KR20210060233A - Pharmaceutical composition for treating or preventing leaky gut syndrome - Google Patents

Abstract

A pharmaceutical composition and health functional food of the present invention contain an Oxya sinuosa extract, thereby having an excellent preventive, therapeutic or alleviating effect on leaky gut syndrome. An objective of the present invention is to provide the pharmaceutical composition and health functional food comprising the Oxya sinuosa extract having a preventive, therapeutic or alleviating effect on leaky gut syndrome.

Description

Pharmaceutical composition for treating or preventing leaky gut syndrome}

The present invention relates to a pharmaceutical composition for preventing or treating leaky intestinal syndrome.

Recently, research on the function of natural resources has been actively conducted in connection with the development of new drugs, and research on insects, which occupy a large number of species on the planet, is being activated. Since ancient times, insects have been frequently used as medicinal herbs, and about 30 species are used as useful insect resources. However, in order to use insects for food and medicine, since they are classified as hate foods, clear data on efficacy must be supported.

Rice grasshopper (Oxya chinensis) has long been used as food in Asia, and is registered as an edible insect by the Korea Food and Drug Administration (KFDA), and is traditionally known as a famine-fighting insect, coughing, pertussis, asthma, bronchitis, and paralysis. , And has been used to treat seizures. However, systematic studies on useful substances contained in rice locusts are still insufficient, and little is known about the effect of treating intestinal leaks in particular.

Korean Patent Publication No. 10-2019-0003305

An object of the present invention is to provide a pharmaceutical composition and a health functional food comprising a rice locust extract having a preventive, therapeutic or ameliorating effect for intestinal leak syndrome.

1. Rice locust (Oxya chinensis) a pharmaceutical composition for the prevention or treatment of leaky intestinal syndrome comprising an extract.

2. In the above 1, the extract is an ethanol extract, the composition.

3. The composition of the above 1, wherein the rice grasshopper extract is an extract of an adult.

4. Rice locust (Oxya chinensis) health functional food for preventing or improving intestinal leak syndrome containing extract.

5. In the above 4, the extract is an ethanol extract, health functional food.

6. In the above 4, wherein the rice grasshopper extract is an extract of adult, health functional food.

The pharmaceutical composition and health functional food of the present invention contains a rice locust extract, and thus has an excellent prevention, treatment, or improvement effect on intestinal leak syndrome.

1 is related to an experiment for selecting a DSS treatment concentration for making a gut leak syndrome model.
2 is related to the toxicity assay experiment of the rice locust extract.
3 to 8 are related to the efficacy assay experiment of the rice locust extract, analysis of the survival rate of fruit flies, morphological analysis of the intestine, Smurf assay for confirming the intestinal barrier function, analysis of promoting differentiation of intestinal stem cells, analysis of intestinal apoptosis and apical region It relates to gene expression level analysis.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating leaky gut syndrome comprising a rice locust extract.

Intestinal leak syndrome is a disease that occurs when a space is formed between cells in the membrane covering the inner wall of the intestine. Intestinal function is reduced and intestinal permeability is increased, which causes absorption of water and nutrients and disorders in the immune system.

The present invention is based on the fact that it was confirmed that the active ingredient contained in the rice locust extract can effectively prevent intestinal leakage as it exerts the effect of enhancing the binding or cohesion between cells in the membrane covering the inner wall of the intestine. The efficacy was specifically verified from the results of confirming the high expression levels of the region genes aPKC and crumbs.

The rice grasshopper may be used in various stages of growth, and in terms of its suitability for food, it may be preferably a rice grasshopper of the adult stage.

The extract may be a hot water extract, a cold needle extract, a reflux extract, a solvent extract, a steam distillation extract, an ultrasonic extract, an eluate or a compressed extract, but contains an appropriate amount of an active ingredient in the rice locust to exert the effect of preventing or treating intestinal leakage syndrome. In terms of, preferably, it may be an ethanol extract as a solvent extract.

The ethanol may be 60 to 80% ethanol, preferably 65 to 75%.

The extraction method is not particularly limited, and according to an embodiment of the present invention, an extract may be prepared through ultrasonic extraction after adding ethanol to the powder and suspending it.

If necessary, the extract may be prepared by filtering the supernatant centrifuged after ultrasonic extraction and further performing concentration under reduced pressure.

Pharmaceutical compositions containing the extracts are formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. It can be used, but is not limited thereto.

Carriers, excipients and diluents that may be contained in the composition containing the extract include lactose, dextrose, sucrose, dextrin, maltodextrin, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, Gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. Not limited. In the case of formulation, it is prepared using diluents or excipients such as generally used fillers, extenders, binders, wetting agents, disintegrants, and surfactants, but is not limited thereto.

Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like, but these solid preparations include at least one excipient, such as starch, calcium carbonate, in the compound. , Sucrose or lactose, gelatin, etc. are mixed and prepared. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of disease, severity, drug activity, Sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or administered in combination with another therapeutic agent, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.

In the pharmaceutical composition of the present invention, the effective amount may vary depending on the age, sex, and body weight of the patient, and generally 1 to 6000 mg, preferably 60 to 600 mg per kg of body weight can be administered once or three times. have. However, since it may increase or decrease according to the route of administration, the severity of the disease, sex, weight, age, etc., the dosage amount does not limit the scope of the present invention in any way.

The present invention provides a health functional food for preventing or improving intestinal leak syndrome comprising a rice locust extract.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or improving intestinal leak syndrome.

The health functional food of the present invention refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body pursuant to the Health Functional Food Act No.6727, and contains nutrients for the structure and function of the human body. It refers to ingestion for the purpose of regulating or obtaining beneficial effects for health purposes such as physiological effects.

The health functional food of the present invention may contain ordinary food additives, and whether it is suitable as a food additive is determined according to the general rules and general test methods of food additives approved by the Food and Drug Administration, unless otherwise specified. It is judged according to the standards and standards.

Examples of the food additives listed above include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as reduced pigment, licorice extract, crystalline cellulose, high color pigment, and guar gum; It includes, but is not limited to, mixed preparations such as sodium L-glutamate preparations, an alkali additive added to noodles, a preservative preparation, and a tar color preparation.

For example, in a health functional food in the form of a tablet, a mixture obtained by mixing the extract with an excipient, a binder, a disintegrant, and other additives is granulated in a conventional manner, and then compression molded by adding a lubricant or the like, or directly forming the mixture. It can be compression molded. In addition, the health functional food in the form of a tablet may contain a mating agent or the like, if necessary.

Among the health functional foods in the form of capsules, hard capsules can be prepared by filling a conventional hard capsule with a mixture of the extract and an additive such as an excipient, and a soft capsule is a mixture obtained by mixing the extract with an additive such as an excipient. It can be prepared by filling in a capsule base such as. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.

Ring-shaped health functional foods can be prepared by molding a mixture of the above extracts, excipients, binders, disintegrants, etc. by conventionally known methods, and can be coated with white sugar or other coating agents as needed, or starch , You can also coat the surface with a material such as talc.

Health functional foods in the form of granules can be prepared in granular form by a mixture of the extract, excipients, binders, disintegrants, and the like by a conventionally known method, and may contain flavoring agents, flavoring agents, and the like, if necessary.

The health functional foods are beverages, meat, chocolate, foods, sweets. It may be pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements.

The health functional food may be orally applied as a nutritional supplement, and its application form is not particularly limited. For example, in the case of oral administration, the daily intake is preferably 5000 mg or less, more preferably 2000 mg or less, and most preferably 500 to 1500 mg or 650 mg per day. When formulated into capsules or tablets, one tablet may be administered with water once a day.

Detailed descriptions related to the rice grasshopper, its extract, and intestinal leak syndrome are as described above.

Hereinafter, examples will be described in detail in order to describe the present invention in detail.

Experiment method

1. Preparation of rice locust extract

After drying, 10 mL of 70% ethanol per 1 g of pulverized rice locust powder was added and suspended, followed by extraction (80J, 6 times at intervals of 10 seconds) using an ultrasonic crusher. After that, after standing at room temperature for 30 minutes, the supernatant was centrifuged (3,000 rpm, 5 minutes) and filtered using a 0.45 μm syringe filter, concentrated under reduced pressure using a speed vacuum, and dried. % It was dissolved in DMSO (dimethylsulfoxide) and used in the experiment.

2. Breeding of fruit flies

Drosophila used in the experiment was a wild type w ¹¹¹⁸ variety, and was used in the experiment while rearing it on a yeast corn flour agar sucrose medium, which is a standard medium. The stock of fruit flies was maintained at 19°C, and the fruit flies used in all experiments were 3 to 5 days old adults and were reared at 29°C to perform the experiment.

3. Evaluation of the survival rate of fruit flies and oral administration of samples

The survival rate of Drosophila was evaluated for 5 days using 25 Drosophila males and females (50 in total). DSS (dextran sodium sulfate, MP biomedicals, Solon, OH, USA) was used as a substance inducing intestinal damage. DSS and rice locust extract (2 mg/mL) were diluted in minimal medium (5% sucrose), loaded at 0.5 mL increments on 2.5 x 3.75 cm sized chromatography paper, and supplied to Drosophila, with survival rate at 24 hour intervals. Was evaluated. Paper and test tubes loaded with samples were changed daily for 5 days. In the case of DSS-administered fruit flies, a minimum medium not containing each DSS and rice locust extract was used as a control.

4. Intestinal dissection

For intestinal morphological analysis and immunofluorescence staining, it was separated by pulling from the posterior part of the fruit fly using precision tweezers. Immediately after separation, 4% paraformaldehyde (PFA, Biosesang, Korea)/1x PBS (pH 7.4, Biosesang, Korea) solution. Since the female intestine is larger than the male intestine, the female intestine was used for morphological observation and immunofluorescence staining for ease of experimentation.

5. Immunofluorescence staining and microscopic observation

Drosophila intestines were immediately washed with 4% PFA/1x PBS solution for 30 minutes (delta antibody) or 3 hours (pH3, notch antibody) 3 times for 15 minutes with 1x PBS, followed by 0.1% Triton X-100. /0.5% BSA (bovine serum albumin, Sigma, St. Louis, MO, USA) / 1x PBS was blocked with a horse serum solution diluted at a ratio of 1:50 for 2 hours. Blocked intestine is the primary antibody anti-delta (DHSB, monoclonal, 1:20 dilution), anti-pH3 (Cell Signaling Technology, Cell Signaling, Beverly, MA, USA, monoclonal, 1:100 dilution), or anti- Notch (DHSB, monoclonal, 1:20 dilution) was incubated overnight at 4 ℃. Then, it was washed three times with 0.1% Triton X-100/0.5% BSA/1x PBS for 15 minutes, incubated with a secondary antibody, and then washed three times with 0.1% Triton X-100/0.5% BSA/1x PBS. The washed intestine was placed on a slide glass, mounted with an antifade containing DAPI (Vectorshield, Vector Lab, Burlingame, CA, USA), and the expression level of each factor was observed and evaluated with a fluorescence microscope.

6. Smurf Assay

In order to evaluate the degree of damage to the intestine, 1% FCF brilliant blue dye (Sigma, St. Louis, MO, USA) solution was supplied 4 days after oral administration of the intestinal damage substance and rice grasshopper extract, and then supplied on the 5th day. Drosophila with the entire body of the fruit flies stained blue was defined as smurf+, and the number of dyed fruit flies was counted.

7. DSS treatment concentration setting for making intestinal leak syndrome model

As a result of supplying 3, 5, 7, 9% DSS for 5 days to select an appropriate concentration of DSS to induce intestinal damage in Drosophila, the survival rate of the 7% and 9% DSS-treated groups was 80% from the 4th day. It decreased to below, and on the 5th day, a low survival rate of 40% or less was confirmed. However, there was no difference in the survival rate of Drosophila due to the difference in concentration of 7% and 9%, and it was confirmed that the survival rate of DSS of 5% or less was 60% or more on the 5th day of administration (FIG. 1). Based on these results, the concentration of DSS for evaluating the intestinal damage recovery efficacy of the insect extract was selected to be 7%, with a survival rate of about 50% or less of fruit flies on the 5th day of administration.

Experiment result

1. Toxicity test of rice locust extract

As a result of confirming the survival rate after administration of the rice locust extract at a concentration of 2 mg/mL to Drosophila for 5 days, it was found that the survival rate of 90% or more was observed 5 days after oral administration of the sample (FIG. 2 ). Therefore, it was found that the rice locust extract (OCA) itself used in this study was not toxic to fruit flies.

2. Efficacy test of rice locust extract

(1) Analysis of the survival rate of fruit flies

As a result of confirming whether the OCA extract has the effect of improving the survival rate of fruit flies with intestinal damage by oral administration of 7% DSS, the rice locust extract increased the survival rate of fruit flies administered orally with 7% DSS by more than 26%, and administration 5 As a result of evaluating the significance of the survival rate of Drosophila on the first day, it was found that the toxicity of Drosophila by DSS was significantly reduced with a P-value <0.05 (FIG. 3).

(2) Intestinal morphological analysis

According to the previously reported study, it is known that the length and thickness of the intestine isolated from mice or rats administered with DSS are shorter and thinner than in the normal group. Therefore, in this study, changes in the intestinal morphology (length) of Drosophila, which were orally administered with DSS or DSS, rice locust extract, were confirmed. As a result, the length of the intestine of Drosophila administered with 7% DSS alone was shorter than that of the normal Drosophila group, and as a result of oral administration of the rice locust extract with DSS, the length of the intestine was increased by an average of about 1000 μm (FIG. 4 ).

(3) Intestinal stem cell differentiation promotion analysis

After dissecting the intestine of Drosophila to check whether the rice locust extract, which improves the survival rate of Drosophila administered orally administered DSS, improves the differentiation ability of intestinal stem cells, the intestinal stem cell marker Delta and the cell proliferation marker, the phosphorylated histone 3 (phosphorylated Histone3; pH3) was subjected to immunostaining analysis. As a result, cells stained with Delta and pH3 were increased by DSS, and by oral administration of rice locust extract, Delta decreased by about 317, and pH3 decreased by about 10 (FIG. 5). According to existing research results, it is known that when intestinal damage is induced by DSS, the proliferation and differentiation of intestinal stem cells increases to restore the damaged intestine. However, based on the results of this study, when DSS and rice locust extract are administered orally at the same time, the rice locust extract does not promote the differentiation of intestinal stem cells to recover intestinal damage, and that the rice locust extract directly inhibits the intestinal damage action of DSS. Is estimated.

(4) Analysis of apoptosis pattern in the intestine

In this study, a TUNEL assay was conducted to confirm the effect of rice grasshopper extract on apoptosis when intestinal damage caused by DSS. As a result, when DSS alone was administered orally, apoptosis increased by 51.7% compared to the control group (5% sucrose alone treatment), but when rice locust extract was administered with DSS, the intestinal cell death by DSS was reduced to 39.8%. It was confirmed (Fig. 6).

(5) apical region gene expression level analysis

Leakage in the intestine in the intestinal leak syndrome is strongly related to the cohesion between cells. It has been found that the apical region complex genes aPKC and crumbs are involved in this intercellular avidity (Colosimo et al., 2010). Therefore, in this study, we also confirmed the expression of aPKC and crumbs genes, which play a key role in the binding ability between intestinal cells. As a result, the expression of both genes was decreased in Drosophila in which intestinal damage was induced by DSS, but when the rice locust extract was administered simultaneously with DSS, the expression of aPKC increased by 1.8 times or more, and the expression of crumbs was expressed by 1.39 times or more. It can be seen that (Fig. 7).

Claims (6)

Rice locust (Oxya chinensis) a pharmaceutical composition for the prevention or treatment of leaky intestinal syndrome comprising an extract.
The composition of claim 1, wherein the extract is an ethanol extract.
The composition of claim 1, wherein the rice grasshopper extract is an extract of an adult.
Rice locust (Oxya chinensis) health functional food for preventing or improving intestinal leak syndrome containing extract.
The health functional food according to claim 4, wherein the extract is an ethanol extract.
The health functional food according to claim 4, wherein the rice grasshopper extract is an extract of an adult.

Images