KR101344055B1 - Composition for improving liver function containing fermented liquor of Codonopsis lanceolata extract as effective component - Google Patents

Abstract

The present invention provides a composition for improving liver function and a health food for improving liver function containing the fermented product of Deodeok extract.

Description

Composition for improving liver function containing fermented liquor of Codonopsis lanceolata extract as effective component}

The present invention relates to a composition for improving liver function containing the fermented product of Deodeok extract as an active ingredient, and more particularly, a composition for improving liver function and a liver food improving composition containing the fermented product of Deodeok extract as an active ingredient. It is about.

In Korea, the mortality rate of liver cancer was 23.4 per 100,000 people in the world, the first in the world, and the chronic liver disease was the third in 28.8 cases. In recent years, the National Statistical Office (NSO) has reported that 56 deaths per 100,000 population in Korea are among the highest causes of liver disease. Alcoholic liver disease caused by excessive drinking, such as fatty liver, alcoholic hepatitis, alcoholic cirrhosis, etc., aggravate the liver damage due to mental stress and smoking, the body's defense and detoxification effect can cause the immune system and cause other diseases. As the damage caused by the liver-related diseases increases, the interest and demand of consumers for the liver functional improvement products are increasing not only in the liver-related therapeutic market but also in the health functional food market.

Codonopsis lanceolata ) is a perennial herb belonging to the genus Campanulaceae, and has been used for ginseng substitutes since its roots are known as larva (Radix Codonopsis lanceolata). In oriental medicine, it is used for Jinhae, expectorant, detoxification, asthma, etc. In addition to its unique flavors, carbohydrates, and proteins, it is rich in minerals and vitamins such as calcium, phosphorus and iron, and has been widely used for food. Examples of the components of the dodec are sterol, N-formylharman, perlolyne, norharman, squalene, a polyacetylene-based substance presumed to be the main component of the fragrance, cyclobutenol, terpenoid, and saponin have been identified.

Studies on deodeok conducted in Korea include the effects of increasing the immune activity of deodeok extract (Seo Jung-sook, 1996, Korean Journal of Food and Nutrition, 9 (4): 379-384) The Korean Journal of Food Science and Technology 23 (3): 311-316) has recently been reported as a functional food material that can enhance immunity in vivo as a study suggesting an action that enhances immunity.

Recently, researches are being conducted on fermenting herbal preparations such as herbal medicines to enhance useful ingredients and to increase the efficacy and absorption rate as functional food materials.

Therefore, the present inventors investigated the improvement of liver function by treating the fermented product of Deodeok extract with hepatocytes damaged by acetaminophen using Lactobacillus brevis , a kind of Lactobacillus. Acetaminophen is a drug widely known as an analgesic or antipyretic. It is a drug that causes chronic hepatitis or liver failure when used in chronic alcohol patients or when overdose (Gyamlani GG, 2002 Crit. Care).

Korean Laid-Open Patent Publication No. 2012-0048801 discloses a buttercup fermentation broth for preventing and improving liver function, and a method for preparing the same, and Korean Patent No. 0631073 discloses a composition for preventing and treating alcoholic liver disease and alcoholic hyperlipidemia including Deodeok extract. It is disclosed, but as for the composition for improving liver function containing the fermented product of the deodeok extract as an active ingredient as in the present invention has not been found.

The present invention is derived from the above requirements, the present invention, but the development of a functional material that can improve the liver function is desired, but these days, a lot of restrictions due to safety problems such as side effects, relatively high safety natural products The present invention was completed by developing a composition for improving liver function by fermenting an induck extract with lactic acid bacteria.

In order to solve the above problems, the present invention provides a composition for improving liver function containing the fermented product of the deodeok extract as an active ingredient.

In addition, the present invention provides a health food for improving liver function containing a fermented product of Deodeok extract.

According to the present invention, the composition of fermented Deodeok extract with lactic acid bacteria is effective for improving liver function, and because it uses a material obtained from natural products, does not cause side effects, and can ensure safety, and functional food industry for improving liver function. It would be a very useful invention.

Figure 1 shows the pH change according to the lactic acid fermentation period of the deodeok extract.
Figure 2 shows the change in acidity according to the lactic acid fermentation period of Deodeok extract.
Figure 3 shows the hepatocyte vitality of mice according to the amount of acetaminophen.
Figure 4 is a result of the cell viability of the liver protective effect of the deodeok extract or fermentation obtained after fermenting these extracts with lactic acid bacteria against cytotoxicity induced by acetaminophen in rat liver cells. Rat hepatocytes were treated with deodeok extracts or fermented products obtained after fermenting these extracts with lactic acid bacteria and 10 ug / mL, and 10 mM acetaminophen for 24 hours. Cell viability was measured by MTT assay.
5 is a result of examining the liver protective effect of the fermented product obtained after the fermentation of Deodeok extract or these extracts with lactic acid bacteria against cytotoxicity induced by acetaminophen in rat liver cells by LDH (lactate dehydrogenase) concentration. Rat hepatocytes were treated with deodeok extracts or fermented products obtained after fermenting these extracts with lactic acid bacteria and 10 ug / mL, and 10 mM acetaminophen for 24 hours. Hepatoprotective effect was measured by LDH analysis.
Figure 6 is a result of investigating the hepatoprotective effect of the deodeok extracts or fermentation products obtained after fermenting these extracts with lactic acid bacteria against the cytotoxicity induced by acetaminophen in rat hepatocytes by ALT (Alanine aminotransferase) concentration. Rat hepatocytes were treated with deodeok extracts or fermented products obtained after fermenting these extracts with lactic acid bacteria and 10 ug / mL, and 10 mM acetaminophen for 24 hours. Hepatoprotective effect was measured by ALT analysis.

In order to achieve the object of the present invention, the present invention provides a composition for improving liver function containing the fermented product of the deodeok extract as an active ingredient.

In the composition according to the embodiment of the present invention, the fermented product of the deodeok extract may be prepared by fermentation after adding lactic acid bacteria to the deodeok extract, the lactic acid bacteria may preferably be a strain of the genus Lactobacillus, more preferably It may be Lactobacillus brevis , but is not limited thereto.

In the composition according to an embodiment of the present invention, the deodeok extract may be a deodeok hydrothermal extract, but is not limited thereto.

The term "extract" in the present invention refers to an active ingredient isolated from a natural product. The extract may be obtained by an extraction process using water, an organic solvent, or a mixed solvent thereof. Lt; RTI ID = 0.0 > formulated < / RTI > The extracted liquid can be used directly or by concentrating and / or drying. When extracted with an organic solvent, methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof is used, and the extract can be extracted at room temperature or warmed under conditions where the active ingredient is not destroyed or minimized. Depending on the organic solvent to be extracted, the degree of extraction and loss of the active ingredient of the medicine may be different, so select an appropriate organic solvent. The extraction method is not particularly limited, and examples thereof include hot water extraction, cold needle extraction, ultrasonic extraction, and reflux extraction.

The solvent extract may further comprise a step of filtering the extract to remove suspended solid particles. Particles may be filtered using cotton, nylon, or the like, or ultrafiltration, freezing, centrifugation, or the like may be used, but is not limited thereto.

When the extract is concentrated, methods such as reduced pressure concentration, reverse osmosis concentration and the like can be used. The post-concentration drying step includes, but is not limited to, freeze drying, vacuum drying, hot air drying, spray drying, vacuum drying, foam drying, high frequency drying, infrared drying and the like.

In the composition according to an embodiment of the present invention, the fermentation of the deodeok extract is preferably

(A) sterilizing for 13 to 17 minutes at 120 ~ 122 ℃ by adding glucose and skim milk powder to the deodeok hot water extract; And (b) after cooling the sterilized additive of step (a), Lactobacillus brevis ( Lactobacillus) brevis ) can be prepared by inoculating the strain and fermentation at 35 ~ 39 ℃ 12-16 days,

More preferably,

(A) sterilizing for 13-17 minutes at 120 ~ 122 ℃ by adding 0.8 ~ 1.2% glucose and 0.8 ~ 1.2% skim milk powder to the deodeok hot water extract; And (b) after cooling the sterilized additive of step (a), inoculated with Lactobacillus brevis strain ( Lactobacillus brevis ) strain can be prepared by fermentation at 35 ~ 39 ℃ 12-16 days,

Most preferably,

(a) adding 1% glucose and 1% skim milk powder to Deodeok hydrothermal extract and sterilizing at 121 ° C. for 15 minutes; And (b) after cooling the sterilized additive of step (a), Lactobacillus brevis ( Lactobacillus) brevis ) can be prepared by inoculating a strain for 14 days at 37 ℃, but is not limited thereto.

In addition, the present invention provides a functional food for improving liver function containing a fermented product by Lactic acid bacteria fermentation of Deodeok extract.

When the fermented product by lactic acid bacterium fermentation of Deodeok extract of the present invention is used as a food additive, the fermented product by lactic acid bacterium fermentation of Deodeok extract may be added as it is or may be used together with other foods or food ingredients, in a conventional method Can be used accordingly. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). Generally, in the preparation of food or beverages, the fermented products of the present invention are added in an amount of up to 15 parts by weight, preferably up to 10 parts by weight relative to the raw materials. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range .

There is no particular limitation on the kind of the food. As a specific example, the fermented product may be used to produce processed foods that improve the shelf life while simultaneously modifying the characteristics of agricultural products, livestock products or aquatic products. Such processed foods include, for example, confectionery, drinks, liquor, fermented foods, canned foods, milk processed foods, meat processed foods, noodles and the like. The sweets include biscuits, pies, breads, candies, jellies, gums, cereals (including dinner utensils such as cereal flakes). Beverages include carbonated drinks, functional hot drinks, juices (eg, apples, pears, grapes, aloes, citrus fruits, peaches, carrots, tomato juices, etc.), Sikhye, and the like. Alcoholic beverages include sake, whiskey, shochu, beer, liquor and fruit wine. Fermented foods include soy sauce, miso, and kochujang. Canned food includes canned seafood (eg, tuna, mackerel, canned meat, canned seashells, etc.), canned animal products (eg beef, pork, chicken, turkey canned, etc.), canned produce (corn, peaches, canned pineapples, etc.). Milk processed foods include cheese, butter, yogurt and the like. Meat processed foods include pork cutlet, beef cutlet, chicken cutlet, sausage, sweet and sour pork, nuggets, nubucki, and the like. And noodles such as sealed packaging raw noodles. In addition, the composition may be used in retort food, soup and the like.

In addition, the fermented product may be used to produce a functional food, health food or health supplement food. Functional food, health food or dietary supplement means a food that provides a bioregulatory function, including a physiologically active ingredient in addition to the nutritional function, the fermented product of the present invention has a variety of specific natural Since the extract is included as an active ingredient, it can be used in the manufacture of functional foods, health foods or health supplements.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

1) Raw materials

Deodeok was purchased from Dongbu Herbal Cooperative Corporation.

2) Lactic Acid Bacteria

Lactobacillus brevis was used for lactic acid bacteria fermentation.

3) Sample preparation method

The manufacturing method for the deodeok extract and lactic acid bacteria fermentation is as follows. That is, 4L of purified water was added to 1 kg of Deodeok and extracted at 100 ° C. for 3 hours, and the concentration was 7.5 brix. Each extract was adjusted to distilled water to a final volume of 1 L to include 1%, 3%, and 5%, respectively, and then sterilized at 121 ° C. for 15 minutes by adding 1% glucose and 1% skim milk powder. 10 ml of lactic acid bacterium culture solution was inoculated into the sterilized extract cooled at room temperature and then fermented at 37 ° C. for 14 days. As a control, sterilized extracts which were not subjected to lactic acid bacteria fermentation were used as they were.

4) General ingredient analysis

General ingredients were analyzed according to the Association of Official Analytical Chemists (AOAC) method. In other words, 5g of each sample is placed in a weighing bottle and dried in a 105 ℃ dry oven until it is weighed. The weight is measured and the content is calculated. Inquiry content is pre-incubated at 250 ° C after about 2g of sample. In the direct ingestion method, the crude protein content was calculated by multiplying the nitrogen content measured by the Kjeldahl method by the nitrogen coefficient of 6.25, and the crude fat content was measured by Soxhlet extraction method after drying the sample. In addition, crude fiber was analyzed by the Hennerberg Stohmann improved AOAC method, and the content of soluble nitrogen was calculated by subtracting moisture, crude ash, crude protein, crude fat, crude fiber content from the total amount.

5) Free Sugar Analysis

The free sugar component was analyzed according to the method of Wilson et al. That is, distilled water was added to 20 g of the sample, ground with a homogenizer, stirred, leached to a constant volume of 100 mL, centrifuged (3,000 rpm, 30 minutes), and the supernatant was collected and filtered (Whatman No. 2). The filtered filtrate was purified by Sep-pak C ₁₈ and then filtered through a 0.45㎛ membrane filter (Millipore Co., USA) was used as an analytical sample for HPLC, the content is calculated by the external standard method and HPLC conditions are shown in Table 1.

6) Organic Acid Analysis

Organic acid analysis was performed in the same manner as the pretreatment of free sugar analysis, and the filtrate extracted was analyzed under the conditions of Table 2 using HPLC.

7) Amino Acid Analysis

A) Amino Acid Analysis

1 g of sample was added to a test tube, 10 mL of 6N-HCl solution was added, and the filtrate obtained by hydrolysis at 110 ° C. for 24 hours was centrifuged. The supernatant was concentrated at 50 ° C. to completely evaporate hydrochloric acid and water, followed by 20 mM HCl (pH 2.2). ) Was filtered to 0.4 mL membrane filter, and the filtrate was derivatized with AccQ-Tag reagent and used as analytical sample for high performance liquid chromatography. Analysis conditions are shown in Table 3, and the amino acid content was calculated by an external standard method by an integrator.

B) free amino acid analysis

Free amino acid analysis of the sample was analyzed by Ohara and Ariyosh's method for filtrate obtained by pretreatment in the same way as free sugar quantification. 25 mg of sulfosalicylic acid was added to 10 ml of the filtrate, which was allowed to stand at 4 ° C. for 4 hours, followed by centrifugation (50000 rpm, 30 minutes) to remove proteins, and the supernatant was filtered through a 0.45 μm membrane filter. The obtained filtrate was derivatized with AccQ-Tag reagent and analyzed by HPLC. The analysis conditions are shown in Table 3.

8) pH measurement

The pH change during the fermentation period of the sample was measured using a pH meter (Orion 940. USA) taking 20 mL of the sample.

9) pH measurement

The total acid content during the fermentation period of the sample was centrifuged to take 10 mL of the supernatant, and 1% phenolphthalein was used as an indicator. After titration with 01 N NaOH solution, the total acid content was multiplied by lactic acid coefficient 0.009 to convert lactic acid.

10) Liver protection and liver function improvement experiment

Collagenase perfusion

Sprague-Dawley (SD) rat hepatocytes were isolated by Berry and Friend's modified two-stage collagenase perfusion method. To obtain hepatocytes, SD rats were anesthetized with ether, sterilized the abdomen with 70% ethanol, and then opened to open the right atrium to remove the blood and to remove the Hank's balanced salt with a 21 gauge syringe in the left ventricle. solution; HBSS) 150 ml was injected into the blood vessel. After removing the blood, 100 ml of Korean herbal equilibrium saline containing collagenase (100 U / ml) was recycled to release the hepatocytes. Remove liver tissue and add 60 ml of oriental equilibrium saline solution in 100 mm culture dish. One blade was obtained by cutting with 11 blades. To remove collagenase in this cell suspension, three times Weymouth MB 752/1 medium (5% fetal bovine serum, 2.0 mg / ml bovine serum albumin, 10) ^-6 M dexamethasone, 10 ^-7 M insulin, 5.32 x 10 ^-2 M L-serine, 4.09 x 10 ^-2 M L-alanine, 2.67 x 10 ^-2 M NaHCO ₃ , 100 IU / ml penicillin Rinse with 100 IU / ml streptomycin, 50 / ml gentamicin sulfate. Thus obtained hepatocytes were diluted to 5 x 10 ⁵ cell / ml and attached to a 96-well plate (96-well plate) coated with collagen (collagen) for 4 hours, and then treated with each sample.

B) Cytotoxicity analysis

The primary liver cells of the rats were treated with samples by incubating in 96 well plates at a concentration of 5 x 10 ⁵ cells / ml. After 24 hours of treatment, formazan was formed for 4 hours by adding 0.5 μg / ml tetrazolium-based colorimetric (MTT). After the reaction, the medium was removed, and formazan was dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured at 540 nm using a microplate reader. Cell viability of each sample was calculated by the untreated group with 100% control.

C) liver function test

Enzymes were quantitatively measured using a rat alanine transaminase (ALT) and rat lactate dehydrogenase (LDH) ELISA kit purchased from BlueGene Co. (Shanghai, China) for liver function tests. .

Example  1. Analysis of general ingredients, free sugars, organic acids and amino acids in Deodeok

1) General ingredient content

Of deodeok used in the present invention General ingredient contents are shown in Table 4. The moisture content was 11.45%, the ash content was 3.04%, and the crude protein content was 13.32%. Crude fat content was 2.67% and crude fiber content was 10.49%.

2) free sugar content

Of deodeok and extract used in the present invention Free sugar content is shown in Table 5. The main sugars were glucose and fructose. Maltose was found to be 1.41% in the dry sample, but was not detected in the sample using the extract.

Sucrose was detected in Deodeok and Deodeok extracts by 5.14% and 0.17%, respectively. The free sugar content of the lactic acid fermentation solution prepared by varying the deodeok extract content was lower than all the free sugars, including the total free sugar content, before the lactic acid bacteria fermentation.

3) organic acid content

Organic acid content of the deodeok used in the present invention is shown in Table 6. The main organic acids detected were malic acid, lactic acid and acetic acid. The total organic acid content according to the extract content was increased in proportion to the amount of extracts added in the test groups not inoculated with lactic acid bacteria. Lactic acid bacteria fermentation, the final product, was confirmed that the total organic acid content decreases as the amount of extract added.

4) amino acid content

A) Amino Acid Content

Amino acid analysis results of the samples used in the present invention are shown in Table 7. A total of 16 amino acids were detected in the sample, and the total content was 9,955.83 mg% of Deodeok and 2,259.91 mg% of Deodeok extract. The major constituent amino acids of deodeok and deodeok extracts were arginine, threonine and glutamic acid.

B) free amino acid content

Free amino acid analysis results of the samples used in the present invention are shown in Table 8. Like the constituent amino acids, a total of 16 amino acids were detected in the sample, and total free amino acid contents were 1,075.41 mg% and Deodeok extract 244.88 mg%. The major free amino acids of Deodeok and Deodeok extracts were arginine and proline, and threonine and glycine were not detected in the extracts.

Example  2. Lactic Acid Bacteria Fermentation of Deodeok Extract pH  Changes and lactic acid bacteria Fermented  PH change

1) pH change by lactic acid fermentation

PH change according to the fermentation period of the samples to which the deodeok extract is added is as shown in FIG. The pH of the deodeok extract was the lowest between 1 and 3 days, after which the constant pH was maintained without significant change. The total pH was lowest with 1% added fermentation, followed by 3% added fermentation and 5% added fermentation.

2) Change in Acidity of Lactic Acid Fermentation

Acidity change in the deodeok extract is shown in FIG. The fermented product added 1% of Deodeok extract showed the highest acidity, and greatly increased from 5 to 11 days of fermentation period and then gradually decreased. Acidity increased up to 7 days of fermentation in 3% fermented and 5% fermented extracts of Deodeok extract, and there was no significant difference thereafter.

Example  3. Lactic Acid Bacteria of Deodeok Extract Fermented  Liver protection and liver function improvement effect

A) Effect of Lactic Acid Bacteria Fermentation of Deodeok Extract on Cytotoxicity of Acetaminophen

Rat primary hepatocytes were treated with acetaminophen at concentrations of 0, 0.5, 2.5, 10, 50 mM and then incubated for 24 hours. As a result of MTT experiment in each well after the reaction, cell viability decreased depending on the concentration. When 10 mM acetaminophen was treated to induce hepatotoxicity, hepatocyte cytotoxicity was induced, and hepatotoxicity was induced with 10 mM acetaminophen, and hepatocellular protective effect and liver function improvement effect experiment were performed (FIG. 3).

In order to confirm the hepatocellular protective effect of the deodeok extract fermented with deodeok extract and lactic acid bacteria, 10 mM acetaminophen and 10 ㎍ / mL of each sample was confirmed to confirm the hepatocyte protective effect due to liver toxicity (Fig. 4). As a result, the cell viability of Deodeok extract was measured to be 125.2%, and the Deodeok extract fermented with lactic acid bacteria was measured to be 132.4%. It was confirmed that the extracts were hepatocellular protection, but the deodeok extracts fermented with lactic acid bacteria increased the hepatocellular protection by 7.2%. These results suggest that the fermentation with lactic acid bacteria rather than extracts strengthened the hepatocellular protective effect.

B) Liver function improvement effect test (LDH)

When 10 mM acetaminophen was toxic to hepatocytes, the secretion of lactate dehydrogenase (LDH) secreted was confirmed to be increased compared to the control (FIG. 5). Deodeok extract was measured as 49.7 ng / mL, but deodeok extract fermented with lactic acid bacteria was measured as 10.0 ng / mL, and when fermented with lactic acid bacteria, the secretion of lactate dehydrogenase was lowered by 39.7 ng / mL. The fermented extract was confirmed to improve the liver function.

C) Liver function improvement effect test (ALT)

ALT of primary rat hepatocytes induced by hepatic toxicity with 10 mM acetaminophen increased (FIG. 6). Deodeok extracts were confirmed to decrease the secretion of ALT by 4.5 ng / mL when fermented with lactic acid bacteria, these results confirmed that the extract fermented with lactic acid bacteria than the extract was superior to liver function.

Claims (6)

After adding glucose and skim milk powder to the deodeok hot water extract, the sterilized additives were cooled at 120-122 ° C for 13-17 minutes, inoculated with Lactobacillus brevis strain and fermented at 35-39 ° C for 12-16 days. Composition for improving liver function containing the fermented product of the prepared Deodeok extract as an active ingredient. delete delete delete delete After adding glucose and skim milk powder to the deodeok hot water extract, the sterilized additives were cooled at 120-122 ° C for 13-17 minutes, inoculated with Lactobacillus brevis strain and fermented at 35-39 ° C for 12-16 days. Health food for improving liver function containing the fermented product of the manufactured Deodeok extract as an active ingredient.

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