WO2021010790A1 - Anticancer composition comprising salvia miltiorrhiza bioconversion extract and red ginseng bioconversion extract - Google Patents

Abstract

The present invention relates to an anticancer composition comprising a Salvia miltiorrhiza bioconversion extract and a red ginseng bioconversion extract. A bioconversion complex extract, which is prepared by subjecting the Salvia miltiorrhiza extract and the red ginseng extract to enzymatic treatment, followed by reaction with a Trichoderma reesei culture, exhibits a superb effect on the inhibition of proliferation of cancer cells, and thus the complex extract can be effectively used in the prevention or treatment of cancer.

Description

Anticancer composition comprising biotransformation extract of short ginseng and red ginseng bioconversion extract

This patent application claims priority to Korean Patent Application No. 10-2019-0086665 filed with the Korean Intellectual Property Office on July 17, 2019, and the disclosures of the patent application are incorporated herein by reference.

The present invention relates to an anticancer composition comprising a Salvia miltiorrhiza biotransformation extract and a red ginseng bioconversion extract, and more particularly, to a Salvia miltiorrhiza extract and a red ginseng extract after enzymatic treatment, Trichoderma Reessay (Trichoderma) reesei) It relates to a biotransformation complex extract having an anticancer effect prepared by reacting a culture solution.

Cancer is counted as the two leading cause of death along with cardiovascular disease in modern society. According to recent news reports, cancer deaths are on the rise. Cancer diseases leading to death were in the order of lung, liver, stomach, pancreas, colon, and breast cancer.

There are three types of chemotherapy currently being performed. The resection of the affected area through surgical therapy, chemotherapy using irradiation, and chemotherapeutic therapy to suppress the cell division cycle are the main ones.

However, since the side effects of general chemotherapy are very severe, patients are having a very difficult time in chemotherapy.

In general, anticancer drugs take into account characteristics such as rapid proliferation and abnormal differentiation of cancer cells, and inhibit them, thereby preventing the proliferation of cancer. The characteristics of these anticancer drugs also act on some normal cells with characteristics similar to those of cancer cells, resulting in side effects that inhibit the growth of normal tissues.

These side effects appear as a variety of symptoms such as hair loss, dry mouth and stomatitis, nausea and vomiting, diarrhea, constipation, neutropenia, and anemia, making it more difficult for patients to fight the disease. Therefore, functional foods that can replace or supplement these chemical drug use methods can be seen as having very important value.

To achieve this purpose, the present inventors first selected red ginseng, which has proven excellent anticancer effect in several studies, among food ingredients registered in the Food Code, and in particular, compound K ( compound K) was mainly used. In addition, Salvia miltiorrhiza, which is widely known for its cardiovascular protective action, and whose anticancer effect has been proven in recent studies, was used.

Saponin, the main physiologically active substance of red ginseng (ginseng), has excellent pharmacological activity, so it is called ginsenoside separately from saponins found in other plants. Ginsenosides are ginsenosides of diol-based (Protopanaxadiol; PPD) and triol-based (Protopanaxatriol; PPT) depending on the type and number of sugars bonded to the R1, R2, and R3 positions in a kind of glycoside with a dammarane skeleton. They are classified as side, etc.

However, in order for ginsenosides to be absorbed into the body, the diol-based ginsenosides are 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol (20-O-β-D-glucopyranosyl-20( S)-protopanaxa-diol, FGM 1, hereinafter referred to as'Compound K'), and the triol-based ginsenoside is 20(S)-protopanaxatriol (20(S)-protopanaxatriol, After being decomposed into FGM 4), it is absorbed into the body to exert its efficacy, but only a small amount is absorbed by heat, acid, pressure, and digestive enzymes. Recent research has shown that ginseng saponins require intestinal microbes to convert into these end products.

In order for saponin taken orally to be converted to compound K and be absorbed, it must be decomposed by intestinal microbes. In a study estimated by investigating human content among the general population, 30% of normal people do not have any bacteria that can metabolize saponin, although the remaining bacteria are present. Among 70%, there are differences in metabolic ability, so only a few people can metabolize both diol-based and triol-based saponins normally.

According to a study published by Kyunghee University College of Pharmacy, a maximum blood concentration of Compound K of 0.2 ng/mL per 1 mg of ginsenoside was shown in an oral intake experiment of 24 healthy male adults, and 15 ng per mg of oral Compound K. The maximum blood concentration of /mL was shown, indicating a difference of about 75 times the compound K blood concentration. From this, it can be seen that the compound K conversion rate of general ginsenoside is very low in the intestine. In addition, it is known that the absorption rate of major ginsenosides such as Rb1 in the body is less than 1%.

In this regard, by already completing fermentation with intestinal microbes from the outside and converting it into a form that can absorb the active ingredients of ginseng regardless of the individual's metabolic function, we developed a compound K-fortified ginseng or red ginseng concentrate that anyone can benefit from. It can be considered very desirable to do.

Dansam is a plant belonging to the genus Salvia of the Lamiaceae family, and more than 110 components have been isolated so far, and studies on various pharmacological activities are ongoing. Salvianolic acid B, among the main ingredients of Dansam, has been evaluated as a substance with various pharmacological activities such as lowering blood pressure, preventing Alzheimer's, antioxidant effect, hepatocyte protection, anticancer effect, and alleviating chronic inflammation. According to a study by German Aventis (Aventis), it has also been reported that sweet ginseng extract containing salbinonic acid B showed superior effects over hypertension drugs. In addition, the Department of Family Medicine at the National University of Singapore has experimentally proved the inhibitory effect of single ginseng on liver cancer and induction of hepatocellular apoptosis (Apoptosis). According to studies by many other researchers, the inhibitory effect of salvianolic acid A on breast cancer, It has anticancer effect of tanshinone.

There is no precedent for the processing type using the bioconversion technology of Danseng. In particular, the process of performing a complex bioconversion reaction by mixing with red ginseng is a new manufacturing method. In addition, the complex reaction of a specific ratio of red ginseng and sweet ginseng is expected to improve the solubility of compound K generated during the red ginseng reaction, thereby increasing the absorption rate in the body, and there is a need for research and development on treatment methods for various diseases using these.

Accordingly, the present inventors confirmed that the contents of compound K and salbinonic acid B are high in the bioconversion complex extract prepared by reacting the microbial culture medium after enzyme treatment with extracts of Salvia miltiorrhiza and red ginseng, and excellent anticancer efficacy. I did.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for anticancer comprising a bioconversion extract and red ginseng bioconversion extract.

Another object of the present invention is to provide a method for preparing a pharmaceutical composition for anticancer comprising a biotransformation extract and red ginseng biotransformation extract comprising the following steps:

Enzymatic treatment step of preparing a sweet ginseng enzyme reaction product and a red ginseng enzyme reaction product by adding a brewing enzyme to the sweet ginseng extract and the red ginseng extract; And

A bioconversion step of reacting a microbial culture solution with the enzyme reaction product of Dansam and the enzyme reaction of red ginseng to prepare a bioconversion extract and a bioconversion extract of red ginseng.

Another object of the present invention is to provide a food composition for improving cancer comprising a biotransformation extract of short ginseng and a biotransformation extract of red ginseng.

Another object of the present invention is to provide a method for preparing a food composition for improving cancer comprising the biotransformation extract and red ginseng biotransformation extract comprising the following steps:

Enzymatic treatment step of preparing a sweet ginseng enzyme reaction product and a red ginseng enzyme reaction product by adding a brewing enzyme to the sweet ginseng extract and the red ginseng extract; And

A bioconversion step of reacting a microbial culture solution with the enzyme reaction product of Dansam and the enzyme reaction of red ginseng to prepare a bioconversion extract and a bioconversion extract of red ginseng.

Another object of the present invention relates to the use of the biotransformation extract and red ginseng biotransformation extract for preventing or treating cancer.

The present invention relates to an anticancer composition comprising a Salvia miltiorrhiza biotransformation extract and a red ginseng biotransformation extract, and the present inventors show a high content of compound K and salbinonic acid B in the composition according to the present invention. , It was confirmed that the anticancer efficacy is also excellent.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention is a pharmaceutical composition for anticancer comprising a bioconversion extract and red ginseng bioconversion extract.

The biotransformation extract and red ginseng bioconversion extract may be mixed after preparing the extract by performing a bioconversion process, respectively, and those prepared by performing a bioconversion process to extracts of sweet ginseng and red ginseng may be used.

The term "biotransformation or bioconversion" as used herein refers to a process of converting an added substance into a chemically modified form by using the physiological function of an organism, and specifically, a function of a living body or a living body possessed by a living body. It refers to a technology that uses the function of a catalyst to produce new neoplastic products or to replace existing chemical products synthesized and produced by the existing chemical synthesis process with neoplastic products.

The cancer may be selected from the group consisting of lung cancer, melanoma, gastric cancer, esophageal cancer, breast cancer, ovarian cancer, sarcoma, renal cell cancer, prostate cancer, gastrointestinal stromal tumor, and pancreatic cancer, but is not limited thereto.

The composition may be prepared by adding a microbial culture solution to the enzyme reaction product of sweet ginseng and the enzyme reaction of red ginseng, and the microbial culture solution is Trichoderma reesei , Aspergillus oryzae , Bifidobacterium Longum ( Bifidobacterium longum ) or Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) may be a culture medium of one or more strains selected from the group consisting of, for example, it may be a culture medium of Trichoderma reessay strain, but limited thereto. It does not become.

The term "enzyme reactant" in the present specification refers to a result of performing the enzyme treatment process presented in the present invention.

The single ginseng and red ginseng complex enzyme reactant may be prepared by adding a brewing enzyme to the ginseng extract and red ginseng extract, and at least one enzyme selected from the group consisting of pectinase and arabinase, or expressing the same It may be prepared by adding the strain to the sweet ginseng extract and the red ginseng extract, for example, pectinase and arabinase, or may be prepared by adding a strain expressing the same to the sweet ginseng extract and the red ginseng extract, but limited thereto. It does not become.

The term "brewing enzyme" as used herein refers to an enzyme used in the production process of alcoholic beverages produced by a fermentation process, and more specifically means fermentation enzymes that can be used in a low temperature fermentation process. Specific examples of commercially available brewing enzymes include Gluc Gin, Ginjou-koji, and tome-koji.

The brewing enzyme of the present invention is 1 to 10 parts by weight, more specifically 2 to 8 parts by weight, more specifically 3 to 7 parts by weight, and even more specifically, based on 100 parts by weight of the result obtained through the previous implementation steps. By adding 4 to 6 parts by weight, it can be reacted at a temperature relatively higher than room temperature. Specifically, for example, it can be reacted at a temperature of 25°C to 55°C, 30°C to 55°C, 35°C to 55°C, 40°C to 55°C, 45°C to 55°C, and 48°C to 52°C. The reaction time is suitably about 30 minutes to several hours, specifically, for example, 30 minutes to 6 hours, more specifically 1 hour to 5 hours, and even more specifically 2 hours to 4 hours.

In one embodiment of the present invention, the sweet ginseng and red ginseng complex enzyme reactant may be prepared by adding pectinase and arabinase, or Aspergillus niger var strain to the sweet ginseng extract and red ginseng extract.

The single ginseng and red ginseng complex enzyme reactants are 1:9 to 9:1, 1:9 to 7:3, 1:9 to 6:4, 1:9 to 5:5, 3: It may be included in 7 to 9:1, 3:7 to 7:3 or 3:7 to 6:4, for example, it may be included in 3:7 to 5:5, but is limited thereto no.

The sweet ginseng and red ginseng complex enzyme reactants can be prepared by mixing the enzyme reactants obtained by performing an enzyme treatment process on the sweet ginseng extract and the red ginseng extract, respectively, and by performing an enzyme treatment process on the complex extract obtained by mixing the sweet ginseng extract and the red ginseng extract. It can also be manufactured.

The Dansam extract is 30 to 80% (w/w), 30 to 75% (w/w), 30 to 70% (w/w), 30 to 65% (w/w), 30 to 60% (w /w), 30 to 55% (w/w), 40 to 80% (w/w), 40 to 75% (w/w), 40 to 70% (w/w), 40 to 65% (w /w) or 40 to 60% (w / w) of ethanol may be obtained as a solvent, for example, 40 to 55% (w / w) of ethanol may be obtained as a solvent, It is not limited thereto.

The red ginseng extract is 30 to 80% (w / w), 30 to 75% (w / w), 30 to 70% (w / w), 30 to 65% (w / w), 30 to 60% (w /w), 30 to 55% (w/w), 40 to 80% (w/w), 40 to 75% (w/w), 40 to 70% (w/w), 40 to 65% (w /w) or 40 to 60% (w / w) of ethanol may be obtained as a solvent, for example, 40 to 55% (w / w) of ethanol may be obtained as a solvent, It is not limited thereto.

The composition may include salvianolic acid B and compound K.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used at the time of formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It does not become.

The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, such as intrathecal administration, intravenous administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, intrasternal administration, intratumoral administration, intranasal administration , Intracranial administration, intracranial administration, intrapulmonary administration, and rectal administration may be administered, but are not limited thereto.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity of the patient, Usually, the skilled practitioner can easily determine and prescribe the effective dosage (pharmaceutically effective amount) for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.0001-100 mg/kg.

In the present specification, the term "pharmaceutically effective amount" means an amount sufficient to prevent or treat the aforementioned diseases.

In the present specification, the term “prevention” means preventing or protective treatment of a disease or disease state. As used herein, the term “treatment” refers to reduction, suppression, sedation or eradication of a disease state.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the art. Or it can be made by incorporation into a multi-dose container. At this time, the dosage form may be prepared in various ways, such as internal medicines and injections, and may be in the form of solutions, suspensions, or emulsions in oil or aqueous media, or in the form of extracts, powders, suppositories, powders, granules, tablets or capsules, and may be in the form of a dispersant or stable. Topics may additionally be included.

Another aspect of the present invention is a method for preparing an anticancer pharmaceutical composition comprising the biotransformation extract and red ginseng biotransformation extract comprising the following steps:

Enzymatic treatment step of preparing a sweet ginseng enzyme reaction product and a red ginseng enzyme reaction product by adding a brewing enzyme to the sweet ginseng extract and the red ginseng extract; And

A bioconversion step of reacting a microbial culture solution with the enzyme reaction product of Dansam and the enzyme reaction of red ginseng to prepare a bioconversion extract and a bioconversion extract of red ginseng.

The method may further include an inactivation step of inactivating the brewing enzyme after the enzyme treatment step. Conversion of compound K can be prevented by performing the inactivation step as described above.

The brewing enzyme may be one or more enzymes selected from the group consisting of pectinase and arabinase, and instead of the brewing enzyme, a strain expressing one or more enzymes selected from the group consisting of pectinase and arabinase is added to treat enzymes. You can do it.

The cancer may be selected from the group consisting of lung cancer, melanoma, gastric cancer, esophageal cancer, breast cancer, ovarian cancer, sarcoma, renal cell cancer, prostate cancer, gastrointestinal stromal tumor, and pancreatic cancer, but is not limited thereto. .

In preparing the Danseng extract, the roots of Danseng are taken and crushed after washing and drying with purified water at room temperature, but at this time, it is preferable to have a size of between 40 and 50 mesh. This is because the smaller the particle size, the easier extraction is, but the filtration process becomes difficult.

It is preferable to use 50% ethanol of 10 to 20 times the weight of the crushed powder as the solvent. Alternatively, the purified water extraction step and the ethanol extraction step may be sequentially performed, but it does not significantly affect the final yield. In addition, it is preferable to perform extraction at a temperature between 55 and 60°C. This is because some of the pharmacological ingredients of Dansam are weak to heat. The extract can be used by concentrating under reduced pressure to 30 brix under conditions not exceeding 60°C.

In preparing the red ginseng extract, red ginseng, red ginseng and white ginseng may be used, and it is preferable to use 4 years or more of ginseng. When extracting red ginseng, it is known that ethanol of 10 to 20 times the weight of red ginseng is used, so it is preferable to use ethanol of 20 times the weight of red ginseng in order to maximize the extraction yield of ginsenoside. Ethanol extraction may be performed three times, but is preferably carried out two times.

The enzymatic treatment step may be prepared by adding pectinase and arabinase, or a strain expressing pectinase and arabinase at the same time, to a sweet ginseng extract and a red ginseng extract, but is not limited thereto.

In one embodiment of the present invention, the sweet ginseng and red ginseng complex enzyme reactant may be prepared by adding pectinase and arabinase, or Aspergillus niger var strain to the sweet ginseng extract and red ginseng extract.

In order to perform the enzyme treatment step, purified water may be added so as to be the same as the weight at the time of ethanol extraction, but is not limited thereto. The dosage of the enzyme may be 1 to 2% of the weight of the sweet ginseng or red ginseng powder, and it is preferable not to exceed 3%.

The enzyme treatment step is 20 to 50 ℃, 20 to 45 ℃, 20 to 42 ℃, 28 to 50 ℃, 28 to 45 ℃, 28 to 42 ℃, 32 to 50 ℃, 32 to 45 ℃, 32 to 42 ℃, It may be performed at 40 to 50°C, or 40 to 45°C, for example, 40 to 42°C, but is not limited thereto.

Since salbinonic acid B is vulnerable to heat, when the enzyme treatment step is performed at a temperature of 60°C or higher, its amount may decrease rapidly, and thus, when the enzyme treatment step is performed at a low temperature condition of 60°C or less, the yield can be increased. However, the reaction time may take a long time under a temperature condition of less than 40°C.

The enzyme treatment step is 16 to 36 hours, 16 to 32 hours, 16 to 30 hours, 16 to 25 hours, 18 to 36 hours, 18 to 32 hours, 18 to 30 hours, 18 to 25 hours, 20 to 36 hours, It may be performed for 20 to 32 hours or 20 to 30 hours, for example, 20 to 25 hours, but is not limited thereto.

In mixing the sweet ginseng and red ginseng complex enzyme reactants, it is preferable to sufficiently agitate them so that they are completely mixed. The weight ratio of the sweet ginseng extract and the red ginseng extract is 1:9 to 9:1, 1:9 to 7:3, 1:9 to 6:4, 1:9 to 5:5, 3:7 to 9:1, 3: 7 to 7:3 or 3:7 to 6:4 may be included, for example, may be included in 3:7 to 5:5, but is not limited thereto.

In the biotransformation step, the microbial culture medium may be a culture medium of one or more strains selected from the group consisting of Trichoderma reessay, Aspergillus oris, Bifidobacterium longum, or Lactobacillus rhamnosus, for example For example, it may be a culture medium of the Trichoderma Reessay strain, but is not limited thereto.

Microbial culture solution may be added 1 to 5 parts by weight, and reacted at 40 to 45°C for 50 to 80 hours, and then at 70 to 100°C for 40 to 80 minutes with respect to 100 parts by weight of the Dansam and Red Ginseng complex enzyme reactants. While heating may be performed to terminate the bioconversion process, it is not limited thereto.

Another aspect of the present invention is a food composition for improving cancer comprising a biotransformation extract of short ginseng and a biotransformation extract of red ginseng.

The single ginseng bioconversion extract and the red ginseng bioconversion extract may be mixed after preparing an extract by performing a bioconversion process, respectively, and those prepared by performing a bioconversion process of a single ginseng and red ginseng extract may be used.

The single ginseng bioconversion extract and red ginseng bioconversion extract may be prepared by adding a microbial culture solution to a single ginseng enzyme reaction product and a red ginseng enzyme reaction product.

The cancer may be selected from the group consisting of lung cancer, melanoma, gastric cancer, esophageal cancer, breast cancer, ovarian cancer, sarcoma, renal cell cancer, prostate cancer, gastrointestinal stromal tumor, and pancreatic cancer, but is not limited thereto. .

The sweet ginseng and red ginseng complex enzyme reactant may be prepared by adding a brewing enzyme to the sweet ginseng extract and red ginseng extract, and at least one enzyme selected from the group consisting of pectinase and arabinase, or a strain expressing the same may be used as a sweet ginseng extract and It may be prepared by adding to the red ginseng extract, for example, pectinase and arabinase, or a strain expressing the same may be added to the sweet ginseng extract and the red ginseng extract, but is not limited thereto.

In one embodiment of the present invention, the sweet ginseng and red ginseng complex enzyme reactant may be prepared by adding pectinase and arabinase, or Aspergillus niger strain to the sweet ginseng extract and red ginseng extract.

The single ginseng and red ginseng complex enzyme reactants are 1:9 to 9:1, 1:9 to 7:3, 1:9 to 6:4, 1:9 to 5:5, 3: It may be included in 7 to 9:1, 3:7 to 7:3 or 3:7 to 6:4, for example, it may be included in 3:7 to 5:5, but is limited thereto no.

The sweet ginseng and red ginseng complex enzyme reactants can be prepared by mixing the enzyme reactants obtained by performing an enzyme treatment process on the sweet ginseng extract and the red ginseng extract, respectively, and by performing an enzyme treatment process on the complex extract obtained by mixing the sweet ginseng extract and the red ginseng extract. It can also be manufactured.

The Dansam extract is 30 to 80% (w/w), 30 to 75% (w/w), 30 to 70% (w/w), 30 to 65% (w/w), 30 to 60% (w /w), 30 to 55% (w/w), 40 to 80% (w/w), 40 to 75% (w/w), 40 to 70% (w/w), 40 to 65% (w /w) or 40 to 60% (w / w) of ethanol may be obtained as a solvent, for example, 40 to 55% (w / w) of ethanol may be obtained as a solvent, It is not limited thereto.

The red ginseng extract is 30 to 80% (w / w), 30 to 75% (w / w), 30 to 70% (w / w), 30 to 65% (w / w), 30 to 60% (w /w), 30 to 55% (w/w), 40 to 80% (w/w), 40 to 75% (w/w), 40 to 70% (w/w), 40 to 65% (w /w) or 40 to 60% (w / w) of ethanol may be obtained as a solvent, for example, 40 to 55% (w / w) of ethanol may be obtained as a solvent, It is not limited thereto.

The microbial culture medium may be a culture medium of one or more strains selected from the group consisting of Trichoderma reessay, Aspergillus oris, Bifidobacterium longum, or Lactobacillus rhamnosus, for example, Trichoderma It may be a culture medium of the Reessay strain, but is not limited thereto.

Another aspect of the present invention is a method for preparing a food composition for improving cancer comprising the biotransformation extract and red ginseng biotransformation extract comprising the following steps:

Enzymatic treatment step of preparing a sweet ginseng enzyme reaction product and a red ginseng enzyme reaction product by adding a brewing enzyme to the sweet ginseng extract and the red ginseng extract; And

A bioconversion step of reacting a microbial culture solution with the enzyme reaction product of Dansam and the enzyme reaction of red ginseng to prepare a bioconversion extract and a bioconversion extract of red ginseng.

The method may further include an inactivation step of inactivating the brewing enzyme after the enzyme treatment step. Conversion of compound K can be prevented by performing the inactivation step as described above.

The brewing enzyme may be one or more enzymes selected from the group consisting of pectinase and arabinase, and instead of the brewing enzyme, a strain expressing one or more enzymes selected from the group consisting of pectinase and arabinase is added to treat enzymes. You can do it.

The cancer may be selected from the group consisting of lung cancer, melanoma, gastric cancer, esophageal cancer, breast cancer, ovarian cancer, sarcoma, renal cell cancer, prostate cancer, gastrointestinal stromal tumor, and pancreatic cancer, but is not limited thereto. .

The enzymatic treatment step may be performed by adding pectinase and arabinase, or a strain expressing pectinase and arabinase at the same time to the sweet ginseng extract and the red ginseng extract, but is not limited thereto.

In one embodiment of the present invention, the sweet ginseng and red ginseng complex enzyme reactant may be prepared by adding pectinase and arabinase, or Aspergillus niger var strain to the sweet ginseng extract and red ginseng extract.

The enzyme treatment step is 20 to 50 ℃, 20 to 45 ℃, 20 to 42 ℃, 28 to 50 ℃, 28 to 45 ℃, 28 to 42 ℃, 32 to 50 ℃, 32 to 45 ℃, 32 to 42 ℃, It may be performed at 40 to 50°C, or 40 to 45°C, for example, 40 to 42°C, but is not limited thereto.

The enzyme treatment step is 16 to 36 hours, 16 to 32 hours, 16 to 30 hours, 16 to 25 hours, 18 to 36 hours, 18 to 32 hours, 18 to 30 hours, 18 to 25 hours, 20 to 36 hours, It may be performed for 20 to 32 hours or 20 to 30 hours, for example, 20 to 25 hours, but is not limited thereto.

The single ginseng and red ginseng complex enzyme reactants are 1:9 to 9:1, 1:9 to 7:3, 1:9 to 6:4, 1:9 to 5:5, 3: It may be included in 7 to 9:1, 3:7 to 7:3 or 3:7 to 6:4, for example, it may be included in 3:7 to 5:5, but is limited thereto no.

The microbial culture medium may be a culture medium of a strain of the genus Trichoderma, for example, one selected from the group consisting of Trichoderma reessay, Aspergillus oris, Bifidobacterium longum, or Lactobacillus rhamnosus It may be a culture solution of the above strain, for example, may be a culture solution of the Trichoderma Reessay strain, but is not limited thereto.

According to another aspect of the present invention, the present invention provides a method for preventing or treating cancer comprising administering to a subject a pharmaceutical composition comprising the aforementioned biotransformation extract and red ginseng biotransformation extract as active ingredients. .

As used herein, the term "administering" or "administering" refers to a subject by directly administering a therapeutically or prophylactically effective amount of a composition of the present invention to a subject (individual) suffering from or likely to suffer from the subject disease. It means that the same amount is formed in the body.

The "therapeutically effective amount" of the composition means an amount of the composition sufficient to provide a therapeutic or prophylactic effect to a subject to which the composition is to be administered, and includes a "prophylactically effective amount".

In addition, the term "subject (individual)" as used herein is a mammal including humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons and rhesus monkeys. . Most specifically, the subject of the present invention is a human.

Since the method for preventing or treating cancer of the present invention is a method comprising administering a pharmaceutical composition, which is an aspect of the present invention, descriptions of overlapping contents are omitted in order to avoid excessive redundancy in the present specification. .

The present invention relates to a composition for anticancer comprising a Salvia miltiorrhiza bioconversion extract and a red ginseng bioconversion extract, and a Trichoderma reesei culture solution after enzymatic treatment in the Dansam extract and red ginseng extract Since the biotransformation complex extract prepared by reacting has an excellent effect on inhibiting the proliferation of cancer cells, it can be effectively used for the prevention or treatment of cancer.

1 is a diagram showing a manufacturing process of a bioconversion complex extract according to an embodiment of the present invention.

2 is a graph showing high-performance liquid chromatography (HPLC) data of a compound K content analysis experiment of a bioconversion complex extract according to an embodiment of the present invention.

Figure 3 is a graph showing the MTT analysis (assay) experiment results for the inhibitory effect of the lung cancer cell line activity of the biotransformation complex extract according to an embodiment of the present invention according to the composition ratio of sweet ginseng and red ginseng.

4 is a graph showing the experimental results of a colony formation assay on the inhibitory effect on the activity of lung cancer cell lines of a bioconversion complex extract according to an embodiment of the present invention according to the composition ratio of sweet ginseng and red ginseng.

5A is a graph showing the results of flow cytometry cell counting (FACC) on the inhibitory effect of biotransformation complex extracts according to an embodiment of the present invention according to the composition ratio of sweet ginseng and red ginseng.

5B is a graph showing the results of flow cytometry cell counting (FACC) on the inhibitory effect on lung cancer cell line activity of the biotransformation complex extract according to an embodiment of the present invention according to the composition ratio of sweet ginseng and red ginseng.

5C is a graph showing the results of flow cytometry cell counting (FACC) on the inhibitory effect on lung cancer cell line activity of the biotransformation complex extract according to an embodiment of the present invention according to the composition ratio of sweet ginseng and red ginseng.

5D is a graph showing the results of flow cytometry cell counting (FACC) on the inhibitory effect of the biotransformation complex extract according to an embodiment of the present invention according to the composition ratio of single ginseng and red ginseng.

5E is a graph showing the results of flow cytometry cell counting (FACC) on the inhibitory effect on lung cancer cell line activity of the biotransformation complex extract according to an embodiment of the present invention according to the composition ratio of sweet ginseng and red ginseng.

5F is a graph showing the results of flow cytometry cell counting (FACC) on the inhibitory effect of the bioconversion complex extract according to an embodiment of the present invention according to the composition ratio of single ginseng and red ginseng.

5G is a graph showing the results of flow cytometry cell counting (FACC) on the inhibitory effect on lung cancer cell line activity of the biotransformation complex extract according to an embodiment of the present invention according to the composition ratio of sweet ginseng and red ginseng.

5H is a graph showing the results of flow cytometry cell counting (FACC) on the inhibitory effect on lung cancer cell line activity of the bioconversion complex extract according to an embodiment of the present invention according to the composition ratio of sweet ginseng and red ginseng.

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Throughout this specification, "%" used to indicate the concentration of a specific substance is (weight/weight)% for solids/solids, (weight/volume)% for solids/liquids, and Liquid/liquid is (vol/vol)%.

Example 1: Preparation of biotransformation complex extract

1-1. Preparation of enzyme reaction product of Danshen

50 kg of the roots of Danseng were purchased, the residual soil was removed with purified water, washed, and then crushed by drying with warm air at 40 to 50°C. With respect to 45 kg of the crushed ginseng root powder, 900 L of 50% ethanol was added and extracted for 72 hours at 55°C to prepare an ethanol extract, which was concentrated under reduced pressure to 30 brix without filtration.

Then, purified water was added to dilute 10 times, and Gluc gin (manufacturer: Higusi Moyashi, enzyme type: Pectinase and Arabinase, enzyme producing microorganism: Aspergillus niger var) was added 2 to 3% by weight of the diluted solution. The reaction was carried out at 50 to 55° C. for 24 hours.

The reaction product was heated at 90° C. for 1 hour to inactivate the residual enzyme, and the reaction product was concentrated under reduced pressure to 30 brix. After analyzing the content of the physiologically active substance salvianolic acid B in the concentrate, it was diluted with purified water so that the final concentration was 2 mg/ml.

1-2. Preparation of red ginseng enzyme reactant

It was carried out in the same manner as in Example 1-1, but was prepared using red ginseng or red ginseng instead of sweet ginseng. In the concentrate, it was diluted with purified water so that the final concentration of Rb1 was 2 mg/ml.

1-3. Preparation of Trichoderma Reessay culture solution

After incubating the Trichoderma reesei strain in a 30°C liquid medium (YM broth: Yeast Extract 3 g, Malt Extract 3 g, Peptone 5 g, Dextrose 10 g dissolved in 1 liter of purified water) for 24 hours , The supernatant was taken through centrifugation, salting out was performed once, and the obtained reaction solution was filtered (pore size 0.22 μm) to obtain a culture solution.

1-4. Preparation of bioconversion complex extracts of sweet and red ginseng

The enzyme reaction products of Examples 1-1 and 1-2 were mixed in various weight ratios of 0:10, 3:7, 4:6, 5:5, 6:4, 7:3 and 10:0. Thereafter, 2 parts by weight of the culture solution of Trichoderma reesei of Example 1-3 was added to 100 parts by weight of the enzyme reaction products of sweet ginseng and red ginseng, followed by reaction at 40 to 45° C. for 72 hours to bioconvert. Thereafter, the reaction solution was heated at 90° C. for 1 hour to inactivate the residual enzyme and terminate the bioconversion process. The prepared single ginseng and red ginseng bioconversion complex extract was cooled to 40 to 50°C and then filtered using perlite. The filtrate was concentrated under reduced pressure to 70 brix to obtain a final product (hyperfree).

Example 2: Confirmation of compound K content of bioconversion complex extracts of sweet ginseng and red ginseng

High-performance liquid chromatography under the conditions shown in Table 1 below by diluting the complex extracts for bioconversion of sweet ginseng and red ginseng prepared according to an embodiment of the present invention, and the complex extracts that have not undergone any of the enzyme treatment and bioconversion processes, respectively. The content of compound K was compared through (high-performance liquid chromatography, HPLC).

Column	Kromasil 100-5-C18
Analytical time	70 min.
Mobile phase	Water:Acetonitrile (80:20~50:50)
Detection wavelength	203 nm

As can be seen in FIG. 2, compound K was not identified in the enzyme reaction product when the bioconversion process was not performed, and 17.3 mg/g of the compound in the bioconversion complex extract of sweet ginseng and red ginseng which was subjected to the bioconversion process through the above method. The K content was confirmed.

Example 3: Confirmation of the inhibitory effect on lung cancer cell line activity in the biotransformation complex extract of sweet ginseng and red ginseng

After dispensing 15,000 lung cancer cell lines (H460, ATCC) per well into a 24-well plate in advance, and then incubating for 24 hours using DMEM medium in a CO ₂ incubator, Examples of cultured lung cancer cells Samples with different compositions of sweet ginseng and red ginseng prepared in 1-4 were treated.

At this time, the samples for each step were diluted 50 times, and 0.5 ml (9.5 ml of medium) was added to each well, and further cultured for 24 hours. The media components of each well were discarded and the remaining cells were expressed as percentages of the cell viability of the other experimental groups based on the cell viability of the control group using the MTT assay.

As can be seen in Fig. 3 and Table 2, when administered to the lung cancer cell line H460 a single ginseng and red ginseng biotransformation complex extract showed strong toxicity.

Dansam: Red ginseng	Control	0:10	3:7	4:6	5:5	6:4	7:3	10:0
Cell viability (%)	100	88.5	73	43	53.5	65	78.5	95

As shown in the chart above, the lung cancer cell inhibitory activity was different for each specific ratio, and particularly, when the ratio was 4:6, the lung cancer cell inhibitory activity was remarkable.

Example 4: Confirmation of the effect of inhibiting colony generation of lung cancer cell lines in the biotransformation complex extract of sweet ginseng and red ginseng

The anticancer effect of the bioconversion complex extract of sweet ginseng and red ginseng was confirmed through colony formation assay and flow cytometry cell counting (FACC).

Specifically, 200 lung cancer cell lines (H460, ATCC) were previously dispensed on a 60 mm plate, and then cultured for 24 hours using DMEM medium in a CO ₂ incubator, and the above-described step-by-step samples were treated on the cultured lung cancer cells. . At this time, each step sample was diluted 50 times, administered 0.5 ml to each well (9.5 ml of medium), and further cultured for 1 week. At this time, the medium was changed every 72 hours.

After one week, the medium components of each well were discarded, washed with PBS, and fixed with methanol and acetic acid (MeOH: acetic acid = 3: 1) for 15 minutes. After that, it was stained with 0.5% crystal violet for 15 minutes at room temperature.

As can be seen in Figure 4, the anticancer effect of the biotransformation complex extract of sweet ginseng and red ginseng showed similar colony-producing inhibitory activity at almost the same ratio as in Example 3, and FACC analysis was also performed based on this.

After dispensing 10,000 lung cancer cell lines (H460, ATCC) on a 60 mm plate in advance, and incubating for 24 hours using DMEM medium in a CO ₂ incubator, the above-described step-by-step samples were treated on the cultured lung cancer cells. At this time, each step sample was diluted 50 times, administered to each well, and cultured for 72 hours.

The media components of each well were discarded and washed with PBS, followed by FACC analysis. At this time, the nuclei of all cells were stained with PI (Propidium Iodide), and Annexin V stained using Invitrogen™ Annexin V, Alexa Fluor™ 488 conjugate.

As can be seen in FIGS. 5A to 5H, the apoptosis pattern of the H460 cancer cell line was different according to the mixing ratio of the biotransformation complex extract of sweet ginseng and red ginseng, and in the ratio of 4:6, 5:5, 6:4 Considering the amount of annexin V, it can be seen that it induces apoptosis of the H460 cancer cell line at a remarkably high rate. Therefore, it can be seen that the bioconversion complex extract of sweet ginseng and red ginseng has excellent anticancer effect.

Claims (16)

1. Dansam ( Salvia miltiorrhiza ) bioconversion extract and red ginseng (red ginseng) a pharmaceutical composition for anticancer comprising a bioconversion extract.
2. The pharmaceutical composition of claim 1, wherein the bioconversion extract and red ginseng bioconversion extract are prepared by adding a microbial culture solution to the enzyme reaction product and the enzyme reaction product of red ginseng.
3. The method of claim 2, wherein the microbial culture medium consists of Trichoderma reesei , Aspergillus oryzae , Bifidobacterium longum , or Lactobacillus rhamnosus . It is a culture medium of one or more strains selected from the group, pharmaceutical composition.
4. The pharmaceutical composition of claim 2, wherein the enzyme reaction product of sweet ginseng and the enzyme reactant of red ginseng are prepared by adding a brewing enzyme to the extract of sweet ginseng and red ginseng.
5. The pharmaceutical composition of claim 1, wherein the composition comprises a single ginseng bioconversion extract and a red ginseng bioconversion extract in a weight ratio of 1:9 to 9:1.
6. The pharmaceutical composition of claim 5, wherein the composition comprises a bioconversion extract of short ginseng and a bioconversion extract of red ginseng in a weight ratio of 3:7 to 7:3.
7. A method for preparing an anticancer pharmaceutical composition comprising the following steps: Salvia miltiorrhiza biotransformation extract and red ginseng biotransformation extract:

   Enzymatic treatment step of preparing a sweet ginseng enzyme reaction product and a red ginseng enzyme reaction product by adding a brewing enzyme to the sweet ginseng extract and the red ginseng extract; And

   A bioconversion step of reacting a microbial culture solution with the enzyme reaction product of Dansam and the enzyme reaction of red ginseng to prepare a bioconversion extract and a bioconversion extract of red ginseng.
8. The method of claim 7, wherein the enzyme treatment step is performed at 20 to 50°C.
9. The method of claim 7, wherein the enzyme treatment step is performed for 16 to 36 hours.
10. The method of claim 7, wherein the composition comprises a bioconversion extract of short ginseng and a bioconversion extract of red ginseng in a weight ratio of 1:9 to 9:1.
11. The method of claim 10, wherein the composition comprises a biotransformation extract of short ginseng and a bioconversion extract of red ginseng in a weight ratio of 3:7 to 7:3.
12. The method of claim 7, wherein the microbial culture medium is a culture medium of a strain of the genus Trichoderma .
13. Dansam ( Salvia miltiorrhiza ) bioconversion extract and red ginseng (red ginseng) a food composition for improving cancer comprising a bioconversion extract.
14. The food composition for cancer improvement according to claim 13, wherein the bioconversion extract and red ginseng bioconversion extract are prepared by adding a microbial culture solution to the enzyme reaction product and the enzyme reaction product of red ginseng.
15. The method of claim 14, wherein the microbial culture medium is composed of Trichoderma reesei , Aspergillus oryzae , Bifidobacterium longum , or Lactobacillus rhamnosus . It is a culture medium of one or more strains selected from the group, cancer improvement food composition.
16. A method of preparing a food for cancer improvement comprising the following steps: Salvia miltiorrhiza biotransformation extract and red ginseng biotransformation extract:

    Enzymatic treatment step of preparing a sweet ginseng enzyme reaction product and a red ginseng enzyme reaction product by adding a brewing enzyme to the sweet ginseng extract and the red ginseng extract; And

    A bioconversion step of reacting a microbial culture solution with the enzyme reaction product of Dansam and the enzyme reaction of red ginseng to prepare a bioconversion extract and a bioconversion extract of red ginseng.

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