KR102328203B1 - Fermented Orostachys japonicus water extract and method of manufacturing the same to improve fuction of dameged liver by oxidative stress - Google Patents

Abstract

The present invention relates to a Wasong extract for improving liver damage caused by oxidative stress, and more particularly, to a food composition for improving liver damage due to oxidative stress containing the Wasong fermented extract as an active ingredient.
In addition, the present invention relates to a method for producing a Wasong extract, comprising the steps of: drying the Wasong at 30 ° C. for 5 days at a low temperature; powdering the dried wasong; Lactobacillus plantarum ( Lactobacillus plantarum ) and fruit juice medium were added to the wasong powder to ^{inoculate 0.5 ml of a strain culture solution maintaining a concentration of 10 6} to 10 ⁷ cells/ml, and then stand still in an incubator at 30° C. and a shaker incubator. It comprises the step of culturing.
Accordingly, it may have the effect of protecting the liver from oxidative stress caused by tBHP and improving the function of the liver.

Description

Wasong fermented extract and method of manufacturing the same to improve function of dameged liver by oxidative stress}

The present invention relates to a fermented Wasong extract capable of improving liver damage caused by oxidative stress and a method for preparing the same, and more particularly, to oxidative stress caused by active oxygen, etc. using a fermented product of the Wasong extract. To prevent liver damage or to improve the damaged liver by Wasong fermented extract and a manufacturing method thereof.

The liver is an important organ in the human body that is responsible for a number of metabolic processes, such as metabolizing carbohydrates, amino acids, proteins, and fats and decomposing toxic substances that enter the body. There are many causes of liver damage, including alcohol, smoking, viral infections, and poison poisoning. In particular, tert-butyl hydroperoxide (hereinafter referred to as 'tBHP') is generated by reactive oxygen species (ROS), which causes lipid peroxidation and oxidative damage to the liver.

Oxidative stress is a cause of typical diseases that appear in humans living in a rapidly changing modern society, and is caused by pollutants and pollutants, wrong eating habits, or mental stress. In particular, tBHP is widely known as a substance that directly induces oxidative stress, and generates reactive oxygen species (ROS) in cells, which act as a basic cause of various diseases and are mediators of aging, cancer, and immunosuppression. plays a role Recently, various studies on ROS have been reported, and in particular, many studies on oxidative stress on human organs such as liver and kidneys have been conducted.

The present inventors confirmed that the Wasong extract and the Wasong fermented extract are effective against liver damage caused by oxidative stress and completed the present invention.

KR 10-0900343 B1 (2009. 05. 25.)

An object to be solved in the present invention is to provide a food composition capable of preventing or improving liver damage caused by oxidative stress using wasong, a natural material.

The composition according to the present invention for solving the above problems provides a Wasong extract for improving liver damage due to oxidative stress and a method for preparing the same, and has its technical characteristics.

The present invention is a fermented broth fermented with Lactobacillus plantarum by obtaining an extract from Wasong as a main material, and can have the effect of protecting the liver from oxidative stress caused by tBHP and improving the liver function.

1 is a graph showing the cytotoxicity results for HepG2 hepatocytes of Wasong extract (OJD) and Wasong fermentation extract (FOJD)
Figure 2 is a graph showing the results of inhibition of cytotoxicity of HepG2 hepatocytes induced by t-BHP by Wasong extract (OJD) and Wasong fermentation extract (FOJD);
Figure 3 is a graph showing the effect of Wasong extract (OJD) and Wasong fermentation extract (FOJD) on the production of reactive oxygen species in HepG2 hepatocytes
Figure 4 is a graph showing the effect of Wasong extract (OJD) and Wasong fermentation extract (FOJD) on GSH production of HepG2 hepatocytes
5 is a graph showing the effect of Wasong extract (OJD) and Wasong ferment extract (FOJD) on MDA production of HepG2 hepatocytes
6 to 19 are graphs showing the gene expression inhibition effect related to oxidative stress of Wasong extract (OJD) or Wasong fermentation extract (FOJD)

The terms or words used in the present specification and claims conform to the technical idea of the present invention based on the "principle that the inventor can appropriately define the concept of a term in order to best describe his invention" It should be interpreted as the meaning and concept that

Therefore, the embodiments described in this specification and the configurations shown in the drawings are only the most preferred embodiments of the present invention, and do not represent all the technical ideas of the present invention, so various equivalents that can replace them at the time of the present application It should be understood that there may be water and variations.

Wasong ( Orostachys japonicus) is a perennial herbaceous plant of the sedumaceae family called rock brush. It is used as a herbal medicine in Korea and Asian countries to treat various diseases including liver disease. A total of 31 chemical components, including flavonoids, triterpenes, and steroids, were identified as the components present in Wasong.

Lactobacillus plantarum ( Lactobacillus plantarum ) is a lactic acid bacillus performing homozygous or heterozygous fermentation, and is commonly found in the intestinal tract of animals, including humans, and in the fermentation process of dairy products or vegetables. Microorganisms of the genus Lactobacillus maintain the pH of the intestine to be acidic, suppressing the reproduction of harmful bacteria such as E. coli and Clostridium, and improving diarrhea and constipation, as well as vitamin synthesis, anticancer action, and lowering of serum cholesterol. It is known to play a role. Acidophillin produced by lactic acid bacilli is known to inhibit the growth of Shigella, Salmonella, Staphylococcus aureus, Escherichia coli, and the like. In addition, it inhibits the proliferation of diarrhea-causing bacteria and normalizes the intestinal flora to stop diarrhea.

Example 1: Preparation of Wasong Extract

After thoroughly washing the wasong purchased at the oriental medicine room in Gyeongdong Market, Seoul, use an ultrafine particle grinder (Jet Mill, Ducksan, SiHeung, Korea) to break it into fine powder, and then add 70% ethanol to 100g/L for 5 hours each. After extraction under reflux three times and cooling, it was filtered with Whatman #10 filter paper. The filtrate thus obtained was again finally filtered using a filter of 0.25 uM. The filtered extract was concentrated under reduced pressure at 50° C. or less to prepare a Wasong extract.

Example 2: Preparation of Wasong Fermented Extract

After washing the wasong purchased at the oriental medicine store in Gyeongdong Market, Seoul, dry it at 30°C for 5 days at a low temperature using a dryer (HDG-222, HyunDaeEnetech, HwaSung, Korea), and then use an ultra-fine particle grinder (Jet Mill, Ducksan, SiHeung, Korea) 5 g was weighed by subdividing into fine powder using

After inoculating 0.5 ml of the strain culture medium maintaining the concentration of ^{10 6} ~ 10 ⁷ cells/ml by adding liquid fermented Lactobacillus plantarum and fruit juice medium to Wasong powder, incubator at 30℃ and shaking incubator was politically cultured in Samples were shaken intermittently during fermentation.

Experimental Example 1. Measurement of polyphenol and flavonoid content

1-1. experimental process

The content of polyphenols and flavonoids contained in the Wasong extract of Example 1 and the Wasong fermented extract of Example 2 was measured. In the case of the Wasong fermented extract of Example 2, the content was measured according to the number of fermentation days (1-5 days).

1-2. Experiment result

As shown in Table 1, it can be seen that the polyphenol content was higher in the case of the Wasong fermented extract (FOJD) fermented for 1 day compared to the Wasong extract (OJD). However, as the number of fermentation days increased, the polyphenol content decreased.

In the case of flavonoids, it can be seen that there is a clear difference between before and after fermentation.

Experimental Example 2. Analysis of major components of Wasong extract and fermented Wasong extract

2-1. experimental process

The main component content analysis of the Wasong extract of Example 1 and the Wasong fermented extract of Example 2 was performed. The main components detected were gallic acid, catechin, epicatechin, epigallocatechin gallate (EGCG), epicatechin gallate, and isoquercetin.

2-2. Experiment result

As shown in Table 2, it can be seen that only catechin and epicatechin content increased with the passage of fermentation time, and it can be seen that other components decreased as the fermentation progressed. In addition, it can be seen that the contents of catechin and epicatechin increased after fermentation than before fermentation.

Experimental Example 3. DPPH and ABTS radical scavenging activity analysis

3-1. experimental process

DPPH and ABTS radical scavenging activity of the Wasong extract of Example 1 and the Wasong fermented extract of Example 2 was analyzed. As a positive control, ascorbic acid was used.

3-2. Experiment result

As shown in Table 3, in the case of Wasong extract (OJD), it can be seen that even with a smaller amount than ascorbic acid, the DPPH radical scavenging ability is excellent, and the ABTS radical scavenging ability is excellent at almost half.

In the case of Wasong fermented extract (FOJD), it can be seen that the capacity of the extract required for scavenging DPPH and ABTS radicals increases as the fermentation period increases.

Experimental Example 4. Evaluation of the effect of Wasong fermented extract on hepatocyte survival

4-1. experimental process

HepG2 hepatocytes were treated with different concentrations of wasong extract (OJD) and fermented wasong extract (FOJD) at a concentration of 0-500 μg/mL, then tBHP was added and reacted for 4 hours.

4-2. Experiment result

As shown in Figure 1, the Wasong extract (OJD) showed little cytotoxicity in all concentration ranges, but the Wasong fermented extract (FOJD) showed little cytotoxicity up to a concentration of 250㎍/㎖, It can be seen that the concentration shows cytotoxicity.

And, as shown in FIG. 2, cytotoxicity was induced in the tBHP-treated experimental group, thereby reducing the survival rate of hepatocytes by about 60-80%. However, the experimental group treated with the Wasong extract (OJD) and the Wasong fermented extract (FOJD) had high cell viability in a dose-dependent manner (5 ~ 50 μg/ml), and the cell viability in the section pretreated with the sample at 7.8 μg/ml this was the highest

In addition, the cell viability of the experimental group treated with the Wasong ferment extract (FOJD) was higher than the cell viability of the experimental group treated with the Wasong extract (OJD).

Experimental Example 5. Evaluation of the effect of Wasong fermented extract on the generation of reactive oxygen species

5-1. experimental process

When the HepG2 hepatocytes were treated with the ferment extract, the amount of reactive oxygen species was measured using dichlorodihydrofluorescein diacetate (DCFH-DA) to evaluate the effect on the generation of reactive oxygen species. HepG2 hepatocytes induced by oxidative stress with tBHP at 1 x 10 ⁶ cells/ml were treated with 20uM DCFH-DA and cultured for 30 minutes. The culture medium was centrifuged at 2500 rpm for 10 minutes to recover the supernatant, and the fluorescence value of dichlorofluorescein (DCF) generated by measuring at an excitation wavelength of 485 nm and an emission wavelength of 523 nm using a spectrofluorometer was measured. measured.

5-2. Experiment result

As shown in FIG. 3, DCF fluorescence value due to the generation of reactive oxygen species was decreased in a concentration-dependent manner in the experimental group treated with the Wasong ferment extract (FOJD) than in the experimental group treated with the Wasong extract (OJD). In particular, it was confirmed that when 25-100 ㎍ / ㎖ of Wasong fermented extract (FOJD) was treated, the inhibitory effect on the production of reactive oxygen species was excellent.

Experimental Example 6. Evaluation of the effect of Wasong fermented extract on GSH production

6-1. Experiment preparation

The GSH (glutathione) concentration of HepG2 hepatocytes induced by oxidative stress with tBHP to induce hepatitis was measured by Ellman's method. 0.4% sulfosalicylic acid was added to 1 x 106 cells/ml of HepG2 hepatocytes, and the supernatant was recovered after centrifugation at 2500 rpm for 10 minutes. 0.3 ml of the recovered supernatant was taken, 2.7 ml of a disulfide reagent was added, and left at room temperature for 20 minutes. Absorbance was measured at 412 nm and quantified using a standard calibration curve.

6-2. Experiment result

As shown in FIG. 4, the concentration of GSH increased in a concentration-dependent manner in the experimental group treated with the Wasong ferment extract (FOJD) than in the experimental group treated with the Wasong extract (OJD). In particular, it was confirmed that the GSH increasing effect was excellent when 100 ㎍ / ㎖ of Wasong fermented extract (FOJD) was treated.

Experimental Example 7. Evaluation of the effect of Wasong fermented extract on MDA production

7-1. experimental process

The following experiment was performed to measure malondialdehyde (MDA), an indicator of lipid peroxidation. HepG2 hepatocytes, which induced hepatitis by inducing oxidative stress with tBHP, were cultured at 37°C in a 5% carbon dioxide incubator. Then, after centrifugation at 2500 rpm for 10 minutes, the supernatant was removed, and 0.37% (w/v) thiobarbituric acid (TBA) and 15% (w/v) trichloride were added to the pellet. A 0.25 M hydrochloric acid (HCl) solution containing trichloroacetic acid (TCA) was added and heated to 80° C. for 15 minutes. After adding the same amount of butanol and centrifuging at 10,000 rpm for 5 minutes, the supernatant was recovered and absorbance was measured at a wavelength of 530 nm.

7-2. Experiment result

As shown in FIG. 5 , the concentration of MDA was decreased in a concentration-dependent manner in the experimental group treated with the Wasong ferment extract (FOJD) than in the experimental group treated with the Wasong extract (OJD). In particular, it was confirmed that the MDA production inhibitory effect was excellent when 100 μg/ml of Wasong ferment extract (FOJD) was treated.

Experimental Example 8. Analysis of mRNA levels of factors related to oxidative stress

8-1. experimental process

Factors increased when oxidative stress occurred were analyzed at the mRNA level. The genes to be analyzed are AMPKα-1 (5' adenosine monophosphate-activated protein kinase-1), AMPKα-2 (5' adenosine monophosphate-activated protein kinase-2), ACC-1 (acetyl-CoA carboxylase 1), SOD-1 (superoxide dismutase 1), SOD-2 (superoxide dismutase 2), CAT (catalase), GPX (glutathione peroxidase), and the like.

8-2. experimental process

1) In the AMPKα-1 gene, a large amount of mRNA was transcribed according to the presence of tBHP as shown in FIG. 6 , and the transcription amount was rapidly decreased when the concentration of Wasong extract (OJD) was 12.5 μg/ml.

2) In the AMPKα-1 gene, a large amount of mRNA was transcribed according to the presence of tBHP as shown in FIG. 7 , and the transcription amount was rapidly reduced when the concentration of Wasong ferment extract (FOJD) was 125 μg/ml.

3) In the AMPKα-2 gene, a large amount of mRNA was transcribed according to the presence of tBHP as shown in FIG. 8, and when the concentration of Wasong extract (OJD) was 6.25 μg/ml, the transcription amount sharply decreased, and 12.5 μg The expression level at the time of /ml converged to 0.

4) In the AMPKα-2 gene, a large amount of mRNA was transcribed according to the presence of tBHP as shown in FIG. 9, and when the concentration of Wasong ferment extract (FOJD) was 31.3 μg/ml, the transcription amount was rapidly reduced.

5) In the ACC-1 gene, a large amount of mRNA was transcribed according to the presence of tBHP as shown in FIG. 10, and the concentration of Wasong extract (OJD) was reduced to half when 3.13 μg/ml, and 12.5 μg/ml. The expression level at the time of ml converged to 0.

6) As shown in FIG. 11, a large amount of mRNA was transcribed in the ACC-1 gene according to the presence of tBHP, and when the concentration of Wasong ferment extract (FOJD) was 31.3 μg/ml, the transcription amount was rapidly decreased.

7) In the SOD-1 gene, a large amount of mRNA was transcribed according to the presence of tBHP as shown in FIG. 12, and the concentration of Wasong extract (OJD) was reduced to 1/3 level when 3.13 μg/ml, 12.5 The expression level at the time of μg/ml converged to 0.

8) As shown in FIG. 13 , a large amount of mRNA was transcribed in the SOD-1 gene according to the presence of tBHP, and when the concentration of Wasong ferment extract (FOJD) was 62.5 μg/ml, the transcription amount was rapidly reduced.

9) In the SOD-2 gene, a large amount of mRNA was transcribed according to the presence of tBHP as shown in FIG. 14, and the concentration of Wasong extract (OJD) was decreased to 1/3 level when 3.13 μg/ml, 12.5 The expression level at the time of μg/ml converged to 0.

10) As shown in FIG. 15 , a large amount of mRNA was transcribed in the SOD-2 gene according to the presence of tBHP, and when the concentration of Wasong ferment extract (FOJD) was 62.5 μg/ml, the transcription amount was reduced to half.

11) In the CAT gene, a large amount of mRNA was transcribed according to the presence of tBHP as shown in FIG. 16 , and the concentration of Wasong extract (OJD) was rapidly decreased when 3.13 μg/ml and 12.5 μg/ml. The expression level converged to 0.

12) In the CAT gene, a large amount of mRNA was transcribed according to the presence of tBHP as shown in FIG. 17, and when the concentration of Wasong ferment extract (FOJD) was 31.3 μg/ml, the transcription amount was reduced to half.

13) In the GPX gene, a large amount of mRNA was transcribed according to the presence of tBHP as shown in FIG. 18, and the concentration of Wasong extract (OJD) was rapidly decreased when 3.13 μg/ml and 12.5 μg/ml. The expression level converges to almost zero.

14) As shown in FIG. 19 , a large amount of mRNA was transcribed in the GPX gene according to the presence of tBHP, and when the concentration of Wasong fermented extract (FOJD) was 62.5 μg/ml, the transcription amount was reduced to half.

Claims (4)

delete delete delete Drying the wasong at 30° C. for 5 days at a low temperature to prepare a dried wasong;
preparing a wasong powder by powdering the dried wasong;
Lactobacillus plantarum ( Lactobacillus plantarum ) and fruit juice medium were added to the wasong powder to ^{inoculate 0.5 ml of a strain culture solution maintaining a concentration of 10 6} ~ 10 ⁷ cells/ml, and then incubate 4 in an incubator at 30° C. and a shaking incubator. Preparing a Wasong fermented extract by stationary culture for one day; and
Preparing a food composition containing 100 μg/ml of the Wasong ferment extract based on ^{1 x 10 6} cells/ml of HepG2 hepatocytes induced by oxidative stress to induce hepatitis;
A method for preparing a food composition for preventing hepatitis due to oxidative stress.

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