KR102227963B1 - Antiangiogenic composition comprising bioconverted herbal composition, and its preparation method - Google Patents

Abstract

The present invention relates to a composition for inhibiting angiogenesis and a method for preparing the same, comprising a bioconversion composition obtained by bioconversion of a herbal composition comprising Sukjihwang, Baekjak, Cheongung and Angelicae into Aspergillus Kawachi culture solution as an active ingredient. The bioconversion composition of the present invention is excellent in the effect of inhibiting angiogenesis as confirmed by the effect in tube formation analysis, cell invasion analysis, and in vivo angiogenesis model.

Description

[Antiangiogenic composition comprising bioconverted herbal composition, and its preparation method}

The present invention relates to a composition for inhibiting angiogenesis including a bioconverted herbal composition and a method for preparing the same, and more particularly, a bioconversion of a herbal composition including Sukjihwang, Baekjak, Chungoong and Angelicae into Aspergillus Kawachi culture solution It relates to a bioconversion composition and a method for preparing the same.

Angiogenesis is the growth of new blood vessels, which is a process mainly dependent on migration, proliferation and tube formation by capillary endothelial cells. During angiogenesis, endothelial cells escape from the stationary phase and proliferate rapidly, and various angiogenic factors are involved in the angiogenesis process. It is understood that angiogenesis occurs by inducing proliferation of non-self-restricted endothelial cells and surrounding endothelial cells due to a breakdown of the fine balance between pro-angiogenic factors and anti-angiogenic factors during pathology. Angiogenesis (angiogenesis) is essential for a variety of processes, including organ growth, embryonic development, and wound healing, but is also associated with a variety of pathological conditions. Angiogenesis occurs in ocular neovascularization diseases, arthritis, skin diseases, tumors, etc., and a method of inhibiting angiogenesis is an effective treatment capable of inhibiting and delaying the progression of these diseases.

To treat such malignant diseases, a number of anti-angiogenic agents have been developed.Examples of such agents control the interaction between vascular endothelial growth factor (VEGF) and its receptor (VEGFR), which is the main process that controls angiogenesis in humans. Two agents, 2-methoxyestradiol (2-ME2), a natural metabolite of estradiol, which has unique anti-tumor and anti-angiogenic properties, and angiostatin, a proteolytic cleavage fragment of plasminogen and collagen XVIII, respectively. Novel angiogenesis inhibitors such as endostatin.

Such anti-angiogenic agents have problems in that their success rate is limited when systemically administered, and exhibits considerable toxicity, including thrombocytopenia, leukopenia, and hemoptysis.

Samultang is a medicinal mixture of Sukjihwang (熟地黃), Baek peony, Cheongung, and Angelica (當歸). . Samul-tang is a drug used for blood hemostatic and blood clots, and is used for menstrual impurities, infertility, menopausal disorders, addiction to pregnancy, and postpartum symptoms. Samultang was first recorded in the topic of national defense (和劑局方) in China, and there are prescription records in Donguibogam, Mimunbogam (醫門寶鑑) and Jejungsinpyeon (濟衆新編) in Korea.

Korean Patent Registration No. 10-0500298 Korean Patent Publication No. 10-2016-0072932 Korean Patent Publication No. 10-2006-0039036 Korean Patent Publication No. 10-1084446

An object of the present invention is to provide a composition obtained by bioconversion of a herbal composition containing herbal medicinal herbs used in Samultang, including Sukjihwang, Baekjak, Cheongung and Angelicae, and a composition having no toxicity and excellent angiogenesis inhibitory ability, and a method for preparing the same.

In order to achieve the above object, in the present invention,

It provides a composition for inhibiting angiogenesis, comprising as an active ingredient a bioconversion composition obtained by bioconversion of a herbal composition comprising Sukjihwang, Earl, Chungoong and Angelicae into Aspergillus Kawachi culture.

The herbal composition is preferably containing 10 to 40% by weight of sukjihwang, 10 to 40% by weight of the earl, 10 to 40% by weight of cheonggung and 10 to 40% by weight of Angelicae. It is particularly preferred that the herbal composition is mixed with Sukjihwang, Baekjak, Cheongung and Angelicae in a weight ratio of 1:1:1:1.

The Aspergillus Kawachi culture solution,

Adding 8 to 12 ml of distilled water based on 10 g of bran, crushing and mixing so that the bran absorbs a small amount of water, and sterilizing;

Cooling after the sterilization, inoculating Aspergillus kawachi, and incubating at room temperature for 2 to 4 days;

After the cultivation, adding and mixing 45 to 55 ml of sodium phosphate buffer stock having a concentration of 10 mM based on 10 g of bran, and immersing for 5 to 10 hours at 4° C. to obtain a cultured sample;

Filtering the cultured sample to separate the filtrate; And

It is preferable that it is prepared by a method comprising the step of centrifuging the filtrate and then filtering the supernatant to obtain Aspergillus Kawachi culture.

In addition, the present invention,

Obtaining a herbal composition by mixing Sukjihwang, Earl Peony, Cheongung and Angelicae;

Extracting the herbal composition by mixing 70% ethanol in a weight ratio of 1: 2 to 4;

Filtering after the extraction to obtain a filtrate;

Concentrating the filtrate under reduced pressure and freeze-drying to obtain an extract powder;

Mixing distilled water to the extract powder at a weight ratio of 1:5 to 10 and performing sound wave treatment;

Mixing the Aspergillus Kawachi culture solution to the sonicated one at a weight ratio of 100:50 to 200 and culturing with shaking; And

It provides a method for preparing a bioconversion composition for inhibiting angiogenesis, comprising the step of obtaining a bioconversion composition by concentrating under reduced pressure after the culture.

The manufacturing method may further include the step of obtaining a powdery bioconversion composition by concentrating under reduced pressure and then lyophilizing.

In the above manufacturing method, the herbal composition preferably contains 10 to 40% by weight of Sukjihwang, 10 to 40% by weight of Earl, 10 to 40% by weight of cnidium and 10 to 40% by weight of Angelicae.

In the manufacturing method, the Aspergillus Kawachi culture solution,

Adding 8 to 12 ml of distilled water based on 10 g of bran, crushing and mixing so that the bran absorbs a small amount of water, and sterilizing;

Cooling after the sterilization, inoculating Aspergillus kawachi, and incubating at room temperature for 2 to 4 days;

After the cultivation, adding and mixing 45 to 55 ml of sodium phosphate buffer stock having a concentration of 10 mM based on 10 g of bran, and immersing for 5 to 10 hours at 4° C. to obtain a cultured sample;

Filtering the cultured sample to separate the filtrate; And

It is preferable that the filtrate is centrifuged and then the supernatant is filtered to obtain Aspergillus Kawachi culture.

In the present invention, the bioconversion composition obtained by bioconverting the herbal composition including Sukjihwang, Earl, Chungoong, and Angelicae into Aspergillus Kawachi culture solution was confirmed by the effect of tube formation analysis, cell invasion analysis, and in vivo angiogenesis model. As described above, the effect of inhibiting angiogenesis is excellent. In particular, the effect of inhibiting angiogenesis is superior compared to the case of using the raw materials of Samultang (Chungung and Angelicae) alone, and the effect of inhibiting angiogenesis is excellent even compared to a non-bioconverted herbal composition. Therefore, the composition for inhibiting angiogenesis comprising the bioconversion composition of the present invention as an active ingredient can be effectively used for the prevention and treatment of diseases related to angiogenesis.

1 is a result of confirming the effect of biotransformation on the degree of tube formation.
2 is a result of confirming the effect of biotransformation on cell invasion induced by fibronectin.
3 is a result of confirming the effect of biotransformation on the cell invasion induced by fibronectin in the extract of Chunkyung and Angelicae extract.
4 is a result of confirming the effect of biotransformation on an angiogenesis model in vivo.

The composition for inhibiting angiogenesis of the present invention comprises, as an active ingredient, a bioconversion composition obtained by bioconverting a herbal composition including Sukjihwang, Baekjak, Cheongung, and Angelicae into Aspergillus Kawachi culture solution.

In the present invention, "Bio Conversion" is defined as a process of strengthening a desired substance from an existing biomaterial using an enzymatic function of a microorganism.

Hereinafter, the present invention will be described in detail.

In the composition for inhibiting angiogenesis of the present invention, the herbal composition is obtained by mixing Sukjihwang, Baekjak, Chungoong, and Angelicae. Preferably, 10 to 40% by weight of Sukjihwang, about 10 to 40% by weight of Count, 10 to 40% by weight of Chungo and 10 to 40% by weight of Angelicae are mixed. As the herbal composition, particularly preferably, Samultang in which Sukjihwang, Baekjakjak, Cheongung, and Angelicae are mixed in a weight ratio of 1:1:1:1 may be used.

In the herbal composition mixed in the same ratio as described above, 70% ethanol is mixed and extracted in a weight ratio of 1:2-4. Extraction is preferably extracted 2 to 3 times by heating for 2 to 4 hours at a temperature of 80 to 100 °C.

After extraction, it is filtered to obtain a filtrate. At this time, it is more preferable to filter using filter paper.

The filtrate is concentrated under reduced pressure to remove the solvent and freeze-dried to obtain an extract powder.

Distilled water is mixed with the extract powder at a weight ratio of 1:5 to 20, followed by sonication.

Aspergillus Kawachi ( Aspergillus kawachii ) culture solution is mixed at a weight ratio of 100:50~200 and cultured with shaking. It is preferable to incubate for 10 to 15 hours in a shaking incubator.

After the cultivation, it is concentrated under reduced pressure to obtain the bioconversion composition of the present invention.

If necessary, the bioconversion composition may be lyophilized to obtain a powdery bioconversion composition.

The Aspergillus Kawachi culture solution is preferably prepared as follows.

Add 8 to 12 ml of distilled water based on 10 g of bran, crush and mix so that the bran absorbs a small amount of water, and sterilize. Cool the sterilized sample, inoculate Aspergillus kawachi, and incubate for 2 to 4 hours at room temperature. Then, diluted and prepared sodium phosphate buffer stock (1M) to a final concentration of 10 mM was added to each of 45 to 55 mL based on 10 g of bran, mixed, and incubated overnight at 4°C. The cultured sample is filtered to separate the filtrate, the separated filtrate is centrifuged, and the supernatant is filtered again to obtain the Aspergillus Kawachi culture solution of the present invention.

The obtained Aspergillus Kawachi culture solution is stored at 4°C until use.

The bioconversion composition of the present invention was found to have excellent effects of inhibiting angiogenesis in tube formation analysis, cell invasion analysis, and in vivo angiogenesis model, as confirmed in the examples below.

The present invention will be described in more detail through the following examples. These examples are illustrative of the present invention, and the scope of the present invention is not limited thereto.

<Example 1>

Angelica, Cheongung, Peony and Sukjihwang were purchased from Omnihub. Each herbal medicine was mixed with 70% ethanol of 3 times the weight of the herbal medicine, heated for 3 hours, and extracted twice. After extraction, it was filtered with filter paper, the filtrate was concentrated under reduced pressure to remove the solvent, and freeze-dried to prepare an extract powder.

To a 250 ml Erlenmeyer flask, 10 g of bran and 10 ml of distilled water were added, crushed and mixed so that the bran absorbs a small amount of water, and then sterilized. The sterilized sample was cooled, inoculated with Aspergillus kawachi, and incubated at room temperature for 3 hours. After incubation, sodium phosphate buffer stock (1M) was diluted to obtain a final concentration of 10 mM, 50 ml each was added to each flask, mixed, and incubated overnight at 4°C. The cultured sample was wrapped in a nonwoven fabric to separate the culture solution, the separated culture solution was centrifuged, and the supernatant was filtered through a filter to obtain Aspergillus Kawachi culture solution. The obtained Aspergillus Kawachi culture was stored at 4°C until use.

To 100 g of the lyophilized extract powder obtained above, 1 L of distilled water was added and mixed, followed by sonication. 1 L of the Aspergillus Kawachi culture solution was added to 1 L of the sonicated extract, followed by shaking culture overnight in a shaking incubator, concentrated under reduced pressure, and freeze-dried to obtain a bioconversion composition powder.

< Comparative example 1>

Angelica, Cheongung, Peony and Sukjihwang were purchased from Omnihub. Each herbal medicine was mixed with 70% ethanol of 3 times the weight of the herbal medicine, heated for 3 hours, and extracted twice. After extraction, it was filtered with filter paper, the filtrate was concentrated under reduced pressure to remove the solvent, and freeze-dried to prepare an extract powder.

<Comparative Example 2>

Chungoong was bioconverted in the same manner as in Example 1 to obtain a bioconverted Chungoung extract powder.

<Comparative Example 3>

Chungoong was extracted in the same manner as in Comparative Example 1 to obtain a Chungoung extract powder that was not bioconverted.

<Comparative Example 4>

Angelicae was bioconverted in the same manner as in Example 1 to obtain a bioconverted Angelicae extract powder.

<Comparative Example 5>

Angelicae was extracted in the same manner as in Comparative Example 1 to obtain an Angelicae extract powder that was not bioconverted.

<Experimental Example 1>

Tube formation assay

In order to confirm the effect of the bioconversion composition on angiogenesis, a tube formation test was performed as follows.

SVEC4-10 cells (purchased from ATCC) were used as cells, and the bioconversion composition powder of Example 1 and the non-bioconversion composition powder of Comparative Example 1 were used as samples. No treatment was applied to the control.

After addition of 50 µl of Matrigel to a 96-well plate, it was allowed to stand at 37° C. for 30 minutes for polymerization. SVEC4-10 cells were aliquoted into 3×10 ⁴ cells/well, and extract samples at concentrations of 0 μg/ml, 25 μg/ml, 50 μg/ml, and 100 μg/ml were added, followed by pretreatment for 30 minutes. The pretreated SVEC4-10 cells were seeded on Matrigel and cultured for 3 hours at _{37° C. and 5% CO 2.}

The results of confirming the tube formation degree of the cultured cells are shown in FIG. 1. In FIG. 1, C represents an untreated control, SMT represents a case of treating the non-bioconverted composition of Comparative Example 1, and BSMT represents a case of treating the biotransformation composition of Example 1.

As shown in the results of FIG. 1, when the bioconversion composition was treated, it was confirmed that the degree of tube formation was lower than when the non-bioconverted composition was treated at all treatment concentrations.

<Experimental Example 2>

Cell invasion assay, Transwell migration assay

The effect of the bioconversion composition on the cell invasion induced by fibronectin (FN) was confirmed by the following transwell migration assay.

SVEC4-10 cells (purchased from ATCC) were used as cells, and the bioconversion composition powder of Example 1 and the non-bioconversion composition powder of Comparative Example 1 were used as samples.

Transwells were coated with 5 μg/ml fibronectin and left overnight at 4°C. The coated transwells were blocked with 1% BSA for 60 minutes and washed with PBS. After washing, 3×10 ⁴ cells/well of SVEC4-10 cells were pretreated with each extract at concentrations of 0 μg/ml, 50 μg/ml, and 100 μg/ml for 30 minutes, and then dispensed into the upper chamber. Incubated for 16 hours at ℃, 5% CO _{2 conditions.} The cells of the cultured transwell were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. As a control, a transwell coated with 2% BSA and left overnight at 4°C was used.

The results of confirming the invaded cell rate are shown in FIG. 2.

As shown in the results of Figure 2, in the cell invasion induced by fibronectin (FN), the biotransformation composition (BSMT) significantly reduced the cell invasion, unlike the non-biotransformed composition (SMT), and thus treated with only 2% BSA. It showed a similar pattern.

<Experimental Example 3>

Cell invasion assay

The effect of the extracts of Chunkyung and Angelicae on the cell invasion induced by fibronectin (FN) was confirmed as follows.

As a sample, the bioconverted Cnidium extract of Comparative Example 2, the non-bioconverted Cnidium extract of Comparative Example 3, the bioconverted Angelicae extract of Comparative Example 4, and the non-bioconverted Angelicae extract of Comparative Example 5 were used. Was experimented in the same manner as in Experimental Example 2.

The results of confirming the invaded cell rate are shown in FIG. 3.

As shown in the results of FIG. 3, the bioconverted Cnidium extract and Angelicae extract in the cell invasion phase induced by fibronectin (FN) did not show any difference in the cell invasion phase and the non-bioconverted Cnidium extract and Angelicae extract.

<Experimental Example 4>

In vivo Matrigel plug analysis ( in vivo Matrigel plug assay)

In the in vivo angiogenesis model, the effect of the bioconversion composition of the present invention on the hemoglobin content was confirmed as follows.

As for the mice, 7-week-old C57BL/6 mice were purchased and used from Coretech, Inc., and the bioconversion composition powder of Example 1 and the non-bioconversion composition powder of Comparative Example 1 were used as samples.

To remove bubbles after mixing the bioconversion composition powder and the non-bioconversion composition powder in a Matrigel mixture (Matrigel 200μg/ml, VEGF 300ng/ml, heparin 50U) at a concentration of 100µg/ml, respectively, at 4°C. It was left on ice. The prepared mice were anesthetized and hairs on both sides were removed, and then the Matrigel mixture was inserted subcutaneously at 200µl for each site. The control was not treated with the Matrigel mixture.

After leaving for 1 week, the mouse was dissected to check the shape and color of Matrigel.

In addition, a part of Matrigel was cut and placed in a tube containing 500 µl DW (+ 0.2% heparin), left overnight in a refrigerator, and centrifuged at 15,000 rpm for 20 minutes. 100 µl of the centrifuged supernatant was mixed with 100 µl of a Drabskin kit solution, and the concentration of hemoglobin was measured at 540 nm.

The results are shown in FIG. 4.

As shown in the results of Figure 4, in the in vivo angiogenesis model, the VEGF-treated group showed a high hemoglobin content, and when the bioconversion composition was treated, the hemoglobin content was significantly lower than when the non-bioconverted composition was treated.

Claims (9)

As an active ingredient, a bioconversion composition obtained by bioconversion of an extract of a herbal composition including Sukjihwang, Earl, Cheongung, and Angelicae into Aspergillus Kawachi culture solution,
The extract of the herbal composition is extracted by mixing 70% ethanol in a weight ratio of 1: 2 to 4 in a herbal composition obtained by mixing Sukjihwang, Baekjak, Cheongung, and Angelicae, followed by filtration, concentration, and freeze-drying to obtain an extract powder, This extract powder was mixed with distilled water at a weight ratio of 1:5 to 20 and subjected to sound wave treatment,
The biotransformation is characterized in that shaking culture by mixing Aspergillus Kawachi culture solution in a weight ratio of 100:50 ~ 200 to the extract of the herbal composition subjected to the sonication treatment, the composition for inhibiting angiogenesis. The method of claim 1,
The herbal composition is a composition for inhibiting angiogenesis, characterized in that it comprises 10 to 40% by weight of Sukjihwang, 10 to 40% by weight of Earl, 10 to 40% by weight of cnidium and 10 to 40% by weight of Angelicae. The method of claim 1,
The herbal composition is a composition for inhibiting angiogenesis, characterized in that the mixture of Sukjihwang, Baekjak, Chungoong and Angelicae in a weight ratio of 1:1:1:1. The method according to any one of claims 1 to 3,
The Aspergillus Kawachi culture solution,
Adding 8 to 12 ml of distilled water based on 10 g of bran, crushing and mixing so that the bran absorbs a small amount of water, and sterilizing;
Cooling after the sterilization, inoculating Aspergillus kawachi, and incubating for 2 to 4 hours at room temperature;
After the incubation, adding 45 to 55 ml of sodium phosphate buffer stock having a concentration of 10 mM based on 10 g of bran, mixing, and incubating at 4° C. overnight to obtain a cultured sample;
Filtering the cultured sample to separate the filtrate; And
A composition for inhibiting angiogenesis, characterized in that it is prepared by a method comprising the step of obtaining an Aspergillus Kawachi culture solution by centrifuging the filtrate and filtering the supernatant. delete Obtaining a herbal composition by mixing Sukjihwang, Earl Peony, Cheongung and Angelicae;
Extracting the herbal composition by mixing 70% ethanol in a weight ratio of 1: 2 to 4;
Filtering after the extraction to obtain a filtrate;
Concentrating the filtrate under reduced pressure and freeze-drying to obtain an extract powder;
Mixing distilled water to the extract powder at a weight ratio of 1:5 to 20 and performing sound wave treatment;
Mixing the Aspergillus Kawachi culture solution to the sonicated one at a weight ratio of 100:50 to 200 and culturing with shaking; And
A method for producing a bioconversion composition for inhibiting angiogenesis, comprising the step of obtaining a bioconversion composition by concentrating under reduced pressure after the culture. The method of claim 6,
The herbal composition is characterized in that it comprises 10 to 40% by weight of Sukjihwang, 10 to 40% by weight of the earl, 10 to 40% by weight of cheonggung and 10 to 40% by weight of Angelicae, a method for producing a bioconversion composition. The method of claim 6,
The method for producing a bioconversion composition, further comprising the step of obtaining a powdery bioconversion composition by lyophilizing after concentration under reduced pressure. The method according to any one of claims 6 to 8,
The Aspergillus Kawachi culture solution,
Adding 8 to 12 ml of distilled water based on 10 g of bran, crushing and mixing so that the bran absorbs a small amount of water, and sterilizing;
Cooling after the sterilization, inoculating Aspergillus kawachi, and incubating for 2 to 4 hours at room temperature;
After the incubation, adding 45 to 55 ml of sodium phosphate buffer stock having a concentration of 10 mM based on 10 g of bran, mixing, and incubating at 4° C. overnight to obtain a cultured sample;
Filtering the cultured sample to separate the filtrate; And
A method for producing a bioconversion composition, characterized in that it is prepared by a method comprising the step of obtaining Aspergillus Kawachi culture by filtering the supernatant after centrifuging the filtrate.

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