KR101835227B1 - Composition for preveting or treating ocular disease containing rhynchosia nulubilis - Google Patents

Abstract

The present invention relates to a composition for preventing or treating eye diseases including a Rhynchosia volubilis extract as an active ingredient. According to the present invention, various eye diseases can be effectively prevented or treated by an excellent antioxidant activity of the Rhynchosia volubilis extract.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or treating eye diseases,

The present invention relates to a composition for preventing and treating eye diseases and a functional food composition comprising an extract of Panax ginseng as an active ingredient.

Ocular diseases are caused by various factors such as oxidative stress, inflammation, bacteria, external stimuli, aging, etc. In recent years, eyes have been overused due to frequent use of smart phones, TVs and computers, The incidence of diseases is increasing.

Rhynchosia nulubilis is a kind of black bean, whose skin is black and its size is much larger than that of black beans. It is called "ryeomtae" because it looks like a snowball. Seomok-tae has been widely used for medicines as well as for edible and medicinal purposes, and is known to be useful for the prevention and treatment of various adult diseases such as diabetes, hypertension, arteriosclerosis and heart disease. Therefore, many studies are being conducted to apply Seomyeongtae to various diseases.

Korean Patent No. 10-0550358

It is intended to provide a pharmaceutical composition and a functional food composition for preventing or treating eye diseases, which comprises an extract of Seomyeok-myeon as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing and treating eye disease and a functional food composition for preventing and improving eye diseases, which comprises an extract of Panax ginseng extract as an active ingredient.

In one embodiment of the present invention, the Saemari root extract may reduce the oxidative stress of the eye.

In one embodiment of the present invention, the ocular disease may be selected from the group consisting of dry eye syndrome, blepharitis, keratitis, retinitis, and optic neuritis.

In one embodiment of the present invention, the Saemari root extract may be a hot-water extract.

The composition comprising the extract of the present invention of the present invention can promote tear production and is effective for the treatment of dry eye syndrome. In addition, the antioxidant activity of Seomyeongae extract can be applied to the prevention or treatment of various ocular diseases caused by oxidative stress.

1 is a flowchart illustrating a method of manufacturing a hot water extract of Seomunjang in an embodiment of the present invention.
FIG. 2 shows an experimental schedule of the treatment of Seomyeonhak extract in rats according to one embodiment of the present invention.
Fig. 3ds is a graph showing the result of measuring fluorescence area of eyeballs after the treatment of Seomoko extract of one example of the present invention .
FIG. 4 is a graph showing the results of STT test after the treatment experiment of Seomyeok-myeon extract in the embodiment of the present invention.
FIG. 5 is a graph showing the results of capsaicin test after the treatment of the extract of Seomyeok-myeon extract in the examples of the present invention.
6 is a graph showing the antioxidant activity of the extract of Seomyeonoaki in the example of the present invention.
FIG. 7 is a graph showing antioxidant activity of the extract of Seomyeok-myeon in the embodiment of the present invention.
FIG. 8 is a graph showing the results of the antioxidation FIG.
FIG. 9 is a Western blot result showing the antioxidative activity of the Seomyeok extract in the examples of the present invention.
10 is a Western blot result showing the antioxidative activity of the extract of Seodo extract in the examples of the present invention.
Figure 11 is a Western blot result showing the antioxidant activity of the extract of Seomunjeok in the examples of the present invention.
12 is a Western blot result showing the antioxidative activity of the extract of Seomyeonjang extract in the examples of the present invention.
13 is an image showing the result of H & E staining of an animal eye in the example of the present invention. A) Control B) Vehicle C) Pre-low D) Pre-high E) Post-low F) Post-high G) Positive.
14 is a graph showing the results of measuring the thickness of the epithelial layer of the eyeball in the embodiment of the present invention.
Fig. 15 shows the results of measurement of the number of cells in epithelial tissues in the examples of the present invention.
FIG. 16 is an image of a lacrimal gland cell area measured in a lacrimal gland according to an embodiment of the present invention. A) Control B) Vehicle C) Pre-low D) Pre-high E) Post-low F) Post-high G) Positive. Left: Results of H & E staining, right: Results of image processing of tear cell area.
17 is a graph (b) showing the result of measuring the area of the lacrimal gland cell in the lacrimal gland in the example of the present invention.
18 is a graph showing the results of measurement of cytokine levels of (a) eyeball and (b) serum in the examples of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, detailed description of known techniques well known to those skilled in the art may be omitted. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear. Also, terminologies used herein are terms used to properly represent preferred embodiments of the present invention, which may vary depending on the user, intent of the operator, or custom in the field to which the present invention belongs.

Therefore, the definition of these terms should be based on the contents throughout this specification. Throughout the specification, when an element is referred to as "comprising ", it means that it can include other elements as well, without excluding other elements unless specifically stated otherwise.

The present invention relates to a pharmaceutical composition for preventing and treating eye disease and a functional food composition for preventing and improving eye diseases, which comprises an extract of Seomyeok-myeon as an active ingredient.

The Saemarius tincture extract may include any method that can be modified by those of ordinary skill in the art, and may be extracted by, for example, hot water extraction, organic solvent extraction, or the like.

As an example, the Saemari root extract can be prepared according to the process shown in Fig. Concretely, the pressure is extracted, and the water is then pulverized and washed, and diluted with about 20 times of water. It is then subjected to hot water extraction and filtration to finally obtain a Pseudomonas sp. Extract.

The above embodiments can be pre-treated to improve yield, efficacy, etc. prior to pressure extraction.

The hot water extraction may be performed at a temperature of 80 to 300 ° C, for example, 100 to 300 ° C, 150 to 300 ° C, 200 to 300 ° C, 250 to 300 ° C, 80 to 250 ° C, 80 to 200 ° C, Or at a temperature of 80 to 100 ° C, but is not limited thereto.

The extract of the present invention may be contained in a pharmaceutically effective amount, for example, 0.1 to 700 μg / mL, 0.1 to 500 μg / mL, 0.1 to 250 μg / mL, 0.1 to 100 μg / mL, 0.1 ML, 10-700 μg / mL, 50-700 μg / mL, 100-700 μg / mL, 250-700 μg / mL, or 10-700 μg / mL, ~ 650 μg / mL can be used.

The extract of the present invention may be used in combination with a physiologically active substance. For example, the above-mentioned extract of Lycoris sieboldii can be used in combination with cinnamon extract and the like.

The pharmaceutical composition may comprise one or more pharmaceutically acceptable carriers, excipients or diluents.

The "pharmaceutically effective amount" as used herein refers to an amount sufficient for the physiologically active component to be administered to an animal or a human to exhibit a desired physiological or pharmacological activity. However, the pharmaceutically effective amount may be appropriately changed depending on the severity of symptoms, the age, body weight, health condition, sex, administration route and treatment period of the patient.

The term "pharmaceutically acceptable" as used herein means physiologically acceptable and does not generally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to a human. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

The compositions of the present invention may also be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal, and may be formulated as a variety of compositions for oral or parenteral administration . ≪ / RTI > The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

The composition according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous or muscular, and the dose of the active ingredient may be varied depending on various factors such as route of administration, age, sex, And the like. In addition, the composition of the present invention may be administered in combination with a known compound capable of raising the desired effect.

Furthermore, the anti-obesity composition according to the present invention can be used not only as a pharmaceutical composition as described above, but also as a health functional food. For example, it can be easily used as a raw material for food, an ingredient, a food additive, a functional food or a beverage.

The term " food " means a natural product or a processed product containing one or more nutrients, preferably a state of being able to be eaten directly through a certain degree of processing, , Food additives, functional foods and beverages.

Foods to which the food composition can be added include, for example, various foods, beverages, gums, tea, vitamin complexes, and functional foods. In addition, it is also possible to use special nutritive foods (eg crude oil, milk, infant food, etc.), meat products, fish products, tofu, jelly, noodles (eg ramen, (Such as fermented milk, cheese, etc.), processed foods, kimchi, pickled foods (various kinds of kimchi, etc.), sauces, confectionery (eg, snacks), candies, chocolate, gums, ice cream, But not limited to, beverages (e.g., fruit drinks, vegetable beverages, beverages, fermented beverages, etc.), natural seasonings (e.g., ramen soup, etc.). The food, beverage or food additive may be prepared by a conventional production method.

The term " functional food " or " health functional food " refers to a food group imparted with added value to function or express the function of the food to a specific purpose by physical, biochemical or biotechnological techniques, Defensive rhythm control, prevention and restoration of disease, and the like, and it can be a health functional food. The functional food may include a food-acceptable food-aid additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of functional foods.

The health supplements may be in the form of powders, granules, tablets, capsules or drinks, although not limited thereto.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are for illustrating the present invention specifically, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of Seomyeongdae Extract

The extract of P. monocotus was prepared according to the process shown in Fig.

Seomyeongtae was obtained from Omni Hub (Daegu, Korea). After washing, Seomoktabi was pressure-extracted with a hand-held pressureless extractor (KSNP-BL180-240) and crushed with Pin Mil (Pin cracher). The pulverized material was washed twice, dried and then pulverized again. The pulverized material was heated at 100 占 폚 for 4 hours with 20 times of water. Then, the mixture was filtered three times with a filter cloth, and finally filtered with a filter paper to prepare a Pseudomonas sp. Extract. The filtrate was refrigerated until use.

Experimental Example 1. Animal Model Experiment

Animal experiments were conducted according to the experimental schedule as shown in Fig.

Rats (SD species, 4 weeks old, 100-110 g, male) (purchased from Hyosung Animal Co.) were all 8 groups and 8 rats per group were experimented. Pre-high dose (250 mg / 100 g rat weight) was administered to high-dose group, and pre-low dose (250 mg / 100 g rat weight) (65mg / 100g rat weight), low pre-low dose (65mg / 100g rat weight), low-dose pre-low dose (65mg / 100g rat weight) Omega 3 (85 mg / 100 g rat weight) was administered as a positive control group. The pre-group was administered to the oral cavity for 8 days after the 7th day of adaptation. Pre-Group induced ocular dryness induced ocular dryness on the 14th day and secondary ocular dryness on the 21st day. The Post Group administered the extracts of Seomyeok - tae with concentration of the first eye dryness induction on the 7th day after adaptation, followed by the second eye dry induction on the 14th day and animal sacrifice on the 37th day. Dry eye induction was performed by injecting 15 μl of Freund's Adjuvant complete, which was purchased from Sigma in the same way, into the lacrimal gland using a dedicated syringe. Animals were sacrificed on day 37 and then subjected to STT, visual and capsaicin tests.

First, 40 μl of alkaline 0.5% eye drops were dropped on the fluorescein paper, and the area of the eyeball fluorescence was observed in the dark room using innotech INV-150 after 20 seconds, Rasband, National Institutes of Health, USA). The results are shown in FIG. The larger the degree of damage due to ocular dryness, the wider the area in which the fluorescent material is observed. As shown in Fig. 3, the fluorescent area of the vehicle was 6.962 ± 1.01, post-low was 5.892 ± 0.42, post-high was 3.129 ± 0.32, pre-low was 1.18 ± 0.19 and pre- high group showed a small fluorescence area.

The results of the area of the paper wetted with tears are shown in FIG. 4, and the STT (schirmer tear test) method is used to measure the amount of tear by standardized sterilizing strips paper. As shown in Figure 4, the amount of tears in the eye was 4.91 ± 0.45 in the vehicle, 6.31 ± 0.42 in the post-low, 7.08 ± 0.35 in the post-high, 6.42 ± 0.31 in the pre-low, 6.91 ± 0.31 in the pre- 0.19, respectively, and the amount of tear was increased compared to the induced group.

Capsaicin behavioral assessment was performed to assess the degree of eye damage. The increase in the number of wiping around the eyes is understood to be the degree of damage. 20 μl of capsaicin (15 mg / 1 ml) was administered to the eye to measure the number of times the eye area of the rat was wiped for 3 minutes. The results are shown in FIG. The mean pre-treatment group was 32.67 ± 1.20, post-low was 30.33 ± 0.88, post-high was 31.34 ± 0.89, pre-low was 26.34 ± 0.66 and pre-high was 28.00 ± 0.57. Respectively.

Experimental Example 2 Evaluation of Antioxidative Activity

DPPH radical scavenging activity

The DPPH radical scavenging activity of Seomyeongae extract was measured.

The hydrazyl of DPPH (Diphenylpicryl hydrazyl) used in this experiment has easily absorbed hydrogen atoms because the nitrogen atom is in an unstable state, and reacts with the antioxidant material to accept the hydrogen atoms, thereby losing its color. 0.2 mM DPPH solution (99.5% ethanol) was added to 0.1 ml (control: 99.5% ethanol) of the concentrated solution of each substance in Seomok-tae prepared at seven concentrations of 10, 20, 40, 80, 160, 320 and 640 μg / ml. After shaking for 10 seconds with a Vortex mixer, incubation was carried out at 37 ° C for 30 minutes. The absorbance was then measured at 517 nm using an x-Mark micro spectrophotometer reader.

L-ascorbic acid was prepared at 6 concentrations of 10, 20, 40, 80, 160, 320, and 640 μg / mL (99.5% ethanol) as positive control drugs. The antioxidative activity of each sample was expressed as% antioxidant activity (electron donating ability) against DPPH, and the results are shown in FIG. As can be seen from FIG. 6, the antioxidant activity was increased in a concentration-dependent manner and the activity was about 50% at 640 μg / mL.

Nitric oxide measurement

0.4 mL / g of PRO-PREP (INTRON.) With homogenized cornea samples of the animal model of Experimental Example 1 was mixed and reacted at 4 ° C for 30 minutes. After centrifugation at 13,000 rpm, supernatant was collected and Nitric oxide was measured. 100 μl of supernatant and 100 μl of Griess reagent [1% (w / v) sulfanilamide, 0.1% (w / v) naphtylethylenediamine in 2.5% (v / v) phosphoric acid] After mixing for 10 min on 96-well plates, absorbance was measured at 540 nm using an x-Mark micro spectrophotometer reader. Standard concentration curves were obtained by stepwise dilution of sodium nitrite (NaNO ₂ ). FIG. 7 shows the result of measurement of nitric oxide, and it can be confirmed that the Nitric oxide is shown in the animal model treated with the Seomyeok-sae extract.

Measurement of Xanthine oxidase inhibitory activity

Xanthine oxidase acts as a rate-limiting enzyme in the terminal oxidation of all purines and is an enzyme that acts as a source of oxidizing agents such as superoxide radicals and hydrogen peroxide. The superoxide anion inhibition by xanthine / xanthine oxidase enzyme is shown by superoxide anion scavenging activity and xanthine oxidase enzyme inhibition and is biologically important by inhibiting the formation of free radicals.

0.1 ml of sample solution and 0.2 ml of 2 mM xanthine dissolved in 0.6 ml of 0.1 M potassium phosphate buffer at pH 7.5 were added, and 0.1 ml of 0.2 unit / ml xanthine oxidase was added. The mixture was reacted at 37 ° C for 5 minutes, and 1 N HCl ml was added to terminate the reaction, and the absorbance of uric acid produced in the reaction solution was measured at 292 nm. Xanthin oxidase inhibitory activity was expressed as the absorbance reduction rate of the sample solution added group and no addition group. The superoxide radical scavenging effects of 10, 20, 40, 80, 160, 320, and 640 μg mL ^-1 of RNE were compared using BHA (butylated hydroxy anisole) as a positive control. As can be seen from FIG. 8, the extracts of S. epidermidis showed superoxide radical scavenging activity in a concentration-dependent manner and showed an elimination effect of 55% or more at a concentration of 640 μg mL ^-1 .

Western blot

Western blotting was performed to confirm the expression of Nrf2, iNOS, VEGF, and NF-κB, which are signal transduction proteins related to inflammation, antioxidation, and neovascularization in the ocular and optic nerve glands. The results are shown in FIGS. The expression of Nrf2 in the eye was statistically significantly decreased at pre-low (1.29 ± 0.03) and post-low (1.14 ± 0.02) compared to vehicle group (1.66 ± 0.09) (P <0.05) in the pre-low (1.18 ± 0.02) compared to the control (0.12) (FIG. 9). The expression of iNOS was significantly lower in the pre-low (1.97 ± 0.06), post-high (1.56 ± 0.08) and post-low (0.92 ± 0.05) than the vehicle group (2.95 ± 0.15) , P &lt; 0.01) (Fig. 10).

Expression of NF-κB in the lacrimal gland was significantly reduced in Pre-high (1.49 ± 0.02) and Post-high (1.52 ± 0.04) compared to vehicle group (2.56 ± 0.07) (P <0.05, P <0.01) (Fig. 11) compared with the pre-high (1.00 ± 0.04) and post-high (1.06 ± 0.09) Nrf was significantly lower in pre-high (0.99 ± 0.09), pre-low (1.17 ± 0.02) and post-high (1.01 ± 0.07) than vehicle group (1.99 ± 0.06) 0.01) (Fig. 12).

Thus, the expression of Nrf2, iNOS, VEGF, and NF-κB, which are anti-inflammatory, antioxidant, and angiogenic signaling proteins, is significantly lower in the high and low dose groups than in the vehicle group, And ophthalmic diseases by controlling the anti-inflammatory, antioxidant, and neoangiogenic signal transduction by the extract mixture of the present invention in ophthalmic diseases. In addition, Nrf2, which enters the nucleus, is activated by binding with the antioxidant response element (ARE), and the Fos gene is expressed. The c-Fos gene, which is the Fos gene, binds to the c-Jun protein and promotes the transcription of the inflammatory cytokine gene , Which is thought to be the result of decreased expression of IL-1β and IL-6, which are proinflammatory cytokines in the treated group. In accordance with this fact, it is known that the activity of eye and lacrimal glands is lower than that of vehicle group there was.

Experimental Example 3. Results of H & E staining of lacrimal gland and eyeball

The thickness of the epithelial layer of the eye of the animal model of Experimental Example 1 was measured through H & E staining. The corneal epithelium was measured using ImagePro viewing software at 8x magnification at 400x magnification. The measurement of corneal epithelial layer was performed based on the presence of limbus in the transition between the cornea and sclera. Epithelial thickness of the eye was measured by H & E staining of the eyes. There was a significant difference in the thickness of the epithelium layer compared with vehicle. The pre-high epithelium thickness (mean 17.46 μm) was significantly higher than vehicle epithelium thickness (mean 12.37 μm) (Fig. 13 and 14).

The number of cells in the epithelium was measured by counting the number of cells on the basis of the formal nucleus using Image J64 (Wayne Rasband, National Institutes of Health, USA), and excluded the cells that could not be calculated. Each measurer measured the number of cells in a blind fashion. The number of cells in the epithelial layer in the animal group was shown in FIG. 15. Compared with the vehicle, the number of cells in the pre-high group was significantly higher than that in the vehicle group (mean 39.5 cells / mm) -high number of cells (mean 27.25 cells / mm) decreased remarkably.

The lacrimal gland cell area comparison in the lacrimal gland was performed using Image J64 (Wayne Rasband, National Institutes of Health, USA). Each lacrimal gland was sampled and the same area was collected and measured. The measurement results are shown in FIGS. 16 and 17. As compared with the control group (1 ± 0.05), the vehicle group (0.39 ± 0.07) was slightly smaller than that of the control group There was a significant change in pre-high (0.78 ± 0.04) (p <0.01) and a statistically significant change in other groups (p <0.05).

Experimental Example 4. Measurement of cytokine

The ocular and serum cytokines of the animal model of Experimental Example 1 were measured.

The proinflammatory cytokine assay was performed by measuring the 450 nm absorbance of the microplate reader using the R & D kit using serum from the supernatant after centrifugation and blood drawn from the supernatant obtained from homogenate and from the abdominal vein. The concentrations (pg / mL) of IL-6, IL-1β, and TNF-α in eye tissues and serum were calculated based on the quantitative curve of the standard solution and the results are shown in FIG. Specifically, IL-6 in eyes and sera was significantly lower than that of group (445.23 ± 23.12) (495.48 ± 53.85), Pre-low (47.42 ± 23.71) And post-high (90.87 ± 14.31) (81.5 ± 6.45) post-low (97.85 ± 8.35) (123.5 ± 7.61) TNF-α was significantly higher in the pre-low (40.04 ± 12.23) (28.89 ± 8.99), pre-high (22.17 ± 13.9) and post-low And post-high (34.41 ± 3.19) (34.21 ± 12.56), respectively. IL-1β was significantly higher in the pre-low (32.07 ± 0.60) (31.67 ± 1.94), pre-high (30.46 ± 0.92) and post-low (32.67 ± 0.69) and post-high (32.08 ± 3.43) (37.93 ± 6.75), respectively. The IL-6, IL-1β, and TNF-α levels of the inflammatory cytokines IL-1β and TNF-α, which have been studied for ocular dryness and eye disease, are statistically significantly lower than those of the control group at both high and low concentrations there was. From this, it can be confirmed that the extract of the present invention has an anti-inflammatory effect by increasing the secretion of inflammatory cytokine upon induction of ocular dryness, and thus it can be effective for ocular dryness and eye disease prevention or treatment.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (5)

A pharmaceutical composition for the prevention or treatment of ophthalmic diseases selected from the group consisting of dry eye syndrome, eyebrows, keratitis, retinitis and optic neuritis, which comprises Seomunghae extract as an active ingredient. The method according to claim 1,
Wherein said extract is for reducing oxidative stress in the eye. delete The method according to claim 1,
Wherein the extract is a hot-water extract. Wherein the active ingredient is selected from the group consisting of dry eye syndrome, eyebrows, keratitis, retinitis, and optic neuritis.

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