KR102158752B1 - A pharmaceutical composition comprising a Allomyrina dichotoma extract and a health functional food - Google Patents

Abstract

A pharmaceutical composition and health functional food of the present invention contain an Allomyrina dichotoma extract, thereby providing excellent prevention, treatment or alleviation effect on leaky gut syndrome.

Description

A pharmaceutical composition comprising a Allomyrina dichotoma extract and a health functional food}

The present invention relates to a pharmaceutical composition and health functional food comprising a longevity beetle extract having a preventive, therapeutic or ameliorating effect on 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, such as slugs, silkworms, cicada hulls, cordyceps and centipedes, are used as useful insect resources.

Longevity beetle (scientific name: Allomyrina dichotoma) is an insect belonging to the Coleoptera and Family Dynastidae. As folk remedies are known that slugs, the larvae of longevity beetles, are effective in liver disease, the tendency to use them as health supplements is increasing. In addition, it has been reported that the protein isolated from the longevity beetle shows a strong inhibition of activity against bacteria, and the lectin extracted from the longevity beetle exhibits strong anticancer activity and can expect anticancer effect. In addition, a paper confirming the hepatoprotective effect on rats that induced hepatotoxicity with carbon tetrachloride was reported. However, while longevity beetles and their larvae are widely used in home remedies, research data on their physiological function is very insufficient. In order to use insects for food and medicine, they are classified as hate foods, so clear data on efficacy must be supported. Currently, there are no reports on anti-inflammatory using longevity beetles, and there are reports of swallowtail butterflies, warts, and ladybugs in anti-inflammatory patents using similar insects, but these are not equipped with a current breeding system.

Korean Patent No. 1935153

An object of the present invention is to provide a pharmaceutical composition and health functional food comprising a longevity beetle extract having a preventive, therapeutic or ameliorating effect on leaky intestinal syndrome.

1. Longevity beetle ( Allomyrina dichotoma ) intestinal leak syndrome (leaky gut syndrome) prevention or treatment pharmaceutical composition containing an extract.

2. The composition of 1 above, wherein the extract is an ethanol extract.

3. The composition of the above 1, comprising an extract of longevity beetle larva (larva).

4. Longevity beetle ( Allomyrina dichotoma ) intestinal leak syndrome (leaky gut syndrome) prevention or improvement health functional food containing the extract.

5. In the above 3, the extract is an ethanol extract health functional food.

6. In the above 3, health functional food containing the extract of longevity beetle larva (larva).

The pharmaceutical composition and health functional food of the present invention contains a longevity beetle extract, and thus has an excellent prevention, treatment or improvement effect on leaky gut syndrome.

1 is related to an experiment for selecting a DSS treatment concentration for making a leaky gut syndrome model.
Figure 2 is related to the toxicity assay experiment of beetle extract.
Figures 3 to 9 are related to the efficacy assay experiment of the beetle extract, viability analysis of fruit flies, intestinal morphological analysis, Smurf assay for confirming intestinal barrier function, intestinal stem cell differentiation promotion analysis, intestinal apoptosis analysis and E -It is related to the analysis of Cadherin expression level.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating leaky gut syndrome comprising an extract of longevity beetle ( Allomyrina dichotoma ).

Leaky intestinal syndrome is a disease that occurs when a space is formed between cells in the membrane covering the inner wall of the intestine, and the function of the intestine decreases, and the permeability of the intestine increases, causing 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 long-sleeved beetle extract can effectively prevent intestinal leakage as it exerts the effect of enhancing the binding or cohesiveness 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 level of E-Cadherin.

The longevity beetle may have various growth stages, and may preferably be a larval stage of the longevity beetle in terms of its suitability for food.

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 includes an appropriate amount of the active ingredient in the longevity beetle so as to exhibit the effect of preventing or treating intestinal leakage syndrome. In that aspect, preferably, it may be an ethanol extract as a solvent extract.

The pharmaceutical composition containing the extract is 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 according to a conventional method. 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. . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, 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, glycerogelatin, and the like may be used.

The present invention provides a health functional food for preventing or improving leaky gut syndrome, including a longevity beetle ( Allomyrina dichotoma ) 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 controlling 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 for food additives approved by the Food and Drug Administration, unless otherwise specified. It is judged according to standards and standards.

Examples of the items listed in the food additive process 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 a sodium L-glutamate preparation, an alkali additive for 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 a lubricant is added thereto and compression molding, or the mixture is directly 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 capsule-type health functional foods, hard capsules can be prepared by filling a mixture of the extract with additives such as excipients in a conventional hard capsule, and soft capsules include gelatin with a mixture of the extract and additives such as excipients. It can be prepared by filling in a capsule base such as. The soft capsules 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 extract, 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.

The health functional food 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.

Detailed descriptions related to the longevity beetle, its extract, and leaky gut syndrome are as described above.

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

Experiment method

1. Preparation of beetle extract

After drying, pulverized longevity beetle (larva step; larva) was suspended by adding 10 mL of 70% ethanol per 1 g of powder, followed by extraction (80J, 6 times every 10 seconds) using an ultrasonic crusher. Thereafter, 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. % DMSO (dimethylsulfoxide) was dissolved 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. Drosophila stock (stock) was maintained at 19 ℃, the fruit flies used in all experiments were carried out while culturing at 29 ℃ as a 3 to 5 days old adults.

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

The survival rate of fruit flies was evaluated for 5 days using 25 male and female (50 total) Drosophila. DSS (dextran sodium sulfate, MP biomedicals, Solon, OH, USA) was used as a substance inducing intestinal damage. DSS and insect 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 fruit flies, with survival rate at 24 hour intervals. Was evaluated. Paper and test tubes loaded with samples were replaced daily for 5 days. In the case of DSS-administered fruit flies, a minimal medium not containing each drug and insect 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 female intestine is larger than male intestine, 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) for 15 minutes 3 times 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 anti-fade 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 intestinal wall, 4 days after oral administration of intestinal injuries and each insect sample, 1% FCF brilliant blue dye (Sigma, St. Louis, MO, USA) was supplied and supplied on the 5th day. In the Drosophila, the whole body of Drosophila stained blue was defined as smurf+, and the number of Drosophila stained was counted.

7. DSS concentration and administration time setting for intestinal leak syndrome model production

(1) concentration

As a result of supplying 3, 5, 7, 9% DSS for 5 days to select an appropriate concentration of DSS to induce intestinal injury 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 between 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 on the 5th day of administration.

(2) administration time

Since the expression level of transcriptome changes over time after treatment with a specific substance, in order to obtain a large amount of the target specific expression gene for each specific inducer, it is necessary to set a time for transcript analysis after administration of the substance. Therefore, in order to obtain a large amount of genes involved in the maintenance of intestinal homeostasis during intestinal injury, representative genes of several signaling pathways (JAK-STAT, EGFR, AMP, JNK, Wingless) known to regulate the division and differentiation of intestinal stem cells. After selection, the gene expression patterns were confirmed by setting the time before and after 20 hours, which is estimated to be the time affected by the division and differentiation of intestinal stem cells in Drosophila after administration of the damaging substance in the previous studies. Most genes related to the regulation of intestinal stem cells showed a pattern of increasing expression over time after administration of each damaging substance. We confirmed that the expression of the genes selected by us increased within 24 hours after treatment of the injured substance under the criteria that each gene expression was expressed more than 1.5 times compared to the control group and differentiated expression when treated with a specific substance compared to normal. In addition, we confirmed that the length and thickness of the Drosophila intestine were changed by each injured substance, and the time for the Drosophila intestine to change significantly for all damaged substances was 18-20 hours and 48-72 hours.

Experiment result

1. Toxicity test of beetle extract

As a result of confirming the survival rate after administration of 2 mg/mL concentration of longevity beetle extract (ADL) alone to Drosophila for 5 days, it was found that the survival rate of 80% or more was observed 5 days after oral administration of the sample (FIG. 2 ). Therefore, it was found that the extract of the beetle used in this study was not toxic to fruit flies.

2. Efficacy test of beetle extract

(1) Analysis of the survival rate of fruit flies

As a result of confirming whether the longevity beetle extract (ADL) has the effect of improving the survival rate of fruit flies with intestinal damage (leaking intestines) by oral administration of 7% DSS, the result of confirming that the longevity beetle extract is about the survival rate of fruit flies administered orally with 7% DSS It was increased by more than 20%, and as a result of evaluating the significance of the survival rate of Drosophila on the 5th day of administration, it was found that the toxicity of Drosophila by DSS was significantly reduced with a P-value <0.05 (Fig. 3; significance test student-t test) , * P value <0.05).

(2) Intestinal morphological analysis

According to a previously reported study, it is known that the length and thickness of the intestine isolated from the mice or rats administered with DSS are shorter and thinner than in the normal group. Therefore, in this study, the change in the morphology (length) of the intestine of Drosophila was confirmed with DSS or DSS with oral beetle extract. As a result, the intestine length of the fruit flies administered with 7% DSS alone was shorter than that of the normal Drosophila group, and as a result of oral administration of the beetle extract with DSS, the intestine length increased by the length of the normal group (Fig. 4; Significance test student-t test, * P value <0.05).

(3) Smurf Assay for confirmation of intestinal barrier function

Although the exact mechanism of causing damage to the intestinal tissue of DSS is not known, it has been reported that DSS induces damage to the intestinal tissue by destroying the basement membrane organization of the intestinal tissue. Therefore, a Smurf assay was performed to determine the relationship between the effect of enhancing the survival rate of Drosophila beetle extract with the presence or absence of barrier tissue destruction. As shown in Fig. 5, the fruit flies stained with blue all over the body were considered Smurf+ and the number was counted. As a result, compared to the untreated group (1%), the proportion of Drosophila (Smurf+) stained whole body increased significantly to 31% in the 7% DSS alone oral administration group. It was confirmed that the ratio of the number of Smurf+ Drosophila was significantly reduced when DSS and the beetle extract were administered simultaneously (Fig. 6; significance test student-t test, * P value <0.05). Therefore, it is presumed that the beetle extract increased the survival rate of fruit flies by inhibiting the destruction of the basal membrane tissue of the intestinal tissue by DSS.

(4) Intestinal stem cell differentiation promotion analysis

In order to determine whether the extract of Longevity beetle, which improves the survival rate of fruit flies administered with DSS orally, improves the differentiation ability of intestinal stem cells, after dissecting the intestine of Drosophila, the intestinal stem cell marker Delta and the cell proliferation marker phosphorylation Immunostaining analysis was performed on the resulting histone 3 (phosphorylated Histone3; pH3). As a result, the number of cells stained with Delta and pH3 increased by DSS, and the number was decreased by oral administration of a beetle extract, which was similar to that of the control group (Fig. 7; Scale bar = 50 μm). According to the 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, if DSS and Longevity beetle extract are administered orally at the same time, longevity beetle extract promotes differentiation of intestinal stem cells and does not recover intestinal damage, and longevity beetle extract directly inhibits the intestinal damage of DSS. It is presumed to inhibit.

(5) Analysis of intestinal apoptosis pattern

In this study, a TUNEL assay was conducted to confirm the effect of the extract of Longevity beetle on apoptosis during 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 administered with DSS and Longevity beetle extract, 29.1% was caused by DSS. It was confirmed that intestinal cell death was reduced (Fig. 8; significance test student-t test, * P value <0.05).

(6) E-Cadherin expression level analysis

As a result of confirming the expression of the E-cadherin gene, which plays a key role in leaky gut syndrome, the expression of E-cadherin was reduced in Drosophila induced intestinal damage by DSS, but E-cadherin extract was administered simultaneously with DSS. It was found that the expression amount of cadherin was overexpressed three times or more (Fig. 9; significance test student-t test, * P value <0.05), which increases the binding force between cells constituting the intestine, thereby preventing leakage of the intestinal barrier. It implies that it is possible to prevent or treat leak syndrome.

Claims (6)

Longevity beetle ( Allomyrina dichotoma ) intestinal leak syndrome (leaky gut syndrome) prevention or treatment pharmaceutical composition comprising an extract.
The composition of claim 1, wherein the extract is an ethanol extract.
The composition of claim 1, wherein the longevity beetle extract is an extract of larva.
Longevity beetle ( Allomyrina dichotoma ) intestinal leak syndrome (leaky gut syndrome) prevention or improvement health functional food containing the 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 longevity beetle extract is an extract of larva.

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