Compositions and methods for treating, preventing, and alleviating respiratory symptoms or diseases

Abstract

The present invention provides a method for producing a processed parsnip extract with improved physiological activity compared to raw parsnip extract, the method comprising pre-treating raw parsnips to produce aged parsnips, and extracting the aged parsnips. Furthermore, the present invention provides a composition comprising the parsnip extract, and a method using the composition to promote respiratory health and / or prevent, alleviate, or treat respiratory symptoms or diseases.

Description

Technical Field

[0001] The present invention relates to a method for producing a parsley extract with improved biological activity, a composition containing the parsley extract produced by the production method, and a method for enhancing the health of the respiratory tract using the composition.

Background Art

[0002] Mammals including humans suffer from various respiratory symptoms and diseases due to genetic or environmental factors, and there is an urgent need for new solutions to effectively prevent, improve, or treat such respiratory symptoms and diseases.

[0003] J Clin Invest, 111: 291-297, 2003;N Engl J. Med. 2001;44(5): 350-62;Toshio Hirano, Cytokine molecular biology, World Science, 2002;Am J Respir Crit Care Med, 2013, 187: 347-365;Am J Respir Crit Care Med, 2009, 180:396-406;Biol Pharm Bull, 2012, 35:1752-1760;Am J Respir Crit Care Med, 1997 155, 1441-1447;Am J Physiol Lung CellMol Physiol, 2010, 298:L262-L269;Am J Respir Cell Mol Biol, As reported in literature such as 2013, 48:531-539;Eur Respir J, 1998, 12:1200-1208;LoS One. 2014 May 15;9(5): e97784;Respir Res. 2015 May 22;16:59.;J Immunol Res. 2017;2017:6710278;Respirology. 2016 Apr;21(3): 467-75;Am J Respir Crit Care Med 154(1): 76-81, 1996, research on bronchial asthma and chronic obstructive pulmonary disease (COPD) is actively being conducted. However, there are very few functional foods or drugs that can effectively prevent, improve, or treat bronchial asthma and chronic obstructive pulmonary disease. [Overview of the project]

[0004] This invention provides a method for producing a parsnip extract with improved physiological activity compared to the raw parsnip by pre-treating raw parsnips to produce aged parsnips and then extracting the aged parsnips. Furthermore, it provides a composition containing the improved physiological activity parsnip extract produced by this method. It also provides a method for using this composition to promote respiratory health and / or prevent, improve, or treat respiratory symptoms or diseases.

[0005] One aspect of the present invention provides a composition for promoting respiratory health. More specifically, one aspect of the present invention provides a composition for promoting respiratory health that contains, as an active ingredient, an extract of processed parsnip having a total polyphenol content greater than that of unprocessed natural (raw) parsnip. The promotion of respiratory health may include the prevention, improvement, or treatment of diseases, disorders, conditions, or symptoms related to respiratory diseases. The respiratory diseases may include bronchial asthma and chronic obstructive pulmonary disease. The processed parsnip extract of the present invention may have a total polyphenol content that is preferably at least 1.5 times, more preferably 2 times, and even more preferably 3 times higher than that of raw parsnip.

[0006] According to one embodiment of the present invention, raw parsnips are heat-dried at a specific temperature for a specific period of time until the color of the raw parsnips changes to brown or black, and then an extraction step is added to obtain an extract. As an example, the specific temperature can be adjusted to a temperature between approximately 50°C and approximately 70°C, and the specific period can be adjusted to a period between approximately 10 and approximately 30 days. As another example, the specific temperature can be adjusted to a temperature between approximately 55°C and approximately 65°C, and the specific period can be adjusted to a period between approximately 10 and approximately 20 days. As yet another example, the specific temperature can be adjusted to a temperature between approximately 70°C and approximately 90°C, and the specific period can be adjusted to a period between approximately 3 and approximately 10 days. As yet another example, the specific temperature can be adjusted to a temperature between approximately 85°C and approximately 90°C, and the specific period can be adjusted to a period between 3 and 10 days. According to other embodiments, the specific temperature and specific period can be adjusted to such an extent that the color of the original parsnip changes to brown or black, and / or that the total polyphenol content is greater than the total polyphenol content of the original parsnip.

[0007] Another aspect of the present invention provides a method for producing the extract. More specifically, another aspect of the present invention provides a method for producing a processed aged parsnip extract containing a total polyphenol content greater than (for example, at least twice) the total polyphenol content of unprocessed natural (raw) parsnips. The production method is characterized by comprising the steps of: heating and drying raw parsnips at a specific temperature for a specific period of time; adding alcohol, water, or a mixture thereof to the thus heated and dried parsnips; filtering the supernatant; and removing unwanted components from the filtered supernatant. According to one embodiment of the present invention, the heating and drying step may be carried out to a degree sufficient to change the color of the raw parsnips to brown or black and to increase the total polyphenol content to a level greater than the total polyphenol content of the raw parsnips. The aforementioned aspects of the present invention, other aspects, and embodiments of the present invention are described in further detail below. [Brief explanation of the drawing]

[0008] [Figure 1]This diagram illustrates the production process of parsnip extract according to one embodiment of the present invention. [Figure 2] This graph shows the change in total polyphenol content of parsnip extract according to one embodiment of the present invention. AP10D, AP20D, and AP30D refer to parsnip extracts obtained by pre-treating (aging or processing) raw parsnips for 10, 20, and 30 days, respectively, while raw parsnips refer to extracts obtained without such pre-treatment. [Figure 3] This graph shows the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability of parsnip extract according to one embodiment of the present invention. AP10D, AP20D, and AP30D refer to parsnip extracts obtained by pre-treating (aging or processing) raw parsnips for 10, 20, and 30 days, respectively, while raw parsnips refer to extracts obtained without such pre-treatment. [Figure 4] This example demonstrates the cytotoxicity of a parsnip extract according to one embodiment of the present invention against human-derived bronchial epithelial cells (BEAS-2B). AP20D refers to the parsnip extract obtained by pre-treating (aging or processing) raw parsnips for 20 days before extraction. MTT refers to 2,5-diphenyl-2H-tetrazolium bromide. PBS refers to phosphate-buffered saline. [Figure 5] This document demonstrates the efficacy of parsnip extract against acrolein-induced inflammation according to one embodiment of the present invention. NEG refers to the negative control group, POS refers to the positive control group, and AP20D refers to the parsnip extract obtained by pre-treating (aging or processing) raw parsnips for 20 days before extraction. The P-value was calculated through a step-wise t-test. [Figure 6]This experimental design was created to confirm that a parsnip extract according to one embodiment of the present invention has anti-inflammatory efficacy in mice exposed to acrolein. The negative control group was treated with a basal diet (AIN-93G) only, the positive control group was treated with acrolein along with the basal diet, and the test group was treated with a basal diet, a parsnip extract according to one embodiment of the present invention (AP20D), and acrolein. [Figure 7] This study demonstrates the effect of parsnip extract according to one embodiment of the present invention on the expression level of inflammatory cytokines in the blood. The negative control group was treated with a basal diet (AIN-93G) only, the positive control group was treated with acrolein along with the basal diet, and the test group was treated with a basal diet, parsnip extract according to one embodiment of the present invention (AP20D), and acrolein. The p-values ​​were calculated using a step-wise t-test. * indicates a p-value less than 0.05, ** indicates a p-value less than 0.01, and *** indicates a p-value less than 0.001. [Figure 8] This study demonstrates the effect of parsnip extract according to one embodiment of the present invention on the expression levels of TNF-alpha (TNF-α) messenger RNA (mRNA) and interleukin-1 beta (IL-1β) messenger RNA. The negative control group was treated with only the basal diet (AIN-93G), the positive control group was treated with the basal diet and acrolein, and the test group was treated with the basal diet, parsnip extract according to one embodiment of the present invention (AP20D), and acrolein. The p-value was calculated using a step-wise t-test. [Figure 9] This shows the results of H&E staining reactions on lung tissue treated with parsnip extract according to one embodiment of the present invention. The negative control group was treated with only the basal diet (AIN-93G), the positive control group was treated with the basal diet and acrolein, and the test group was treated with the basal diet, parsnip extract according to one embodiment of the present invention (AP20D), and acrolein. [Figure 10]This shows the results of the periodic acid-Schiff (PAS) staining reaction on lung tissue treated with parsnip extract according to one embodiment of the present invention. The negative control group was treated with only the basal diet (AIN-93G), the positive control group was treated with the basal diet and acrolein, and the test group was treated with the basal diet, parsnip extract according to one embodiment of the present invention (AP20D), and acrolein. [Figure 11] This diagram illustrates the production process of parsnip extract according to another embodiment of the present invention. [Figure 12A] This is the standard line used to measure the DPPH radical scavenging ability of parsnip extract according to other embodiments of the present invention. [Figure 12B] This graph shows the DPPH radical scavenging ability of parsnip extract samples according to other embodiments of the present invention. Test group A refers to the group treated with the parsnip extract obtained in Example 1 of the present invention, test group B refers to the group treated with the parsnip extract obtained in Example 6 of the present invention, and the control group refers to the group treated with an extract obtained from raw parsnips without undergoing a pretreatment (maturation) process. [Figure 13A] This is the standard line used to measure the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid);ABTS radical scavenging ability of parsnip extract according to other embodiments of the present invention. [Figure 13B] This graph shows the ABTS radical scavenging ability of parsnip extracts according to other embodiments of the present invention. Test group A refers to the group treated with the parsnip extract obtained in Example 1 of the present invention, test group B refers to the group treated with the parsnip extract obtained in Example 6 of the present invention, and the control group refers to the group treated with an extract obtained from raw parsnips without undergoing a pretreatment (maturation) process. [Figure 14] These are the results of a cytotoxicity test of parsnip extract on normal human lung cells according to another embodiment of the present invention. [Figure 15] The lung tissue protective efficacy of parsnip extract according to another embodiment of the present invention is demonstrated. [Modes for carrying out the invention]

[0009] As used herein, the term "parsnip" refers to parsnip (Pastinaca sativa).

[0010] To aid in understanding the fundamental concepts of the present invention, the invention will be described below with reference to embodiments illustrated in the drawings, and specific terms will be used in such description. However, such description should not be understood to impose any limitations on the scope of the invention.

[0011] When the term "about" as used in the specification of this invention is used in relation to a value or range, it should be interpreted that the value or range has a certain degree of variability, for example, it may have variability of 10%, 5%, or 1%.

[0012] When the term "substantially" as used in the specification of the present invention is used in relation to a value or range, that value or range should be interpreted as having a certain degree of variability, for example, it may have variability of up to 90%, up to 95%, or up to 99%.

[0013] As used in the specification of this invention, the terms "one," "one," or "it" should be interpreted as including one or more unless otherwise explicitly defined in the context. The term "or" as used in the specification of this invention should be interpreted in an inclusive sense unless otherwise specifically defined.

[0014] The syntax or terms used in the specification of the present invention are only used for the purpose of explaining the present invention unless otherwise specifically defined, and the content of the invention shall not be restrictively interpreted by such syntax or terms. The titles of each section used in the specification of the present invention are only used for the purpose of assisting in the understanding of the present invention, and the content of the present invention shall not be restrictively interpreted by the titles of each section. The information related to the title of a section may be within the section or outside the section. Furthermore, all published documents, patent gazettes, etc. included as references in the specification of the present invention shall be regarded as being included in the content of the specification of the present invention. If the above-mentioned documents and gazettes are inconsistent with the content of the specification of the present invention, the above-mentioned documents and gazettes shall be regarded as supplementing the specification of the present invention, and if the inconsistency is irreconcilable, the specification of the present invention shall take precedence. I. Parsnip Extract

[0015] In one aspect, the present invention provides a parsnip extract having improved physiological activity compared to the original parsnip. According to one embodiment, there is provided a parsnip extract produced by pretreating the original parsnip to obtain an aged parsnip or a processed parsnip and then extracting the aged or processed parsnip. The parsnip extract obtained from the aged parsnip has higher physiological activity than the original parsnip.

[0016] There is a report (Plant Foods for Human Nutrition (2020) 75:292-297) on the process of pretreating the original parsnip, but that process takes too much time (4 weeks) until the process is completed and requires maintaining a high level of humidity (95%) together with heat treatment at a high temperature (80°C), so there are limitations in applying it to the industrial site. There is an urgent need for a new method of pretreatment under milder process conditions in a shorter period.

[0017] According to one embodiment of the present invention, parsnip extract with significantly improved physiological activity compared to parsnip can be obtained by pre-treating raw parsnips under relatively mild process conditions in a relatively short time.

[0018] According to one embodiment of the present invention, raw parsnips are pre-treated to produce aged parsnips. The raw parsnips can be cut before pre-treatment. The shape (e.g., circular, spherical, rectangular, cubic, etc.) and size (e.g., large, medium, small, etc.) of the resulting cut pieces can be appropriately adjusted as needed. The temperature, time, and / or pressure can be appropriately determined during the pre-treatment process.

[0019] For example, heating can be performed to approximately 40-90 degrees, 45-85 degrees, 50-80 degrees, 55-75 degrees, or 60-70 degrees. However, the above temperature ranges are merely examples, and the heating temperature can be appropriately adjusted as needed. The heating can be performed for an appropriate period of time. For example, heating can be performed for approximately 1-30 days, 5-25 days, 10-20 days, or 13-17 days. However, the above heating period ranges are merely examples, and the heating period can be appropriately adjusted as needed. The pressure conditions during heating can be appropriately adjusted. For example, it can be performed under atmospheric pressure or high pressure. However, the above pressure conditions are merely examples, and the pressure conditions can be appropriately adjusted as needed. The above heating and drying process can be performed wet or dry. If necessary, wet drying and dry drying can be appropriately combined.

[0020] After the pretreatment and maturation are complete, either water alone, alcohol alone, or a mixture of water and alcohol may be added. The type of alcohol may be appropriately selected as needed. Preferably, ethanol may be selected. The alcohol concentration may be appropriately adjusted as needed. Preferably, about 10-45% alcohol can be used. More preferably, about 15-40% alcohol can be used. Even more preferably, 20-30% alcohol can be used. However, these ranges of alcohol concentration are merely examples and may be appropriately adjusted as needed. Preferably, water, alcohol, or a water / alcohol mixture may be used in larger quantities than the original (e.g., 2, 3, 4, 5, 6 times the original amount).

[0021] Subsequently, the mixture is extracted by shaking with a stirrer. The extraction temperature and time can be appropriately selected as needed. For example, extraction can be performed at room temperature for 24 hours. After extraction, the supernatant is filtered, and additional extraction can be performed if necessary. All the supernatants obtained during the extraction process are combined, concentrated under reduced pressure to remove any remaining organic solvents, and treated with nitrogen to remove any remaining water. II. Composition

[0022] In another aspect, the present invention provides compositions for the prevention, improvement (alleviation), and treatment of respiratory diseases, disorders, conditions, and symptoms, comprising a parsnip extract having improved physiological activity compared to the original parsnip.

[0023] As used in the specification of the present invention, the term “composition” includes extracts obtained by the methods described above, or other substances obtained directly or indirectly from such extracts. The term “composition” includes combinations of one or more such extracts and one or more substances obtained from such extracts, combinations of two or more such extracts, or combinations of two or more substances obtained from such extracts.

[0024] As used in the specification of this invention, the term “composition” includes a mixture of a pharmaceutically or therapeutically active ingredient (ingredient) and one or more other ingredients, the other ingredients may be chemically or biologically active or inactive. Such ingredients may include, but are not limited to, carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, excipients, and auxiliaries.

[0025] Any pharmaceutically acceptable carrier, stabilizer, diluent, dispersant, suspending agent, thickener, excipient, and auxiliary agent known to those skilled in the art as being usable in pharmaceutical compositions may be appropriately selected and used in the compositions of the present invention. As used in the specification of the present invention, the term “acceptable,” when used in relation to a pharmaceutical preparation, composition, or component, means that it does not have a sustainably adverse effect on the overall health of the person being treated. As used in the specification of the present invention, the term “carrier” refers to a chemical or biological substance that can facilitate the uptake of therapeutically active ingredients into cells or tissues. Examples of pharmaceutically acceptable carriers include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginic acid, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0026] Suitable excipients may include water, pharmaceutically acceptable organic solvents such as paraffin (e.g., petroleum fractions), vegetable oils (e.g., peanut or sesame oil), monofunctional or polyfunctional alcohols (e.g., ethanol or glycerol), carriers such as natural mineral powders (e.g., kaolin, clay, talc, chalk), carriers such as synthetic mineral powders (e.g., highly dispersed silicic acid and silicates), sugars (e.g., cane sugar, lactose, and glucose), emulsifiers (e.g., lignin, waste sulfite solution, methylcellulose, starch, and polyvinylpyrrolidone), and lubricants (e.g., magnesium stearate, talc, stearic acid, and sodium lauryl sulfate).

[0027] The compositions according to the present invention may be in the form of solids, liquids, or gases (aerosols). For example, these may be in the form of tablets (or coated tablets) made of coridone or shellac, gum arabic, talc, titanium dioxide, or sugar, capsules (gelatin), solutions (aqueous or aqueous ethanol solutions), syrups containing the active substance, emulsions or inhalable powders (made of a mixture of various sugars such as lactose or glucose, salts, and these excipients), and aerosols (inhalation solutions containing propellants or inhalation solutions without propellants). Furthermore, the compositions of the present invention may be formulated for sustained or sustained release. III. Method

[0028] In another aspect, the present invention provides a method for administering a composition containing a parsnip extract with enhanced physiological activity to a subject or patient in order to improve the respiratory health of the subject or patient and / or prevent, improve, or treat respiratory symptoms, conditions, or diseases.

[0029] As used in the specification of this invention, the terms “to treat,” “to treat,” or “to treat” mean a method of alleviating, reducing, improving, suppressing, curing, discontinuing, or preventing a disease, disorder, condition, or symptom. The term also refers to a method of alleviating, reducing, improving, suppressing, curing, discontinuing, or preventing the underlying cause of a disease, disorder, condition, or symptom.

[0030] As used in the specification of this invention, the terms "subject" or "patient" include mammals and non-mammals. Examples of mammals include, but are not limited to, humans, chimpanzees, apes, monkeys, cattle, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, etc.

[0031] As used in the specification of the present invention, the terms “administer” or “to administer” the composition mean to provide the composition and / or its prodrug to a person in need of treatment.

[0032] As used in the specification of this invention, the terms “effective dose” or “therapeutic effective dose” mean an amount sufficient to improve, to some extent, any symptom or condition of the disease being treated. The result of such improvement means a reduction and / or mitigation of the signs, symptoms, or causes of the disease, or a modification of the biological system in other desirable ways. For example, in the therapeutic use of the compositions of this invention, “effective dose” means an amount sufficient to clinically significantly reduce the symptoms of the disease. An appropriate “effective” dose in individual cases may be determined using various techniques, such as dose-elevation studies.

[0033] Furthermore, such compositions may be administered alone or in combination with one or more additional functional or therapeutic formulations. Methods of administration of such compositions may include, but are not limited to, intravenous administration, inhalation, oral administration, rectal administration, parenteral administration, intravitreal administration, subcutaneous administration, intramuscular administration, intranasal administration, cutaneous administration, topical administration, ophthalmic administration, buccal administration, tracheal administration, bronchial administration, sublingual administration, or optic nerve administration. The method of administration of the composition is determined by the severity of the symptoms. The dosage of the active ingredient in the composition may vary depending on the route of administration, the severity of the disease, the patient's age, sex, and weight, and the composition may be administered, for example, once or several times a day, but is not limited thereto.

[0034] The compositions of the present invention may be administered in any known dosage form, including, but not limited to, tablets, capsules, or elixirs for oral administration; suppositories for rectal administration; sterile solutions or suspensions for parenteral or intramuscular administration; and lotions, gels, ointments, or creams for topical administration. In one embodiment of the present invention, the compositions of the present invention may be put into dosage forms in the form of tablets, pills, capsules, liquids, inhalants, nasal spray solutions, suppositories, solutions, gels, emulsions, ointments, eye drops, or ear drops.

[0035] While other factors may be considered, the therapeutically effective dose generally varies depending on the type and severity of the disease, the subject's age and relative health, the efficacy of the administered compound, the method of administration, and the desired treatment. The required dosage will vary depending on the method of administration, the state of the specific disease being treated, and the desired therapeutic effect.

[0036] The foregoing and other embodiments and the scope of their applicability will be apparent to those ordinary skill in the art in light of the description of the present invention. The following embodiments are presented only as examples of various embodiments and should not be construed as limiting the present invention in any way. [Examples]

[0037] The present invention will be described in more detail below through the examples. These examples are provided solely to illustrate the present invention in more detail, and a person with ordinary skill in the art to which the present invention pertains will fully understand that these examples do not limit the gist or scope of the present invention. Example 1: Preparation of Parsnip Extract Sample A

[0038] The outer skin of the parsnips was removed, washed, and cut, and divided into 200g portions. The divided parsnips were dried / heat-treated in a drying chamber maintained at 55-65°C for more than 10 days. 30% alcohol (or a mixture of alcohol and water) was added to the prepared parsnip sample. Extraction was carried out at room temperature (20-25°C) for 24 hours in a stirred incubator (125 rpm). The supernatant was filtered through filter paper (Whatman No. 4). The parsnips remaining on the filter paper were extracted once more under the same conditions, and the supernatant was obtained again and combined. The organic solvent was removed from the final obtained extract using a rotary vacuum concentrator, and residual water was removed using nitrogen to prepare parsnip extract sample A. The overall manufacturing process is shown in Figure 1. Example 2: Evaluation of the antioxidant properties of parsnip extract sample A 1. Measurement of total polyphenol content

[0039] Parsnip extract sample A, prepared in Example 1, was prepared at concentrations of 2.5 mg / mL, 5.0 mg / mL, and 10.0 mg / mL. 100 μL of each sample solution was reacted with 200 μL of 2% Na2CO3 for 3 minutes. Then, 10 μL of 50% Folin-ciocalteu reagent was added and reacted for 3 minutes, after which the absorbance was measured at 720 nm. A blank was prepared by reacting a mixture of sample solvent (15% DMSO) and 2% Na2CO3 for 3 minutes, and a control was prepared by adding 50% Folin-ciocalteu reagent to this mixture and reacting for 3 minutes. After calculating the total polyphenol content, it was shown against a calibration curve using gallic acid.

[0040] As shown in Figure 2, the processed (aged) parsnips of the present invention showed a total polyphenol content approximately twice as high as that of raw parsnips from as early as the 10th day. Processed parsnips aged for 20 days and processed parsnips aged for 30 days had total polyphenol content at a level equivalent to or higher than that of red ginseng. The difference between the processed parsnip extract aged for 20 days and the processed parsnip extract aged for 30 days was not significant. Overall, the total polyphenol content of the processed parsnips produced by the method of this embodiment was significantly higher than that of raw parsnips (Figure 2). For example, the processed parsnip extract aged for 20 days or more had a total polyphenol content equivalent to or higher than that of commercially available red ginseng. Thus, processed parsnips with a high level of total polyphenol content could be obtained in a relatively short time. 2. DPPH radical scavenging ability

[0041] To evaluate the antioxidant properties of parsnips that have undergone drying and heat processing, a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability test was performed along with the analysis of total polyphenol content. Parsnip extract samples prepared in Example 1 were prepared at concentrations of 2.5 mg / mL, 5.0 mg / mL, and 10.0 mg / mL. 20 μL of each sample solution was mixed with 80 μL of 0.3 mM DPPH (2,2-diphenyl-1-picrylhydrazyl) reagent, and after dark reaction at room temperature for 1 hour, the absorbance was measured at 517 nm. A blank was prepared by reacting 20 μL of sample solvent (10% DMSO) with 80 μL of 100% ethanol for 1 hour, and a control was prepared by reacting 20 μL of sample solvent with 80 μL of 0.3 mM DPPH reagent for 1 hour. The antioxidant activity was calculated using the following formula and is shown below. Antioxidant activity (%) = 100 × (1 - (Sample OD - Blank OD) / (Control OD - Blank OD))

[0042] As shown in Figure 3, the processed parsnips of the present invention showed DPPH radical scavenging ability approximately three times higher than that of raw parsnips from as early as the 10th day (based on 5 mg and 10 mg / mL concentration samples). Processed parsnips aged for 20 days and processed parsnips aged for 30 days showed superior DPPH radical scavenging ability compared to red ginseng. Similar to the results of total polyphenol content analysis, the difference between processed parsnips aged for 20 days and processed parsnips aged for 30 days was not significant. Overall, the DPPH radical scavenging ability of processed parsnips according to the examples of the present invention was significantly higher than that of raw parsnips. For example, processed parsnip extracts aged for 20 days or more showed superior scavenging ability compared to commercially available red ginseng (Figure 3). Thus, processed parsnips with such high scavenging ability could be obtained in a relatively short time. Example 3: Evaluation of the therapeutic, mitigating, and preventive efficacy of parsnip extract sample A against respiratory inflammatory responses (In Vitro experiment) 1.Cytotoxicity analysis

[0043] Based on the results of Example 2, it was observed that the physiological activity of processed parsnips did not increase linearly even after additional drying and heat treatment following a certain period (e.g., 20 days). In subsequent experiments, experiments were conducted to determine the maximum concentration of processed parsnip extract that did not induce cytotoxicity after a 20-day maturation process. To embody a similar respiratory inflammation suppression response, human bronchial epithelial cells (BEAS-2B human bronchial epithelial cells) were cultured at 37°C and under 5% CO2 saturation conditions. For cytotoxicity experiments, the cultured cells were dispensed into 96-well plates at a rate of 5 × 10⁴ cells / cm² and stabilized for 12 hours, after which they were treated with processed parsnip extracts of various concentrations (here, processed parsnip extracts heated and dried for 20 days were used). Parsnip extract was used to treat cells at concentrations of 0 mg / mL (i.e., phosphate-buffered saline instead of parsnip), 0.625 mg / mL, 1.25 mg / mL, 2.5 mg / mL, 5 mg / mL, and 10 mg / mL. After 24 hours of treatment, cell viability was examined by MTT analysis, and the highest concentration that did not affect cell viability (in this case, 2.5 mg / mL) was found (Figure 4). 2. Evaluation of the prophylactic and therapeutic efficacy of acrolein-induced inflammatory responses.

[0044] Acrolein is an indicator substance for air pollution and is a highly reactive aldehyde. Acrolein is known to induce oxidative stress within cells, as well as to bind to various macromolecules (e.g., DNA or proteins) to induce inflammatory responses and cell death. Based on the research results described above, we investigated at the cellular level whether processed parsnip extract, matured for 20 days under non-toxic conditions, has efficacy against acrolein-induced inflammation. More specifically, human-derived bronchial epithelial cells (BEAS-2B cells) were treated with processed parsnip extract according to the present invention's examples, and then treated with 160 μM acrolein. Gene expression of signaling pathways associated with inflammation was analyzed. As shown in Figure 5, in the group treated with acrolein, the gene expression of inflammation-related cytokine markers (TNF-α, IL-6, IL-1β) and p53, which is associated with cell death, all significantly increased. Conversely, in the group treated with processed parsnip extract (2.5 mg / mL), these inflammatory and cell death responses were significantly reduced (Figure 5).

[0045] Similarly, we also verified at the cellular level whether the processed parsnip extract according to the examples of the present invention has significant therapeutic efficacy against acrolein-induced inflammation under non-toxic conditions. More specifically, human-derived bronchial epithelial cells (BEAS-2B cells) were first treated with 160 μM acrolein, then treated with the processed parsnip extract, and gene expression of the inflammation-related signaling pathway was analyzed. Although not shown here, results similar to those in Figure 5 were obtained. Example 4: Evaluation of the therapeutic, mitigating, and preventive efficacy of parsnip extract sample A against respiratory inflammatory response (In Vivo experiment) 1. Analysis of inflammatory markers

[0046] Based on the results of the aforementioned cell experiments, efficacy analysis on respiratory inflammation markers was performed using C57BL6 mice. More specifically, processed parsnip extract according to the examples of the present invention was orally administered to C57BL6 male mice at a dose of 10 mg / kg bw for 2 weeks, and acrolein was administered intranasally (nasal installation; 10 μg / kg bw) for 5 days starting from day 9. On day 14, male mice were sacrificed and analyzed for related inflammation markers (Figure 6). 2. Cytokine protein content analysis

[0047] The protein content of various pro-inflammatory cytokines present in the blood samples obtained in the aforementioned animal experiments was analyzed. The inflammatory cytokines in the blood shown in Figure 7 are mostly known as markers closely related to anti-inflammatory mechanisms. As can be seen from Figure 7, it was confirmed that the expression level of inflammatory cytokines induced by acrolein administration was significantly reduced by treatment with the processed parsnip extract according to the examples of the present invention (Figure 7). 3. Analysis of inflammatory gene expression levels in lung tissue

[0048] In addition to the systemic inflammatory response confirmed in Figure 7, lung tissue was extracted from the animals to analyze the local inflammatory response, and the expression levels of TNF-α and IL-1β, two genes crucial to the inflammatory mechanism, were measured. The anti-inflammatory efficacy was clearly reproduced in the group administered parsnip extract processed according to the present invention's examples, even in the localized lung inflammatory response (Figure 8). 4. H&E staining analysis of lung tissue

[0049] Lung tissue was stained with H&E and histologically analyzed. As shown in Figure 9, the positive control group showed a considerably greater degree of bronchial lesions in the lung tissue compared to the negative control group, but the group treated with the processed parsnip extract according to the present invention showed a reduction in the incidence of bronchial lesions in the lung tissue. 5. PAS staining analysis of lung tissue

[0050] Lung tissue was stained with PAS to confirm the degree of mucus accumulation in the lung tissue. As shown in Figure 10, mucus accumulation in the lung tissue of the positive control group was significantly increased compared to the negative control group, but this phenotype was mitigated in mice that ingested the processed parsnip extract according to the present invention. While a considerable amount of mucus accumulated in the lung tissue of the positive control group compared to the negative control group, very little mucus accumulated in the lung tissue of the group treated with the processed parsnip extract according to the present invention.

[0051] Similar to what was done in cell models, we also verified whether the processed parsnip extract of the present invention has significant therapeutic efficacy against acrolein-induced inflammation in animal models under non-toxic conditions. More specifically, C57BL6 male mice were first treated with acrolein, then subsequently treated with the processed parsnip extract according to the examples of the present invention, followed by analysis of related inflammatory markers, cytokine protein content, and lung tissue. Although not shown here, results similar to those in Figures 7 to 10 were obtained. Example 6: Preparation of Parsnip Extract Sample B

[0052] Similar to Example 1, raw parsnips were peeled, washed, and cut, and divided into 200g portions. The divided parsnips were placed in a drying chamber maintained at 85-90°C and dried / heat-treated for 6 days. 30% alcohol (or a mixture of alcohol and water) was added to the parsnips thus prepared. Extraction was carried out at room temperature (20-25°C) for 24 hours. The supernatant was filtered through filter paper (Whatman No. 4). The parsnips remaining on the filter paper were extracted once more under the same conditions, and the supernatant was obtained again and combined. The primary organic solvent was removed from the final obtained extract using a rotary vacuum concentrator, and residual water was removed using nitrogen to prepare parsnip extract sample B. The overall manufacturing process is shown in Figure 11. Example 7: Evaluation of antioxidant properties of parsnip extract samples A and B 1. DPPH radical scavenging ability

[0053] A DPPH radical scavenging test was performed to evaluate the antioxidant properties of the processed parsnip extract. Parsnip extract prepared in Example 6 was prepared at concentrations of 2.5 mg / mL, 5.0 mg / mL, and 10.0 mg / mL. 20 μL of each sample solution was mixed with 180 μL of 0.2 mM DPPH reagent (2,2-diphenyl-1-picrylhydrazyl), and after a dark reaction at room temperature for 30 minutes, the absorbance was measured at 517 nm.

[0054] For the comparison group, vitamin C (0-100 ug / mL) was reacted with the DPPH reagent under the same conditions as the samples. A standard curve (Figure 12A) was created using the measured absorbance values, and the DPPH radical scavenging ability was confirmed by comparing it with the concentration of vitamin C. As shown in Figure 12B, the DPPH radical scavenging ability of processed parsnip extract sample A and processed parsnip extract sample B according to the present invention was superior to that of raw parsnip extract that had not undergone maturation treatment. 2. ABTS radical scavenging ability

[0055] Antioxidant activity was measured using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). ABTS and AAPH were dissolved in 100 mL of PBS at concentrations of 2.5 mM and 1 mM, respectively, and heated at 70°C for 1 hour, followed by cooling at room temperature for 20-30 minutes. Standard solutions were prepared by sequentially diluting vitamin C from 100 μg / mL to 0 μg / mL. Sample solutions were sequentially diluted so that the absorbance at 734 nm was between 0.65 and 0.9. 10 μL of each standard solution and sample solution were placed in a 96-well plate, 190 μL of ABTS solution was added, and the plate was allowed to react in the dark at room temperature for 10 minutes. Based on the absorbance measured at 734 nm, the radical scavenging activity of the samples was expressed in terms of vitamin C concentration using the equation of the standard line (Figure 13A). As shown in Figure 13B, the ABTS radical scavenging ability of processed parsnip extract sample A and processed parsnip extract sample B according to the present invention was superior to that of unaged raw parsnip extract. Example 8: Evaluation of the therapeutic, mitigating, and preventive efficacy of parsnip extract sample B against respiratory inflammatory responses (In Vitro experiment) 1.Cytotoxicity analysis

[0056] To evaluate whether the antioxidant capacity of the processed parsnip extract according to the present invention acts as a protective agent for lung cells, efficacy evaluation was performed on human normal lung cells (human primary bronchial / tracheal epithelial cells). Cells were cultured at 37°C in a 5% CO2 environment in DMEM / F-12 medium supplemented with 10% FBS and 1% penicillin-streptomycin. After inoculating 96-well plates with cells at a density of 5 × 10⁴ cells / well, adhesion and growth were induced for 24 hours. Parsnip extract was prepared at concentrations of 0, 25, 50, 100, 200, and 400 μg / mL, and 100 μL of each extract was added to the wells, and cells were cultured for 24 hours. MTT solution was prepared in PBS at a concentration of 5 mg / mL, and 10 μL was added to each well to a final concentration of 0.5 mg / mL, after which the cells were cultured for 4 hours. After removing the culture medium and dissolving the formazan crystals with 100 μL of DMSO, the absorbance was measured at 570 nm and corrected for background absorbance at 630 nm. Cell viability was calculated as a percentage relative to the control group, and all experiments were repeated three times. Data were expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism through one-way analysis of variance (ANOVA) and Tukey's post-hoc test, with p-values ​​< 0.05 considered statistically significant. As illustrated in Figure 14, processed parsnip extract sample B according to the present invention showed cytotoxicity at a concentration of 400 ug / mL. 2. Protective efficacy against lung cells from LPS-induced inflammatory toxicity.

[0057] A concentration that did not exhibit cytotoxicity was selected, and subsequent experiments were carried out. One group of cells was treated with LPS at a concentration of 20 ug / mL for 24 hours to induce inflammatory toxicity. On the other hand, the other group of cells was treated simultaneously with processed parsnip extract sample B according to the present invention and LPS. After 24 hours, cell viability was measured by MTT analysis (Figure 15). As can be seen from Figure 15, processed parsnip extract B according to the present invention had the efficacy to protect human normal lung cells from inflammatory toxicity induced by LPS.

[0058] Similarly, we also verified at the cellular level whether the processed parsnip extract according to the present invention's examples has significant therapeutic efficacy against LPS-induced inflammation under non-toxic conditions. More specifically, human primary bronchial / tracheal epithelial cells were first treated with 20 ug / mL of LPS to induce inflammation, and then treated with the processed parsnip extract according to the present invention's examples. Gene expression in the inflammation-related signaling pathway was then analyzed. Although not shown here, results similar to those in Figure 15 were obtained.

Claims

1. A composition for promoting respiratory health, containing an extract of processed parsnips, which has a higher total polyphenol content than natural parsnips, as an active ingredient.

2. The composition according to claim 1, characterized in that the promotion of respiratory health includes the prevention, improvement, or treatment of diseases, disorders, conditions, or symptoms related to respiratory diseases.

3. The composition according to claim 2, characterized in that the respiratory disease is bronchial asthma.

4. The composition according to claim 2, characterized in that the respiratory disease is chronic obstructive pulmonary disease (COPD).

5. The composition according to claim 1, characterized in that the processed parsnip extract is obtained by heating and drying raw parsnips within a specific temperature range for a specific period of time, and then extracting the parsnips after they have been aged until their color changes to brown or black.

6. The composition according to claim 5, characterized in that the specified temperature is within the range of 50°C to 65°C, and the specified period is a period of 10 to 20 days.

7. The composition according to claim 5, characterized in that the specified temperature is within the range of 85°C to 90°C, and the specified period is a period of 3 to 10 days.

8. The composition according to claim 1, characterized in that the processed parsnip extract has a total polyphenol content that is at least twice as high as that of raw parsnip.

9. A method for producing an extract of processed parsnip, which has a higher antioxidant capacity than natural parsnip, wherein the production method comprises the steps of: heat-drying raw parsnip at a specific temperature for a specific period of time; adding alcohol, water, or a mixture thereof to the heat-dried parsnip; filtering the supernatant; and removing unwanted components from the filtered supernatant.

10. The method according to claim 9, characterized in that the heating and drying step is carried out until the color of the original parsnip changes to brown or black.

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