KR20240040487A - Extraction method to obtain Platycodin D and Polygalacin D rich P. grandiflorum extract - Google Patents

Abstract

The present invention relates to a platycodin d and polygalaxin d extract method with an increased content of platycodin d and polygalaxin d. the extract of the present invention contains a high content of platycodin d and polygalaxin d to have anti-inflammatory, antioxidant, and mucus hypersecretion alleviating effects, thereby helping to alleviate respiratory-related diseases.

Description

Bellflower root extraction method with increased platycodin D and polygalacin D content {Extraction method to obtain Platycodin D and Polygalacin D rich P. grandiflorum extract}

The present invention relates to a method of extracting bellflower root with increased contents of platycodin D and polygalaxin D.

Bellflower root is a representative Korean medicinal crop that has been growing throughout Korea since ancient times, and is effective against inflammation (K. J. Jang et al., 2013), cancer (Yim, Hwang, Liang, & Ma, 2016), and oxidative stress (J.-R. It is known to be effective in relation to respiratory health (Jang, Hwang, & Lim, 2011) and respiratory health (J. Ryu et al., 2014), and is widely used for edible and medicinal purposes. In particular, as the severity of various modern respiratory hazards such as fine dust and viruses increases day by day, the consumption of processed balloon flower products is increasing.

Bellflower root is known to be rich in various types of saponins such as Platycodin D, Platycodin D3, Platycosied E, and Polygalacin D. Accordingly, many studies on the biological efficacy of bellflower root point out the saponin component in bellflower root as the main factor.

Among them, platycodin D is a major bellflower saponin and is known to have an expectorant effect on bronchial mucus secretion (Shin, Lee, Lee, Choi, & Ko, 2002), and polygalaxin D is known to induce the death of lung cancer cells. There is a report (Seo, Y.S et al., 2018).

However, despite the biological efficacy of platycodin D and polygalaxin D in relation to respiratory health, research on the conditions for extracting extracts with increased contents of both platycodin D and polygalaxin D from bellflower is insufficient. .

Accordingly, the present invention uses response surface analysis to explore the optimal extraction conditions for producing an extract with simultaneously increased platycodin D and polygalaxin D content from bellflower root, and to investigate the various biological effects of bellflower root extract extracted under the corresponding conditions. The present invention was completed by confirming the above.

This patent was carried out as an agent for the Gangwon-do order, and the research project name is 'Standardization and trial production of food raw materials for neck health using bellflower, a wild plant in Pyeongchang,' and the research period is 2020.08.01. ~ 2021.09.30.

Korean Patent No. 10-1880934 (July 17, 2018) discloses a method for producing a pharmaceutical composition containing bellflower saponin concentrate extracted from bellflower root. Korean Patent Publication No. 10-2020-0045982 (2020.05.06.) discloses a pharmaceutical composition for preventing or treating liver disease and improving liver function containing a standardized bellflower extract containing bellflower saponin or a membrane-separated bellflower saponin extract. It is disclosed regarding health functional foods.

One object of the present invention is to extract dried pulverized bellflower root using a solvent extraction method, which uses 0-4% (v/v) ethanol as an extraction solvent at 50-80°C. To provide a method for producing bellflower extract, characterized by extraction for 6 to 18 hours.

Another object of the present invention is to provide a bellflower extract obtained by adding 0-4% (v/v) ethanol to bellflower root and extracting it at 50-80°C for 6-18 hours.

Another object of the present invention is to provide a use of the bellflower extract according to the present invention.

The present invention extracts dried pulverized bellflower root using a solvent extraction method, which uses 0 to 4% (v/v) ethanol as an extraction solvent and extracts at 50 to 80°C for 6 to 18 hours. It provides a method for producing bellflower extract, characterized in that:

The present invention provides a bellflower extract obtained by adding 0-4% (v/v) ethanol to bellflower root and extracting it at 50-80°C for 6-18 hours.

The present invention is a food composition for improving throat-related or inflammatory diseases containing as an active ingredient a bellflower extract obtained by adding 0-4% (v/v) ethanol to bellflower root and extracting it at 50-80°C for 6-18 hours. provides.

At this time, the throat-related disease may be, for example, any one selected from sore throat, cough due to excessive secretion of bronchial mucus, chronic cough, tonsillitis, hoarseness, voice disorder, epiglottitis, sore throat, laryngobronchitis, and phlegm.

At this time, the inflammatory disease may be, for example, any one selected from pharyngitis, tonsillitis, epiglottitis, laryngobronchitis, hepatitis, esophagitis, gastritis, dermatitis, arthritis, enteritis, vaginitis, pneumonia, cholangitis, cholecystitis, and pancreatitis.

The present invention is a product for the prevention or treatment of throat-related or inflammatory diseases containing as an active ingredient a bellflower extract obtained by adding 0-4% (v/v) ethanol to bellflower root and extracting it at 50-80°C for 6-18 hours. A pharmaceutical composition is provided.

At this time, the throat-related disease may be, for example, any one selected from sore throat, cough due to excessive secretion of bronchial mucus, chronic cough, tonsillitis, hoarseness, voice disorder, epiglottitis, sore throat, laryngobronchitis, and phlegm.

At this time, the inflammatory disease may be, for example, any one selected from pharyngitis, tonsillitis, epiglottitis, laryngobronchitis, hepatitis, esophagitis, gastritis, dermatitis, arthritis, enteritis, vaginitis, pneumonia, cholangitis, cholecystitis, and pancreatitis.

The present invention provides an anti-inflammatory and antioxidant cosmetic composition containing as an active ingredient a bellflower extract obtained by adding 0-4% (v/v) ethanol to bellflower root and extracting it at 50-80°C for 6-18 hours.

The extract of the present invention contains high levels of platycodin D and polygalaxin D and has anti-inflammatory, antioxidant, and mucus hypersecretion alleviating effects, which can help improve respiratory-related diseases.

Figure 1 is a diagram showing the optimal extraction range of platycodin D (PD) and polygalaxin D (PGD).
Figure 2 is a diagram confirming the change in expression of MUC5AC mRNA, a factor related to bronchial mucus hypersecretion, according to treatment with different platycodin D concentrations.
Figure 3 is a diagram confirming changes in secretion of TNF-α, an inflammation-related protein, according to treatment at different polygalaxin D concentrations.
Figure 4 is a diagram confirming changes in secretion of IL-6, an inflammation-related protein, according to treatment at different polygalaxin D concentrations.
Figure 5 is a diagram confirming the change in expression of MUC5AC mRNA, a factor related to bronchial mucus hypersecretion, according to treatment at different concentrations of the bellflower extract (PGE) of the present invention.
Figure 6 is a diagram confirming the change in secretion of TNF-α, an inflammation-related protein, according to treatment of the bellflower extract of the present invention.
Figure 7 is a diagram confirming the change in secretion of IL-6, an inflammation-related protein, according to treatment with the bellflower extract of the present invention.
Figure 8 shows the results of confirming the antioxidant efficacy of the bellflower extract of the present invention according to treatment at different concentrations.

The present invention extracts dried pulverized bellflower root using a solvent extraction method, which uses 0-4% (v/v) ethanol as an extraction solvent and extracts at 50-80°C for 6-18 hours. It provides a method for producing bellflower extract, characterized in that:

More preferably, extraction is performed at 50 to 65°C for 6 to 18 hours using 0 to 4% (v/v) ethanol as an extraction solvent. At this time, ethanol is preferably added in an amount of 5 to 20 times the weight of the dried bellflower root.

According to one embodiment of the present invention, when bellflower root is extracted under the above-mentioned conditions during solvent extraction, an extract with effectively increased content of platicodin D and polygalacin D can be obtained. there was.

In the production method of the present invention, the bellflower extract preferably contains 2.0 mg/g or more of platycodin D and polygalaxin D, respectively, relative to the dry weight of the bellflower extract, and more preferably platycodin D and polygalaxin D. The content of D and polygalaxin D may each be 3.0 mg/g or more, and more preferably, the content of platycodin D and polygalaxin D may each be 4.0 mg/g or more.

On the other hand, response surface analysis is a regression analysis method that assumes a quadratic model and finds the optimal conditions for factors that give the optimal response value from the change pattern of response variables according to changes in factor levels. When several factors affect the properties of the product, This is a useful method. In the present invention, by using response surface analysis, optimal conditions for extracting extracts with high platycodin D and polygalaxin D were found.

The bellflower extract of the present invention can be commercialized by further including the step of sterilizing after concentration.

Preferably, the concentration can be concentrated to 10 to 50 brix, and more preferably, it can be concentrated to 15 to 30 brix. More preferably, the concentration can be performed in two stages, and centrifugation can be added between the two stages of concentration. The centrifugation may be preferably performed at 2,500 to 5,000 RPM for 1 to 10 minutes at 30°C or lower, and more preferably at 3,500 to 4,500 RPM for 3 to 7 minutes at 5 to 30°C.

Preferably, the sterilization may be performed at 80 to 100°C for 50 to 70 minutes.

In the present invention, the grinding can be done using any known method, and there is no limitation on the grinding method. However, it would be preferable to grind it to a known size that is known to be suitable for solvent extraction.

Meanwhile, the present invention provides a bellflower extract obtained by adding 0-4% (v/v) ethanol to bellflower root and extracting it at 50-80°C for 6-18 hours. At this time, this is because it is possible to obtain a bellflower extract that effectively increases the content of platicodin D and polygalacin D at the same time.

Meanwhile, according to one embodiment of the present invention, it has been confirmed that the bellflower extract of the present invention has anti-inflammatory, antioxidant, and mucus hypersecretion alleviating effects. Therefore, the purpose of the present invention is to provide the uses of the bellflower extract described below.

Accordingly, the present invention is a treatment for improving throat-related or inflammatory diseases containing as an active ingredient a bellflower extract obtained by adding 0-4% (v/v) ethanol to bellflower root and extracting it at 50-80°C for 6-18 hours. Provides a food composition.

At this time, the throat-related disease may be, for example, any one selected from pharyngitis, cough due to excessive secretion of bronchial mucus, chronic cough, tonsillitis, hoarseness, voice disorder, epiglottitis, sore throat, laryngobronchitis, and phlegm, but inflammation and Throat diseases caused by excessive mucus secretion may be included without limitation.

At this time, the inflammatory disease may be, for example, any one selected from pharyngitis, tonsillitis, epiglottitis, laryngobronchitis, hepatitis, esophagitis, gastritis, dermatitis, arthritis, enteritis, vaginitis, pneumonia, cholangitis, cholecystitis, and pancreatitis, It is not limited thereto and may include all diseases accompanied by an inflammatory response without limitation.

In the food composition of the present invention, the content of bellflower root extract is not particularly limited and may vary depending on the condition of the administration subject, the type of specific disease, the degree of progression, etc. If necessary, it can also be included in the total amount of the food.

Food compositions of the present invention include, for example, noodles, gums, dairy products, ice cream, meat, grains, caffeinated beverages, general beverages, chocolate, bread, snacks, confectionery, candy, pizza, jelly, alcoholic beverages, alcohol, and vitamin complexes. and other health supplements, but is not necessarily limited thereto.

When the food composition of the present invention is used in the form of a food additive, it can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods.

In addition, “food composition” of the present invention encompasses health functional foods, and the term “health functional foods” refers to raw materials or ingredients that have functionality useful to the human body according to Act No. 6727 on Health Functional Foods. It refers to food manufactured and processed using , and “functional” means ingestion for the purpose of controlling nutrients for the structure and function of the human body or obtaining useful effects for health purposes such as physiological effects.

The food composition of the present invention may contain additional ingredients that are commonly used to improve smell, taste, vision, etc. For example, it may include biotin, folate, pantothenic acid, vitamins A, C, D, E, B1, B2, B6, B12, niacin, etc. Additionally, it may contain minerals such as chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), zinc (Zn), iron (Fe), and calcium (Ca). Additionally, it may contain amino acids such as cysteine, valine, lysine, and tryptophan. In addition, preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dihydroacetate, etc.), colorants (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), disinfectants (bleaching powder, high-level bleaching powder, Sodium hypochlorite, etc.), swelling agents (alum, D-potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (greasing), coating agents, antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.) , seasonings (MSG monosodium glutamate, etc.), sweeteners (dulcine, cyclemate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), food additives such as gum base, foam suppressants, solvents, improvers, etc. can be added. The additives can be selected depending on the type of food and used in an appropriate amount.

In addition, the present invention is a method for preventing or inflammatory diseases related to the throat, comprising as an active ingredient a bellflower extract obtained by adding 0-4% (v/v) ethanol to bellflower root and extracting it at 50-80° C. for 6-18 hours. Provided is a pharmaceutical composition for treatment.

At this time, the throat-related disease may be, for example, any one selected from pharyngitis, cough due to excessive secretion of bronchial mucus, chronic cough, tonsillitis, hoarseness, voice disorder, epiglottitis, sore throat, laryngobronchitis, and phlegm, but inflammation and Throat diseases caused by excessive mucus secretion may be included without limitation.

At this time, the inflammatory disease may be, for example, any one selected from pharyngitis, tonsillitis, epiglottitis, laryngobronchitis, hepatitis, esophagitis, gastritis, dermatitis, arthritis, enteritis, vaginitis, pneumonia, cholangitis, cholecystitis, and pancreatitis, It is not limited thereto and may include all diseases accompanied by an inflammatory response without limitation.

As used in the present invention, the term “prevention” refers to any action that suppresses or delays the onset of a throat-related disease or inflammatory disease by administering the pharmaceutical composition according to the present invention.

As used in the present invention, the term “treatment” refers to any action in which symptoms caused by a neck-related disease or inflammatory disease are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

The bellflower extract of the present invention may contain one or more active ingredients that exhibit the same or similar functions in addition to the above ingredients.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier in addition to the bellflower extract.

The type of carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art can be used. Non-limiting examples of the carrier include lactose, dextrose, sucrose, sorbitol, mannitol, saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, etc. You can. These may be used alone or in combination of two or more types.

In addition, the pharmaceutical composition of the present invention can be used by adding other pharmaceutically acceptable additives, such as antioxidants, excipients, diluents, buffers or bacteriostatic agents, if necessary, as well as surfactants, binders, fillers, extenders, wetting agents, and disintegrants. , it can be used by additionally adding a dispersant or lubricant.

In the pharmaceutical composition of the present invention, bellflower extract may be included in an amount of 0.00001% to 99.99% by weight, preferably 0.1% to 90% by weight, more preferably 0.1% by weight, based on the total weight of the pharmaceutical composition. It may be included in an amount of from 0.1% to 50% by weight, more preferably from 0.1% to 50% by weight, but is not limited thereto and may vary depending on the condition of the administration subject, the type of specific disease, the degree of progression, etc. If necessary, it may also be included in the total content of the pharmaceutical composition.

That is, the pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time, and/or administration route of the pharmaceutical composition, and the desired effect to be achieved by administration of the pharmaceutical composition Type and degree of reaction, type of subject to be administered, age, weight, general health condition, symptoms or severity of disease, gender, diet, excretion, drugs used simultaneously or simultaneously with the subject, other composition ingredients, etc. It may vary depending on various factors including and similar factors well known in the pharmaceutical field, and a person skilled in the art can easily determine and prescribe an effective dosage for the desired treatment. For example, the daily dosage of the pharmaceutical composition of the present invention is about 0.01 to 1,000 mg/kg, preferably 0.1 to 100 mg/kg, and can be administered once or in divided doses several times a day.

The pharmaceutical composition of the present invention may be administered once a day, or may be administered in several divided doses. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Considering all of the above factors, it can be administered in an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The pharmaceutical composition of the present invention can be used in combination with various methods such as hormone therapy and drug therapy to prevent or treat neck-related diseases or inflammatory diseases.

The term "administration" used in the present invention means introducing the pharmaceutical composition of the present invention into a patient by any appropriate method, and the administration route and mode of administration of the pharmaceutical composition of the present invention may be independent, respectively. As long as the pharmaceutical composition can reach the relevant area, it can be administered by any route and method of administration without particular limitations.

The pharmaceutical composition can be administered by oral or parenteral administration, and can be formulated and used in various dosage forms suitable for oral or parenteral administration.

Non-limiting examples of preparations for oral administration using the pharmaceutical composition of the present invention include oily suspensions, troches, lozenges, tablets, aqueous suspensions, preparation powders, granules, emulsions, hard capsules, and soft capsules. Capsules, syrups, or elixirs may be used.

In order to formulate the pharmaceutical composition of the present invention for oral administration, binders such as sorbitol, mannitol, starch, amylopectin, cellulose lactose, saccharose or gelatin; lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax; excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch can be used, and fragrances, syrups, sweeteners, etc. can also be used. Furthermore, in the case of capsules, in addition to the above-mentioned substances, a liquid carrier such as fatty oil can be additionally used.

Parenteral administration methods of the pharmaceutical composition of the present invention include intramuscular administration, transdermal administration, intravenous administration, intraperitoneal administration, or subcutaneous administration. Methods include applying the composition to the diseased area, spraying it, or inhaling it. It can also be used, but is not limited to this.

Non-limiting examples of parenteral preparations using the pharmaceutical composition of the present invention include injection solutions, suppositories, ointments, powders for application, oils, powders for respiratory inhalation, aerosol preparations for sprays, creams, etc.

To prepare the pharmaceutical composition of the present invention for parenteral administration, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, topical preparations, etc. can be used, and the non-aqueous solutions and suspensions include olive oil, etc. Vegetable oil, propylene glycol, polyethylene glycol, and injectable esters such as ethyl oleate can be used.

When the pharmaceutical composition of the present invention is formulated as an injection solution, the pharmaceutical composition of the present invention is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is then administered in ampoules or vials for unit administration. It can be formulated.

When the pharmaceutical composition of the present invention is formulated as an aerosol, a propellant or the like can be mixed with additives to disperse the water-dispersed concentrate or wet powder.

When the pharmaceutical composition of the present invention is formulated into ointments, oils, creams, powders for application, external skin preparations, etc., animal oil, vegetable oil, wax, paraffin, polyethylene glycol, silicone, bentonite, silica, talc, starch, trimethylamine, etc. It can be formulated using Kant, cellulose derivatives, zinc oxide, etc. as carriers.

In addition, the present invention provides an anti-inflammatory and antioxidant cosmetic composition containing as an active ingredient a bellflower extract obtained by adding 0-4% (v/v) ethanol to bellflower root and extracting it at 50-80° C. for 6-18 hours. do.

The anti-inflammatory refers to the prevention or improvement effect on skin inflammation, and the skin inflammation is, for example, selected from atopic dermatitis, contact dermatitis, seborrhoea, and acne. It may be any one of the following, but is not limited thereto and may include without limitation all skin inflammatory diseases accompanied by an inflammatory reaction that occurs in the skin.

Meanwhile, in the present invention, the bellflower extract is preferably contained in an amount of 0.0001 to 30.0% by weight based on the total weight of the cosmetic composition. More preferably, it is contained in 0.01 to 10% by weight based on the total weight of the cosmetic composition. If the content of bellflower extract is less than 0.0001% by weight, the anti-inflammatory and antioxidant effects are minimal, and if it exceeds 30.0% by weight, the effect does not increase significantly as the content increases.

Meanwhile, the ingredients included in the cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions in addition to the bellflower extract of the present invention as an active ingredient, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances. Contains common auxiliaries such as, and carriers.

The cosmetic composition of the present invention can be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing products. , may be formulated into oils, packs, massage creams, sprays, etc., but are not limited thereto. More specifically, it can be manufactured in the form of softening lotion, nourishing lotion, nourishing cream, massage cream, essence, cleansing cream, cleansing foam, cleansing water, pack, spray, or powder.

When the formulation of the cosmetic composition of the present invention is a paste, cream or gel, the carrier ingredient may include animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide. This can be used.

When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solubilizing agent, or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, and propylene. There are fatty acid esters of glycol, 1,3-butylglycol oil, glycerol aliphatic esters, polyethylene glycol or sorbitan.

When the formulation of the cosmetic composition of the present invention is a suspension, the carrier component includes water, a liquid diluent such as ethanol or propylene glycol, and a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester. , microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant, etc. can be used.

When the formulation of the cosmetic composition of the present invention is powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the cosmetic composition is a spray, chlorofluorohydride may be used as a carrier ingredient. May contain propellants such as carbon, propane/butane or dimethyl ether.

When the formulation of the cosmetic composition of the present invention is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, Fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester can be used.

When the cosmetic composition of the present invention is a soap, a surfactant-containing cleansing formulation, or a surfactant-free cleansing formulation, it can be applied to the skin and then wiped off, removed, or washed with water. As a specific example, the soap is liquid soap, powdered soap, solid soap, and oil soap, the surfactant-containing cleansing formulation is cleansing foam, cleansing water, cleansing towel, and cleansing pack, and the surfactant-free cleansing formulation is cleansing cream. , cleansing lotion, cleansing water, and cleansing gel, but are not limited thereto.

Hereinafter, the contents of the present invention will be described in more detail through the following examples or experimental examples. However, the scope of the present invention is not limited to the following examples or experimental examples, and includes modifications of the technical idea equivalent thereto.

[ Example One: platycodin D and polygalaxin [Manufacture of bellflower extract with simultaneously increased D content]

1. Setting of response surface analysis conditions

To perform response surface analysis, an equal interval difference is required between the values of one variable. Accordingly, as shown in Table 1 below, the three independent variables: ethanol concentration (X1, 0~40% (v/v); extraction temperature (X2, 50~80℃); extraction time (X3, 6~18 hours) Condition values were coded as -1, 0, and 1.

RSM analysis conditions variable -One 0 One Ethanol ratio ( EtOH fraction, %(v/v) 0 20 40 Temperature (°C) 50 65 80 Extraction time (hour) 6 12 18

2.Box- Behnken Optimization of extraction conditions using response surface analysis based on experimental methods

Response surface analysis was performed to explore the optimal extraction conditions for platycodin D and polygalaxin D, the active substances of bellflower root. In the Box-behnken design, Box-behnken experimental conditions were designed to extract bellflower extract with improved contents of platycodin D and polygalaxin D, the active ingredients of bellflower root, for each extraction temperature, extraction time, and composition of extraction solvent. and was repeated three times in random order.

Afterwards, the content of platycodin D and polygalaxin D in the extract was analyzed, converted to content per dry weight of bellflower root, and set as dependent variables. The designed Box-behnken experiment conditions and their experimental and predicted values are shown in Table 2 below.

Box-behnken experimental conditions and their experimental and predicted values Independent variables Platycodin D Responses Polygalacin D Responses EtOH Fraction
(%) Temperature
(℃) extraction time
(hour) Actual value Predicted value Actual value Predicted value One 20 50 6 2.06 2.18 2.18 2.06 2 0 50 12 5.53 5.28 5.28 5.53 3 40 50 12 1.72 1.63 1.63 1.72 4 20 50 18 1.80 2.02 2.02 1.80 5 0 65 6 4.68 4.80 4.80 4.68 6 40 65 6 1.83 1.80 1.80 1.83 7 20 65 12 2.35 2.55 2.55 2.74 8 20 65 12 2.74 2.55 2.55 2.35 9 20 65 12 2.58 2.55 2.55 2.58 10 0 65 18 4.84 4.87 4.87 4.84 11 40 65 18 2.18 2.05 2.05 2.18 12 20 80 6 2.13 1.91 1.91 2.13 13 0 80 12 4.48 4.58 4.58 4.48 14 40 80 12 2.17 2.42 2.42 2.17 15 20 80 18 2.50 2.37 2.37 2.50

As a result of the experiment, the optimal conditions were 0% (v/v) EtOH, 50°C, 11.84 hours (Figure 1). Figure 1 is a diagram showing the optimal extraction range of platycodin D (PD) and polygalaxin D (PGD).

It was confirmed that there was a slight difference between the experimental and predicted values of the dependent variables (contents of platycodin D and polygalaxin D) analyzed according to the relevant experimental design.

Meanwhile, in order to determine the effect of the model and set independent variables on the contents of platycodin D and polygalaxin D, the results of ANOVA were confirmed, and the results are shown in Tables 3 and 4 below.

Results of RSM model ANOVA analysis for platycodin D Source SS C.E. S.E. F-value p-value Model 22.70 2.55 0.16 33.20 0.0006 X _One -Ethanol concentration 16.93 -1.45 0.10 222.90 < 0.0001 X ₂ -Temperature 0.00 0.02 0.10 0.04 0.8419 X ₃ -Time 0.05 0.08 0.10 0.64 0.4611 X _One X ₂ 0.56 0.37 0.14 7.37 0.0421 X _One X ₃ 0.01 0.05 0.14 0.11 0.7521 X ₂ X ₃ 0.10 0.16 0.14 1.29 0.3076 X _One ² 4.40 1.09 0.14 57.94 0.0006 X ₂ ² 0.11 -0.17 0.14 1.38 0.2924 X ₃ ² 0.26 -0.27 0.14 3.42 0.1236 Residual 0.38 Lack of fit 0.30 2.66 0.2846 R ² 0.98 Adjusted R ² 0.95

*SS: Sum of squares, CE: Coefficient estimate, SE: Standard error

RSM model ANOVA analysis results for polygalaxin D Source SS C.E. S.E. F-value p-value Model 17.48 0.78 0.24 10.88 0.0086 X _One -Ethanol concentration 9.83 -1.1 0.15 55.11 0.0007 X ₂ -Temperature 0.70 -0.30 0.1 3.92 0.1046 X ₃ -Time 0.11 0.12 0.15 0.60 0.4745 X _One X ₂ 1.98 0.70 0.21 11.10 0.0207 X _One X ₃ 0.00 0.032 0.21 0.02 0.8858 X ₂ X ₃ 0.02 0.076 0.21 0.13 0.7343 X _One ² 4.34 1.08 0.22 24.32 0.0044 X ₂ ² 0.00 -0.026 0.22 0.01 0.9108 X ₃ ² 0.27 -0.27 0.22 1.52 0.2721 Residual 0.89 Lack of fit 0.84 9.83 0.0938 R ² 0.95 Adjusted R ² 0.86

*SS: Sum of squares, CE: Coefficient estimate, SE: Standard error

As a result of the experiment, as in the Box-behnken experiment above, in the ANOVA results, the p-value of the model was less than 0.05, and the p-value of lack of fit was more than 0.05, confirming that the prediction model was significant.

3. Bellflower extract platycodin D and polygalaxin Analysis of D content

Platicodin D and polygalaxin D contents were confirmed using high-performance liquid chromatography (HPLC) analysis. All experiments were repeated three or more times, and the dependent variable was converted based on dry weight.

In the response surface analysis, the polynomial regression analysis equation for the dependent variables, extraction yield, platycodin D, and polygalaxin D, on the independent variables, ethanol concentration, extraction temperature, and extraction time, is as shown in Equation 1 below. The unit of extraction yield is %, and the unit of the remaining dependent variables is mg/g dry platycodon.

[Equation 1]

As a result of the experiment, the expected value of platycodin D in bellflower extract extracted under optimal extraction conditions derived through response surface analysis was found to be 5.30 mg/g, and the actual measured value was confirmed to be 5.63 mg/g. In addition, the expected value of polychalaxin D was 3.95 mg/g, and the actual measured value was confirmed to be 4.68 mg/g.

According to the above results, the suitability of the model derived from this experiment is very high, and the optimal condition for obtaining bellflower extract with simultaneously increased contents of platycodin D and polygalaxin D is 0~65℃ at 50~65℃. It was confirmed that solvent extraction was performed with 4% ethanol for 6 to 18 hours.

4. platycodin D and polygalaxin Preparation of D simultaneous enhancement bellflower extract

Dried bellflower root (Sejong Agricultural Products) was powdered using a mill powder tech® and filtered through a stainless steel sieve with a diameter of 500㎛.

Afterwards, 10 ml of solvent (bellflower root: solvent = 1:10) was added to 1 g of bellflower root powder and extracted under the optimal extraction conditions of 0% (v/v) EtOH, 50°C, 11 hours according to Box-behnken design conditions. did. The obtained extract was filtered through a 0.45㎛ filter (Advantec).

Afterwards, the filtrate was concentrated under reduced pressure and freeze-dried. The dried bellflower root powder was weighed to determine the extraction yield, which was then freeze-dried and used in the following experiment.

The extraction yield was approximately 23% (w/w).

[ Experiment example One: platycodin D and polygalaxin D Efficacy evaluation of a single substance]

1. Evaluation of efficacy in preventing bronchial mucus hypersecretion

To confirm the efficacy of platycodin D as a single substance in improving respiratory diseases, a human-derived bronchial epithelial cell line (NCI-H292) used in respiratory-related research was used. The degree of prevention of mRNA overexpression of MUC5AC, a bronchial mucus hypersecretion protein used as an indicator related to respiratory health, was confirmed by treating NCI-H292 with PMA to induce overexpression.

Specifically, after treatment with platycodin D at a concentration of 1 to 10 μM for 30 minutes, treatment with PMA 10 ng/ml for 24 hours, and total RNA was isolated using the Total RNA Extraction kit (MGmed) according to the kit's protocol. did. 20 μl of total reaction solution was prepared to contain 1.5 μg of total RNA.

Afterwards, it was reverse transcribed into cDNA using the cDNA Synthesis kit (Takara Bio) according to the kit's protocol, and qRT-PCR was performed using IQ Sybr Green SuperMix (Biorad,). The reaction was carried out at 95°C for 10 minutes, 44 cycles of 10 seconds at 95°C, and then at 65°C for 30 seconds. Additionally, β-actin was used as an internal standard.

As a result of the experiment, it was confirmed that treatment with platycodin D suppressed mRNA overexpression of MUC5AC in a concentration-dependent manner (Figure 2). Figure 2 is a diagram confirming the change in expression of MUC5AC mRNA, a factor related to bronchial mucus hypersecretion, according to treatment with different platycodin D concentrations.

2. Confirmation of inflammation prevention efficacy

To confirm the efficacy of polygalaxin D in controlling inflammation/immune function as a single substance, a mouse-derived macrophage cell line (RAW264.7) was used. RAW264.7 was treated with LPS to induce hypersecretion of TNF-α and IL-6 proteins, which are used as indicators of inflammation, and the alleviating effect of polygalaxin D treatment was confirmed.

Specifically, polygalaxin D was treated at a concentration of 1 to 50 μM for 1 hour, and then LPS 10 ng/ml was treated for 24 hours. The medium was obtained, centrifuged, and the supernatant was obtained.

Afterwards, ELISA was performed using the mouse TNF-α ELISA kit (R&D systems) and the mouse IL-6 ELISA kit (R&D Systems) according to the kit's protocol, and the absorbance was measured at 450 nm and 570 nm. .

As a result of the experiment, it was confirmed that hypersecretion of TNF-α and IL-6 proteins induced by LPS treatment was suppressed in a concentration-dependent manner by treatment with polygalaxin D (Figures 3 and 4). Figures 3 and 4 show changes in secretion of inflammation-related proteins, TNF-α and IL-6, respectively, according to treatment with polygalaxin D concentration.

[ Experiment example 2: Example Efficacy evaluation of bellflower extract prepared in 1]

1. Evaluation of efficacy in preventing bronchial mucus hypersecretion

The experiment was performed in the same manner as in Experimental Example 1, except that instead of platycodin D, bellflower extract was treated at a concentration of 100 to 800 μg/ml for 30 minutes.

As a result of the experiment, it was confirmed that the mRNA overexpression of MUC5AC was suppressed in a concentration-dependent manner by treatment with bellflower extract containing high levels of platycodin D and polygalaxin D obtained under optimal extraction conditions (FIG. 5). Figure 5 is a diagram confirming the change in expression of MUC5AC mRNA, a factor related to bronchial mucus hypersecretion, according to treatment at different concentrations of the bellflower extract (PGE) of the present invention.

2. Confirmation of inflammation prevention efficacy

The experiment was performed in the same manner as in Experimental Example 1, except that instead of polygalaxin D, bellflower extract was treated at a concentration of 100 μg/ml for 1 hour and then LPS 10ng/ml was treated for 18 or 24 hours, respectively. did.

As a result of the experiment, it was confirmed that hypersecretion of TNF-α and IL-6 proteins induced by LPS treatment was suppressed by treatment with bellflower extract high in platycodin D and polygalaxin D obtained under optimal extraction conditions (Figs. 6 to 6) 7). Figures 6 and 7 are diagrams confirming changes in secretion of inflammation-related proteins TNF-α and IL-6, respectively, according to treatment with the bellflower extract of the present invention.

3. Confirmation of antioxidant efficacy

ABTS analysis was performed to measure antioxidant activity using bellflower root extract extracted under optimal extraction conditions. For this purpose, 20 ml of a mixed solution of 7.4mM ABTS and 2.45mM potassium persulfate was prepared and stored in a dark room at room temperature for 16 hours.

The ABTS solution was diluted with distilled water until the absorbance at 734 nm was 0.70 ± 0.02. 180㎕ of ABTS solution was mixed with 20㎕ of bellflower extract at a concentration of 100-800㎍/㎖, reacted for 10 minutes in the dark at room temperature, and absorbance was measured at 734㎚.

As a result of the experiment, it was confirmed that treatment with bellflower extract containing high platycodin D and polygalaxin D obtained under optimal extraction conditions showed radical scavenging activity in a concentration-dependent manner (FIG. 8). Figure 8 shows the results of confirming the antioxidant efficacy of the bellflower extract of the present invention according to treatment at different concentrations.

Claims (9)

The dried pulverized bellflower root is extracted using a solvent extraction method.
The solvent extraction method is,
A method for producing bellflower extract, characterized in that extraction is performed at 50-80°C for 6-18 hours using 0-4% (v/v) ethanol as an extraction solvent.
Bellflower extract obtained by adding 0-4% (v/v) ethanol to bellflower root and extracting at 50-80°C for 6-18 hours.
A food composition for improving throat-related or inflammatory diseases containing as an active ingredient a bellflower extract obtained by adding 0-4% (v/v) ethanol to bellflower root and extracting it at 50-80°C for 6-18 hours.
According to paragraph 3,
The neck-related diseases are:
A food composition selected from the group consisting of sore throat, cough due to excessive secretion of bronchial mucus, chronic cough, tonsillitis, hoarseness, voice disorder, epiglottitis, sore throat, laryngobronchitis, and phlegm.
According to paragraph 3,
The inflammatory disease is
A food composition selected from pharyngitis, tonsillitis, epiglottitis, laryngobronchitis, hepatitis, esophagitis, gastritis, dermatitis, arthritis, enteritis, vaginitis, pneumonia, cholangitis, cholecystitis, and pancreatitis.
A pharmaceutical composition for the prevention or treatment of throat-related or inflammatory diseases containing as an active ingredient a bellflower extract obtained by adding 0-4% (v/v) ethanol to bellflower root and extracting it at 50-80°C for 6-18 hours.
According to clause 6,
The neck-related diseases are:
A pharmaceutical composition selected from the group consisting of sore throat, cough due to excessive secretion of bronchial mucus, chronic cough, tonsillitis, hoarseness, voice disorder, epiglottitis, sore throat, laryngobronchitis, and phlegm.
According to clause 6,
The inflammatory disease is
A pharmaceutical composition selected from pharyngitis, tonsillitis, epiglottitis, laryngobronchitis, hepatitis, esophagitis, gastritis, dermatitis, arthritis, enteritis, vaginitis, pneumonia, cholangitis, cholecystitis, and pancreatitis.
An anti-inflammatory and antioxidant cosmetic composition containing bellflower root extract as an active ingredient, obtained by adding 0-4% (v/v) ethanol to bellflower root and extracting it at 50-80°C for 6-18 hours.