COMPOSITION COMPRISING BEETLE LARVA OR EXTRACT THEREOF AS ACTIVE INGREDIENT FOR IMPROVING BOWEL MOVEMENT FUNCTION

Abstract

The present invention relates to a composition comprising a beetle larva or an extract thereof for improving a bowel movement function or for alleviating constipation.

Claims

1. A composition comprising a beetle larva or an extract thereof for improving a bowel movement function.

2. The composition of claim 1, wherein the beetle larva extract is a solvent extract or an enzyme extract.

3. The composition of claim 2, wherein the solvent is an extract by water, an organic solvent, or a mixed solvent thereof.

4. The composition of claim 3, wherein the organic solvent is alcohol having 1 to 4 carbon atoms.

5. The composition of claim 2, wherein the enzyme extract is prepared by decomposing a protein by protease.

6. The composition of claim 1, wherein the improvement of the bowel movement function is an increase in fecal weight, an increase in water content in feces, an increase in gastrointestinal transport capacity, or an increase in length and area of intestinal mucosa.

7. A composition comprising a beetle larva or an extract thereof for improving constipation.

8. The composition of claim 7, wherein the constipation is transient constipation, atonic constipation, spastic constipation, bowel movement impaired constipation, or organic constipation.

9. A health functional food comprising beetle larva or an extract thereof for improving constipation.

10. A method of improving a bowel movement function in a subject in need thereof comprising administering an effective amount of the composition according to claim 1 to the subject.

11. The method of claim 10, wherein the beetle larva extract is a solvent extract or an enzyme extract.

12. The method of claim 11, wherein the solvent is an extract by water, an organic solvent, or a mixed solvent thereof.

13. The method of claim 12, wherein the organic solvent is alcohol having 1 to 4 carbon atoms.

14. The method of claim 11, wherein the enzyme extract is prepared by decomposing a protein by protease.

15. The method of claim 10, wherein the improvement of the bowel movement function is an increase in fecal weight, an increase in water content in feces, an increase in gastrointestinal transport capacity, or an increase in length and area of intestinal mucosa.

16. A method of improving constipation in a subject in need thereof comprising administering an effective amount of the composition according to claim 7 to the subject.

17. The method of claim 16, wherein the constipation is transient constipation, atonic constipation, spastic constipation, bowel movement impaired constipation, or organic constipation.

Description

DESCRIPTION OF DRAWINGS

[0087] FIG. 1 is a diagram illustrating results of measuring fecal weight and water content in feces to determine an effect of administration of a beetle larva extract on a fecal index. [N(Normal): normal control; C(Control): loperamide alone; PC (Positive control): Psyllium husk 120 mg/kg; J120: loperamide+beetle larva enzyme extract 120 mg/kg; J500: loperamide+beetle larva enzyme extract 500 mg/kg; J′120: loperamide+beetle larva ethanol extract 120 mg/kg, an error bar represents a standard error, and n=5]

[0088] FIG. 2 is a diagram illustrating results of measuring digestive tract mobility to determine an effect of administration of a beetle larva extract on a gastrointestinal transport capacity. [N (Normal): normal control; C (Control): loperamide alone; PC (Positive control): Psyllium husk 120 mg/kg; J120: loperamide+beetle larva enzyme extract 120 mg/kg; J500: loperamide+beetle larva enzyme extract 500 mg/kg; J′120: loperamide+beetle larva ethanol extract 120 mg/kg, an error bar represents a standard error, and n=5]

[0089] FIG. 3 is a diagram illustrating results of staining a colon tissue with hematoxylin-eosin to determine an effect of administration of a beetle larva extract on intestinal digestive movement.

BEST MODE

[0090] Hereinafter, the present invention will be described in detail by Examples and Experimental Examples. However, these Examples and Experimental Examples are only illustrative the present invention, and the scope of the present invention is not limited to these Examples and Experimental Examples.

Preparation Example 1

[0091] Preparation of Beetle Larva Enzyme Extract

[0092] (1) Step of Controlling Diet of Beetle Larva

[0093] Allomyrina dichotoma larva was used as beetle larva. The 5 to 6-month-old beetle larva dieted on sawdust was subjected to a process of changing a wheat flour diet before use and excreting an excrement while fasting. The beetle larva after 48-hour fasting was washed and frozen-stored.

[0094] (2) Step of Hydrolyzing, Sterilizing, and Wet-Crashing Beetle Larva

[0095] 100 parts by weight of the pretreated raw material was added with 1900 parts by weight of water and then sterilized at 100° C. for 30 minutes. Thereafter, the beetle larva was crushed with a homogenizer at 6000 rpm for 10 minutes to obtain a liquid form that was easily treated with enzymes.

[0096] (3) Step of Treating Beetle Larva Solution with Protease to Decompose Protein

[0097] The crushed beetle larva solution was added with 0.1% of protease, Protamex, and treated at a temperature of 60° C. for 1 hour and then the enzyme was inactivated at 100° C. for 10 minutes.

[0098] (4) Step of Filtering Beetle Larva Extract

[0099] The protease-treated product was filtered under reduced pressure through 1 μm glass fiber filter paper, and then only a filtrate was taken.

[0100] (5) Step of Concentrating Beetle Larva Extract

[0101] The filtrate was concentrated with a decompression concentrator to prepare a concentrate of 10 brix or more.

[0102] (6) Step of Powdering Beetle Larva Extract

[0103] The concentrate was lyophilized and powdered, and specifically, sequentially maintained at −45° C. for 6 hours, at −20° C. for 21 hours, at −15° C. for 11 hours, and at −5° C. for 11 hours and then thawed, and then powdered by removing moisture.

Preparation Example 2

[0104] Preparation of Beetle Larva Ethanolextract

[0105] (1) Step of Controlling Diet of Beetle Larva

[0106] Allomyrina dichotoma larva was used as beetle larva. The 5 to 6-months-old beetle larva dieted on sawdust was subjected to a process of changing a wheat flour diet before use and excreting an excrement while fasting. The beetle larva after 48-hour fasting was washed and frozen-stored.

[0107] (2) Step of Drying and then Crushing Beetle Larva

[0108] The frozen-stored Allomyrina dichotoma larva was washed and then dried at 90° C. for 24 hours. The dried raw material was finely crushed with a crusher for easy extraction.

[0109] (3) Step of Extracting Beetle Larva Powder by Adding Ethanol

[0110] 100 parts by weight of the pretreated raw material was added with 900 parts by weight of ethanol and then extracted at 25° C. and 100 rpm for 24 hours.

[0111] (4) Step of Filtering Beetle Larva Extract

[0112] The extract was filtered under reduced pressure through 1 μm glass fiber filter paper, and then only a filtrate was taken.

[0113] (5) Step of Concentrating Beetle Larva Extract

[0114] The filtered extract was concentrated with a decompression concentrator to prepare a concentrate of 10 brix or more.

[0115] (6) Step of Powdering Beetle Larva Extract

[0116] The concentrate was lyophilized and powdered, and specifically, sequentially maintained at −45° C. for 6 hours, at −20° C. for 21 hours, at −15° C. for 11 hours, and at −5° C. for 11 hours and then thawed, and then powdered by removing moisture.

Experimental Example

[0117] Confirmation of Bowel Movement Function of Beetle Larva Extract

Experiment Example 1: Breeding of Experimental Animals and Design of Experiments

[0118] This study was conducted in accordance with the policies and regulations of the Institutional Animal Care and Use committee (SEMI-19-008) of Southeast Medi-Chem Institute. As experimental animals used in the experiment, a 7-week-old ICR mouse model (male) was received from Hana Bio (Seongnam, Gyeonggi-do, Korea) and quarantined, acclimatized and bred in an animal kennel (Animal Facility Registration Certificate: No. 412) and then experimented. During breeding, a lighting time was set to a 12-hour (07:00 to 19:00) cycle, and a diet and water were freely ingested.

[0119] First, 5 ICR mice (7 weeks old, male) were placed in each group, and the experimental groups were classified into a total of 6 groups. The experimental groups were divided into a normal control group (N), a loperamide administered group (C), a Psyllium husk administered group (PC), a loperamide+low concentration enzyme extract group (120 mg/kg) (J120), a loperamide+high concentration enzyme extract group (500 mg/kg) (J500), and a loperamide+low concentration ethanol extract group (120 mg/kg) (J′120).

[0120] The samples were orally administered for a total of 7 days according to the contents in Table 1, and on day 7 of administration, all the groups except for the normal control group were orally administered with 5 mg/kg of loperamide (in saline) to induce constipation. For a feed, solid feed for the experimental animals (Hana Bio, Seongnam, Gyeonggi-do, Korea) was provided, and changes in body weight was measured twice a week.

TABLE-US-00001 TABLE 1 Group Experiment Note Control group 1 (N) Administration of saline No administration of Loperamide Control group 2 (N) Saline Control group 3 (PC) Psyllium husk 120 mg/kg Example 1 (S120) Preparation Example 1 120 mg/kg Examp1e 2 (S500) Preparation Example 1 500 mg/kg Example 3 (S′120) Preparation Example 2 120 mg/kg

[0121] The measured experimental results were represented as means and standard errors, and the significance test of each group was statistically processed using an anova t-test in the Statview program.

Experiment Example 2: Changes in Fecal Index by Beetle Larva Extract

[0122] The feces of the experimental animals were collected after administration of loperamide (5 mg/kg, body weight) on day 6 of sample administration, and the weight and number of feces per subject were measured. In order to check the water content in feces, a change amount was calculated by comparing a weight immediately after collection with a weight after drying at 105° C. for 48 hours.

Water content(%)=[(weight before drying−weight after drying)/weight before drying]×100

[0123] The results were shown in Table 1 and FIG. 1.

TABLE-US-00002 TABLE 2 After administration of loperamide The number of Fecal Water Group subjects weight (g) content (%) Control group 1 (N) 5 0.373# 37.99* Control group 2 (C) 5 0.093 25.01 Control group 3 (PC) 5 0.163 30.03 Example 1 (S120) 5 0.21* 44.35* Example 2 (S500) 5 0.206* 43.68* Example 3 (S′120) 5 0.092 41.00*

[0124] Table 2 shows the experimental results as means and standard errors. Significance was represented for each group as follows:*compared with P<0.05 C group, # compared with P<0.001 C group.

[0125] N (Normal): normal control; C (Control): loperamide alone administered group; PC (Positive control): Psyllium husk 120 mg/kg; J120: loperamide+beetle larva enzyme extract 120 mg/kg; J500: loperamide+beetle larva enzyme extract 500 mg/kg; J′120: loperamide+beetle larva ethanol extract 120 mg/kg

[0126] As shown in Table 2 and FIG. 1, the water content in feces showed a significant decrease in group C causing constipation, compared to group N without causing constipation, but in all of groups J120, J500, and J′120 administered with the beetle larva extract, the water content in feces was significantly high as compared with group C, and higher than that of the normal control group (group N).

[0127] The fecal weight showed a significant decrease in group C causing constipation, compared to group N without causing constipation, but in the groups J120 and J500 administered with the of the beetle larva enzyme extract, the fecal weight was not significantly decreased, and a decrease in fecal weight was smaller than that in group PC administered with Psyllium husk.

[0128] As a result, it was confirmed that the beetle larva extract prevented the decrease in fecal weight and improved the bowel movement activity by increasing the water content in feces, so that it was confirmed that an effect of preventing or improving constipation was excellent.

Experiment Example 3: Changes in Digestive Tract Mobility Rate by Beetle Larva Extract

[0129] In order to observe an effect of a beetle larva extract on a gastrointestinal transport capacity, the digestive tract mobility rate was evaluated. The digestive tract mobility rate may be evaluated by measuring the length of movement of an administered indicator material out of the entire length of the small intestine at a certain time, and in this experiment, loperamide, which exhibited a gastrointestinal transport inhibition effect, was used as a drug for causing constipation, and phenol red was used as an indicator material.

[0130] Specifically, 5 mg/kg of loperamide was orally administered after 1 hour of the administration of the beetle larva extract. After 30 minutes of loperamide administration, 0.5% phenol red (in 1.5% methylcellulose) serving as an indicator was orally administered, and autopsy was performed after 20 minutes to measure the total intestinal length and a movement distance of phenol red. The measurement results were analyzed using the following equation, which was calculated as a percentage of the movement distance of phenol red with respect to the total intestinal length. The analysis results were shown in Table 3 and FIG. 2.

Digestive tract mobility rate (%)=(movement distance of Phenol red/total intestinal length)×100

TABLE-US-00003 TABLE 3 The number Digestive tract Group of subjects mobility rate (%) Contro group 1 (N) 5 49.0# Contro group 2 (C) 5 21.1 Contro group 3 (PC) 5 28.8* Example 1 (S120) 5 33.7# Example 2 (S500) 5 34.9# Example 3 (S′120) 5 31.5*

[0131] 1) Table 3 shows the experimental results as means and standard errors. Significance was represented for each group as follows:*compared with P<0.05 C group, # compared with P<0.001 C group.

[0132] 2) N (Normal): normal control; C (Control): loperamide alone administered group; PC (Positive control): Psyllium husk 120 mg/kg administered group; J120: loperamide+beetle larva enzyme extract 120 mg/kg; J500: loperamide+beetle larva enzyme extract 500 mg/kg; J′120: loperamide+beetle larva ethanol extract 120 mg/kg

[0133] As shown in Table 3 and FIG. 2, the digestive tract mobility rate was significantly reduced in group C causing constipation, compared to group N, whereas it was confirmed that in the beetle larva extract administered group, the digestive tract mobility rate was significantly increased compared to group C (p<0.05, p<0.001). Furthermore, it was shown that in the beetle larva extract administered group, the digestive tract mobility rate was higher than that of group PC administered with the Psyllium husk extract.

[0134] From this, it was confirmed that the beetle larva extract activated the digestive movement of the intestine, and as a result, it was confirmed that the beetle larva extract can be effectively applied to the prevention or improvement of constipation.

Experiment Example 4: Confirmation of Structural Changes in Colon Tissue by Beetle Larva Extract

[0135] To determine an effect of the beetle larva extract on the colon tissue, the colon tissue was stained with Hematoxylin & Eosin (H&E).

[0136] Specifically, the colon tissue extracted from each experimental group was immobilized in 41 paraformaldehyde immediately after extraction, washed with water, dehydrated, transparent, and penetrated, and then embedded with paraffin. The prepared paraffin block was micro-sectioned and sliced, and then stained with H&E staining. The stained slides were photographed after observation with an optical microscope (Nikon, E600, Japan), and the results were shown in FIG. 3.

[0137] As illustrated in FIG. 3, it was observed that the length and area of the mucous membrane were significantly reduced in group C causing constipation, compared to non-treated group N, and it was confirmed that in the beetle larva extract administered group, the length and area of the mucous membrane were increased compared to group C.

[0138] From this, it was confirmed that the beetle larva extract could help the digestive movement of the intestine, and as a result, it was confirmed that the beetle larva extract can be effectively applied to the prevention or improvement of constipation.

[0139] Considering the above results, the beetle larva extract showed significant increases in fecal weight, water content in feces, digestive tract mobility rate, and length and area of the mucous membrane compared to a loperamide-administered control group, so that the very excellent effect of improving the bowel movement function and improving intestinal motility was exhibited. Meanwhile, clear dose dependence was observed in 120 and 500 mg/kg administered groups, and the beetle larva enzyme extract 120 mg/kg administered group helped the absorption of water in the intestine at the level equivalent to or higher than that of Psyllium husk set as a positive control, so that the effective dose of the beetle larva extract was determined around 120 mg/kg.