KR101528527B1 - Anti-angiogenic composition comprising Rhizopus sp. - Google Patents

Abstract

The present invention relates to a composition for inhibiting angiogenesis containing a Rhizopus genus strain, and more particularly, to a composition for inhibiting angiogenesis, which comprises a Rhizopus genus, a culture of the strain, a fermented product of the strain and extracts thereof A composition for inhibiting angiogenesis, and a composition for preventing or treating angiogenesis-related diseases. The composition according to the present invention can be effectively used for preventing or treating angiogenesis-related diseases by inhibiting angiogenesis without side effects.

Rizophus, angiogenesis inhibitor

Description

An anti-angiogenic composition comprising Rhizopus sp.

The present invention relates to a composition for inhibiting angiogenesis containing a Rhizopus genus strain, and more particularly, to a composition for inhibiting angiogenesis, which comprises a Rhizopus genus, a culture of the strain, a fermented product of the strain and extracts thereof A composition for inhibiting angiogenesis, and a composition for preventing or treating angiogenesis-related diseases.

Cancer is a chronic disease that is caused by abnormality of cellular proliferation regulation by inherent (genetic) and post-hoc (environmental) factors. Once treated with surgery, radiation and chemotherapy, It is the greatest disease of mankind that causes pain to the patient and leads to death. Recent advances in life sciences have led to an increased understanding of the etiology and mechanism of cancer, and new therapies for cancer promptly detect cell proliferative disorders and inhibit cancer progression before metastasis to malignant tumors or other tissues There is a growing interest in so-called cancer prevention.

Angiogenesis is essential in a variety of physiological processes such as embryonic development, wound healing, and tissue or organ regeneration (Kuwano M. et al., Intern . Med , 40: 565-572, 2001). However, continued non-regulatory angiogenesis is known to cause rheumatoid arthritis, diabetic retinitis, solid tumors, angiogenesis-related diseases, such as hemangiomas, and psoriasis (Andre T. et al, Rev Med Interne, 19:... 904- 9134, 1998).

This angiogenesis process promotes the growth of endothelial cells by tumor cytokines, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), the step of decomposing extracellular matrix proteins by metalloprotease , Cell membrane adhesion molecules, endothelial cell differentiation, and endothelial cell migration mediated by tube formation (Bussolino F. et al., Trends Biochem . Sci., 22: 251-256,1997). Thus, inhibiting the process may be effectively used for the treatment of cancer or other diseases associated with angiogenesis.

Cortisone which inhibits the growth of endothelial cells as a substance inhibiting angiogenesis as described above, medroxyprogesterone acetate which inhibits the production of plasminogen activator by endothelial cells, PUMA which inhibits endothelial cell and tube-growth proliferation Gyrillic acid derivatives, polysaccharide sulfate SD-4152, which inhibits the proliferation and migration of endothelial cells, and angiostatic steroids such as retinoic acid, which responds to modification of endothelial cell differentiation. However, some of the above-mentioned agents for inhibiting angiogenesis are not a complete remedy for clinically inhibiting angiogenesis because of the problems of stability due to strong side effects and the rest of them exhibiting insufficient effects.

On the other hand, Rhizopus sp. Strain has been reported to have a physiological activity promoting action and an aging-inhibiting action. In particular, glucoamylase derived from Rizophus sp. Strain can produce glucose directly from starch, Starch and other cereal raw materials can be easily saccharified into glucose. Thus, the Rizophus genus strain is generally used for the preparation of traditional Chinese liquor or Takju. It is also used in the production of glucoamylase for mass production of glucose and in the production of lipase, which is widely used in pharmaceuticals and foods. The present inventors have previously found that a strain (Rizophus sp. Strain KSD-815 (KFCC), which is superior to glucoamylase activity among the surviving strains by treating various mutant sources such as UV, NTG and EMS in the existing glucoamylase producing strains, -11279)), and the cultured extract of the Rizophus genus strain was firstly found to have a therapeutic or preventive effect on gastritis and peptic ulcer, and was patented and registered (Registration No. 10-0616914).

The present inventors have studied another physiological activity of a strain of Rizopus sp. And discovered that the strain has an angiogenesis inhibitory effect, thus completing the present invention.

It is therefore an object of the present invention to provide a new use of the Rizophus genus.

In order to achieve the above object, the present invention provides a composition for inhibiting angiogenesis, which comprises as an active ingredient any one selected from the group consisting of Rhizopus sp. Strain, culture of the strain, fermented product of the strain and extracts thereof to provide.

In order to accomplish still another object of the present invention, the present invention provides an angiogenesis-related disease comprising an effective component selected from the group consisting of a Rhizopus genus strain, a culture of the strain, a fermented product of the strain, Or a pharmaceutically acceptable salt thereof.

In the present invention, it has been confirmed that the Rhizopus sp. Strain has an inhibitory effect on angiogenesis. Therefore, the composition according to the present invention containing any one selected from the group consisting of Rizophus sp. Strain, the culture of the strain, the fermented product of the strain and the extract thereof can be used as a composition for inhibiting angiogenesis and an angiogenesis- Can be very effectively used in compositions for preventing or treating diseases.

The Rizophus genus refers to KSD-815 (Accession No .: KFCC-11279) in Risofus oryzae.

In addition, the culture of the present invention includes a culture solution obtained by culturing the strain of the genus Rizophus in a liquid medium or a solid medium, or a pellet obtained by centrifuging the culture medium. The cultivation of Rhizopus sp. Strain can be carried out using a culture medium known in the art, for example, a PDA medium or the like, and the culture can be carried out at 20 to 32 ° C for 2 to 10 days.

The fermentation product of the present invention is not particularly limited, but may be prepared by fermenting the fermentation product according to a method known in the art using a culture solution obtained by culturing the strain of the genus Rizophus in a liquid medium or a solid medium. For example, a culture broth obtained by culturing a strain of the genus Rizophus in a liquid medium or a solid medium is dried, pulverized, and then added with glutinous rice, corn starch and water, fermented at 20 to 27 ° C for 3 to 9 days, (See Example 3-2).

The extract of the present invention can be obtained by adding the culture or fermentation product of Rizophus sp. Strain to water or an organic solvent solution, stirring the mixture at room temperature for 10 to 30 hours, and then filtering. The filtrate can be concentrated under reduced pressure and lyophilized. The organic solvent solution is preferably 10 to 100% organic solvent, preferably 80% organic solvent. Also, the organic solvent refers to an organic solvent having 1 to 6 carbon atoms, and may include, but is not limited to, methanol, ethanol, propanol, isopropanol, butanol, isoamyl alcohol, hexane, ethyl acetate or a mixture thereof. In the present invention, the culture or the fermentation product of Rizophus sp. Strain was extracted with water, butanol and ethyl acetate as extraction solvents (see Examples 1 and 3).

CAM (chorioallantonic membrane) analysis was performed to measure the angiogenesis inhibitory effect of the water, butanol and ethyl acetate extracts of the cultures, and the inhibition rate was 50 to 70%, which showed excellent angiogenesis inhibitory effect. In particular, the water extract of the culture was treated by concentration to measure the angiogenesis inhibitory effect, and as the concentration increased, the angiogenesis inhibitory effect was more excellent (see Example 2).

In addition, CAM (chorioallantonic membrane) analysis was performed to measure the angiogenesis inhibitory effect of the ethyl acetate, butanol and water extracts of the above fermented product, and the inhibition rate was 50 to 81.2%, which is an excellent angiogenesis inhibitory effect. In particular, the water extract of the fermented product was treated by concentration to measure angiogenesis inhibitory effect, and as the concentration increased, the angiogenesis inhibitory effect was more excellent (see Example 3).

The water and butanol extracts of the cultures were treated with human vascular endothelial cells to inhibit tubular structure formation (see Example 4).

(VEGF, bFGF, TNF-a, HIF-1a) were obtained by RT-PCR (reverse transcription polymerase chain reaction) and electrophoresis after treating the water extract of the culture with human breast cancer MDA- And GAPDH) were measured. As a result, it was confirmed that as the concentration of the extract increased, the expression level decreased. (See Example 5)

In addition, since the minimum lethal dose (LD ₅₀ ) of the culture extract of the Rizophus genus strain is 2000 mg / kg or more, biostability is very high.

Accordingly, the present invention provides a composition for inhibiting angiogenesis, which comprises an activity of inhibiting angiogenesis by a strain of the genus Rizophus, a culture of the strain, a fermented product of the strain, or an extract thereof.

The present invention also provides a composition for preventing or treating an angiogenesis-related disease containing Rhizopus sp., A culture of the strain, a fermented product of the strain, and an extract thereof, and having activity to inhibit angiogenesis. Such angiogenesis-related diseases include, but are not limited to, rheumatoid arthritis, diabetic retinitis, solid tumors, hemangiomas, psoriasis, and angiogenesis-related cancers.

The composition of the present invention refers to a pharmaceutical composition or a food composition.

The pharmaceutical composition of the present invention comprises as an active ingredient any one selected from the group consisting of Rizophus sp. Strain, culture of the strain, fermented product of the strain, and extract thereof. The Rhizopus sp. Strain contained in the pharmaceutical composition of the present invention may be a live cell. It is preferably preserved so as not to lose its activity by being cultured and concentrated by a conventional method known in the art and freeze / freeze-dry.

The pharmaceutical composition of the present invention may be administered orally or parenterally in various dosage forms at the time of actual clinical administration and diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, . ≪ / RTI > Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like. Such a solid preparation may be prepared by mixing at least one excipient selected from the group consisting of Rhizopus sp. Strain, culture of the strain, extract of the culture, and coenzyme produced using the strain, for example, starch, calcium carbonate calcium carbonate, sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate may also be used. Formulations for oral administration include suspensions, solutions, emulsions, syrups, lyophilisers, suppositories, and the like. Among them, liquid preparations may contain various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like in addition to water and liquid paraffin which are simple diluents commonly used. As a base for the suspension, propylene glycol, polyethene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a base for suppositories, witepsol, macrorol, tween 61, cacao butter, laurin, glycerogelatin and the like can be used. The dose is determined in consideration of the skill in the art depending on the age, sex, etc. of the patient. The effective dose of the Rizophus genus strain or the cultured and fermented extract of the present invention is 1 to 5000 mg / kg / day, preferably 5 to 2000 mg / kg / day. The pharmaceutical composition according to the present invention can be administered one to three times a day.

The food composition of the present invention comprises A culture of the strain, a fermented product of the strain, and an extract thereof, as an active ingredient. The ingredient of the Rizophus genus strain contained in the food composition of the present invention may be contained in an amount of 0.001 to 50% by weight, preferably 0.01 to 5% by weight, based on the total weight of the food composition. The content may be used in accordance with an effective amount of the pharmaceutical composition, but may be less than the above range for long-term consumption for health promotion and control. However, since the strain of the genus Rizophus is not toxic, it can be used in an amount exceeding the above range. There is no particular restriction on the type of food mentioned above, and it may be a general processed food, a fermented food or a saccharified food. Examples of foods that can be added to the food composition of the present invention include meat, candy, snacks, confectionery, noodles, gums, dairy products (milk, ice cream, cheese, etc.) (Such as carbonated drinks, alcoholic beverages, tea, and drinks), alcoholic beverages, reproductive foods, and souvenirs. The food composition provided in the present invention can be used as a functional food, a health supplement food or a special nutrition food showing an angiogenesis inhibitory effect.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Risofus  Cultivation and Extract Production of Escherichia coli

100 μl of a KSD-815 (KFCC-11279) seed culture medium and 0.1% HCl were added to 100 g of sterilized wheat bran in Rissofus oryzae cultured on a horse chest culture medium and cultured at 30 ° C for 3 days. Thereafter, the culture was pulverized. 8,000 g of the pulverized culture was added to 10 L of 80% methanol aqueous solution, and the mixture was stirred for 3 hours at room temperature for 24 hours. The mixture was filtered through filter paper and the filtrate was concentrated under reduced pressure to obtain about 1,227 g of an extract. The obtained extract was suspended in 3 L of water and repeated 3 times with 3 L of ethylacetate to obtain about 231 g of ethyl acetate extract by successive fractionation and the aqueous portion was washed with 2.5 L of n-butanol ) To obtain about 114 g of butanol extract and 870 g of water extract.

≪ Example 2 >

Risofus  Inhibitory effect of extracts

<2-1> of Rizzo crispus spp extracts CAM (Chorioallantonic Measurement of angiogenesis inhibitory effect by membrane analysis

The embryos were grown for 3 days in an incubator maintained at a humidity of 90% and a temperature of 37 캜, and 3 ml of albumin was obtained using a hypodermic needle (18-gauge) by piercing. When it was doubled for 4 days, a round window was taken out to peel off the outer membrane of CAM, and then the extract prepared in Example 1 was applied to each thermogen on a thermanox coverslip (Gibco BRL) at a rate of 4.5 days. The applied coverslip was placed on embryo CAM of 3 ~ 5 mm in diameter and incubated for another 1.5 days. Intralipose (Green Cross) was injected to observe angiogenesis inhibition using a dissecting microscope. (avascular zone). The control group was treated with 0.1% DMSO (dimethyl sulfoxide) in a coverslip and positive

(retinoic acid, 1 ㎍ / CAM), which is known to inhibit angiogenesis, was used as a control. The results are shown in Table 1 after 1.5 days.

Inhibitory effect of extracts of Rhizopus sp. Strain on angiogenesis through CAM analysis Treatment concentration
(/ / CAM) The number of surviving eggs in a total of 20 boats The number of vessels that inhibit angiogenesis % Inhibition Control group
(0.1% DMSO) 13 0 0 Positive control group
(Retinoic acid) One 13 9 69.9 Ethyl acetate extract 100 15 9 60.0 Butanol extract 100 16 8 50.0 Water extract 100 13 9 69.2

Inhibition rate (%) = (number of angiogenesis inhibited eggs / number of survived eggs) * 100

As shown in Table 1, the positive control group showed an inhibitory effect of angiogenesis of 69.9%, while the inhibitory rate of 60.0%, 50.0% of butanol extract and 69.2% of water extract, respectively, were obtained when ethyl acetate extract was treated. Therefore, it was found that the extract of Rizophus sp. Strain of the present invention has excellent angiogenesis inhibitory effect and can be effectively used as a composition for inhibiting angiogenesis.

<2-2> Measurement of inhibitory effect of concentration of angiogenesis by CAM (chorioallantonic membrane) analysis of water extract of Rizophus genus

The water extract of Rhizopus sp. Strain having the best inhibitory effect on angiogenesis in the above Example <2-1> was treated and analyzed in the same manner as in Example <2-1> at concentrations of 1, 10 and 100 μg / The results are shown in Table 2.

Inhibitory effect of water extract of Rhizopus sp. Strain on angiogenesis through CAM analysis Treatment concentration
(/ / CAM) The number of surviving eggs in a total of 20 boats The number of vessels that inhibit angiogenesis % Inhibition Control group
(0.1% DMSO) 13 One 7.69 Positive control group
(Retinoic acid) One 12 8 66.7 Water extract of Rhizopus sp. Strain One 12 6 50.0 10 13 9 69.2 100 15 11 73.3

Inhibition rate (%) = (number of angiogenesis inhibited eggs / number of survived eggs) * 100

As shown in Table 2, the positive control group showed an angiogenesis inhibitory effect of 66.7%, whereas the water extract of Rizopus sp. Strain showed the highest inhibitory effect of 73.3% when treated with 100 μg / CAM, and 10 μg / CAM, the inhibitory effect was 69.2% and 50.0% when treated with 1 ㎍ / CAM, respectively.

&Lt; Example 3 >

Risofus  Preparation of Traditional Yakju Using Traditional Strains and Measurement of Angiogenesis Inhibitory Effect of Yakju Extracts

&Lt; 3-1 & gt ; Preparation of crude enzyme preparation

The wheat bran without added moisture was added to the bran, and the culture medium containing 0.1% hydrochloric acid of 60% by weight and 40% of Rizophus sp. Strain was mixed well and cultured at 28 ~ 30 ° C for 3 days. The culture was dried to about 10% And crushed.

<3-2> Preparation of traditional Yakju and production of Yakju extract

The untreated glutinous rice was immersed in water for about 1 hour and then pulverized. The above glutinous rice was supplemented with corn starch, the coenzyme produced in Example <3-1>, the pressed yeast (Ottogi), various medicinal materials (ginseng, gugija, kagurai, licorice, , White sachet) and water were added, and the resulting mixture was fermented at about 20 to 27 ° C for 3 to 9 days, and then concentrated under reduced pressure to give ethyl acetate, butanol and water extracts in the same manner as in Example 1.

<3-3> CAM of traditional Yakju extract ( Chorioallantonic Measurement of angiogenesis inhibitory effect by membrane analysis

The concentrate, ethyl acetate, butanol and water extract of 100 / / CAM prepared in Example <3-2> were analyzed by the same method as in Example <2-1>, and the results are shown in Table 3 Respectively.

Inhibition of angiogenesis by extract of traditional Yakju from CAM analysis Treatment concentration
(/ / CAM) The number of surviving eggs in a total of 20 boats The number of vessels that inhibit angiogenesis % Inhibition Control group
(0.1% DMSO) 13 0 0 Positive control group
(Retinoic acid) One 13 9 69.9 Traditional Chinese condiment concentrate 100 18 13 72.2 Ethyl acetate extract 100 16 9 56.3 Butanol extract 100 14 7 50.0 Water extract 100 16 13 81.2

Inhibition rate (%) = (number of angiogenesis inhibited eggs / number of survived eggs) * 100

As shown in the above Table 3, the positive control group showed an inhibitory effect of angiogenesis of 69.9%, while the conventional concentrates and extracts showed a high inhibitory effect of about 50-81.2%.

<3-4> CAM of water extract of traditional Yakju ( Chorioallantonic membrane) analyzing the concentrations angiogenesis inhibitory effect measured by

The water extract of the traditional Chinese cabbage having the best inhibitory effect on angiogenesis was treated at the concentrations of 1, 10 and 100 μg / CAM in the same manner as in Example <2-3> Table 4 shows the results.

Angiogenesis inhibition effect of water extracts of traditional Yakju by CAM analysis Treatment concentration
(/ / CAM) The number of surviving eggs in a total of 20 boats The number of vessels that inhibit angiogenesis % Inhibition Control group
(0.1% DMSO) 13 One 7.69 Positive control group
(Retinoic acid) One 12 8 66.7 Traditional Chinese water extract One 12 7 58.3 10 15 11 73.3 100 14 11 78.6

Inhibition rate (%) = (number of angiogenesis inhibited eggs / number of survived eggs) * 100

As shown in Table 4, the positive control group showed an inhibitory effect of angiogenesis of 66.7%, whereas the water extract of traditional Chinese medicine showed a high inhibitory effect of about 58.3% to 78.6%.

<Example 4>

Risofus  Inhibitory Effect of Extracts of the Genus Strain on Tubular Structure

(Matrigel, BD, CA. USA), a solid gel of mouse basal membrane protein, was dissolved in EBM-2 (hydrocortisone, hEGF, FBS, VEGF, hFGF-b, R3-IGF-1, ascorbic acid (Cambrex Bio Science, Walkersville, Inc) medium (Gentamycin / Amphotericin-B) medium was added to a 24-well plate, and the mixture was gelled at 37 ° C for 30 minutes. Human vascular endothelial cells Human umbilical vein endothelial cells, HUVECs) were subcultured at a concentration of 5 × 10 ⁴ per well. Thereafter, the butanol and water extract prepared in Example 1 were treated at a concentration of 50 μg / ml and cultured at 37 ° C. for 24 hours. The tubular structure formation was observed under a microscope to be shown in FIG.

As shown in FIG. 1, the control group (the group not treated with the extract) normally formed a complete tubular structure. However, when the water extract of Rhizopus sp. Strain was treated, a tubular structure of a specific type was not formed. An incomplete tubular structure was formed. Therefore, it was found that the extract of Rhizopus sp. Strain had an activity of inhibiting the formation of tubular structure.

&Lt; Example 5 >

Risofus  Of the genus strain Angiogenic factor  Expression inhibitory effect

The expression of VEGF, bFGF, TNF-a, and HIF-1a, which are angiogenic factors, was measured to confirm the inhibitory effect of the extract of Rizophus sp. Strain on angiogenic factor expression.

Human breast cancer MDA-MB-468 cells treated with water extract of Rizophus strain prepared in Example 1 for 24 hours at 0, 0.1, 1, 10, and 100 占 퐂 / ml were inoculated into 1 ml of Dulbeco's phosphate buffered saline) and 1 ml of Trizol (Invitrogen, Madison, Wis.) reagent was added thereto. Then, total RNA was isolated according to a method known in the art and suspended in 20 μl of DEPC (diethyl pyrocarbonate) treated distilled water .

The first strand cDNA was synthesized from the total RNA by RT-PCR (RT-PCR). Specifically, 1 to 5 μg of total RNA was reacted with oligo-dT at 65 ° C. for 10 minutes, and then 100 units of M-MLV reverse transcriptase (Super-Bio Co. Ltd.) and 4 μl of 5 × buffer 50 mM DTT, 15 mM magnesium chloride, pH 8.9) and 4 μl of 2.5 mM dNTP, and the reaction volume was adjusted to 20 μl at 37 ° C The reaction was carried out for 60 minutes and the reaction was carried out at 70 ° C for 10 minutes in order to inactivate the enzyme.

 PCR was performed using AccuPower PCR Premix (Bioneer, Korea) according to a method known in the art using 1 쨉 l of the synthesized cDNA and 10 pmol of the primer shown in Table 5 below. Specifically, the total reaction volume is 20 μl. Denaturation is carried out at 94 ° C. for 60 seconds, annealing at 54 ° to 58 ° C. for 60 seconds, extension at 70 ° C. for 60 seconds 30-35 times PCR was performed. The resulting product was subjected to electrophoresis on 1.5% agarose gel, stained with EtBr (ethidium bromide), and observed under UV conditions. The results are shown in FIG.

As shown in FIG. 2, it was confirmed that the expression amounts of VEGF, bFGF, TNF-a, and HIF-Ia, which are angiogenic factors, decreased as the water extract prepared in Example 1 was administered more. Therefore, it was found that the extract of Rhizopus sp. Strain can be effectively used as a composition for inhibiting angiogenesis by inhibiting the expression of angiogenic factors.

Primer sequences of angiogenic factors Angiogenic factor Primer sequence SEQ ID NO: VEGF sense 5'-GCC TCG CCT TGC TGC TCT ACC TC-3 ' One Antisense 5'-GTT CTG TCG ATG GTG ATG GTG TG-3 ' 2 bFGF sense 5'-ACT TCA AGG ACC CCA AGC GG-3 ' 3 Antisense 5'-GCT CTT AGC AGA CAT TGG AA-3 ' 4 TNF-a sense 5'-AAG CCT GTA GCC ATG TTG TAG C-3 ' 5 Antisense 5'-GAA GAC CCC TCC CAG ATA GAT G-3 ' 6 HIF-1a sense 5'-CTC AAA GTC GGA CAG CCTCA-3 ' 7 Antisense 5'-CCC TCG AGT AGG TTT CTG CT-3 ' 8 GAPDH sense 5'-CCT GGC CAA GGT CAT CCA T-3 ' 9 Antisense 5'-GCC ATG TAG GCC ATG AGG T-3 ' 10

&Lt; Preparation Example 1 &

Preparation of agents for inhibiting angiogenesis using Rhizopus sp. Strain

<1-1> Preparation of tablets

250 mg of the culture extract of Rhizopus sp. Strain prepared in Example 1 was mixed with 260 mg of lactose for use in excipient, 35 mg of avicel (microcrystalline cellulose), 15 mg of sodium starch glycolate disintegration aid, and L-HPC low-hydrosyprophylcellulose) in a U-shaped mixer and mixed for 20 minutes. After mixing was completed, 10 mg of magnesium stearate was further added as a lubricant and mixed for 3 minutes. The tablets were prepared by tabletting and film coating after the quantitative test and the humidity test.

&Lt; 1-2 & gt ; Preparation of capsules

450 mg of the culture extract of Rhizopus sp. Strain prepared in Example 1 and 50 mg of lactose were mixed and filled in hard gelatine capsules to prepare capsules.

&Lt; 1-3 & gt ; Preparation of syrup preparation

An appropriate amount of white sugar was dissolved in a certain amount of water, and thereto were added 80 mg of paraoxymethyl benzoate and 16 mg of paraoxypropyl benzoate as a preservative. Then, 4.5 g of the culture extract of Rizophus strain prepared in Example 1 was added, and the additives were completely dissolved while maintaining the water temperature at 60 캜. After cooling the solution, distilled water was added to make a final volume of 150 ml to prepare a 3% syrup.

&Lt; Preparation Example 2 &

Production of food for inhibiting angiogenesis using Rhizopus sp. Strain

<2-1> Production of Reproduction

Soybean embryo fermentation powders were prepared by adding various kinds of grains (brown rice, barley, corn, mung bean, yulmu, sesame and red bean) powders, seaweed (seaweed, seaweed and kelp) powder, fruit and vegetables (kale, aloe and carrot), mushroom ) Powder and the culture extract of the strain of the genus Rizophus prepared in Example 1 were mixed to produce a reproductive product. The specific composition is shown in Table 6 below.

ingredient 1 capsule Soybean embryo fermentation powder 25 Brown rice powder 10 Barley powder 5 Corn powder 3 Mung bean powder 3 Yulmu powder 3 Sesame powder 3 Red bean powder 3 Kim powder 5 Seaweed Powder 8 Kelp powder 12 Kale powder 5 Aloe powder 3 Carrot powder 2 Shiitake mushrooms 4 Gongji mushroom 4 Culture extract of Rizophus genus 2 total 100 (% by weight)

<2-2> Production of Kochujang

Glutinous rice was soaked in water and then ground with powder. KSD-815 (activity: sp 3,000 ~ 5,000) was leached in 2 ~ 4 L of warm water and filtered through 1 ~ 10 wt% of Rissofus oryzae of the amount of glutinous rice powder (0.5 ~ 1.5 kg). The leachate of the Rizophus genus strain was added to the glutinous rice flour and then sacrificed at 40 to 60 DEG C (glutinous rice paste production). Meju flour and red pepper powder were mixed in glutinous rice paste. Then, it was seasoned with salt, put in a jar and aged for about one month.

<2-3> Manufacture of Sikhye

KSD-815 (potency: sp 3,000 ~ 5,000) was leached in 1 to 10% by weight of Rispus oryzae in 1 to 10% by weight of the total weight of rice cakes, for 1 to 2 hours and then filtered. 200 g of rice gruel were mixed with maltose and leachate of Escherichia coli strain. Thereafter, the mixture was saccharified at 50 to 70 ° C for 3 to 8 hours. When saccharification was completed, it was sterilized by autoclave, and ginger, pear and the like were added at the time of sterilization, sterilized and stored in the refrigerator.

&Lt; 2-4 >

25 to 35% by weight of brown rice, 10 to 15% by weight of other ingredients (pumpkin, radish, ginger, pear, etc.) are added to a pressure cooker Steamed rice was prepared. Thereafter, KSD-815 (potency: sp 3,000 ~ 5,000) and 2 to 4 times as much water as the steamed rice were added to the above-mentioned steamed rice in 1 to 10% by weight of the total weight of the steamed rice, and then saccharified. When the saccharification was completed, it was filtered through a fine mesh. The filtered saccharified liquid was dissolved in a weak fire for 10 to 20 hours to prepare a tonic.

FIG. 1 is a photograph showing microscopic examination of the inhibitory effect on the tubular structure formation of water and butanol extract of Rizophus spp.

A. Control (group not treated with the extract of Rizophus sp. Strain)

The water extract of B. risottus strain treated with 50 / / CAM

Butanol extract of C. risottus strain treated with 50 / / CAM

FIG. 2 is a graph showing the results of the RT-PCR (reverse transcription polymerase chain reaction) and electrophoresis of the water extract of Rizophus sp. Strain to inhibit angiogenic factor expression.

<110> KOOKSOONDANG BREWERY CO., LTD. <120> Anti-angiogenic composition comprising Rhizopus sp. <130> NP07-0081 <160> 10 <170> Kopatentin 1.71 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> VEGF-sense-primer <400> 1 gcctcgcctt gctgctctac ctc 23 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> VEGF-antisense-primer <400> 2 gttctgtcga tggtgatggt gtg 23 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> bFGF-sense-primer <400> 3 acttcaagga ccccaagcgg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> bFGF-antisense-primer <400> 4 gctcttagca gacattggaa 20 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> TNFa-sense primer <400> 5 aagcctgtag ccatgttgta gc 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> TNFa-antisense-primer <400> 6 gaagacccct cccagataga tg 22 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HIF1a-sense-primer <400> 7 ctcaaagtcg gacagcctca 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HIF1a-antisense-primer <400> 8 ccctcgagta ggtttctct 20 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-sense-primer <400> 9 cctggccaag gtcatccat 19 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-antisense-primer <400> 10 gccatgtagg ccatgaggt 19

Claims (5)

A composition for inhibiting angiogenesis, which comprises an extract of at least one substance selected from a culture of a strain KSD-815 (Accession No .: KFCC-11279) in Rhizopus oryzae and a fermentation product of said strain. An extract of at least one substance selected from a culture of a strain of KSD-815 (accession number: KFCC-11279) and a fermentation product of said strain in Rispops oryzae, Rheumatoid arthritis, diabetic retinitis, solid tumors, hemangiomas, psoriasis and cancer. The composition according to claim 1 or 2, wherein the extract is obtained by extracting the culture or fermentation product of the Rizophus genus strain with water or an organic solvent having 1 to 6 carbon atoms. 4. The composition according to claim 3, wherein the organic solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, isoamyl alcohol, hexane and ethyl acetate. delete

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