Composition and method for treating, preventing, and alleviating respiratory symptom or disease

Abstract

The present invention provides a method for preparing a parsnib extract having improved physiological activity compared to the original parsnib by pretreating the original parsnib to make aged parsnib, followed by extraction from the aged parsnib. Furthermore, the present invention provides a composition comprising a parsnib extract with improved physiological activity, prepared by the preparation method, and also provides a method for enhancing respiratory health and / or preventing, alleviating, and treating respiratory symptoms or diseases by using the composition.

Description

[0001] Compositions and methods for treating, preventing, or improving respiratory symptoms or diseases Technical field The present invention relates to a method for preparing a parsnip extract with improved physiological activity, a composition comprising a parsnip extract prepared by the preparation method, and a method for promoting respiratory health using the composition. Background Art Mammals, including humans, suffer from various respiratory symptoms or 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 or diseases.

[0002] 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 Cell Mol Physiol, 2010, 298:L262-L269; As reported in the literature (Am J Respir Cell Mol Biol, 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 underway. However, there are virtually no functional foods or drugs that effectively prevent, improve, or treat bronchial asthma and chronic obstructive pulmonary disease. Summary of the Invention The present invention provides a method for producing a parsnip extract having improved physiological activity compared to that of the raw parsnip, comprising preprocessing raw parsnip to produce a ripened parsnip and extracting the ripened parsnip. Furthermore, a composition comprising the parsnip extract having improved physiological activity produced by the method is provided.In addition, methods for promoting respiratory health and / or preventing, improving, or treating respiratory symptoms or diseases using the composition are provided. 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, comprising 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 preventing, improving, or treating diseases, disorders, conditions, or symptoms associated with respiratory diseases. The respiratory diseases may include bronchial asthma or chronic obstructive pulmonary disease. The processed parsnip extract of the present invention may preferably have a total polyphenol content greater than that of raw parsnip by at least 1.5 times, more preferably by at least 2 times, and even more preferably by at least 3 times. According to one embodiment of the present invention, raw parsnip is heat-dried at a specific temperature for a specific period of time to the extent that the color of the raw parsnip changes to brown or black, and then an extraction process is performed to obtain an extract. As an example, the specific temperature may be adjusted to a temperature between about 50°C and about 70°C, and the specific period may be adjusted to a period between about 10 and about 30 days. As another example, the specific temperature may be adjusted to a temperature between about 55°C and about 65°C, and the specific period may be adjusted to a period between about 10 and about 20 days. As another example, the specific temperature may be adjusted to a temperature between about 70°C and about 90°C, and the specific period may be adjusted to a period between about 3 and about 10 days. As another example, the specific temperature may be adjusted to a temperature between about 85°C and about 90°C, and the specific period may be adjusted to a period between 3 and 10 days.According to another embodiment, the specific temperature and the specific period can be adjusted so that the color of the raw parsnip turns brown or black and / or so that the total polyphenol content is greater than the total polyphenol content of the raw parsnip. Another aspect of the present invention provides a method for preparing the extract. More specifically, another aspect of the present invention provides a method for preparing a processed aged parsnip extract comprising a total polyphenol content greater than (for example, at least twice or more) the total polyphenol content of an unprocessed natural (raw) parsnip. The method is characterized by comprising the steps of heating and drying the raw parsnip at a specific temperature for a specific period of time, adding ethanol, water, or a mixture thereof to the thus heated and dried parsnip, filtering the supernatant, and removing unnecessary components from the filtered supernatant. According to one embodiment of the present invention, the heat-drying step may be performed to a degree sufficient to change the color of the raw parsnip to brown or black and also to increase the total polyphenol content to be greater than the total polyphenol content of the raw parsnip. The above aspects of the present invention, other aspects, and embodiments of the present invention are described in more detail below. Brief Description of the Drawings Figure 1 is a diagram illustrating a process for producing a parsnip extract according to one embodiment of the present invention. Figure 2 is a graph showing changes in the total polyphenol content of a parsnip extract according to one embodiment of the present invention. AP 10D, AP 20D, and AP 30D refer to parsnip extracts obtained by pretreating (aging or processing) raw parsnip for 10 days, 20 days, and 30 days, respectively, and raw parsnip refers to an extract obtained without such pretreatment. FIG. 3 is a graph showing the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of a parsnip extract according to one embodiment of the present invention.AP 10D, AP 20D, and AP 30D refer to parsnip extracts obtained by pretreating (aging or processing) raw parsnip for 10, 20, and 30 days, respectively, and raw parsnip refers to an extract obtained without such pretreatment. Figure 4 shows the cytotoxicity of parsnip extracts according to one embodiment of the present invention against human-derived bronchial epithelial cells (BEAS-2B). AP 20D refers to a parsnip extract obtained by pretreating (aging or processing) raw parsnip for 20 days and then extracting. MTT refers to 2,5-diphenyl-2H-tetrazolium bromide. PB refers to phosphate buffered saline. FIG. 5 shows the efficacy of a parsnip extract according to an embodiment of the present invention on acrolein-induced inflammation. NEG refers to a negative control group, POS refers to a positive control group, and AP 20D refers to a parsnip extract obtained by pretreating (ripening or processing) raw parsnip for 20 days and then extracting it. The P value is calculated through a step-wise t-test. FIG. 6 is an experimental design designed to confirm whether a parsnip extract according to an embodiment of the present invention has an anti-inflammatory effect in rats exposed to acrolein. The negative control group was treated with only a basal diet (AIN-93G), the positive control group was treated with acrolein together with the basal diet, and the test group was treated with the basal diet, the parsnip extract according to an embodiment of the present invention (AP 20D), and acrolein. FIG. 7 shows the effect of a parsnip extract according to an embodiment of the present invention on the expression level of inflammatory cytokines in the blood.The negative control group was treated with only the basal diet (AIN-930), the positive control group was treated with the basal diet together with acrolein, and the test group was treated with the basal diet, the parsnip extract (AP 20D) according to an embodiment of the present invention, and acrolein. The P value is a value calculated through a step-wise t-test. * means a p value of less than 0.05, ** means a p value of less than 0.01, and *** means a p value of less than 0.001. Figure 8 shows the effect of the parsnip extract according to an embodiment of the present invention on the expression levels of tumor necrosis factor-alpha (TNF-a) messenger RNA (mRNA) and interleukin-1 beta (IL-10) 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 together with acrolein, and the test group was treated with the basal diet, a parsnip extract (AP 20D) according to an embodiment of the present invention, and acrolein. ? The values ​​are calculated through a step-wise t-test. Figure 9 shows the results of H&E staining reactions for lung tissue treated with a parsnip extract according to an 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 together with acrolein, and the test group was treated with the basal diet, a parsnip extract (AP 20D) according to an embodiment of the present invention, and acrolein. Figure 10 shows the results of Periodic acid-Schiff (PAS) staining reactions for lung tissue treated with a parsnip extract according to an 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 together with acrolein, and the test group was treated with the basal diet, a parsnip extract (AP 20D) according to one embodiment of the present invention, and acrolein. Figure 11 is a schematic diagram of the manufacturing process of a parsnip extract according to another embodiment of the present invention.FIG. 12A is a standard line used to measure the DPPH radical scavenging activity of a parsnip extract according to another embodiment of the present invention, and FIG. 12B is a graph showing the DPPH radical scavenging activity of a parsnip extract sample according to another embodiment of the present invention. Test group A refers to a group treated with the parsnip extract obtained in Example 1 of the present invention, test group B refers to a group treated with the parsnip extract obtained in Example 6 of the present invention, and the control group refers to a group treated with an extract obtained from raw parsnip without undergoing a pretreatment (ripening) process. FIG. 13A is a standard line used to measure the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity of a parsnip extract according to another embodiment of the present invention, and FIG. 13B is a graph showing the ABTS radical scavenging activity of a parsnip extract according to another embodiment of the present invention. Test group A refers to a group treated with the parsnip extract obtained in Example 1 of the present invention, test group B refers to a group treated with the parsnip extract obtained in Example 6 of the present invention, and the control group refers to a group treated with an extract obtained from raw parsnip without undergoing a pretreatment (ripening) process. FIG. 14 shows the results of a cytotoxicity test on human normal lung cells of a parsnip extract according to another embodiment of the present invention. Figure 15 illustrates the lung tissue protective efficacy of a parsnip extract according to another embodiment of the present invention. Detailed Description of the Invention To facilitate understanding of the basic concept of the present invention, the present invention will be described below with reference to embodiments illustrated in the drawings, and specific terminology will be used in such description. However, such description should not be construed as imposing any limitations on the scope of the present invention.When the term "approximately" as used herein is used in connection with a value or range, it should be interpreted that the value or range has a certain degree of variability, for example, it can have a variability of within 10%, within 5%, or within 1%. When the term "substantially" as used herein is used in connection with a value or range, it should be interpreted that the value or range has a certain degree of variability, for example, it can have a variability of within 90%, within 95%, or within 99%. The terms "a," "an," or "the" as used herein should be interpreted to include one or more unless the context clearly defines otherwise. The term "or" as used herein should be interpreted in an inclusive sense unless specifically defined otherwise. The phraseology or terms used in the present invention are used only for the purpose of describing the present invention unless specifically defined otherwise, and the content of the invention should not be construed as being limited by such phraseology or terms. The section titles used in this specification are solely for the purpose of facilitating understanding of the present invention, and should not be construed as limiting the contents of the present invention. Information related to a section title may be located within or outside of the relevant section. Furthermore, all publications, patent publications, etc. incorporated by reference in this specification shall be deemed to be included in the contents of this specification. If the above documents or publications are inconsistent with the contents of this specification, the above documents and publications shall be considered supplementary to the present specification, and if the inconsistency cannot be reconciled, the present specification shall take precedence.

[0003] I. Parsnip Extract In one aspect, the present invention provides a parsnip extract having improved physiological activity compared to raw parsnip. According to one embodiment, a parsnip extract is provided, which is prepared by pretreating raw parsnip to produce aged parsnip or processed parsnip and extracting the aged or processed parsnip. The parsnip extract obtained from the aged parsnip has higher physiological activity than that of the raw parsnip. Although there is a report on a process for pretreating raw parsnip (Plant Foods for Human Nutrition (2020) 75:292-297), the process takes too long (4 weeks) to complete and requires heat treatment at high temperature (80.0) and maintenance of a high level of humidity (95%), which limits its application to industrial sites. A novel method for pretreating under milder process conditions in a shorter period of time is urgently needed. According to one embodiment of the present invention, a parsnip extract having significantly improved physiological activity compared to the raw parsnip can be obtained by pretreating raw parsnip under relatively mild processing conditions within a relatively short period of time. According to one embodiment of the present invention, a ripened parsnip is prepared by pretreating raw parsnip. The raw parsnip can be cut before pretreating. The shape (e.g., circle, sphere, rectangular parallelepiped, cube, etc.) and size (e.g., large, medium, small, etc.) of the cut result can be appropriately adjusted as needed. The temperature, time, and / or pressure can be appropriately determined during the pretreatment process. For example, the parsnip can be heated to about 40 to 90 degrees, 45 to 85 degrees, 50 to 80 degrees, 55 to 75 degrees, or 60 to 70 degrees. However, the above temperature ranges are only examples, and the heating temperature can be appropriately adjusted as needed. The heating can be performed for an appropriate time. For example, it can be heated for about 1 to 30 days, 5 to 25 days, 10 to 20 days, or 13 to 17 days.However, the above heating period ranges are only examples, and the heating period can be appropriately adjusted as needed. The pressure conditions during the heating can be appropriately adjusted. For example, it can be performed under atmospheric pressure or high pressure. However, the above pressure conditions are only examples, and the pressure conditions can be appropriately adjusted as needed. In addition, the above heating and drying process can be performed in a wet or dry manner. If necessary, wet drying and dry drying can be appropriately combined. After the pretreatment aging is complete, only water, only alcohol, or a mixture of water and alcohol is added. The type of alcohol can be appropriately selected as needed. Ethanol can be preferably selected. The alcohol concentration can be appropriately adjusted as needed. Preferably, about 10 to 45% alcohol can be used. More preferably, about 15 to 40% alcohol can be used. Even more preferably, 20 to 30% alcohol can be used. However, these alcohol concentration ranges are only examples, and can be appropriately adjusted as needed. Preferably, water, alcohol, or a mixture of water and alcohol can be used in an amount greater than that of the original material (e.g., 2 times, 3 times, 4 times, 5 times, 6 times, etc.). After that, shaking extraction is performed in 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 supernatants obtained during the extraction process are combined, concentrated under reduced pressure to remove the residual organic solvent, and treated with nitrogen to remove the residual moisture.

[0004] II. Composition In another aspect, the present invention provides a composition for preventing, improving (alleviating), and treating respiratory diseases, disorders, conditions, and symptoms, comprising a parsnip extract having improved physiological activity compared to raw parsnip. The term "composition" as used in the present specification includes the extract obtained by the method described above, or other water obtained directly or indirectly from the extract. The term "composition" includes a combination comprising at least one of the above extracts and at least one of the waters obtained from the extract, a combination comprising at least two of the above extracts, or a combination comprising at least two of the waters obtained from the extract. The term "composition" as used herein includes a mixture of a pharmaceutically or therapeutically active ingredient (ingredient) and one or more other ingredients, which 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. Any pharmaceutically acceptable carrier, stabilizer, diluent, dispersant, suspending agent, thickener, excipient and auxiliaries known to those skilled in the art to be usable in pharmaceutical compositions may be appropriately selected and used in the compositions of the present invention. The term "acceptable" as used herein, when used in connection with a pharmaceutical preparation, composition, or component, means not having a persistently detrimental effect on the overall health of the subject to be treated. The term "carrier" as used herein refers to a chemical or biological material that can facilitate the incorporation of therapeutically active ingredient(s) into cells or tissues.Examples of pharmaceutically acceptable carriers include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginic acid, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. Examples of suitable excipients may include water, pharmaceutically acceptable organic solvents such as paraffin (e.g., petroleum fractions), vegetable oils (e.g., peanut or sesame oil), mono- 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). The composition according to the present invention may be in the form of a solid, a liquid, or a gas (aerosol). For example, they may be in the form of tablets (or coated tablets), capsules (gelatin), solutions (aqueous or aqueous ethanolic solutions), syrups containing the active substance, emulsions (of various sugars, salts and mixtures of these excipients, such as lactose or glucose), or inhalable powders, and aerosols (propellant-containing or propellant-free inhalation solutions) made of Kollidon or shellac, gum arabic, talc, titanium dioxide or sugar. The compositions of the present invention may also be formulated for sustained or sustained release.

[0005] III. Method In another aspect, the present invention provides a method for improving respiratory health and / or preventing, improving, or treating respiratory symptoms, conditions, or diseases in a subject or patient by administering to the subject or patient a composition comprising a parsnip extract having enhanced physiological activity. As used herein, the terms "treating," "treating," or "treatment" refer to a method for ameliorating, alleviating, improving, suppressing, curing, stopping, or preventing a disease, disorder, condition, or symptom. The terms also refer to a method for ameliorating, alleviating, improving, suppressing, curing, stopping, or preventing the underlying cause of the disease, disorder, condition, or symptom. The terms "subject" or "patient" as used herein include mammals and non-mammals. Examples of mammals include, but are not limited to, humans, chimpanzees, apes, monkeys, cows, horses, sheep, goats, dogs, rabbits, dogs, cats, guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. As used herein, the term "administering" or "administering" of the composition means providing the composition of the present invention and / or a prodrug thereof to a subject in need of treatment. The term "effective amount" or "therapeutically effective amount" as used herein means, for example, an amount sufficient to improve to some extent any symptom or condition of a disease being treated. The result of such improvement is that the signs, symptoms, or causes of the disease are reduced and / or alleviated, or the biological system is altered in some other desirable manner. For example, an "effective amount" in the therapeutic use of the composition of the present invention means an amount that clinically significantly reduces disease symptoms. The appropriate "effective" amount in an individual case can be determined using various techniques, such as dose-escalation studies.Furthermore, these compositions may be administered alone or in combination with one or more additional functional or therapeutic agents. Methods of administration of these 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, dermal 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 according to the severity of the symptoms. Furthermore, the dosage of the active ingredient in the composition may vary depending on the route of administration, the severity of the disease, the age, sex, and body weight of the patient, and the composition may be administered, for example, once or several times a day, but is not limited thereto. The composition 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 composition of the present invention may be formulated in the form of tablets, pills, capsules, liquids, inhalants, nasal spray solutions, suppositories, solutions, gels, emulsions, ointments, eye drops, or ear drops. The therapeutically effective amount will generally vary depending on the type of disease, the severity of the disease, the age and relative health of the subject, the potency of the compound administered, the route of administration, and the desired treatment, among other factors. The required dosage will vary depending on the route of administration, the specific disease state to be treated, and the desired therapeutic effect. The above and other embodiments and the scope of their applicability will be apparent to those skilled in the art to which the present invention pertains in light of the description of the present invention.The following examples are provided merely as illustrations of various embodiments, and the present invention should not be construed as being limited thereto in any way. EXAMPLES Hereinafter, the present invention will be described in more detail through examples. These examples are provided merely to explain the present invention in more detail, and it will be fully understood by those skilled in the art that the spirit or scope of the present invention is not limited by these examples. Example 1: Preparation of parsnip extract sample A The outer skin of raw parsnips was removed, washed, cut, and portioned into 200 g. The portioned parsnips were placed in a drying chamber maintained at 55 to 65°C and dried / heat-treated for more than 10 days. 30% ethanol (or a mixture of ethanol and water) was added to the prepared parsnip sample. Extraction was performed in a shaking incubator (125 rpm) at room temperature (20 to 25°C) for 24 hours. The supernatant was filtered through a filter paper (Whatman No. 4). The parsnip remaining on the filter paper was extracted once more under the same conditions, and the supernatant was obtained once more and combined. The organic solvent was removed from the final obtained extract using a rotary vacuum evaporator, and the residual moisture was removed using nitrogen to prepare parsnip extract sample A. The overall manufacturing process is illustrated in Fig. 1. Example 2: Evaluation of the antioxidant properties of parsnip extract sample A.

[0006] 1. Measurement of total polyphenol content Sample A of the parsnip extract prepared in Example 1 was prepared at concentrations of 2.5 mg / mL, 5.0 mg / mL, and 10.0 mg / mL. 100 |iL of each concentration sample solution and 200 |lL of 2% Na2CO3 were reacted for 3 minutes. Then, 10 |iL of 50% Folin-ciocalteu reagent was added, reacted for 3 minutes, and the absorbance was measured at 720 nm. A mixture of sample solvent (15% DMSO) and 2% Na2CO3 reacted for 3 minutes was used as a blank, and a mixture reacted for 3 minutes with 50% Folin's reagent added thereto was used as a control. The total polyphenol content was calculated and expressed against a calibration curve using gallic acid. As shown in FIG. 2, the processed (aged) parsnips of the present invention showed a total polyphenol content that was about twice as high as that of the raw parsnip as early as the 10th day, and the processed parsnips aged for 20 days and those aged for 30 days had total polyphenol contents that were equivalent to or higher than those of red ginseng. The difference between the processed parsnip extract aged for 20 days and those aged for 30 days was not significant. In summary, the total polyphenol content of the processed parsnips produced by the method according to the present example was significantly higher than that of the raw parsnips (FIG. 2). For example, the processed parsnip extract aged for 20 days or more had a total polyphenol content that was equivalent to or higher than that of commercially available red ginseng. It was possible to obtain processed parsnips having such a high total polyphenol content in a relatively short period of time.

[0007] 2. DPPH radical scavenging ability In order to evaluate the antioxidant properties of parsnips subjected to dry heat processing, a 2,2-diphenyl- 1 -picrylhydrazyl (DPPH) radical scavenging ability test was performed in addition to an analysis of the total polyphenol content. The 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 the sample solutions of each concentration and 80 μL of 0.3 mM DPPH (2,2-diphenyl- 1-picrylhydrazyl) reagent were mixed, and the mixture was incubated in the dark at room temperature for 1 hour, after which the absorbance was measured at 517 nm. A mixture of 20 |iL of sample solvent (10% DMSO) and 80 |iL of 100% ethanol, reacted for 1 hour, was used as a blank, and a mixture of 20 |iL of sample solvent and 80 |iL of 0.3 mM DPPH reagent, reacted for 1 hour, was used as a control. The antioxidant activity was calculated using the following formula.

[0008] Sample 0D — Blank 0D Antioxidant activity (%) = 100 X Control OD — Blank OD As shown in Figure 3, the processed parsnip of the present invention showed DPPH radical scavenging activity that was about 3 times higher than that of the raw parsnip as early as the 10th day (based on 5 mg and 10 mg / mL concentration samples), and the processed parsnip aged for 20 days and the processed parsnip aged for 30 days showed DPPH radical scavenging activity that was significantly higher than that of red ginseng. Similar to the results of the total polyphenol content analysis, the difference between the processed parsnip aged for 20 days and the processed parsnip aged for 30 days was not significant. In summary, the DPPH radical scavenging activity of the processed parsnip according to the embodiment of the present invention was significantly higher than that of the raw parsnip. For example, the processed parsnip extract aged for more than 20 days showed a level of scavenging activity that was significantly higher than that of the red ginseng sold on the market (Figure 3). Processed parsnip with such a high level of scavenging activity could be obtained in a relatively short period of time. Example 3: Evaluation of the therapeutic, alleviating, and preventive effects of parsnip extract sample A on respiratory inflammatory responses (In vitro experiment)

[0009] 1. Cytotoxicity analysis Based on the results of Example 2 above, it was observed that the physiological activity of processed parsnip did not increase linearly even after an additional dry heat treatment was performed after a certain period of time (e.g., 20 days). In the subsequent experiment, an experiment was conducted to establish the maximum concentration that did not induce cytotoxicity for the processed parsnip extract that had undergone a 20-day aging process. In order to similarly implement the respiratory inflammation inhibition response, human-derived bronchial epithelial cells (BEAS-2B human bronchial epithelial cells) were cultured at 37°C and 5% CO2 saturated conditions. The cultured cells were 5 x 10 for cytotoxicity testing. 4 cells / cm 2After dispensing into 96-well plates under the conditions of and stabilizing for 12 hours, various concentrations of processed parsnip extract (here, processed parsnip extract heat-dried for 20 days was used) were treated. The parsnip extract was treated 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 confirmed by MTT assay, and the highest concentration (here, 2.5 mg / mL) that did not affect cell viability was found. (Fig. 4).

[0010] 2. Evaluation of preventive and therapeutic efficacy against inflammatory responses induced by acrolein Acrolein is an air pollution indicator and a highly reactive aldehyde. Acrolein is known to induce oxidative stress within cells and to bind to various macromolecules (e.g., DNA or proteins) to induce inflammatory responses and cell death. Based on the above-mentioned research results, the efficacy of a processed parsnip extract aged for 20 days under non-toxic conditions against acrolein-induced inflammation was verified at the cellular level. More specifically, human-derived bronchial epithelial cells (BEAS-2B cells) were treated with the processed parsnip extract according to the present invention, followed by treatment with 160 pIM of acrolein. The gene expression of the signaling pathway related to inflammation was analyzed. As shown in Fig. 5, in the group treated with acrolein, both markers of inflammation-related cytokines (TNF-α, IL-6, IL-10) and the gene expression of p53, which is related to cell death, significantly increased. In contrast, in the group treated with the processed parsnip extract (2.5 mg / mL), these inflammatory and cell death responses were significantly reduced (Fig. 5). Similarly, the efficacy of the processed parsnip extract according to the present invention to significantly treat inflammation induced by acrolein was verified at the cellular level under non-toxic conditions. More specifically, human-derived bronchial epithelial cells (BEAS-2B cells) were first treated with 160 pIM of acrolein and then treated with the processed parsnip extract, and then the gene expression of the inflammation-related signaling pathway was analyzed. Although not shown here, similar results to those in Fig. 5 were obtained. Example 4: Evaluation of the therapeutic, alleviating, and preventive efficacy of parsnip extract sample A on respiratory inflammatory responses (in vivo experiment)

[0011] 1. Analysis of inflammation markers Based on the results of the above cell experiment, an efficacy analysis on respiratory inflammation markers was performed using C57BL6 mice. More specifically, the processed parsnip extract according to the present invention was orally administered to C57BL6 male mice at 10 mg / kg bw for 2 weeks, and acrolein was administered intranasally (nasal installation; 10 pg / kg bw) for 5 days starting on the 9th day. On the 14th day, the male mice were sacrificed and analysis of related inflammation markers was performed (Fig. 6). 2. Analysis of cytokine protein content The protein content of various pro-inflammatory cytokines present in the blood samples obtained in the above animal experiment was analyzed. Most of the blood inflammatory cytokines shown in Fig. 7 are known to be markers closely related to anti-inflammatory mechanisms. As can be seen in Fig. 7, it was confirmed that the expression level of inflammatory cytokines induced by the administration of acrolein was significantly reduced by the treatment with the processed parsnip extract according to the present invention (Fig. 7).

[0012] 3. Analysis of inflammatory gene expression in lung tissue In addition to the systemic inflammatory response confirmed in Fig. 7, lung tissues of the animals were extracted to analyze local inflammatory responses, and the expression levels of TNF-α and IL-1P, two genes key to the inflammatory mechanism, were measured. In the local lung inflammatory response, the anti-inflammatory efficacy was clearly reproduced in the group administered the extract of the processed parsnip according to the present invention (Fig. 8).

[0013] 4. H&E staining analysis of lung tissue Histological analysis was performed by H&E staining of lung tissue. As shown in Fig. 9, in the positive control group, bronchial lesions in lung tissue were significantly induced compared to the negative control group, but in the group treated with the processed parsnip extract according to the present invention, the occurrence of bronchial lesions in lung tissue was reduced.

[0014] 5. PAS staining analysis of lung tissue The extent of mucus accumulation in the lung tissue was confirmed by PAS staining of lung tissue. As shown in Fig. 10, mucus accumulation in the lung tissue of the positive control group was significantly increased compared to the negative control group, but the phenotype was alleviated in the mice that consumed the processed parsnip extract according to the example of the present invention. In the positive control group, mucus accumulated significantly in the lung tissue compared to the negative control group, but in the group treated with the processed parsnip extract according to the example of the present invention, mucus accumulated very little in the lung tissue. As was done in the cell model, it was also verified in an animal model that the processed parsnip extract of the present invention has a significant effect in treating inflammation induced by acrolein under conditions that do not show toxicity. More specifically, C57BL6 male mice were first treated with acrolein, and then subsequently treated with the processed parsnip extract according to the example of the present invention, followed by analysis of inflammatory markers, cytokine protein content, and lung tissue analysis. Although not shown here, similar results to those in FIGS. 7 to 10 were obtained. Example 6: Preparation of parsnip extract sample B As in Example 1, raw parsnips were peeled, washed, cut, and portioned into 200 g portions. The portioned parsnips were placed in a drying chamber maintained at 85 to 90°C and dried / heated for 6 days. 30% ethanol (or a mixture of ethanol and water) was added to the parsnips thus prepared. Extraction was performed at room temperature (20-25°C) for 24 hours. The supernatant was filtered through a filter paper (Whatman No. 4). The parsnips remaining on the filter paper were extracted once more under the same conditions, and the supernatants were obtained once more and combined. The primary organic solvent was removed from the final obtained extract using a rotary vacuum evaporator, and the residual moisture was removed using nitrogen to prepare parsnip extract sample B. The overall manufacturing process is illustrated in Fig. 11.Example 7: Evaluation of antioxidant properties of parsnip extract samples A and B.

[0015] 1. DPPH radical scavenging activity To evaluate the antioxidant properties of the processed parsnip extract, a DPPH radical scavenging activity test was performed. The parsnip extract prepared in Example 1 was prepared at concentrations of 2.5 mg / mL, 5.0 mg / mL, and 10.0 mg / mL. 20 pL of each concentration sample solution was mixed with 180 pL of 0.2 mM DPPH reagent (2,2-diphenyl-1 -picrylhydrazyl), incubated at room temperature for 30 minutes in the dark, and then the absorbance was measured at 517 nm. For the control group, vitamin C (0-100 ug / mL) was reacted with the DPPH reagent under the same conditions as the sample. A standard curve (Fig. 12A) was created using the measured absorbance values, and the DPPH radical scavenging activity was confirmed by comparing it with the concentration of vitamin C. As shown in Fig. 12B, the DPPH radical scavenging activity of processed parsnip extract sample A according to the present invention and the DPPH radical scavenging activity of processed parsnip extract sample B according to the present invention were superior to the DPPH radical scavenging activity of raw parsnip extract that had not undergone aging treatment. 2. ABTS radical scavenging activity

[0016] 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 to concentrations of 2.5 mM and 1 mM, respectively, and heated at 70 T for 1 h and then cooled to room temperature for 20–30 min. Standard solutions were prepared by serially diluting vitamin C from 100 pg / mL to 0 pg / mL. Sample solutions were serially diluted so that the absorbance at 734 nm was between 0.65 and 0.9. 10 pL of each standard solution and sample solution were placed in a 96-well plate, and 190 pL of ABTS solution was added, followed by dark reaction at room temperature for 10 min. Based on the results of measuring the absorbance at 734 nm, the radical scavenging activity of the sample was converted into the vitamin C concentration using the equation of the standard line (Fig. 13A). As shown in Fig. 13B, the ABTS radical scavenging activity of the processed parsnip extract sample A according to the present invention and the ABTS radical scavenging activity of the processed parsnip extract sample B according to the present invention were superior to the ABTS radical scavenging activity of the raw parsnip extract that had not been aged. Example 8: Evaluation of the therapeutic, alleviating, and preventive efficacy of parsnip extract sample B on respiratory inflammatory responses (In Vitro experiment)

[0017] 1. Cytotoxicity analysis In order to evaluate whether the antioxidant activity of the processed parsnip extract according to the present invention acts as a lung cell protective activity, efficacy evaluation was performed using human primary bronchial / tracheal epithelial cells. The cells were cultured at 37°C in a 5% CO2 environment by adding 10% urea and 1% penicillin-streptomycin to DMEM / F-12 medium. 5 x 10 cells were seeded in a 96-well plate. 4After seeding cells at a density of 10 cells / well, attachment and growth were induced for 24 h. Parsnip extracts were prepared at concentrations of 0, 25, 50, 100, 200, and 400 pg / mL, and 100 pL of each concentration of extract was added to the wells, and the cells were cultured for 24 h. MTT solution was prepared at a concentration of 5 mg / mL in PBS, and 10 pL was added to each well to obtain a final concentration of 0.5 mg / mL, and the cells were cultured for 4 h. The medium was removed, and 100 pL of DMSO was added to dissolve the formazan crystals, and the absorbance was measured at 570 nm and corrected for the background absorbance at 630 nm. Cell viability was calculated as a percentage compared to the control group, and all experiments were performed in triplicate, and the data were expressed as the mean and standard deviation. Statistical analysis was performed using GraphPad Prism using one-way analysis of variance (ANOVA) and Kiki's post-hoc test, and a p value < 0.05 was considered statistically significant. As shown in Fig. 14, sample B of the processed parsnip extract according to the present invention exhibited cytotoxicity at a concentration of 400 ug / mL.

[0018] 2. Efficacy in protecting lung cells from LPS-induced inflammatory toxicity. The subsequent experiments were performed by selecting a concentration that did not exhibit cytotoxicity. One group of cells was treated with LPS at a concentration of 20 ug / mL for 24 hours to induce inflammatory toxicity. Meanwhile, another group of cells was simultaneously treated with the processed parsnip extract sample according to the present invention and LPS. After 24 hours, cell viability was measured using an MTT assay (Fig. 15). As can be seen in Fig. 15, the processed parsnip extract B according to the present invention had the efficacy in protecting human normal lung cells from LPS-induced inflammatory toxicity. Similarly, it was verified at the cellular level whether the processed parsnip extract according to the present invention has the efficacy in significantly treating LPS-induced inflammation under conditions that do not exhibit toxicity. 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, and the gene expression of the inflammation-related signaling pathway was analyzed. Although not shown here, similar results to those in Fig. 15 were obtained.

Claims

Scope of patent claims 1. A composition for promoting respiratory health, comprising as an active ingredient an extract of processed parsnip having a higher total polyphenol content than natural parsnip.

2. A composition according to the above paragraph 1, characterized in that the promotion of respiratory health includes preventing, improving, or treating a disease, disorder, condition, or symptom related to a respiratory disease.

3. A composition according to the second paragraph, characterized in that the respiratory disease is bronchial asthma.

4. A composition according to the second paragraph, characterized in that the respiratory disease is chronic obstructive pulmonary disease.

5. A composition characterized in that, in the above paragraph 1, the processed parsnip extract is obtained by heat-drying raw parsnip at a specific temperature for a specific period of time, aging the raw parsnip to the extent that the color of the raw parsnip changes to brown or black, and then extracting the raw parsnip.

6. A composition according to the above paragraph 5, characterized in that the specific temperature is a temperature between 50°C and 65°C, and the specific period is a period between 10 and 20 days.

7. A composition according to the above paragraph 5, characterized in that the specific temperature is a temperature between 85°C and 90°C, and the specific period is a period between 3 and 10 days.

8. A composition according to the first paragraph, characterized in that the processed parsnip extract has a total polyphenol content that is at least twice that of the raw parsnip.

9. A method for producing an extract of processed parsnip having a higher antioxidant capacity than natural parsnip, the method comprising the steps of: heating and 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 A method characterized by comprising a step of removing unnecessary components from the filtered supernatant.

10. A method according to claim 9, characterized in that the heating and drying step is performed until the color of the raw parsnip changes to brown or black.

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