KR100845463B1 - Extracting Method of Mushroom Extract, Mushroom Extract Extracted by it and Therapeutic/Preventive Agent for Osteoporosis Containing Mushroom Extract - Google Patents

Abstract

The present invention relates to a method for producing a mushroom extract, a mushroom extract obtained thereby and an osteoporosis prevention and treatment using the same as an active ingredient, washing the mushrooms, putting the mushrooms together with bottled water in an extractor, at a low temperature of 50 ℃ to 70 ℃ It is characterized in that the extraction for at least 3 to 5 hours in a pressureless state. In the case of mushroom extract extracted by the manufacturing method of the present invention because it is extracted under low pressure at low temperature, proteins, chitin, vitamins, minerals, etc., including glutathione to remove harmful oxygen, and phenolic compounds contained differently depending on the type of mushroom (Tannin, Resorcinol), protein polysaccharides, beta glucans, flavone compounds such as quercetin and the like can be kept high without destroying, resulting in osteoblast proliferation effect and some mushroom extracts It has an inhibitory effect on osteoclast proliferation.

Agaricus, Cordyceps, Chaga, Mushroom Extract, Osteoporosis

Description

Extraction Method of Mushroom Extract, Mushroom Extract Extracted by it and Therapeutic / Preventive Agent for Osteoporosis Containing Mushroom Extract}

1 is a graph showing the osteoblast proliferation effect on the Agaricus mushroom extract extracted by the manufacturing method according to the present invention;

Figure 2a and 2b is a graph showing the osteoclast proliferation inhibitory effect on the agaricus mushroom extract extracted by the manufacturing method according to the present invention;

Figure 3 is a graph showing the osteoblast proliferation effect on Cordyceps sinensis extract extracted by the manufacturing method according to the present invention;

4a and 4b are graphs showing the osteoclast proliferation inhibitory effect on Cordyceps sinensis extract extracted by the production method according to the present invention;

Figure 5 is a graph showing the osteoblast proliferation effect on chaga extract extracted by the manufacturing method according to the present invention;

Figure 6 is a graph showing the osteoblast proliferation effect on the mixed solution of agaricus mushroom extract, Cordyceps sinensis extract and chaga mushroom extract extracted by the manufacturing method according to the present invention;

7 is a graph showing the osteoblast differentiation effect of L-ascorbic acid;

8A is a photograph showing CKII expression by L-ascorbic acid treatment;

8B is a graph showing that CKII is associated with osteoblast differentiation effect of L-ascorbic acid;

9A and 9B are photographs showing the inhibitory effect of Ikaros by L-ascorbic acid treatment; And

Fig. 10 is this photograph showing the change of osteoclast differentiation factor by L-ascorbic acid treatment.

The present invention relates to a method for producing a mushroom extract, a mushroom extract obtained thereby, and an agent for preventing and treating osteoporosis using the same as an active ingredient. More specifically, by extracting the mushrooms so as not to destroy the active ingredient from the various mushrooms to have the osteoblast proliferation effect and osteoclast growth inhibition effect.

Osteoporosis is a metabolic bone disease that does not change the chemical composition of bone, and causes bone weakness due to a decrease in bone mass and changes in microstructure, leading to an increased risk of fracture. Bone (bone) is formed by repeating the bone formation process produced by osteoblasts and bone remodeling process in which aged bone is dissolved (bone resorption) by osteoclasts.

Osteoporosis is caused by a balance between gastric bone formation and bone resorption, which is either too fast or too slow to produce absorption, leading to a decrease in bone mass, which reduces bone density and bone strength. It can easily cause fractures.

Research on the development of a cure for osteoporosis has been actively conducted, especially in developed countries that are already entering the world aging society a step further. There is an active development of new drugs that are more effective and have improved safety.

Osteoporosis prevention and treatment is classified into the development of osteoclast-activating drugs that promote bone formation, and osteoclast inhibitors that inhibit osteoclasts that promote bone resorption. In particular, the existing therapeutic agent is mainly to suppress the bone resorption by inhibiting the osteoclasts. However, there is an urgent need to develop a therapeutic agent that actively promotes bone formation to substantially improve the bone formation effect and at the same time minimize the accompanying side effects. This is because, in patients whose bone density has already been reduced below the bone threshold, bone resorption alone cannot lower the risk of fracture, and therefore, the amount of bone must be increased above the fracture threshold.

In addition, therapeutic drugs using hormones such as fluoride, parathyroid hormone, growth factor, and anabolic steroid, which are being developed as therapeutic agents for promoting bone formation, may have a risk of cancer, and functional drugs developed as non-hormonal agents include gastrointestinal disorders. There is a problem that side effects occur.

Therefore, the present inventors can easily obtain from the market, and the mushroom extract has no side effects on the human body has osteoblast proliferation effect to promote bone formation according to the production method, and some mushroom extract also has the effect of inhibiting osteoclast growth. In addition, since L-ascorbic acid, known as a long-term nutritional supplement called vitamin C, has osteoblast differentiation effect, when taken with the above-mentioned mushroom extract, the present invention is confirmed to have an effect on osteoblast differentiation and proliferation. Was completed.

It is a first object of the present invention to provide a method for producing a mushroom extract that promotes proliferation of osteoblasts involved in bone formation and inhibits proliferation of osteoclasts involved in bone resorption.

It is a second object of the present invention to provide a mushroom extract extracted by the above-mentioned mushroom extract manufacturing method.

It is a third object of the present invention to provide an agent for preventing and treating osteoporosis using the above-mentioned mushroom extract as an active ingredient.

A fourth object of the present invention is to provide an agent for preventing and treating osteoporosis in which L-ascorbic acid is added to the mushroom extract.

Mushroom extract manufacturing method according to the present invention to achieve the above object, washing the mushrooms, put the mushrooms in the extractor with bottled water, and extracted for at least 3 to 5 hours in a pressureless state at a low temperature of 50 ℃ to 70 ℃ Characterized in that. Mushroom extract extracted by the manufacturing method according to the present invention because it is extracted under a low pressure at the above-mentioned low temperature, protein, chitin, vitamins, minerals, etc., including glutathione to remove harmful oxygen, depending on the type of mushroom Active phenol compounds (tannin, resorcinol), protein polysaccharides, beta glucans, flavone compounds such as quercetin and the like can be kept high without being destroyed. These active ingredients have osteoblast proliferation effects and some mushroom extracts have osteoclast growth inhibition effects.

Looking at the mushroom extract manufacturing method according to the invention in detail, as follows. The mushroom extract manufacturing method described below was based on the amount that can be taken for 10 days by packing the extracted mushroom extract in a pack of 100cc to 120cc three times a day, and also among the mushrooms, Agaricus mushroom (Agaricus blazei Murill) The amount of the mixture of the extracts of Paecilomyces japonica and Chaga Inonotus obliquus was equivalent to the amount taken three times a day for 10 days.

<Example 1>

First, the case of Agaricus mushroom is demonstrated.

Wash commercially purchased 350 g to 500 g of Agaricus mushrooms, and add Agaricus mushrooms to the extractor with 800 cc of bottled water. The temperature is then adjusted to a low temperature of 50 ° C to 70 ° C and extracted for 3 to 5 hours under no pressure. The extractor generally uses a pressureless seeker extractor used to extract the medicine, and the extractor is preferably able to set the temperature. In the case of Agaricus mushroom extract extracted by this method, since the extraction temperature is not high, the active ingredients can be extracted without damaging the Agaricus mushroom. However, in the case of Agaricus mushrooms, because they are extracted at the low temperature, they cannot be stored for a long time in storage and can be easily damaged. To prevent this, 500cc of bottled water is added back to the extractor in the extracted state and extracted for 1 to 2 hours under no pressure at a high temperature of 85 ° C to 95 ° C. By re-extracting under such high temperature, the mushroom extract is sterilized so that it can remain intact for a long time.

Next, the case of Cordyceps sinensis is demonstrated. Cordyceps sinensis is also extracted by a manufacturing method similar to Agaricus mushroom. 200 g to 300 g of Cordyceps sinensis obtained on the market were washed with water, and 800 cc of bottled water was put together in an extractor and extracted for 3 hours to 5 hours at a low pressure of 50 ° C to 70 ° C. In addition, Cordyceps sinensis also can not be stored for a long time in storage and tends to be easily damaged. To prevent this, 500cc of bottled water is extracted again in the above-mentioned way to prevent this, and it is reapplied for 1 to 2 hours at a high pressure of 85 ℃ to 95 ℃. Will be extracted.

Next, the chaga mushroom will be described. Chaga mushroom is washed with 70g to 85g and put into the extractor with 1200cc to 1300cc of bottled water and extracted for 3 hours to 5 hours at a low pressure of 50 ℃ to 70 ℃. In the case of chaga mushrooms, chaga mushrooms do not need to be extracted again at additional high temperatures since the added capacity is relatively small and does not tend to be easily damaged.

Hereinafter, an experimental example of the osteoblast proliferation effect and the osteoclast growth inhibition effect of each mushroom extract extracted by the above-described manufacturing method will be described.

Experimental Example 1

Figure 1 is a graph showing the osteoblast proliferation effect on the Agaricus mushroom extract extracted by the above-described manufacturing method. Specifically, after diluting Agaricus mushroom extract by concentration and treating it with osteoblasts (HOS cells), it is a graph showing the absorbance measured by MTT assay method. The MTT [3- (4,5-dimethylthiazol-2yl) -2,5-diphenyltetrazoilum bromide] assay is one of the methods for confirming the growth of cells. Forma produced by dehydrogenase of mitochondria in cells It is a method to investigate the proliferation or toxicity of cells by measuring the formazan. Specifically, after incubating for 24 hours in a 96 well plate (well plate), the culture solution was removed, and then cultured for 24 hours further by adding 100 μl of the culture solution together with the mushroom extract, and 25 μl of MTT solution was added thereto. After incubating for 4 hours, 150 μl of DMSO (dimethyl sulfoxide, an organic solvent for dissolving formic acid) was added thereto, mixed until the resulting four-component dissolved, and then absorbance was measured at 540 nm. The higher the absorbance measured, the more proliferating the cells are.

As can be seen in Figure 1 it can be seen that the absorbance increases as the concentration of Agaricus mushroom extract increases. That is, in the case of Agaricus mushroom extract, it can be seen that the proliferation of osteoblasts occurred actively, and it can be seen that it has an osteoblast proliferation effect.

Experimental Example 2

2a and 2b are graphs showing the osteoclast proliferation inhibitory effect on the Agaricus mushroom extract extracted by the above-described manufacturing method. Agaricus mushroom extract extracted by the above-described manufacturing method was diluted by concentration, and treated in osteoclast precursor cell lines (Raw264.7 cells), and then the osteoclast inhibitory effect after 24 hours (Fig. 2a), after 48 hours (Fig. 2b). It was measured by the MTT assay method described above. As can be seen from Figure 2a and Figure 2b Agaricus mushroom extract has the result that the absorbance is lowered as the concentration increases. Therefore, the agaricus mushroom extract can confirm that it has an effect of inhibiting the proliferation of osteoclasts.

Experimental Example 3

In order to measure the osteoblast proliferation effect of Cordyceps sinensis extract using the above-described manufacturing method, the extracted Cordyceps sinensis extract was diluted by concentration, and treated with osteoblast cell lines (HOS cells), and the cell proliferation effect was the same as that of <Experimental Example 1>. It was measured by assay method. The results are shown in FIG. Referring to Figure 3, Cordyceps sinensis extract can be seen that the absorbance increases with increasing concentration, from which it can be seen that it has a proliferation effect of osteoblasts.

Experimental Example 4

In order to measure the osteoclast proliferation inhibitory effect of Cordyceps sinensis extract extracted using the above-described manufacturing method, the extract of Cordyceps sinensis extract was diluted by concentration, and treated with osteoclast precursor cell lines (Raw264.7 cells) for 24 hours. After (Fig. 4a), 48 hours later (Fig. 4b) was measured by the MTT assay method. The results are shown in FIGS. 4A and 4B. As can be seen from Figure 4a and 4b, the absorbance decreases as the concentration of Cordyceps sinensis extract increases, it can be seen from the results that Cordyceps sinensis extract inhibits the growth of osteoclast precursor cells.

Experimental Example 5

In order to measure the osteoblast proliferation effect of chaga extract extracted using the above-described manufacturing method, the extracted chaga extract was diluted by concentration, treated with osteoblast cell line (HOS cells), and the cell proliferation effect was analyzed by MTT assay method. Measured. The results are shown in FIG. In Figure 5, chaga extract does not affect the proliferation of osteoblasts in the case of the concentration of 1/10000, 1/1000, it can be seen that increases the proliferation of osteoblasts at 1/100, 1/10 concentration have.

Experimental Example 6

Osteoblast proliferation was measured for a mixture of three kinds of mushroom extracts, namely Agaricus mushroom extract, Cordyceps sinensis extract and Chaga mushroom extract, which were extracted through the above-described manufacturing method. First, the mixture was diluted by concentration, and treated with osteoblasts (HOS cells), and then the cell proliferation effect was measured by MTT assay method, the results are shown in FIG. As can be seen from Figure 6, the mushroom extract mixed solution extracted by the manufacturing method according to the present invention can be seen that the absorbance increases as the concentration increases, and thus the mushroom extract mixture has the effect of increasing the cell proliferation of osteoblasts It can be seen.

As described above, the agaricus mushroom extract and Cordyceps sinensis extract among mushroom extracts have both osteoblast proliferation effect and osteoclast growth inhibition effect. In addition, chaga extract has a osteoblast proliferation effect at a specific concentration, and in the case of a mixture of these three extracts it can be seen that has a osteoblast proliferation effect.

<Example 2>

In addition, L-ascorbic acid may be added to the mushroom extract extracted by the manufacturing method described above in powder form. This is because L-ascorbic acid has a differentiation effect of osteoblasts as described below. Therefore, the addition of L-ascorbic acid to the mushroom extract has an excellent effect on the prevention and treatment of osteoporosis because it has an effect on both the differentiation and proliferation of osteoblasts. However, L-ascorbic acid may not have osteoblast differentiation effect when taking more than 1000mg per day, it is preferable to take about 100mg to 200mg per day.

Below we look at the differentiation effect of osteoblasts of L-ascorbic acid.

Experimental Example 7

L-ascorbic acid was classified by concentration, i.e., 0.06 mM (milli-mole) and 0.125 mM, and treated for osteoblast cell line (MG63), and ALP (alkaline phosphatase: alkaline phosphatse), which is known as a standard for measuring osteoblast differentiation, It was measured using the substrate reaction. Cell differentiation usually occurs over time, so many people wait for more than six days. Therefore, in this experiment, we confirmed the increase of ALP until 6 days. The results are shown in FIG. 'Control' of Figure 7 represents the osteoblast line without any treatment. Referring to FIG. 7, it can be seen that L-ascorbic acid gradually affects the differentiation of osteoblasts over time, and in particular, in the case of 0.06 mM after 6 days, twice as much as the control. In the case of the degree of 0.125mM, it can be seen that the cells differentiate more than three times compared to the control.

Experimental Example 8

As described above, the reason why L-ascorbic acid increases ALP is to regulate the expression of casein kinase II (CKII), which is known as a cell differentiation factor, to confirm the mechanism of osteoblastic action of L-ascorbic acid. The mechanism of action of related L-ascorbic acid was identified. A protein of osteoblasts treated with L-ascorbic acid was extracted and detected using CKII antibody (Western Blot). The results are shown in FIGS. 8A and 8B. Β-actin expression was indicated to indicate that the experiment was performed by extracting the same amount of protein. 8A and 8B, 'AsA' represents a state in which L-ascorbic acid is treated.

In addition, DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazimidazole) is known as an inhibitor of CKII expression and was tested for an increase in ALP when L-ascorbic acid was treated with DRB treatment ( 8b).

Referring to FIG. 8A, it can be seen that CKII expression is increased by L-ascorbic acid. Also, referring to FIG. 8B, the effect of ALP inhibition by artificial CKII inhibition is the effect of ALP activity by L-ascorbic acid treatment. You can see the change being recovered. In particular, it can be seen that ALP is very suppressed in the DRB alone treatment. From these results, it can be seen that CKII plays an important role in the activity of ALP, which causes osteoblast differentiation, and that the increase of cell differentiation factor of osteoblasts by L-ascorbic acid, that is, ALP, is associated with the expression of CKII in osteoblasts. It can be seen that by increasing the.

Experimental Example 9

In this study, the effect of L-ascorbic acid on Ikaros, a transcription inhibitor that inhibits osteoblast differentiation, was examined to investigate the mechanism of action of L-ascorbic acid on osteoblast differentiation. The experiment was performed by the Western Blot method described above, and the results are shown in FIG. 9A. In addition, the degree of binding of the Ikaros transcription factor to the DNA was confirmed by the EMSA method (electrophoretic mobility shift assay) and the results are shown in Figure 9b. Β-actin expression was indicated to indicate that the experiment was performed by extracting the same amount of protein.

Referring to FIG. 9A, it can be seen that as the concentration of L-ascorbic acid increases, the expression of Ikaros, also known as a cell differentiation inhibitor, is greatly suppressed. Referring to FIG. 9B, not only the expression of Ikaros but also its activity as an Ikaros transcription factor It can be confirmed that it is suppressed.

From the results of Experimental Example 7, Experimental Example 8, and Experimental Example 9, it can be seen that L-ascorbic acid increases ALP activity by increasing CKII expression of osteoblasts and inhibiting a differentiation inhibitor called Ikaros.

Experimental Example 10

In this experiment, RT-PCR (Reverse Transcription Polymerase Chain Reaction) method was used to examine the effect of L-ascorbic acid on osteoclast differentiation factor OPG (osteoprotegerin), which is known to promote osteoclast differentiation. Through the mRNA level was confirmed. The experimental results are shown in FIG. Β-actin expression was indicated to indicate that this experiment was performed by extracting the same amount of RNA.

Referring to FIG. 10, it can be seen that OPG, known as an osteoclast differentiation factor, is greatly reduced with increasing concentration of L-ascorbic acid.

As can be seen from Experimental Examples 7 to 10, it can be seen that L-ascorbic acid has a dual effect of promoting osteoblast differentiation and inhibiting osteoclast differentiation.

As described above, in the case of the mushroom extract prepared by the manufacturing method according to the present invention, since the active ingredients are not damaged, they may have a proliferation effect of osteoblasts and an inhibitory effect on the growth of osteoclasts.

In the present invention, when the mushroom extract and L-ascorbic acid are mixed, the activity of osteoblasts is increased by having the proliferation effect of the osteoblasts by the mushroom extract and the differentiation effect by the L-ascorbic acid. As a result, it is very effective in improving osteoporosis and has excellent efficacy as an agent for preventing and treating osteoporosis.

In addition, when the mushroom extract according to the preparation method of the present invention and L-ascorbic acid are mixed, they have a double inhibitory effect on osteoclast activity and osteoclast activity by having both osteoclast growth inhibition and differentiation inhibition activity. Because it has excellent efficacy as an agent for preventing and treating osteoporosis.

In addition, the mushroom extract according to the production method of the present invention is improved osteoarthritis including osteoporosis, degenerative spinal disease, fifty shoulders, ankylosing spondylitis, rickets, osteomalacia, Paget's disease, etc. It can be used as a functional food for.

Claims (9)

delete delete The Agaricus mushrooms were washed with water, the Agaricus mushrooms were poured into the extractor with bottled water, and extracted at a low pressure of 50 ° C. to 70 ° C. for at least 3 hours to 5 hours, and then the bottled water was added to the extractor at 85 ° C. to 95 ° C. Mushroom extract manufacturing method characterized in that the extraction for 1 hour to 2 hours in a pressureless state at high temperature. After washing the Cordyceps sinensis mushroom, the Cordyceps sinensis mushroom with bottled water in an extractor, and extracting for 3 hours to 5 hours under pressureless state at a low temperature of 50 ℃ to 70 ℃, additionally put the bottled water into the extractor high temperature of 85 ℃ to 95 ℃ Mushroom extract manufacturing method, characterized in that the extraction for 1 hour to 2 hours in a pressureless state. Mushroom extract extracted by the method of any one of claims 3 and 4. An osteoporosis prevention and treatment agent comprising the mushroom extract extracted by any one of claims 3 or 4 as an active ingredient. The method of claim 6, The mushroom extract, Agaricus mushroom extract, Cordyceps sinensis extract and chaga mushroom extract, characterized in that the prevention and treatment of osteoporosis. The method of claim 6, Osteoporosis prevention and treatment, characterized in that the powdered L-ascorbic acid is added to the mushroom extract. The method of claim 7, wherein Osteoporosis prevention and treatment agent, characterized in that the L- ascorbic acid in the form of a powder is added to the mixture.

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