KR101047356B1 - Pharmaceutical compositions in the form of eye drops or gels containing seed extracts of European grapes - Google Patents

Abstract

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 composition according to the present invention can maintain physical and chemical stability for a long time, and thus can be usefully applied in the pharmaceutical field because it can be stored for a long time.

Seed Extract of European Grape, Eye Drop, Eye Drop Gel

Description

Pharmaceutical composition in forms of ophthalmic solution or gel containing Vitis vinifera pip extract}

The present invention is European grape ( Vitis vinifera ) relates to a pharmaceutical composition in the form of eye drops or gels containing a seed extract.

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 about 11 genera of 700 genera, including Vitis vinifera and American Vitis. labrusca ), riverside grapes ( Vitis riparia ), desert grapes ( Vitis rupestris ), winter grapes ( Vitis berladieri ), Vitis coignetiae), Vitis amurensis (Vitis amurensis ) is mainly cultivated for fruit use.

Among them, European grapes ( Vitis extracts from the seeds of vinifera ) include mixtures of (-) epicatechin, proanthocyanidins B1 and B2, (+) catechin, and their polymerization derivatives, which are known as procyanidol 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, European grapes ( Vitis) Oral tablets containing seed extracts of vinifera ) are commercially available as supplements for the treatment of disorders related to retinal and choroidal circulation. However, European grapes ( Vitis) Although vinifera ) seed extracts can be expected to have direct pharmacological effects when administered directly to the eye, no topical topical ophthalmic eye drops or gels have been reported. 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 inventors of the European grape varieties ( Vitis Various studies have been conducted to develop ophthalmic formulations containing seed extracts of vinifera ) as active ingredients, that is, formulations having excellent physical and chemical stability as eye drops or gels. 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. .

Thus, the present invention has excellent stability, European grapes ( Vitis It is an object to provide a pharmaceutical composition in the form of an ophthalmic solution or a gel containing a seed extract of vinifera ).

According to one aspect of the invention, European grapes ( Vitis vinifera ) seed extract; 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 antioxidant content may be 0.01 to 15% by weight based on the total weight of the composition, the active grape grapes ( Vitis) The seed extract of vinifera ) may be 0.1 to 30% by weight based on 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.

In addition, optionally, the European grapes ( Vitis) Seed extract of vinifera ) is (1) (i) crushed European grapes ( Vitis) extracting the seeds of vinifera ) with a mixed solvent of acetone and water having a volume ratio of acetone and water of 3-5 to 1: 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) crushed European grapes ( Vitis) extracting seeds of 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, European grapes ( Vitis) The content of the seed extract of 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.

The European Grape Vitis vinifera ) seed extracts are 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

European grapes ( Vitis) 100 mg of seed extract standard of vinifera ) is precisely weighed and added to isopropanol 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

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 Calculation

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 European grapes ( Vitis Seed extract of vinifera ) may be preferably used by the improved manufacturing method developed by the present inventors (ie, Korean Patent Application No. 10-2008-0087109, filed Sep. 4, 2008).

That is, the European grapes ( Vitis Seed extract of vinifera ) was (a) crushed European grapes ( Vitis). vinifera ) extract from acetone and water with a mixed solvent of acetone and water having a volume ratio of 1: 1 to 2 at room temperature and filtered; (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.

Crushed European Grapes ( Vitis) vinifera seeds are European Vitis vinifera ) can be obtained by washing the bark, seeds and eggplants obtained by compressing them with water and drying them using an oven or the like, and then separating the seeds and grinding them in a conventional manner.

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 precipitate is filtered simply by common grape vine (Vitis vinifera ) seed extract can be isolated easily. 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 European grapes ( Vitis) contained as an active ingredient in the pharmaceutical composition of the present invention Seed extract of vinifera ) is (1) (i) crushed European grapes ( Vitis) vinifera ) extract of acetone and water with a mixed solvent of acetone and water having a volume ratio of 3 to 5: 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) crushed European grapes ( Vitis) extracting seeds of 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) European species grape ( Vitis) comprising the step of mixing the first extract and the second extract vinifera ) can be obtained by a method for producing a seed 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 preparing the first extract, step (i) comprises pulverized European grapes ( Vitis) vinifera ) seed is extracted by acetone and water with a mixed solvent of acetone and water having a volume ratio of 3 to 5: 1, more preferably 4: 1, and filtered. 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) is pulverized European grapes ( Vitis vinifera ) seeds can be extracted with water and filtered; 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 Manufacturing example  One. European species  grape( Vitis vinifera Preparation of Seed Extract

European grapes ( Vitis) vinifera ) was pressed, the shells, seeds and branches were collected, washed with 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 less and approximately 3.5 g of European grapes ( Vitis) vinifera ) seed extract was obtained 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 Manufacturing example  2. European species  grape( Vitis vinifera Preparation of Seed Extract

European grapes ( Vitis) vinifera ) was pressed, the shells, seeds and branches were collected, washed with water and dried in a rotary oven, and the seeds were separated. 1 kg of the obtained seed was pulverized, and then extracted by adding 500 ml of acetone aqueous solution (acetone / water = 8/2, v / v). 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 ) was pressed, the shells, seeds and branches were collected, washed with 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).

European grapes ( Vitis) by mixing the first extract and the second extract vinifera ) seed extract of about 35 g. 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 drops Liquid

Sterile purified water to 800 ml Vitis vinifera (Vitis 10 g of seed extract of 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, 0.5 g of sodium edetate were dissolved and then sterilized. Eye drops were prepared by adding purified water to a final volume of 1,000 ml. (pH 6.0)

Example  2. Eye drops Liquid

Sterile purified water to 800 ml Vitis vinifera (Vitis 10 g of seed extract of 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 and 0.5 g of sodium edate were dissolved and then sterilized. Eye drops were prepared by adding purified water to a final volume of 1,000 ml. (pH 6.0)

Example  3. Eye drops Liquid

Sterile purified water to 800 ml Vitis vinifera (Vitis 10 g of seed extract of vinifera ), 8 g of boric acid, 9 g of borax, 2 g of concentrated glycerin, 15 g of D-sorbitol solution (70%), 1 g of ascorbic acid, 0.5 g of sodium edate, were dissolved, and then sterile purified water. An eye drop liquid was prepared by adding the final volume to 1,000 ml. (pH 6.0)

Example  4. Eye drops Liquid

Sterile purified water to 800 ml Vitis vinifera (Vitis 10 g of seed extract of 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 L-threonine, and 0.5 g of sodium edate were dissolved, followed by sterilization. Eye drops were prepared by adding purified water to a final volume of 1,000 ml. (pH 6.0)

Comparative example  1. Eye drops Liquid

Sterile purified water to 800 ml Vitis vinifera (Vitis 10 g of seed extract of vinifera ), 6.8 g of potassium dihydrogen phosphate, and 5 g of sodium chloride were dissolved, and then adjusted to pH 6.0 by addition of 1N sodium hydroxide solution, and the final volume was adjusted to 1,000 ml by adding sterile purified water. A liquid formulation was prepared. (pH 6.0)

Comparative example  2. Eye drops Liquid

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 drops Liquid

Sterile purified water to 800 ml Vitis vinifera (Vitis 10 g of seed extract of 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, followed by addition of sterile purified water to a final volume of 1,000. Dropping solution was prepared in accordance with ml. (pH 6.0)

Example  5. Eye drops Gel

Vitis in 800 mL of sterile purified water 10 g of the seed extract of vinifera ), 16.5 g of sodium carboxymethylcellulose, 50 g of D-sorbitol solution (70%), 3 g of sodium bisulfite, 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)

Example  6. Eye drops Gel

Vitis in 800 mL of sterile purified water 10 g of the seed extract of vinifera ), 16.5 g of sodium carboxymethyl cellulose, 50 g of D-sorbitol solution (70%), 0.25 g of potassium pyrosulfite, 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)

Example  7. Eye drops Gel

Vitis in 800 mL of sterile purified water 10 g of the seed extract of 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 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)

Example  8. Eye drops Gel

Vitis in 800 mL of sterile purified water 10 g of the seed extract of 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 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)

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 results of the change in properties are shown in Tables 1 and 2 below, and the test results for changes in pH are shown in Table 3.

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 - -

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

※ ND : Not detected

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 1 to Table 3, the composition containing an antioxidant such as ascorbic acid, sodium bisulfite according to the present invention does not have 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 4.

※ 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: Vitis of European species in specimen amount of seed extract of vinifera ) (mg)

-Et: moisture content of standard product (%)

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 4, 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 (12)

delete delete delete delete delete delete As a method of stabilizing a pharmaceutical composition in the form of eye drops or gels containing seed extract of the European species Vitis vinifera as an active ingredient, A stabilizing method characterized by adding an antioxidant selected from the group consisting of ascorbic acid or a salt thereof, sodium hydrogen sulfite, potassium pyrosulfite, and L-threonine or a salt thereof as a stabilizer. 8. The stabilization method according to claim 7, wherein the content of the antioxidant is 0.01 to 15% by weight based on the total weight of the composition. The method of claim 7, 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. 10. The method according to any one of claims 7 to 9, wherein the seed extract of Vitis vinifera has a procyanidolic value (PCV) of 80 to 130; Content of (+) catechin and (−) epicatechin of up to 30%; And a proanthocididine content of 95-105%. 10. The method according to any one of claims 7 to 9, wherein the seed extract of the grape varieties Vitis vinifera is (a) the seed of the pulverized grape varieties Vitis vinifera has a volume ratio of acetone and water of 1 to 1 to 2. Extracting at room temperature with a mixed solvent of phosphorus acetone and water, and 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 the chloroform to the concentrate obtained in step (c) and filtering to obtain a precipitate. 10. The seed extract according to any one of claims 7 to 9, wherein the seed extract of the grape varieties Vitis vinifera is (1) (i) the seed of the ground grape varieties Vitis vinifera is 3 to 3 vol. 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) stabilization method characterized in that the extract obtained by the manufacturing method comprising the step of mixing the first extract and the second extract.