 months 12 months Example 1 25 ± 2 ℃ / Relative Humidity 60 ± 5% 6.0 6.0 6.0 6.0 6.0 40 ± 2 ℃ / relative humidity 75 ± 5% 6.0 6.0 5.9 - - Example 2 25 ± 2 ℃ / Relative Humidity 60 ± 5% 6.0 6.0 6.0 6.0 6.0 40 ± 2 ℃ / relative humidity 75 ± 5% 6.0 6.0 5.8 - - Example 3 25 ± 2 ℃ / Relative Humidity 60 ± 5% 6.0 6.0 6.0 6.0 6.0 40 ± 2 ℃ / relative humidity 75 ± 5% 6.0 6.0 5.9 - - Example 4 25 ± 2 ℃ / Relative Humidity 60 ± 5% 6.0 6.0 6.0 6.0 6.0 40 ± 2 ℃ / relative humidity 75 ± 5% 6.0 5.9 5.8 - - Comparative Example 1 25 ± 2 ℃ / Relative Humidity 60 ± 5% 7.0 6.9 6.7 6.5 6.2 40 ± 2 ℃ / relative humidity 75 ± 5% 7.0 6.7 6.1 - - Comparative Example 2 25 ± 2 ℃ / Relative Humidity 60 ± 5% 6.0 5.9 5.8 5.2 5.0 40 ± 2 ℃ / relative humidity 75 ± 5% 6.0 5.7 4.9 - - Comparative Example 3 25 ± 2 ℃ / Relative Humidity 60 ± 5% 6.0 6.0 6.0 5.9 5.9 40 ± 2 ℃ / relative humidity 75 ± 5% 6.0 5.9 5.7 - - Example 5 25 ± 2 ℃ / Relative Humidity 60 ± 5% 6.0 6.0 6.0 6.0 6.0 40 ± 2 ℃ / relative humidity 75 ± 5% 6.0 6.0 5.9 - - Example 6 25 ± 2 ℃ / Relative Humidity 60 ± 5% 6.0 6.0 6.0 6.0 6.0 40 ± 2 ℃ / relative humidity 75 ± 5% 6.0 6.0 5.8 - - Example 7 25 ± 2 ℃ / Relative Humidity 60 ± 5% 6.0 6.0 6.0 6.0 6.0 40 ± 2 ℃ / relative humidity 75 ± 5% 6.0 6.0 5.9 - - Example 8 25 ± 2 ℃ / Relative Humidity 60 ± 5% 6.0 6.0 6.0 6.0 6.0 40 ± 2 ℃ / relative humidity 75 ± 5% 6.0 6.0 5.8 - - Comparative Example 4 25 ± 2 ℃ / Relative Humidity 60 ± 5% 6.0 6.0 6.0 5.8 5.6 40 ± 2 ℃ / relative humidity 75 ± 5% 6.0 5.9 5.5 - -

As can be seen in Table 2 to Table 4, the composition containing an antioxidant such as ascorbic acid, sodium hydrogen sulfite according to the present invention does not have a significant color change at room temperature and accelerated conditions as well as no precipitate was produced. And there is no pH change. Therefore, it can be seen that the eye drop solution and the gel composition of the present invention have excellent physical stability.

(2) chemical stability

The content of procyanidolic oligomer, which is an indicator, was measured in units of 12 months (6 months). The quantification was performed by calculating the amount (mg) of potential cyanidol chloride in 1 mL of eye drop solution (eye drop gel = 1 g), and the amount (mg) of potential cyanidol chloride in 1 ml (or 1 g) of sample was calculated as follows. The results are shown in Table 5.

※ PCV = 105 X [A (t) X Mt X (100-Et)] / [A (s) X M X (100-E)]

※ Potential cyanidol chloride (%) = [PCV X 7% of sample] / 80

※ Amount of potential cyanidol chloride in 1 mL of sample (gel = 1 g) (mg)

      = [10 mg X potential cyanidol chloride (%)] / 100

 A (t): average absorbance of the reacted sample solution

 A (s): average absorbance of the reacted standard solution

 -Mt: standard dose (mg)

M: amount of seed extract of Vitis vinifera in the sample (mg)

 -Et: moisture content of standard product (%)

Table 5 Amount of potential cyanidol chloride in 1 mL of sample (ophthalmic gel = 1 g) (mg) Storage condition Early 6 months 12 months Example 1 25 ± 2 ℃ / Relative Humidity 60 ± 5% 0.86 0.86 0.85 40 ± 2 ℃ / relative humidity 75 ± 5% 0.86 0.83 - Example 2 25 ± 2 ℃ / Relative Humidity 60 ± 5% 0.86 0.86 0.85 40 ± 2 ℃ / relative humidity 75 ± 5% 0.86 0.84 - Example 3 25 ± 2 ℃ / Relative Humidity 60 ± 5% 0.86 0.86 0.86 40 ± 2 ℃ / relative humidity 75 ± 5% 0.86 0.85 - Example 4 25 ± 2 ℃ / Relative Humidity 60 ± 5% 0.86 0.86 0.83 40 ± 2 ℃ / relative humidity 75 ± 5% 0.86 0.82 - Comparative Example 1 25 ± 2 ℃ / Relative Humidity 60 ± 5% 0.86 0.53 0.31 40 ± 2 ℃ / relative humidity 75 ± 5% 0.86 0.21 - Comparative Example 2 25 ± 2 ℃ / Relative Humidity 60 ± 5% 0.86 0.48 0.21 40 ± 2 ℃ / relative humidity 75 ± 5% 0.86 0.15 - Comparative Example 3 25 ± 2 ℃ / Relative Humidity 60 ± 5% 0.86 0.68 0.51 40 ± 2 ℃ / relative humidity 75 ± 5% 0.86 0.39 - Example 5 25 ± 2 ℃ / Relative Humidity 60 ± 5% 0.86 0.86 0.85 40 ± 2 ℃ / relative humidity 75 ± 5% 0.86 0.84 - Example 6 25 ± 2 ℃ / Relative Humidity 60 ± 5% 0.86 0.86 0.85 40 ± 2 ℃ / relative humidity 75 ± 5% 0.86 0.83 - Example 7 25 ± 2 ℃ / Relative Humidity 60 ± 5% 0.86 0.86 0.86 40 ± 2 ℃ / relative humidity 75 ± 5% 0.86 0.85 - Example 8 25 ± 2 ℃ / Relative Humidity 60 ± 5% 0.86 0.86 0.86 40 ± 2 ℃ / relative humidity 75 ± 5% 0.86 0.83 - Comparative Example 4 25 ± 2 ℃ / Relative Humidity 60 ± 5% 0.86 0.75 0.66 40 ± 2 ℃ / relative humidity 75 ± 5% 0.86 0.57 -

As can be seen in Table 5, the composition containing the antioxidant according to the present invention is chemically stable at room temperature and accelerated conditions without significant change of the indicator material, especially a composition containing ascorbic acid (Example 3 and 7) showed the best chemical stability. Therefore, it can be seen that the eye drop solution and the gel composition of the present invention have excellent chemical stability.

Claims (6)

Seed extract of Vitis vinifera ; And an antioxidant selected from the group consisting of ascorbic acid or salts thereof, sodium hydrogen sulfite, potassium pyrosulfite, and L-threonine or salts thereof. The pharmaceutical composition according to claim 1, wherein the content of the antioxidant is 0.01 to 15% by weight based on the total weight of the composition. The pharmaceutical composition according to claim 1 or 2, wherein the content of the seed extract of the grape varieties Vitis vinifera is 0.1 to 30% by weight based on the total weight of the composition. The seed extract of the grape varieties Vitis vinifera has a procyanidolic value (PCV) of 80-130; Content of (+) catechin and (−) epicatechin of up to 30%; And a proanthocididine content of 95-105%. [3] The mixture of acetone and water according to claim 1 or 2, wherein the seed extract of the grape varieties Vitis vinifera is (a) pulverized seeds of the European grape varieties Vitis vinifera . Extracting at room temperature with a solvent and filtering; (b) distilling the filtrate obtained in step (a) to remove acetone, then saturating sodium chloride and filtering; (c) extracting the filtrate obtained in step (b) with ethyl acetate and concentrating; And (d) adding the chloroform to the concentrate obtained in step (c), followed by filtration to obtain a precipitate. The acetone according to claim 1 or 2, wherein the seed extract of the grape varieties Vitis vinifera is (1) (i) the seeds of the ground grape varieties Vitis vinifera are acetone and water having a volume ratio of 3 to 5: 1. Extracting with a mixed solvent of water and filtering; (ii) concentrating the filtrate obtained in step (i) to remove acetone and filtering; (iii) extracting the filtrate obtained in step (ii) with ethyl acetate; (iv) drying the extract obtained in step (iii) to obtain a first extract; (2) (p) extracting the seeds of crushed Vitis vinifera with water and filtering; (q) extracting the filtrate obtained in step (p) with ethanol and filtering; (r) drying the filtrate obtained in step (q) to obtain a second extract; And (3) an extract obtained by a manufacturing method comprising the step of mixing the first extract and the second extract.