KR102167790B1 - Black ginseng extract with increased anti-cancer component contents, method for manufacturing thereof, and anti-cancer composition comprising the same - Google Patents

Abstract

The present invention relates to a black ginseng extract and a method for preparing the same, and more particularly, to a black ginseng extract having a significantly increased content of anticancer components, a method for preparing the same, and an anticancer composition comprising the same.

Description

Black ginseng extract with increased anti-cancer component contents, method for manufacturing thereof, and anti-cancer composition comprising the same}

The present invention relates to a black ginseng extract and a method for preparing the same, and more particularly, to a black ginseng extract having a significantly increased content of anticancer components, a method for preparing the same, and an anticancer composition comprising the same.

Korea's ginseng has a historical record of being manufactured from about 1,000 years ago, and now it is formed with a global market size of about 3.5 billion dollars, making it a unique traditional herbal medicine or health functional food that is good for domestic venture companies to enter the world market. Ginseng is a perennial, semi-neutral plant belonging to the family Ogapiaceae. Its efficacy is widely known in various medical books in China and Korea, and has been used as a medicinal herb in many fields since ancient times. The ingredients of ginseng are carbohydrates (60-70%) and nitrogen-containing compounds. (12~16%), saponin (3~6%), fat-soluble ingredients (1~2%), ash (4~6%), vitamins (0.005%), etc. The physiological activity of ginseng is a recent systematic pharmacological approach. It has a nourishing tonic effect, ameliorating sexual and reproductive dysfunction, antihypertension and anti-arteriosclerosis, hematopoietic and anemia treatment, blood sugar metabolism and diabetes improvement, anticancer effect, liver It has dysfunction improvement effect, hangover relief effect, parasite infection prevention effect, analgesic anti-inflammatory effect, etc. In modern times, ginseng has been widely studied, and it is known that ginseng contains a large amount of ginsenoside, and in addition to medicines that have been used for a long time, it is applied to foods and is widely used as an ingredient of health food.

According to studies so far, the active ingredient present in ginseng is generally known as ginseng saponin (ginsenoside), and ginseng saponin is the alcoholic OH of R1, R2, R3 of protopanaxadiol or protopanaxatriol, a triterpene. As a compound having a structure in which saccharides such as glucose, rhamnose, xylose, and arabinose are ester-linked, more than 30 species have been identified so far, and anticancer activity, antidiabetic activity, central nervous system suppression, arteriosclerosis and hypertension It is known to exhibit pharmacological effects such as prevention, liver function promotion and hangover removal effect, anti-fatigue and anti-stress action, antioxidant action, anti-inflammatory action, protein synthesis promotion action, and immunity enhancing action.

These ginseng saponins are generally known to exist in fresh ginseng, but some ginseng saponins are known to exist only in processed ginseng. Recently, many studies have been conducted to further increase the content of ginseng saponins present only in these processed ginsengs, and as a result, processed ginsengs such as taeguk ginseng and black ginseng have been developed. . In particular, black ginseng refers to ginseng in which ginseng has turned black after several times of steaming and drying, and ginseng saponin that is basically present through the process of multiplying and drying several times is not lost, and is present only in processed ginseng. Since it is known that the content of ginseng saponin is increased, research on a method for more effectively producing such black ginseng is being actively conducted. However, the problem of black ginseng produced to date is that the content of ginseng saponin contained in black ginseng does not show a significant difference from that of red ginseng, so the content of ginseng saponin contained in black ginseng, especially ginseng present only in processed ginseng. If the content of the saponin component can be significantly increased compared to that of red ginseng, it is expected that the above-described problem of black ginseng can be solved, but no other achievement has been reported yet.

KR 10-2018-0078658A

The present invention has been devised to solve the above-described problems, and an object of the present invention is to provide a black ginseng extract and a method of manufacturing the same, in which the content of anticancer components is significantly increased.

In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, or a food composition for preventing or improving cancer, having a black ginseng extract or a fraction thereof containing a high anticancer component as an active ingredient.

In order to solve the above-described problems, the present invention provides (1) a first jeungsam process for heat-treating ginseng at 55℃ to 75℃ and a second jeungsam process for heat treating ginseng performed at 85℃ to 105℃. Including, but repeating the second jeungsam process 2 to 5 times, preparing a black ginseng having an increased content of the compound represented by the following formula (1) and the compound represented by formula (2); And (2) extracting the black ginseng to prepare a black ginseng extract; It provides a method for producing a black ginseng extract containing an increased content of anti-cancer components.

[Formula 1]

[Formula 2]

According to an embodiment of the present invention, the first jeungsam process may be performed for 5 to 15 minutes, and the second jeungsam step may be performed once for 1.5 to 5.5 hours.

In addition, the step of washing and drying ginseng before step (1); may further include.

In addition, the step (2) is 2-1) repeatedly extracting the mixed solution containing black ginseng and organic solvent 2 to 5 times for 20 to 28 hours at 30 ℃ ~ 50 ℃; And 2-2) preparing a black ginseng extract by concentrating the extracted black ginseng under reduced pressure.

In addition, the extraction in step (2) may be performed through an organic solvent, which is a lower alcohol having 1 to 4 carbon atoms. In addition, the organic solvent may be methanol having a concentration of 90% or more.

In addition, the mixed solution of step 2-1) may contain black ginseng and an organic solvent in a weight ratio of 60:40 to 80:20.

In addition, the present invention comprises the steps of fractionating the black ginseng extract prepared by the above-described preparation method to prepare a fraction of the black ginseng extract containing any one or more of the compound represented by the following formula (1) and the compound represented by formula (2); including It provides a method for producing a black ginseng extract fraction with an increased content of anticancer components.

[Formula 1]

[Formula 2]

According to an embodiment of the present invention, the fraction of the black ginseng extract may be an ethyl acetate fraction.

On the other hand, the present invention is prepared by the above-described manufacturing method, and per 1 g of black ginseng having a moisture content of 15% or less, the content of an anticancer component containing 0.15 mg or more of the compound represented by the following formula 1 and 0.10 mg or more of the compound represented by the formula 2 Provides this increased black ginseng extract.

[Formula 1]

[Formula 2]

In addition, the present invention is a compound represented by the following formula (1) and the extract prepared through the above-described manufacturing method including the compound represented by the following formula (2), or the compound represented by the following formula (1) and the compound represented by the following formula (2) It provides a pharmaceutical composition for preventing or treating cancer comprising a fraction prepared through the above-described manufacturing method including any one or more as an active ingredient.

[Formula 1]

[Formula 2]

According to an embodiment of the present invention, the cancer may be any one selected from the group consisting of ovarian cancer, skin cancer, uterine cancer, liver cancer, and colon cancer.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising a compound represented by the following Formula 1 or a salt thereof or a mixture thereof, or a compound represented by Formula 2 or a salt thereof or a mixture thereof as an active ingredient. .

[Formula 1]

[Formula 2]

In addition, the present invention is an extract prepared through the above-described manufacturing method comprising a compound represented by Formula 1 and a compound represented by Formula 2, or any one or more of a compound represented by Formula 1 and a compound represented by Formula 2 It provides a food composition for preventing or improving cancer comprising the fraction prepared through the above-described manufacturing method including as an active ingredient.

Hereinafter, terms used in the present invention will be described.

As a term used in the present invention, the term "prevention" in the present invention means any action of inhibiting the occurrence of cancer or delaying the onset of cancer by administration of the composition.

In the present invention, the term "treatment" refers to any action in which symptoms caused by cancer are improved or beneficially changed by administration of the composition.

The method for preparing black ginseng extract of the present invention can significantly increase the content of the compounds represented by Chemical Formulas 1 and 2 according to the present invention, which has excellent anticancer activity. Accordingly, it is possible to obtain a black ginseng extract containing a high content of anticancer components, and such black ginseng extract can be usefully applied as an active ingredient of a pharmaceutical composition or food composition having an anticancer action.

1 is a result of performing UPLC-QTOF/MS on the extract fraction of black ginseng ethyl acetate according to an embodiment of the present invention.
2 is a graph showing the inhibition of cancer cell toxicity in various cancer cell lines according to the concentration of the compound represented by Formula 1 according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily implement the present invention. The present invention may be implemented in various different forms, and is not limited to the embodiments described herein.

The manufacturing method of the black ginseng extract according to an embodiment of the present invention includes (1) heat-treating ginseng at 55°C to 75°C, and heat-treating the ginseng performed in the first jeungsam process at 85°C to 105°C. Including the second jeungsam process, but repeating the second jeungsam process 2 to 5 times, preparing black ginseng with an increased content of the compound represented by the following formulas 1 and 2, and (2) extracting black ginseng It includes the step of preparing a black ginseng extract.

[Formula 1]

[Formula 2]

The step (1) is a process of producing black ginseng by increasing ginseng, and includes a first jeungsam process and a second jeungsam process that is repeatedly performed 2 to 5 times after the first jeungsam process.

The first jeungsam process is a process of heat-treating ginseng at 55 to 75°C to increase ginseng, and an apparatus used in a conventional jeungsam process may be used, and as an example, may be performed in a steamer.

The ginseng may have been further washed and dried before the first steaming process. The washing is not limited in the case of a method capable of completely removing foreign substances such as dirt and vinyl from the surface of ginseng, but it is preferable to wash the ginseng surface so as not to scratch the surface.

The washed ginseng may be further subjected to a drying process thereafter, and the drying process may be hot air drying performed at 30 to 40°C for 10 to 15 hours, or natural drying performed for 2 to 3 days.

Thereafter, the dried ginseng is put into the first steaming process and rapidly heated from room temperature, for example, 25°C to 55°C to 75°C, and then heat-treated at that temperature for 5 to 15 minutes, preferably 　10 to 15 minutes. have. At this time, the temperature increase rate is preferably 40 ~ 60 ℃ / min, and when the temperature is raised at a rate slower than the temperature increase rate, it may be difficult to achieve the object of the present invention, such as it may be difficult to significantly increase the content of the target anticancer component. The first jeungsam process may be preferably heat-treated at 60°C to 70°C for 6 to 13 minutes after being rapidly heated. If the temperature and/or time range of the heat treatment of the first jeungsam process is not satisfied, the desired anticancer component, particularly the compounds represented by Formulas 1 and 2, is hardly produced, or even if produced, it may be difficult to significantly increase the content. Etc. It may be difficult to achieve the object of the present invention.

After performing the first jeungsam process, the second jeungsam step may be performed, preferably, a drying step may be further performed before performing the second jeungsam step. The drying process is preferably performed for 20 to 30 hours in a dryer of 30 to 40 ℃.

After the first steaming process, the first steamed ginseng that has gone through the drying process is put into the second steamed ginseng process. The second jeungsam process is a process in which the input ginseng is heated from room temperature, for example, 25°C to 85°C to 105°C, and then heat-treated at the corresponding temperature for 1.5 to 5.5 hours, preferably at 90°C to 100°C. It can be heat treated for 5 hours. If the temperature and/or time range of the heat treatment of the second jeungsam process is not satisfied, the target anticancer component, especially the compounds represented by the above-described formulas 1 and 2, is hardly produced, or even if produced, it is difficult to significantly increase the content. It may be difficult to achieve the object of the present invention, such as. On the other hand, in the second jeungsam process, the heating rate is preferably 15 to 20°C/min, and when the temperature is raised at a rate slower than the heating rate, it is difficult to achieve the object of the present invention, such as it may be difficult to significantly increase the content of the target anticancer component. I can.

The ginseng that has undergone the second jeungsam process may be manufactured into black ginseng by repeating the second jeungsam process 2 to 6 times, preferably 3 to 5 times. If the number of repetitions of the second jeungsam process is less than two, the target anticancer component, in particular, the compounds represented by the above-described formulas 1 and 2, is hardly produced, or even if produced, it may be difficult to significantly increase the content of the present invention. Can be difficult to achieve.

On the other hand, after going through the second steaming process once, the drying process may be further carried out before re-entering the second steaming process of the next session. The drying process is preferably performed for 20 to 30 hours in a dryer of 30 to 40 ℃.

In addition, after the final second steaming process is performed, a complete drying process may be further performed, and the complete drying may be performed in a 40 ~ 60℃ dryer for 20 ~ 30 hours, in a 30 ~ 40℃ dryer for 1 ~ 3 days, or several days. Can be naturally dried. Black ginseng that has been subjected to the final complete drying process may have a moisture content of 15% or less.

Next, as step (2) according to the present invention, a step of extracting black ginseng to prepare a black ginseng extract is performed.

Specifically, step (2) may include 2-1) repeatedly extracting a mixed solution containing black ginseng and an organic solvent, and 2-2) preparing a black ginseng extract by concentrating the extracted black ginseng under reduced pressure.

The step 2-1) is a step of repeatedly extracting a mixed solution containing black ginseng and an organic solvent, and the mixed solution may be repeatedly extracted 2 to 5 times for 20 to 28 hours at 30°C to 50°C, preferably 40 It can be repeatedly extracted 2 to 4 times for 22 to 26 hours at ℃ ~ 50 ℃. If the temperature of the repeated extraction does not satisfy the above range, it may be difficult to achieve the object of the present invention, such as it may be difficult to extract the anticancer component, particularly the compounds represented by Formulas 1 and 2, in a significant amount.

On the other hand, the organic solvent can be used without limitation, as long as it is a material that can be used as an extraction solvent in general, preferably a lower alcohol having 1 to 4 carbon atoms, more preferably one selected from methanol, ethanol, and fermented ethanol It may be more than that, and even more preferably methanol may be used. When methanol is used as the organic solvent, the organic solvent may be methanol at a concentration of 80% or more, preferably methanol at a concentration of 85% or more, and more preferably methanol at a concentration of 90% or more. If the concentration of methanol is less than 80%, it may be difficult to achieve the object of the present invention, such as it may be difficult to extract the anticancer component, particularly the compound represented by the above-described formula 1 in a significant amount.

In addition, the mixed solution may contain the black ginseng and the organic solvent in a weight ratio of 60:40 to 80:20, preferably in a weight ratio of 65:35 to 75:25. If the weight ratio of the black ginseng and the organic solvent is out of the above range, it may be difficult to achieve the object of the present invention, such as it may be difficult to extract the anticancer component, particularly the compound represented by Formula 1, in a significant amount.

In addition, the step 2-2) is a step of preparing a black ginseng extract by concentrating black ginseng under reduced pressure, and the conditions of the vacuum concentration can be used without limitation as long as it is a condition of reduced pressure concentration that can be used normally. It does not specifically limit.

On the other hand, in the present invention, per 1 g of black ginseng having a moisture content of 15% or less prepared by the above-described manufacturing method, the content of the anticancer component containing 0.15 mg or more of the compound represented by the following formula 1 and 0.10 mg or more of the compound represented by the formula 2 Contains increased black ginseng extract.

[Formula 1]

[Formula 2]

Compared to other methods of increasing ginseng such as gujeunggupo, the compound represented by Formula 1 and Formula 2 is contained in a significantly higher content, and specifically, the compound represented by Formula 1 is per 1 g of black ginseng having a moisture content of 15% or less. 0.15 mg or more, preferably 0.17 mg or more, more preferably 0.20 mg or more, even more preferably 0.20 to 0.30 mg of the compound represented by Formula 1, and 0.10 mg or more of the compound represented by Formula 2, Since preferably 0.13 mg or more, more preferably 0.15 mg or more, and even more preferably 0.13 to 0.25 mg are included, the black ginseng extract according to the present invention may be very useful as an active ingredient of an anticancer composition.

On the other hand, the black ginseng extract prepared by the above-described manufacturing method may be further subjected to a fractionation process for separating the anticancer component including the compounds represented by Formulas 1 and 2, through which the compound represented by Formula 1 and the compound represented by Formula 2 A fraction of the black ginseng extract containing all of the compounds represented, a fraction containing the compound represented by Formula 1, and a fraction including the compound represented by Formula 2 obtained by fractionating the fraction including both compounds again through column chromatography, etc. Can be manufactured. The fraction of the black ginseng extract, that is, the black ginseng extract fraction may be preferably an ethyl acetate fraction.

Hereinafter, a method of preparing an ethyl acetate fraction containing the compound represented by Formula 1 and the compound represented by Formula 2 will be described in detail, the black ginseng extract obtained by the above-described method is used as ethyl acetate per 1 kg of black ginseng extract (1.5 ~ 2.5L) / water (1.5 ~ 2.5L) partitioned extraction 3 to 5 times through a fractionation solvent, and then the water layer is again fractionated 1 to 3 times with n-butanol (1.5 ~ 2.5L) and concentrated under reduced pressure to ethyl acetate Fractions, water fractions and n-butanol fractions can be obtained.

On the other hand, the obtained ethyl acetate black ginseng extract fraction contains 0.15 mg or more, preferably 0.17 mg or more, more preferably 0.20 mg or more, or more, per 1 g of black ginseng having a moisture content of 15% or less before extraction. More preferably 0.20 to 0.30 mg may be included, and the compound represented by Formula 2 is 0.10 mg or more, preferably 0.13 mg or more, more preferably 0.15 mg or more, even more preferably 0.13 to 0.25 mg The extract fraction of black ginseng according to the present invention can be very useful as an active ingredient in an anticancer composition.

In addition, the present invention comprises a black ginseng extract including a compound represented by Formula 1 and a compound represented by Formula 2 or a black ginseng extract fraction including any one or more of these compounds, prepared through the above-described methods, as an active ingredient. It includes a pharmaceutical composition for preventing or treating cancer.

The compound represented by Formula 1 may be any one cancer selected from the group consisting of cancer, particularly ovarian cancer, skin cancer, uterine cancer, liver cancer, and colon cancer, and more preferably, may have superior preventive or therapeutic effects for uterine cancer. .

In addition, the compound represented by Chemical Formula 2 may be any one cancer selected from the group consisting of cancer, particularly ovarian cancer, skin cancer, uterine cancer, liver cancer, and colon cancer, and more preferably, the prevention or treatment effect for uterine cancer is more excellent. I can.

In addition, the compound represented by Formula 1 and/or the compound represented by Formula 2 may be provided at a concentration of 20 to 100 μM, but is not limited thereto.

On the other hand, the present invention is to implement a pharmaceutical composition for preventing or treating cancer by including a compound represented by Formula 1 or a salt thereof or a mixture thereof, and/or a compound represented by Formula 2 or a salt thereof or a mixture thereof as an active ingredient. I can. The compound represented by Formula 1 and/or the compound represented by Formula 2 may be isolated from the aforementioned black ginseng extract or black ginseng extract fraction. Specifically, the method of separating the compound represented by Formula 1 and the compound represented by Formula 2 will be described, the separation may be used without limitation, as long as it is a conventional separation method, preferably silica gel column chromatography and octadecyl It can be carried out by any one or more methods selected from the group consisting of lane (ODS) silica gel column chromatography, more preferably silica gel column chromatography and octadecyl silane (ODS) silica gel column chromatography as a series of processes. Can be done. In addition, the manufacturing method may further include purifying the separated product after the separation, but is not limited thereto.

The compound represented by Formula 1 or the compound represented by Formula 2 separated and purified through the above-described method may be included in the form of a pharmaceutically acceptable salt. As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous or phosphorous acid, and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , Sebacate, fumarate, maleate, butin-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, me Toxibenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycol Rate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

In addition, the acid addition salt is a conventional method, for example, by dissolving the compound represented by the formula (1) or the compound represented by the formula (2) in an excessive amount of an aqueous acid solution, and the di-salt is dissolved in a water-miscible organic solvent such as methanol, It can be prepared by precipitation using ethanol, acetone or acetonitrile.

In addition, the compound represented by Formula 1 or Formula 2 of the present invention may be used in the form of a pharmaceutically acceptable metal salt using a base. The alkali metal or alkaline earth metal salt can be obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. In addition, the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

In addition, the pharmaceutical composition according to an embodiment of the present invention may include a pharmaceutically acceptable carrier. The composition including a pharmaceutically acceptable carrier may be in various oral or parenteral formulations.

In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient in one or more compounds, such as starch, calcium carbonate, sucrose, or lactose ( lactose), gelatin, etc. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included. have. Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

In addition, the pharmaceutical composition is selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. It can have any one formulation.

In addition, the pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is for the purpose of preventing cancer, or Recognition of the purpose and, if the purpose is to treat cancer, the degree of cancer incidence, cause, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and Other factors well known in the medical field can be determined. However, for a preferred effect, the pharmaceutical composition according to an embodiment of the present invention is preferably administered at 0.0001 to 100 mg/kg per day, preferably 0.001 to 100 mg/kg, and the compound represented by Formula 1 and/ Alternatively, the compound represented by Formula 2 is preferably administered at 0.0001 to 100mg/kg per day, preferably 0.001 to 10mg/kg. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and can be easily determined by a person skilled in the art.

In addition, the pharmaceutical composition according to an embodiment of the present invention may be administered by various methods known in the art, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration. It may be administered, intrapulmonary administration, or rectal administration, but is not limited thereto.

Next, the present invention provides a food composition for preventing and improving cancer comprising a black ginseng extract or extract fraction prepared by the above-described manufacturing method including a compound represented by Formula 1 and/or a compound represented by Formula 2 as an active ingredient. Include.

That is, for the purpose of preventing and improving cancer, the black ginseng extract or extract fraction prepared by the above-described manufacturing method including the compound represented by Formula 1 and/or the compound represented by Formula 2 may be added to the food composition.

In this case, the extract or fraction may be added as it is, or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, in the manufacture of food or beverage, the extract of the present invention or a fraction thereof may be added in an amount of 0.01 to 10% by weight, preferably 0.1 to 3% by weight of the raw material composition, but is not limited thereto. It may be added by changing it in consideration of the like.

There is no particular limitation on the type of food. Examples of foods include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes. And includes all health foods in the usual sense.

In addition, in the case of a health beverage composition, it may contain various flavoring agents or natural carbohydrates as an additional component, like a conventional beverage. The natural carbohydrates described above may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used. The ratio of the natural carbohydrate may be generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention, but is not limited thereto, and may be appropriately changed according to preference.

In addition, various other nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, used in carbonated beverages It may contain a carbonation agent to be used. Although the proportion of these additives is not very important, it is generally selected from 0.01 to 0.1% by weight based on the total weight of the composition. In addition, it may contain flesh for the manufacture of natural fruit juice, fruit juice beverage and vegetable beverage. The proportion of the flesh is not very important, but it is generally selected from 0.01 to 10% by weight based on the total weight of the composition. These components may be used independently or in combination.

The present invention will be described in more detail through the following examples, but the following examples do not limit the scope of the present invention, which should be interpreted to aid understanding of the present invention.

<Example 1>

(1) Preparation of black ginseng extract

6-year-old ginseng was washed with water and hot air-dried at 35°C for 12 hours, put in a steamer, heated to 65°C at a heating rate of 50°C/min at room temperature, and then performed the first steaming process at 65°C for 10 minutes, Then, after drying in a 35℃ dryer for 1 day, it was put into a steamer, and the temperature was raised from room temperature to 94℃ at a rate of 18℃/min, and then the second steaming process was performed at 94℃ for 3.5 hours, and then in a 35℃ dryer for 1 day. It was dried again. The dried jeungsam ginseng was repeated three times under the same conditions as the second jeungsam step to perform the second jeungsam step for a total of four times, and between the repeated second jeungsam step and the second jeungsam step, a drying step of drying for 1 day in a 35°C dryer was performed. After the final second jeungsam process, the final black ginseng was prepared through a complete drying process for 1 day in a 50°C dryer to achieve a moisture content of 15%.

Subsequently, the mixed solution of the prepared black ginseng with methanol at a concentration of 100% and a weight ratio of 70:30 was repeatedly extracted three times at 35°C for 24 hours, and concentrated under reduced pressure for 5 hours at a pressure of 45 psi at a temperature of 45°C. Black ginseng extract was prepared.

(2) Isolation and purification of the compound represented by Formula 1 from black ginseng extract

The obtained black ginseng extract was compared using thin layer chromatography (TLC), and the extracts were combined to obtain a total concentration of 1.5 kg. The concentrated extract was partitioned and extracted three times with EtOAc (3 L) / H ₂ O (3L), and the H ₂ O layer was partitioned and extracted three times with n-BuOH (2.7 L). Each layer was concentrated under reduced pressure to obtain an EtOAc fraction, an n-BuOH fraction, and an H ₂ O fraction.

The obtained ethyl acetate partitioned extract was subjected to silica gel column chromatography (SiO ₂ column chromatography, hereinafter referred to as'SiO ₂ cc') (Φ14 × 16 cm, CHCl ₃ -MeOH-H ₂ O=15:3:1)→ CHCl _3- MeOH-H ₂ O=7:3:1) was carried out and 100 ml of aliquots were collected to obtain aliquots. The aliquot was confirmed by TLC (CHCl ₃ -MeOH-H ₂ O=9:3:1), similar portions were collected together, and concentrated using a vacuum concentrator to obtain 10 fractions (BGE1 to BGE10).

Then, the compound was separated and purified using the 10 fractions.

First, the first fraction (BGE, 1 g) of the 10 fractions was subjected to Octadecyl Silicas column chromatography; ODS cc, manufactured by Waters; Φ 4×9 cm, MeOH-H ₂ O=3:1) was carried out to obtain a total of 5 fractions (BGE1-1 to BGE1-5).

ODS column chromatography (Φ 2.5 × 7 cm, MeOH-H ₂ O = 3: 1) was performed on the first fraction (BGE1-1-1, 300 mg) of the 5 fractions, and a total of 8 fractions (BGE1) -1-1-1 to BGE1-1-1-8) were obtained, and finally, 0.16 mg of the compound represented by the following formula (2) was separated based on 1 g of black ginseng having a water content of 15% before extraction.

Next, ODS column chromatography (Φ 3 × 10 cm, MeOH-H ₂ O = 4: 1) was performed on the 7th fraction (BGE7, 3.30 g) of the 10 fractions, and a total of 8 fractions (BGE7-1 To BGE7-8) was obtained, and for the BGE7-5 fraction (400 mg), about 100 mg each was dissolved in 1 ml of solvent and injected using prep LC, and CH ₂ Cl ₂ -MeOH (5:1) It was flowed at a flow rate of 1.5 ml per minute at the rate of. At this time, the detector used was a refractive index detector, and the signal was recovered while checking the signal coming out of the detector, and the same fractions were collected by repeating the above process and confirmed by TLC. The compound represented by the following formula (1) was separated by 0.22 mg based on 1 g of black ginseng having a moisture content of 15% before extraction.

[Formula 1]

[Formula 2]

<Comparative Examples 1 to 2>

It was prepared in the same manner as in Example 1, but black ginseng from Gujeunggupo purchased on the market was used instead of the black ginseng prepared by the method of the present invention. Specifically, Comparative Example 1 was used by purchasing Cheonil Korea Ginseng, and Comparative Example 2 using Daedong Ginseng.

<Experimental Example 1>

(1) Experimental Example 1-1. Establishment of UPLC analysis conditions

It was performed using a Waters ACQUITY UPLCTM system (Waters Corp., MA, USA). Thermo Hypersil Gold (2.1 X 100 mm 1.9 um) column was used and 0.1% formic acid was added as a mobile phase (A) and 0.1% formic acid was added as acetonitrile; B) was used. The column oven was kept at 40° C. and the mobile phase consisted of solvent A (0.1% formic acid in water, v/v) and solvent B (0.1% formic acid in acetonitrile, v/v). Optimal LC elution conditions were applied as follows: 0-0.5 min, 15-15% B; 0.5-1 min, 15-20% B; 1-6 min, 20-20% B; 6-13 min, 20-30% B; 13-23 min, 30-35% B; 23-24 min, 35-38% B; 24-27 min, 38-60% B, and 27-31 min, 60-90% B. Flow rate was 0.5 mL/min. A 2 μ aliquot was injected into the column using an auto-sampler.

(2) Experimental Example 1-2. Establishment of Q-TOF/MS conditions

Using a Q-TOF micro mass detector (Waters, Manchester, UK), optimal conditions were established for metabolite analysis of black ginseng in negative and positive modes, respectively. As a result of comparison, various metabolites were effectively analyzed in negative mode, and the analysis conditions are as follows; capillary voltage 3000, cone voltage 40, collision energy 6, desolvation temperature 550℃, source temperature 120℃, cone gas flow 30 L/h, desolvation gas flow 800 L/h. The precursor ion information was collected between 100 and 2,000 m/z.

Components of the samples of Example 1, Comparative Example 1, and Comparative Example 2 were analyzed by the above method (FIG. 1, Table 1). At this time, the sample components of Example 1, Comparative Example 1, and Comparative Example 2 were analyzed using an ethyl acetate fraction as a sample.

Observed time (min) compound Content (mg/g) Example 1 Comparative Example 1 Comparative Example 2 23.4 Formula 2 0.16 0.054 0.06 26.54 Formula 1 0.22 0.021 0.01

1 shows the results of UPLC-QTOF/MS, and Table 1 shows the results of quantification. As can be seen in Table 1, it was found that the content of the compound represented by Formula 1 and the compound represented by Formula 2 in the black ginseng prepared through the manufacturing method of the present invention was significantly increased.

<Experimental Example 2>

In order to confirm the cancer prevention and treatment effects of the compound represented by Formula 1 and the compound represented by Formula 2 isolated and purified through Example 1, cancer cell toxicity assay (Cytotoxicity assay) was performed using various types of cancer cell lines as shown in Table 2 below. ) Performed. Specifically, RPMI-1640 medium containing 10% FBS (fetal bovine serum), 100 mg/mL of streptomycin-penicillin and 200 nM of TPA (12- o -tetradecanoylphorbol-13-acetate) is provided. Each of the cancer cell lines in Table 2 was dispensed into one 94-well plate, 1*?* ⁵ cells, and cultured for 24 hours. Thereafter, the compound represented by Formula 1 and the compound represented by Formula 2, separated and purified through Example 1, were dissolved in 0.1% DMSO solvent, treated by concentration, and cultured for 24 hours. After treatment with 20 µl of MTT solution (5mg/ml), incubate in a CO ₂ incubator for 1 hour, and then measure the absorbance at 450 nm in an ELISA reader to check the degree of cancer cytotoxicity, but at a concentration that reduces cancer cell toxicity by 50% Are shown in Table 2 and FIG. 2 for each cancer cell.

Cancer cell line IC ₅₀ (μM) Formula 1 Formula 2 SK-OV-3 (Human Caucasian ovary adenocarcinoma, ovarian cancer) 74.3 > 100 HepG2 (Human hepatocellular carcinoma, liver cancer) 71.6 > 100 Hela (Human cervix epitheloid carcinoma, uterine cancer) 41.280 > 80 HCT-116 (Human colorectal carcinoma, colon cancer) 75.6 > 100 SK-MEL-5 (Human melanoma cell line, skin cancer) 72.6 > 100

As can be seen from Table 2, the compounds represented by Formula 1 and Formula 2 according to the present invention can inhibit cancer cell toxicity by 50% at a very low concentration against ovarian cancer, liver cancer, uterine cancer, colon cancer and skin cancer. It can be confirmed, and through this, it was found that the anticancer effect was excellent against the cancers.

<Examples 2 to 7 and Comparative Examples 3 to 7>

In the same manner as in Example 1, black ginseng was prepared by changing the presence or absence of the first jeung ginseng process, the temperature conditions of the first jeung ginseng process and the second jeung ginseng process, as shown in Table 3 below. Likewise, the compounds represented by Formula 1 and Formula 2 were isolated.

division Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 The first jeungsam process Temperature(℃) 65 65 65 65 65 65 65 Time(min) 10 3 5 15 17 10 10 2nd jeungsam process Temperature(℃) 94 94 94 94 94 94 94 Time(hr) 3.5 3.5 3.5 3.5 3.5 3.5 3.5 Number of repetitions
(time) 4 4 4 4 4 3 5 Compound represented by Formula 1 (mg/g) 0.22 0.12 0.19 0.20 0.14 0.20 0.20 Compound represented by Formula 2 (mg/g) 0.16 0.10 0.14 0.15 0.10 0.15 0.16

division Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 Comparative Example 7 The first jeungsam process Temperature(℃) 50 80 65 65 Not performed Time(min) 10 10 10 10 2nd jeungsam process Temperature(℃) 94 94 94 94 94 Time(hr) 3.5 3.5 3.5 3.5 3.5 Number of repetitions
(time) 4 4 One 7 4 Compound represented by Formula 1 (mg/g) 0.08 0.09 0.04 0.14 0.05 Compound represented by Formula 2 (mg/g) 0.04 0.09 0.05 0.12 0.03

As can be seen in Tables 3 to 4,

In Comparative Examples 3 and 4, which were unsatisfied with the temperature of the first jeungsam process of the present invention, it was confirmed that the content of the compounds represented by the formulas 1 and 2 was significantly reduced compared to Example 1, and the first jeungsam step was not performed. In the case of Comparative Example 7, it can be seen that the content was produced further lower.

In addition, it can be seen that in Examples 1, 6 and 7 in which the number of the second jeungsam step is within the preferred range of the present invention, the content of the compounds represented by Formulas 1 and 2 was significantly increased compared to Comparative Examples 5 and 6.

[Production Example 1]-Pharmaceuticals

In the preparation process of Example 1, 50 mg of black ginseng ethyl acetate fraction, 400 mg of crystalline cellulose, and 5 mg of magnesium stearate were mixed and then tableted according to a conventional tablet preparation method to prepare tablets.

[Production Example 2]-Food

In the manufacturing process of Example 1, black ginseng ethyl acetate fraction 1000 mg, vitamin A acetate 70 ㎍, vitamin E 1.0 mg, vitamin B1 0.13 mg, vitamin B2 0.15 mg, vitamin B6 0.5 mg, vitamin B12 0.2 μg, vitamin C 10 mg, Biotin 10 μg, nicotinic acid amide 1.7 mg, folic acid 50 μg, calcium pantothenate 0.5 mg, ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, potassium monophosphate 15 mg, calcium dibasic 55 mg, citric acid It can be prepared by mixing 90 mg of potassium, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride, and the mixing ratio may be arbitrarily modified. After mixing the above ingredients according to a general health food manufacturing method, the granules are It was manufacture.

Although an embodiment of the present invention has been described above, the spirit of the present invention is not limited to the embodiment presented in the present specification, and those skilled in the art who understand the spirit of the present invention can add components within the scope of the same idea. Other embodiments may be easily proposed by changing, deleting, adding, etc., but it will be said that this is also within the scope of the present invention.

Claims (11)

(1) Including a first jeungsam process of heat-treating ginseng at 55℃ to 75℃ and a second jeungsam process of heat treating ginseng performed at 85℃ to 105℃, wherein the second jeungsam process is 2 to Repeating 5 times to prepare black ginseng having an increased content of the compound represented by Formula 1 and the compound represented by Formula 2; And
(2) extracting black ginseng to prepare a black ginseng extract; a method for preparing a black ginseng extract with increased content of anticancer ingredients.
[Formula 1]

[Formula 2]

The method of claim 1,
The first jeungsam process is performed for 5 to 15 minutes, and the second jeungsam process is performed for one time of 1.5 to 5.5 hours. The method of claim 1,
Washing and drying the ginseng before step (1); black ginseng extract manufacturing method with an increased content of anti-cancer ingredients further comprising. The method of claim 1,
The extraction in step (2) is a method for producing a black ginseng extract with an increased content of anticancer components, characterized in that the black ginseng is extracted with a lower alcohol having 1 to 4 carbon atoms. Preparing a fraction of the black ginseng extract containing any one or more of the compound represented by the following formula (1) and the compound represented by formula (2) by fractionating the black ginseng extract prepared according to any one of claims 1 to 4; Black ginseng extract manufacturing method with an increased content of anti-cancer components comprising a.
[Formula 1]

[Formula 2]

The method of claim 5,
The black ginseng extract fraction is an ethyl acetate fraction, characterized in that the content of the anticancer component is increased black ginseng extract manufacturing method. Anticancer prepared according to any one of claims 1 to 4, and containing 0.15 mg or more of the compound represented by the following formula (1) and 0.10 mg or more of the compound represented by the formula (2) per 1 g of black ginseng having a water content of 15% or less Black ginseng extract with increased component content.
[Formula 1]

[Formula 2]

An extract prepared according to any one of claims 1 to 4 including a compound represented by the following formula 1 and a compound represented by the following formula 2, or a compound represented by the following formula 1 and a compound represented by the following formula 2 A pharmaceutical composition for preventing or treating cancer comprising the extract prepared according to claim 5 containing any one or more of the compounds as an active ingredient.
[Formula 1]

[Formula 2]

The method of claim 8,
The cancer is a pharmaceutical composition for preventing or treating cancer, characterized in that any one selected from the group consisting of ovarian cancer, skin cancer, uterine cancer, liver cancer and colon cancer. delete An extract prepared according to any one of claims 1 to 4 including a compound represented by the following formula 1 and a compound represented by the following formula 2, or a compound represented by the following formula 1 and a compound represented by the following formula 2 A food composition for preventing or improving cancer comprising the extracted fraction prepared according to claim 5 containing any one or more of the compounds as an active ingredient.
[Formula 1]

[Formula 2]

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