Pharmaceutical composition for treating dermatitis containing marsh radish extract

Abstract

To provide natural medicines for dermatitis with little or no side effects.SOLUTION: A pharmaceutical composition for treating dermatitis contains an ethanol extract of Adenostemma lavenia and a pharmaceutically acceptable carrier or salt.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The technical field relates to pharmaceutical compositions for treating dermatitis that contain extracts of Adenostemma lavenia. [Background technology]

[0002] Atopic dermatitis is one of the most common chronic diseases in the world. According to the Global Burden of Disease study published by the World Health Organization (WHO), at least 23 billion people worldwide suffer from atopic dermatitis, and it is the leading cause of non-fatal skin disease burden. Summary of the Invention [Problem to be solved by the invention]

[0003] Most of the currently available topical medications for atopic dermatitis have side effects. For example, the use of steroids can cause side effects such as skin atrophy, linear atrophy, and telangiectasia. Therefore, natural medicines for dermatitis with little or no side effects remain an urgent development focus. [Means for solving the problem]

[0004] The present disclosure provides a pharmaceutical composition for treating dermatitis, including atopic dermatitis or contact dermatitis, comprising a marsh radish extract and a pharmaceutically acceptable carrier or salt.

[0005] Detailed description will be given in the following embodiments with reference to the accompanying drawings. [Brief explanation of the drawings]

[0006] The present invention can be more fully understood from the following detailed description and examples, taken in conjunction with the accompanying drawings. [Figure 1]Figure 1 shows the results of hematoxylin-eosin staining of dorsal skin tissue sections from mice in the non-sensitized group (Naive), the vehicle-treated group, and the daikon radish extract 0805-treated group in an animal model experiment of 2,4-dinitrochlorobenzene (DNCB)-induced dermatitis. Areas marked with arrows indicate hyperplasia, and areas marked with * indicate inflammation. [Figure 2] Figure 2 shows the epidermal thickness of the dorsal skin of mice in the non-sensitized group, the vehicle-treated group, and the daikon radish extract 0805-treated group in an animal model experiment of 2,4-dinitrochlorobenzene-induced dermatitis. Data are shown as mean ± standard error (Mean ± SEM) (n = 5). Statistical analysis was performed using Student's t-test. Compared to the vehicle-treated group, p < 0.05 indicates a statistically significant difference, *p < 0.05, ***p < 0.001. [Figure 3] Figure 3 shows the total histological scores of the dorsal skin of mice in the non-sensitized group, the vehicle-treated group, and the daikon radish extract 0805-treated group in an animal model experiment of 2,4-dinitrochlorobenzene-induced dermatitis. Data are shown as mean ± standard error (n = 5). Statistical analysis was performed using the Mann-Whitney test. Compared to the vehicle-treated group, p < 0.05 indicates a statistically significant difference, *p < 0.05, **p < 0.01. [Figure 4] FIG. 4 shows photographs of mouse ears from the non-sensitized group, the vehicle-administered group, the daikon radish extract 0805-F-administered group, and the cyclosporine A-administered group on day 30 of an animal model experiment on 2,4-dinitrochlorobenzene-induced dermatitis. [Figure 5]Figure 5 shows the results of measurements of ear thickness in mice in the unsensitized, vehicle-treated, daikon radish extract 0805-F-treated, and cyclosporine A-treated groups on day 30 of an animal model experiment of 2,4-dinitrochlorobenzene-induced dermatitis. Data are shown as mean ± standard error (SEM) (n = 5). Statistical analysis was performed using two-way ANOVA combined with Dunnett's multiple comparison test to compare ear thickness in each group with that in the vehicle-treated group. p < 0.05 indicates a statistically significant difference compared to the vehicle-treated group, and ***p < 0.001 indicates a significant difference. Statistical analysis was performed using Student's t-test to compare ear thickness in the daikon radish extract 0805-F 300 mg / kg-treated group with that in the cyclosporine A-treated group. p<0.05 indicates a statistically significant difference, ###p<0.001. [Figure 6] Figure 6 shows the results of hematoxylin-eosin staining of skin tissue sections from the ears of mice in the non-sensitized group, the vehicle-treated group, the daikon radish extract 0805-F-treated group, and the cyclosporine A-treated group. Areas marked with arrows indicate hyperplasia, and areas marked with * indicate inflammation. [Figure 7] Figure 7 shows the total histological scores of mouse ear skin in the unsensitized, vehicle-treated, daikon radish extract 0805-F-treated, and cyclosporine A-treated groups. Data are presented as mean ± standard error (SEM) (n = 5). Statistical analysis was performed using the Mann-Whitney test to compare the total histological scores of each group with those of the vehicle-treated group. p < 0.05 indicates a statistically significant difference, *p < 0.05, **p < 0.01. Statistical analysis was performed using the Mann-Whitney test to compare the total histological scores of the daikon radish extract 0805-F 300 mg / kg-treated group with those of the cyclosporine A-treated group. p < 0.05 indicates a statistically significant difference, #p < 0.05. [Figure 8] Figure 8 shows the results of measuring serum IgE concentrations in each group in an atopic dermatitis model experiment using NC / Nga mice. Data are shown as mean ± standard error (n = 5). Statistical analysis was performed using Student's t-test. When comparing the group administered with marsh radish extract F4AB with the group administered with its vehicle, p < 0.05 indicates a statistically significant difference, *p < 0.05, **p < 0.01. When comparing the group administered with upadacitinib with the group administered with its vehicle, p < 0.05 indicates a statistically significant difference, #p < 0.05, ##p < 0.01. [Figure 9] FIG. 9 shows photographs of the backs of mice in each group before sacrifice on day 42 of the atopic dermatitis model experiment in NC / Nga mice. [Figure 10A] Figure 10A shows the total clinical severity score of dermatitis on the back of mice administered the marsh radish extract F4AB in an atopic dermatitis model experiment using NC / Nga mice. Data are shown as mean ± standard error (n = 5). Statistical analysis was performed using the Mann-Whitney test. Compared to the vehicle-administered group, p < 0.05 indicates a statistically significant difference, *p < 0.05, **p < 0.01. [Figure 10B] Figure 10B shows the total clinical severity score of dermatitis on the back of mice treated with upadacitinib in an atopic dermatitis model using NC / Nga mice. Data are shown as mean ± standard error (n = 5). Statistical analysis was performed using the Mann-Whitney test. Compared to the vehicle-treated group, p < 0.05 indicates a statistically significant difference, #p < 0.05, ##p < 0.01. [Figure 10C]Figure 10C shows the results of the comprehensive dermatitis clinical severity score (score of skin damage) on the backs of NC / Nga mice in an atopic dermatitis model experiment on day 42 before sacrifice. Data are shown as mean ± standard error (n=5). Statistical analysis was performed using the Mann-Whitney test. When comparing the group administered with marsh radish extract F4AB with the group administered with its vehicle, p<0.05 indicates a statistically significant difference, and ***p<0.001 indicates a statistically significant difference. When comparing the group administered with upadacitinib with its vehicle, p<0.05 indicates a statistically significant difference, and ###p<0.001 indicates a statistically significant difference. [Figure 11] Figure 11 shows the results of hematoxylin-eosin staining of dorsal skin tissue sections from mice in the non-sensitized group, the group administered the marsh radish extract F4AB, and the group administered the vehicle in an atopic dermatitis model experiment using NC / Nga mice. Areas marked with arrows indicate hyperplasia, and areas marked with * indicate inflammation. [Figure 12] Figure 12 shows the results of hematoxylin-eosin staining of dorsal skin tissue sections from unsensitized, upadacitinib-treated, and vehicle-treated NC / Nga mice in an atopic dermatitis model experiment. Areas marked with an * indicate inflammation. [Figure 13] Figure 13 shows the epidermal thickness of the dorsal skin of NC / Nga mice treated with an atopic dermatitis model: unsensitized, pigweed vehicle, pigweed extract F4AB, upadacitinib vehicle, and upadacitinib. Data are shown as mean ± standard error (n=5). Statistical analysis was performed using Student's t-test. When comparing the pigweed extract F4AB-treated group with the vehicle-treated group, p<0.05 indicates a statistically significant difference, and **p<0.01 indicates a statistically significant difference. When comparing the upadacitinib-treated group with the vehicle-treated group, p<0.05 indicates a statistically significant difference, and ##p<0.01 indicates a statistically significant difference. [Figure 14A] Figure 14A shows inflammation scores from histopathological section analysis of the dorsal skin of NC / Nga mice in an atopic dermatitis model experiment. Data are presented as mean ± standard error (Mean ± SEM) (n = 5), and statistical analysis was performed using the Mann-Whitney test. When comparing the group administered with marsh radish extract F4AB with the group administered with its vehicle, p < 0.05 indicates a statistically significant difference, and **p < 0.01 indicates a statistically significant difference. When comparing the group administered with upadacitinib with the group administered with its vehicle, p < 0.05 indicates a statistically significant difference, and ##p < 0.01 indicates a statistically significant difference. [Figure 14B] Figure 14B shows the necrosis scores from histopathological section analysis of the dorsal skin of NC / Nga mice in an atopic dermatitis model experiment. Data are presented as mean ± standard error (Mean ± SEM) (n = 5), and statistical analysis was performed using the Mann-Whitney test. When comparing the group treated with marsh radish extract F4AB with the group treated with its vehicle, p < 0.05 indicates a statistically significant difference, *p < 0.05, **p < 0.01. When comparing the group treated with upadacitinib with the group treated with its vehicle, p < 0.05 indicates a statistically significant difference, #p < 0.05, ##p < 0.01. [Figure 14C] Figure 14C shows the hyperplasia scores from histopathological section analysis of the dorsal skin of NC / Nga mice in an atopic dermatitis model experiment. Data are presented as mean ± standard error (Mean ± SEM) (n = 5), and statistical analysis was performed using the Mann-Whitney test. When comparing the group administered with marsh radish extract F4AB with the group administered with its vehicle, p < 0.05 indicates a statistically significant difference, *p < 0.05, **p < 0.01. When comparing the group administered with upadacitinib with the group administered with its vehicle, p < 0.05 indicates a statistically significant difference, and ##p < 0.01. [Figure 14D]Figure 14D shows the hyperkeratosis scores of histopathological sections of the dorsal skin of NC / Nga mice in an atopic dermatitis model experiment. Data are presented as mean ± standard error (SEM) (n = 5), and statistical analysis was performed using the Mann-Whitney test. When comparing the group administered with marsh radish extract F4AB with the group administered with its vehicle, p < 0.05 indicates a statistically significant difference, *p < 0.05, **p < 0.01. When comparing the group administered with upadacitinib with the group administered with its vehicle, p < 0.05 indicates a statistically significant difference, #p < 0.05, ##p < 0.01. [Figure 14E] Figure 14E shows the skin total histopathological severity score from histopathological section analysis of the dorsal skin of NC / Nga mice in an experimental atopic dermatitis model. Data are presented as mean ± standard error (Mean ± SEM) (n = 5), and statistical analysis was performed using the Mann-Whitney test. When comparing the group administered with marsh radish extract F4AB with the group administered with its vehicle, p < 0.05 indicates a statistically significant difference, and **p < 0.01 indicates a statistically significant difference. When comparing the group administered with upadacitinib with the group administered with its vehicle, p < 0.05 indicates a statistically significant difference, and ##p < 0.01 indicates a statistically significant difference. DETAILED DESCRIPTION OF THE INVENTION

[0007] In the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure. However, it will be apparent that one or more embodiments may be practiced without these specific details. Additionally, for purposes of simplifying the drawings, well-known structures and devices are schematically shown.

[0008] The present disclosure may provide a marsh radish extract, which has the effect of suppressing IL-4 secretion from cells and can at least treat and / or alleviate the symptoms of dermatitis.

[0009] Examples of the dermatitis may include, but are not limited to, atopic dermatitis, contact dermatitis, or any combination thereof. In one embodiment, the dermatitis may be atopic dermatitis. In another embodiment, the dermatitis may be contact dermatitis.

[0010] Furthermore, the above-mentioned symptoms of dermatitis may include, but are not limited to, erythema, bleeding, scarring, dryness, edema, peeling, erosion, inflammation, necrosis, hyperplasia, hyperkeratosis, acanthosis, itching, and the like, or any combination thereof.

[0011] The above-mentioned marsh radish extract may be derived from any organ or part of marsh radish, and is not particularly limited. For example, the above-mentioned marsh radish extract may be derived from the whole marsh radish plant, roots, stems, leaves, flowers, seeds, or any combination thereof.

[0012] Furthermore, the above-mentioned marsh radish extract can be obtained using any extraction solvent and / or any extraction method, as long as the obtained marsh radish extract can treat and / or alleviate the symptoms of dermatitis and is not harmful to humans or animals. For example, the above-mentioned marsh radish extract can be obtained by a single extraction using an extraction solvent, or by multiple extractions using the same or different extraction solvents in sequence. The above-mentioned extraction solvents can include, but are not limited to, water, alcohols, esters, alkanes, etc., or any combination thereof in any ratio. Examples of the above-mentioned alcohols can include, but are not limited to, methanol or ethanol. Examples of the above-mentioned esters include, but are not limited to, ethyl acetate. Examples of the above-mentioned alkanes can include, but are not limited to, n-hexane, cyclohexane, or heptane.

[0013] In one embodiment, the above-mentioned marsh radish extract may be an ethanol extract of marsh radish.

[0014] There is no particular limitation on the method for extracting the above-mentioned ethanol extract of marsh radish, as long as the resulting ethanol extract of marsh radish can treat and / or alleviate the symptoms of dermatitis and is not harmful to the human or animal body.

[0015] In one embodiment, the extraction method for the ethanol extract of Swamp Radish described above may include, but is not limited to, the following steps:

[0016] First, a source of marsh radish plant material is prepared. The source of marsh radish plant material may include, but is not limited to, the whole marsh radish plant, roots, stems, leaves, flowers, seeds, or any combination thereof. In one embodiment, the source of marsh radish plant material may be marsh radish leaves. The source of marsh radish plant material may be directly used as the marsh radish plant material. Alternatively, the source of marsh radish plant material may be pre-treated before being used as the marsh radish plant material. The pre-treatment may include, but is not limited to, drying, slicing, grinding, loading into a filter bag, etc., or any combination thereof in any order.

[0017] The marsh radish plant material can then be subjected to a hot extraction process, followed by a solid-liquid separation process to obtain an extract solution and produce a raffinate of the marsh radish plant material. In the hot extraction process, a first ethanol solvent can be used as the extraction solvent. The extract solution can then be used as the marsh radish ethanol extract.

[0018] In the above-mentioned hot extraction process, the ethanol concentration of the first ethanol solvent is not particularly limited and can be adjusted as necessary. For example, the ethanol concentration of the first ethanol solvent can be adjusted according to the external environment (temperature, humidity, pressure, etc.) during the extraction process, the type of source of the marsh radish plant material (whole plant, roots, stems, leaves, flowers, seeds, etc.), the state of the marsh radish plant material (water content, weight, etc.), the weight ratio of the first ethanol solvent used to the marsh radish plant material, the extraction temperature used, and / or the extraction time performed, but is not limited thereto. In one embodiment, the ethanol concentration of the first ethanol solvent can be, but is not limited to, about 30 to 100 vol%, such as about 40 to 95 vol%, about 45 to 90 vol%, about 50 to 85 vol%, about 60 to 80 vol%, about 70 to 75 vol%, about 30 vol%, about 40 vol%, about 45 vol%, about 50 vol%, about 55 vol%, about 60 vol%, about 65 vol%, about 70 vol%, about 75 vol%, about 80 vol%, about 85 vol%, about 90 vol%, or about 95 vol%. In one embodiment, the ethanol concentration of the first ethanol solvent can be about 95%. Furthermore, in one embodiment, the first ethanol solvent can be, but is not limited to, an aqueous ethanol solution.

[0019] In addition, in the above-mentioned hot extraction process, the weight ratio of the first ethanol solvent to the above-mentioned marsh radish plant material is not particularly limited and can be adjusted as needed. For example, the weight ratio of the first ethanol solvent to the above-mentioned marsh radish plant material can be adjusted depending on, but not limited to, the external environment during the extraction process (external environmental temperature, humidity, pressure, etc.), the type of source of the marsh radish plant material (whole plant, roots, stems, leaves, flowers, seeds, etc.), the state of the marsh radish plant material (water content, weight, etc.), the ethanol concentration of the first ethanol solvent used, the extraction temperature used, and / or the extraction time performed. In one embodiment, the weight ratio of the first ethanol solvent to the marsh radish plant material may be about 5-15:1, such as, but not limited to, about 6-12:1, about 7-11:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, or about 15:1. In a particular embodiment, the weight ratio of the first ethanol solvent to the marsh radish plant material may be about 8:1.

[0020] The extraction temperature used in the above-described hot extraction step is not particularly limited, as long as it is higher than room temperature during operation and can be adjusted as needed. For example, the extraction temperature used in the above-described hot extraction step can be adjusted depending on, but is not limited to, the external environment during the extraction step (e.g., temperature, humidity, pressure of the external environment), the type of source of the marsh radish plant material (whole plant, roots, stems, leaves, flowers, seeds, etc.), the state of the marsh radish plant material (e.g., moisture content, weight), the ethanol concentration of the first ethanol solvent used, the weight ratio of the first ethanol solvent used to the marsh radish plant material, and / or the extraction time. In one embodiment, the temperature used in the above-described hot extraction step may be about 40 to 80°C, for example, about 45 to 75°C, about 50 to 70°C, about 55 to 65°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, or about 80°C. In one particular embodiment, the temperature used in the above-mentioned hot extraction step may be about 60°C.

[0021] Similarly, the time for carrying out the above-mentioned hot extraction step is not particularly limited and can be adjusted as needed. For example, the time for carrying out the above-mentioned hot extraction step can be adjusted depending on, but not limited to, the external environment during the extraction step (such as the temperature, humidity, and pressure of the external environment), the type of source of the marsh radish plant material (such as the whole plant, roots, stems, leaves, flowers, and seeds), the state of the marsh radish plant material (such as water content and weight), the ethanol concentration of the first ethanol solvent used, the weight ratio of the first ethanol solvent used to the marsh radish plant material, and / or the extraction temperature used. In one embodiment, the time for carrying out the above-mentioned hot extraction step may be about 1 to 15 hours, for example, but not limited to, about 2 to 12 hours, about 3 to 10 hours, about 4 to 8 hours, about 5 to 6 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, or about 15 hours. In a specific embodiment, the time for carrying out the above-mentioned hot extraction step may be about 8 hours.

[0022] Furthermore, the solid-liquid separation step is not particularly limited as long as the extraction solution and the raffinate of the marsh radish plant material can be obtained after the hot extraction step. Examples of the solid-liquid separation step include, but are not limited to, centrifugation, filtration, or any combination thereof. In one embodiment, the solid-liquid separation step can be a filtration step.

[0023] In another embodiment, the method for extracting the ethanol extract of marsh radish described above may further include, after the aforementioned steps of subjecting marsh radish plant material to a thermal extraction process and then subjecting the solid-liquid separation process to a drying process for the obtained extract solution to form a dry extract, but is similarly not limited thereto. In addition, the dry extract obtained here can be used as the ethanol extract of marsh radish described above.

[0024] The drying process is not particularly limited, as long as it can dry the extract solution to a dry extract and does not adversely affect the extract solution's efficacy in treating and / or alleviating dermatitis symptoms. For example, the drying process may include, but is not limited to, oven drying, rotary drying, and concentration and drying under reduced pressure. In one embodiment, the drying process may be concentration and drying under reduced pressure.

[0025] In another embodiment, the above-mentioned method for extracting an ethanol extract of radish may further include, after the above-mentioned steps of subjecting radish plant material to a thermal extraction process, followed by a solid-liquid separation process to obtain an extract solution and producing a raffinate of the radish plant material, a step of using the above-mentioned raffinate of the radish plant material as the radish plant material, subjecting the radish plant material to a thermal extraction process, followed by a solid-liquid separation process to obtain an extract solution and producing a raffinate of the radish plant material, repeating the above-mentioned steps at least once, and combining all the obtained extract solutions to obtain a combined extract solution.

[0026] For an explanation of the thermal extraction and solid-liquid separation steps during the repeated steps of subjecting the marsh radish plant material to a thermal extraction step and then a solid-liquid separation step to obtain an extract solution and produce a raffinate of the marsh radish plant material (e.g., the method used, operation parameters / conditions), please refer to the descriptions of the thermal extraction step and solid-liquid separation step described above, and therefore details will not be repeated here.

[0027] In addition, when repeated, the method and operational parameters / conditions (e.g., the ethanol concentration of the first ethanol solvent, the weight ratio of the first ethanol solvent to the daikon plant material, the temperature used in the hot extraction step, and the time for performing the hot extraction step) used for the hot extraction and / or solid-liquid separation step in the step of performing a hot extraction step on the daikon plant material to obtain an extract solution and produce a raffinate of the daikon plant material may be the same or different, as needed or depending on the conditions during operation, and are not particularly limited. In one embodiment, when the process is repeated, the method and operational parameters / conditions used for the thermal extraction and / or solid-liquid separation steps in the step of subjecting the marsh radish plant material to a thermal extraction step, followed by a solid-liquid separation step to obtain an extract solution and producing a raffinate of the marsh radish plant material are the same as the method and operational parameters / conditions used for the thermal extraction and / or solid-liquid separation steps in the step of subjecting the marsh radish plant material to a thermal extraction step, followed by a solid-liquid separation step to obtain an extract solution and producing a raffinate of the marsh radish plant material when the process is performed for the first time.

[0028] Furthermore, the method and operational parameters / conditions (e.g., the ethanol concentration of the first ethanol solvent, the weight ratio of the first ethanol solvent to the radish plant material, the temperature used in the hot extraction step, and the time for performing the hot extraction step) used in each of the steps of repeatedly performing a hot extraction step on the radish plant material, followed by a solid-liquid separation step to obtain an extract solution and produce a raffinate of the radish plant material, may be the same or different depending on the conditions during operation, and are not particularly limited. In one embodiment, the method and operational parameters / conditions (e.g., the ethanol concentration of the first ethanol solvent, the weight ratio of the first ethanol solvent to the radish plant material, the temperature used in the hot extraction step, and the time for performing the hot extraction step) used in each of the steps of repeatedly performing a hot extraction step on the radish plant material, followed by a solid-liquid separation step to obtain an extract solution and produce a raffinate of the radish plant material, are the same.

[0029] In yet another embodiment, the above-described method for extracting an ethanol extract of marsh radish may further include, after the aforementioned step of combining all the obtained extract solutions to obtain a combined extract solution, a step of drying the combined extract solution to obtain a dried extract, but is similarly not limited thereto. The drying step described herein is not particularly limited, as long as it can dry the above-described combined extract solution to obtain a dried extract and does not adversely affect the effect of the combined extract solution in treating and / or alleviating dermatitis symptoms. For example, the drying step described herein may include, but is not limited to, oven drying, rotary drying, and concentrating and drying under reduced pressure. In one embodiment, the drying step described herein may be a step of concentrating and drying under reduced pressure. The dried extract obtained here can be used as the above-described ethanol extract of marsh radish.

[0030] In addition, in one particular embodiment, the above-mentioned extraction method for the ethanol extract of marsh radish may further include, but is not limited to, the following steps after the aforementioned step of subjecting the combined extraction solution to a drying process to obtain a dried extract:

[0031] First, a heating step is performed on the aforementioned dry extract in a second ethanol solvent until the aforementioned dry extract dissolves to form a mixture.

[0032] In the heating step, the ethanol concentration of the second ethanol solvent is not particularly limited and can be adjusted as needed. For example, the ethanol concentration of the second ethanol solvent can be adjusted depending on the external environment (temperature, humidity, pressure, etc.) during the heating step, the state of the dried extract (water content, weight, etc.), the weight ratio of the second ethanol solvent to the dried extract used, the heating temperature used, and / or the heating time performed, but is not limited thereto. In one embodiment, the ethanol concentration of the second ethanol solvent may be, but is not limited to, about 30 to 100 vol%, for example, about 40 to 95 vol%, about 45 to 90 vol%, about 50 to 85 vol%, about 60 to 80 vol%, about 70 to 75 vol%, about 30 vol%, about 40 vol%, about 45 vol%, about 50 vol%, about 55 vol%, about 60 vol%, about 65 vol%, about 70 vol%, about 75 vol%, about 80 vol%, about 85 vol%, about 90 vol%, or about 95 vol%. In one embodiment, the ethanol concentration of the second ethanol solvent may be about 95%. In another embodiment, the second ethanol solvent may be, but is not limited to, an aqueous ethanol solution.

[0033] Furthermore, in the heating step described above, the weight ratio of the second ethanol solvent to the dried extract is not particularly limited and can be adjusted as needed. For example, the weight ratio of the first ethanol solvent to the dried extract can be adjusted depending on the external environment (e.g., temperature, humidity, pressure, etc.) during the heating step, the state of the dried extract (e.g., water content, weight), the ethanol concentration of the second ethanol solvent used, the heating temperature used, and / or the heating time, but is not limited thereto. In one embodiment, the weight ratio of the second ethanol solvent to the dried extract may be about 5 to 15:1, for example, about 6 to 12:1, about 7 to 11:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, or about 15:1, but is not limited thereto. In a specific embodiment, the weight ratio of the second ethanol solvent to the dried extract may be about 8:1.

[0034] Furthermore, the temperature used in the heating step described above is not particularly limited, as long as it is higher than room temperature during operation and can be adjusted as necessary. For example, the temperature used in the heating step described above can be adjusted depending on the external environment (e.g., temperature, humidity, pressure of the external environment) during the heating step, the state of the dried extract (e.g., water content, weight), the ethanol concentration of the second ethanol solvent used, the weight ratio of the second ethanol solvent used to the dried extract, and / or the heating time, but is not limited thereto. In one embodiment, the temperature used in the heating step described above may be about 30 to 70°C, for example, about 35 to 65°C, about 40 to 60°C, about 45 to 55°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, or about 70°C, but is not limited thereto. In a specific embodiment, the temperature used in the heating step described above may be about 50°C.

[0035] Next, after forming the mixture, the mixture is subjected to column chromatography, and at least one fraction having the effect of suppressing IL-4 secretion from cells is collected, or the collected and combined fractions are used as an active fraction. The active fractions obtained here can be used as the above-mentioned ethanol extract of Swamp Radish.

[0036] In the column chromatography described above, there are no particular limitations on the solvent that can be used as the mobile phase, as long as it can produce at least one fraction that has the effect of suppressing cellular IL-4 secretion and is not harmful to the human or animal body. Solvents that can be used as the mobile phase include, but are not limited to, water, alcohols, esters, alkanes, etc., or any combination thereof in any ratio. Examples of the alcohols described above include, but are not limited to, methanol or ethanol. Examples of the esters described above include, but are not limited to, ethyl acetate. Examples of the alkanes described above include, but are not limited to, n-hexane, cyclohexane, or heptane. In one embodiment, a methanol solvent can be used as the mobile phase in the column chromatography described above. In another embodiment, a mixed solvent of n-hexane / ethyl acetate can be used as the mobile phase in the column chromatography described above. The volume ratio of n-hexane to ethyl acetate in the n-hexane / ethyl acetate mixed solvent described above is also not particularly limited, as long as it can produce at least one fraction that has the effect of suppressing cellular IL-4 secretion and is not harmful to the human or animal body. For example, the volume ratio of n-hexane to ethyl acetate in the above-mentioned n-hexane / ethyl acetate mixed solvent may be about 0.1 to 1:1, such as 0.2 to 0.8:1, 0.3 to 0.7:1, 0.4 to 0.5:1, 0.1:1, 0.2:1, 0.25:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, or 1:1, but is not limited thereto.

[0037] The type of the column chromatography is not particularly limited. In one embodiment, the column chromatography may be silica gel column chromatography. In another embodiment, the column chromatography may be high performance liquid chromatography (HPLC), but is not limited thereto.

[0038] In yet another specific embodiment, the method for extracting the above-mentioned ethanol extract of marsh radish may further include, but is not limited to, a step of collecting or combining at least one fraction having the effect of suppressing cellular IL-4 secretion to obtain an active fraction, followed by a step of drying the active fraction to obtain a dried active fraction. Furthermore, the dried active fraction obtained here can be used as the above-mentioned ethanol extract of marsh radish.

[0039] Additionally, in another embodiment of the present disclosure, the above-mentioned marsh radish extract may be a water extract of marsh radish.

[0040] There are no particular limitations on the method for extracting the water extract of marsh radish described above, as long as the resulting water extract of marsh radish can treat and / or alleviate the symptoms of dermatitis and is not harmful to the human or animal body.

[0041] In one embodiment, the extraction method for the water extract of Swamp Radish described above may include, but is not limited to, the following steps:

[0042] First, a source of marsh radish plant material is prepared. The source of marsh radish plant material may include, but is not limited to, the whole marsh radish plant, roots, stems, leaves, flowers, seeds, or any combination thereof. In one embodiment, the source of marsh radish plant material may be marsh radish leaves. The source of marsh radish plant material may be directly used as the marsh radish plant material. Alternatively, the source of marsh radish plant material may be pre-treated before being used as the marsh radish plant material. The pre-treatment may include, but is not limited to, drying, slicing, grinding, loading into a filter bag, etc., or any combination thereof in any order.

[0043] The marsh radish plant material can then be subjected to a pressurized extraction step, followed by a solid-liquid separation step to obtain an extract solution and produce a raffinate of the marsh radish plant material. In the pressurized extraction step, water can be used as the extraction solvent. The extract solution obtained here can also be used as the above-mentioned marsh radish water extract.

[0044] In the pressurized extraction process described above, the weight ratio of the water to the radish plant material is not particularly limited and can be adjusted as needed. For example, the weight ratio of the water to the radish plant material can be adjusted depending on the external environment (e.g., temperature, humidity, pressure, etc.) during the extraction process, the type of source of the radish plant material (whole plant, roots, stems, leaves, flowers, seeds, etc.), the state of the radish plant material (e.g., moisture content, weight), the extraction pressure used, the extraction temperature used, and / or the extraction time, but is not limited thereto. In one embodiment, the weight ratio of the water to the radish plant material may be about 10 to 50:1, for example, about 15 to 45:1, about 20 to 40:1, about 25 to 35:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, or about 50:1, but is not limited thereto. In one particular embodiment, the weight ratio of the water to the marsh radish plant material may be about 30:1.

[0045] The pressure used in the above-mentioned pressurized extraction step is not particularly limited and can be adjusted as necessary. For example, the pressure used in the above-mentioned pressurized extraction step can be adjusted depending on the external environment during the pressurized extraction step (such as the temperature, humidity, and pressure of the external environment), the type of source of the marsh radish plant material (whole plant, roots, stems, leaves, flowers, seeds, and the like), the state of the marsh radish plant material (such as water content and weight), the weight ratio of the water used to the marsh radish plant material, the extraction temperature used, and / or the extraction time performed, but is not limited thereto. In one embodiment, the pressure used in the pressurized extraction step is about 101.4 to 140.0 kPa, for example, about 101.5 to 135.0 kPa, about 101.6 to 130.0 kPa, about 101.8 to 125.0 kPa, about 102.0 to 120.0 kPa, about 102.2 to 115.0 kPa, about 102.4 to 110.0 kPa, about 102.6 to 105.0 kPa, about 102.8 to 104.0 kPa, about 101.5 kPa, about 101.6 kPa, about 101.7 kPa, about 101.8 kPa, about 101.9 kPa, about 102.0 kPa, about 102.1 kPa, about 102.2 kPa, or about 102.25 kPa. a, about 102.3 kPa, about 102.4 kPa, about 102.5 kPa, about 102.6 kPa, about 102.7 kPa, about 102.8 kPa, about 102.9 kPa, about 102.92 kPa, about 102.94 kPa, about 102.96 kPa, about 102.97 kPa, about 102.98 kPa, about 103.0 kPa, about 105.0 kPa, about 107.0 kPa, about 108.0 kPa, about 110.0 kPa, about 112.0 kPa, about 115.0 kPa, about 120.0 kPa, about 125.0 kPa, about 130.0 kPa, about 135.0 kPa, and about 140.0 kPa, but are not limited thereto. In one particular embodiment, the pressure used in the pressurized extraction process described above may be about 102.97 kPa.

[0046] The extraction temperature used in the above-mentioned pressurized extraction step is not particularly limited and can be adjusted as necessary. For example, the extraction temperature used in the above-mentioned pressurized extraction step can be adjusted depending on the external environment during the pressurized extraction step (such as the temperature, humidity, and pressure of the external environment), the type of source of the marsh radish plant material (whole plant, roots, stems, leaves, flowers, seeds, and the like), the state of the marsh radish plant material (such as water content and weight), the weight ratio of the water used to the marsh radish plant material, the extraction pressure used, and / or the extraction time performed, but is not limited thereto. In one embodiment, the extraction temperature used in the above-mentioned pressure extraction step may be, but is not limited to, about 100 to 140°C, for example, about 102 to 135°C, about 105 to 130°C, about 108 to 125°C, about 110 to 120°C, about 100°C, about 101°C, about 103°C, about 105°C, about 110°C, about 112°C, about 115°C, about 120°C, etc. In a particular embodiment, the extraction temperature used in the above-mentioned pressure extraction step may be about 120°C.

[0047] The time for carrying out the above-mentioned pressurized extraction step is not particularly limited and can be adjusted as needed. For example, the time for carrying out the above-mentioned pressurized extraction step can be adjusted depending on the external environment (e.g., temperature, humidity, pressure of the external environment) during the pressurized extraction step, the type of source of the marsh radish plant material (whole plant, roots, stems, leaves, flowers, seeds, etc.), the state of the marsh radish plant material (e.g., moisture content, weight), the weight ratio of water to marsh radish plant material used, the extraction pressure and / or extraction temperature used, etc., but is not limited thereto. In one embodiment, the time for carrying out the above-mentioned pressurized extraction step may be about 5 to 60 minutes, about 10 to 55 minutes, about 15 to 50 minutes, about 20 to 45 minutes, about 25 to 40 minutes, about 30 to 35 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, etc., but is not limited thereto. In one particular embodiment, the time period for carrying out the pressurized extraction step described above may be about 20 minutes.

[0048] In another embodiment, the method for extracting the water extract of marsh radish described above may further include, after the aforementioned steps of subjecting marsh radish plant material to a pressurized extraction process and then subjecting the plant material to a solid-liquid separation process, subjecting the obtained extract solution to a drying process to form a dry extract. In addition, the dry extract obtained here can be used as the water extract of marsh radish described above.

[0049] Similarly, the drying process described herein is not particularly limited as long as it can dry the extract solution described above to form a dry extract and does not adversely affect the extract solution's efficacy in treating and / or alleviating dermatitis symptoms. For example, the drying process described herein may include, but is not limited to, oven drying, rotary drying, concentrating and drying under reduced pressure.

[0050] In another embodiment, the method for extracting the water extract of marsh radish described above may further include, after the aforementioned steps of subjecting marsh radish plant material to a pressurized extraction process, followed by a solid-liquid separation process to obtain an extract solution and forming a raffinate of the marsh radish plant material, a step of, but not limited to, using the raffinate of the marsh radish plant material as the marsh radish plant material, repeating the aforementioned steps of subjecting marsh radish plant material to a pressurized extraction process, followed by a solid-liquid separation process to obtain an extract solution and forming a raffinate of the marsh radish plant material at least once, and combining all the resulting extract solutions to obtain a combined extract solution. The combined extract solution obtained here can also be used as the water extract of marsh radish described above. In a specific embodiment, the aforementioned steps of subjecting marsh radish plant material to a pressurized extraction process, followed by a solid-liquid separation process to obtain an extract solution and forming a raffinate of the marsh radish plant material are repeated three times, and all the extract solutions are combined to obtain a combined extract solution. The combined extract solution obtained here can also be used as the water extract of marsh radish described above.

[0051] When repeated, the pressure extraction step is performed on the marsh radish plant material, followed by a solid-liquid separation step to obtain an extract solution and produce a raffinate of the marsh radish plant material. For descriptions of the pressure extraction step and solid-liquid separation step (e.g., the method used, operation parameters / conditions), please refer to the descriptions of the pressure extraction step and solid-liquid separation step described above, and details will not be repeated here.

[0052] In addition, when repeated, the method and operational parameters / conditions (e.g., weight ratio of water to the radish plant material, pressure used in the pressurized extraction step, temperature used in the pressurized extraction step, time for performing the pressurized extraction step) used in the thermal extraction and / or solid-liquid separation step in the step of performing a hot extraction step on the radish plant material, followed by a solid-liquid separation step to obtain an extract solution and produce a raffinate of the radish plant material, and the method and operational parameters / conditions used in the pressurized extraction and / or solid-liquid separation step in the step of performing a hot extraction step on the radish plant material, followed by a solid-liquid separation step to obtain an extract solution and produce a raffinate of the radish plant material, when performed for the first time, may be the same or different, as needed or depending on the conditions during operation, and are not particularly limited. In one embodiment, when repeated, the method and operational parameters / conditions used in the pressurized extraction step and / or solid-liquid separation step in the step of subjecting the marsh radish plant material to a pressurized extraction step, followed by a solid-liquid separation step to obtain an extract solution and producing a raffinate of the marsh radish plant material are the same as the method and operational parameters / conditions used in the pressurized extraction step and solid-liquid separation step in the step of subjecting the marsh radish plant material to a heat extraction step, followed by a solid-liquid separation step to obtain an extract solution and producing a raffinate of the marsh radish plant material when performed the first time.

[0053] Furthermore, the method and operational parameters / conditions (e.g., the weight ratio of water to the radish plant material, the pressure used in the pressurized extraction step, the temperature used in the pressurized extraction step, and the time for performing the pressurized extraction step) used in each of the steps of repeatedly performing a pressurized extraction step on the radish plant material and then a solid-liquid separation step to obtain an extract solution and produce a raffinate of the radish plant material may be the same or different, as needed or depending on the conditions during operation, and are not particularly limited. In one embodiment, the method and operational parameters / conditions used in each of the pressurized extraction steps and / or each solid-liquid separation step in each of the steps of repeatedly performing a pressurized extraction step on the radish plant material and then a solid-liquid separation step to obtain an extract solution and produce a raffinate of the radish plant material are the same.

[0054] In yet another embodiment, the method for extracting the aqueous extract of marsh radish described above may further include, but is not limited to, a step of drying the combined extract solution to obtain a dried extract after the aforementioned step of combining all the obtained extract solutions to obtain a combined extract solution. The dried extract obtained here can also be used as the aqueous extract of marsh radish described above. The drying process described herein is not particularly limited, as long as it can dry the combined extract solution described above to obtain a dried extract and does not adversely affect the effect of the combined extract solution in treating and / or alleviating dermatitis symptoms. For example, the drying process described herein may include, but is not limited to, oven drying, rotary drying, and concentrating and drying under reduced pressure. In one embodiment, the drying process described herein may be a process of concentrating and drying under reduced pressure.

[0055] Furthermore, in a specific embodiment, the above-mentioned method for extracting a water extract of marsh radish may further include, but is not limited to, a step of subjecting the above-mentioned combined extract solution to a first column chromatography after the above-mentioned step of combining all the obtained extract solutions to obtain a combined extract solution. The above-mentioned first column chromatography may include, but is not limited to, the following steps:

[0056] First, the combined extraction solution is loaded into a column packed with a resin. There are no particular limitations on the type of resin packed into the column, as long as it can adsorb the active ingredients in the combined extraction solution. For example, the type of resin packed into the column can include, but is not limited to, styrene-divinylbenzene-based adsorption resins (e.g., HP20, DIAION).

[0057] The combined extraction solution is then loaded onto a column packed with resin, and the column is eluted with water, and the eluate flowing out of the column is discarded.

[0058] The volume ratio of the water used to elute the column to the combined extraction solution loaded onto the column is not particularly limited and can be adjusted as needed. For example, the volume ratio of the water used to elute the column to the combined extraction solution loaded onto the column can be adjusted depending on the environment during column elution (environmental temperature, humidity, pressure, etc.), the amount of combined extraction solution loaded onto the column, and the condition of the eluate flowing out of the column during column elution, but is not limited thereto. In one embodiment, the volume ratio of the water used to elute the column to the combined extraction solution loaded onto the column may be about 0.5 to 10:1, for example, about 1 to 8:1, about 1.5 to 7:1, about 2 to 6:1, about 2.5 to 5:1, about 3 to 4:1, about 0.5:1, about 1:1, about 2:1, about 2.5:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1, but is not limited thereto. In one particular embodiment, the volume ratio of water used to elute the column and the combined extraction solution loaded onto the column may be 1:1.

[0059] The column is then eluted with water, and the eluate flowing through the column is discarded, after which the column is eluted with a third ethanol solvent, and the eluate flowing through the column is discarded.

[0060] The volume ratio of the third ethanol solvent used to elute the column and the combined extraction solution loaded onto the column is not particularly limited and can be adjusted as needed. For example, the volume ratio of the third ethanol solvent used to elute the column and the combined extraction solution loaded onto the column can be adjusted according to the environment (environmental temperature, humidity, pressure, etc.) during column elution, the amount of combined extraction solution loaded onto the column, the ethanol concentration of the third ethanol solvent, the aforementioned conditions of the eluate flowing out of the column when eluting the column with water, and / or the conditions of the eluate flowing out of the column when eluting the column with the third ethanol solvent, but is not limited thereto. In one embodiment, the volume ratio of the third ethanol solvent used to elute the column to the combined extraction solution loaded onto the column can be, but is not limited to, about 0.5 to 10:1, such as about 1 to 8:1, about 1.5 to 7:1, about 2 to 6:1, about 2.5 to 5:1, about 3 to 4:1, about 0.5:1, about 1:1, about 2:1, about 2.5:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In a particular embodiment, the volume ratio of the third ethanol solvent used to elute the column to the combined extraction solution loaded onto the column can be 1:1.

[0061] The ethanol concentration of the third ethanol solvent used to elute the column is not particularly limited and can be adjusted as needed. For example, the ethanol concentration of the third ethanol solvent can be adjusted according to, but not limited to, the environment during column elution (environmental temperature, humidity, pressure, etc.), the amount of the combined extraction solution loaded onto the column, the volume ratio of the third ethanol solvent used to elute the column to the combined extraction solution loaded onto the column, the aforementioned conditions of the eluate flowing out of the column when eluting the column with water, and / or the conditions of the eluate flowing out of the column when eluting the column with the third ethanol solvent. In one embodiment, the ethanol concentration of the third ethanol solvent may be, but is not limited to, about 30 to 100 vol%, such as about 40 to 95 vol%, about 45 to 90 vol%, about 50 to 85 vol%, about 60 to 80 vol%, about 70 to 75 vol%, about 30 vol%, about 40 vol%, about 45 vol%, about 50 vol%, about 55 vol%, about 60 vol%, about 65 vol%, about 70 vol%, about 75 vol%, about 80 vol%, about 85 vol%, about 90 vol%, or about 95 vol%. In one embodiment, the ethanol concentration of the third ethanol solvent may be about 50%. In another embodiment, the third ethanol solvent may be, but is not limited to, an aqueous ethanol solution.

[0062] The column is then eluted with the third ethanol solvent, and the eluate is discarded. The column is then eluted with the fourth ethanol solvent, and the eluate is collected as a preliminary purified extract solution. The preliminary purified extract solution obtained here can be used as the water extract of marsh radish described above.

[0063] Similarly, the volume ratio between the fourth ethanol solvent used to elute the column and the combined extraction solution loaded onto the column is not particularly limited and can be adjusted as needed. For example, the volume ratio between the fourth ethanol solvent used to elute the column and the combined extraction solution loaded onto the column can be adjusted according to the environment (environmental temperature, humidity, pressure, etc.) during column elution, the amount of combined extraction solution loaded onto the column, the ethanol concentration of the fourth ethanol solvent, the aforementioned conditions of the eluate flowing out of the column by eluting the column with the third ethanol solvent, and / or the conditions when the eluate flowing out of the column by eluting the column with the fourth ethanol solvent, but is not limited thereto. In one embodiment, the volume ratio of the fourth ethanol solvent used to elute the column to the combined extraction solution loaded onto the column can be, but is not limited to, about 0.5 to 10:1, such as about 1 to 8:1, about 1.5 to 7:1, about 2 to 6:1, about 2.5 to 5:1, about 3 to 4:1, about 0.5:1, about 1:1, about 2:1, about 2.5:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In a particular embodiment, the volume ratio of the fourth ethanol solvent used to elute the column to the combined extraction solution loaded onto the column can be 1:1.

[0064] The ethanol concentration of the fourth ethanol solvent used to elute the column is not particularly limited and can be adjusted as needed. For example, the ethanol concentration of the fourth ethanol solvent can be adjusted according to, but not limited to, the environment during column elution (environmental temperature, humidity, pressure, etc.), the amount of the combined extraction solution loaded onto the column, the volume ratio of the fourth ethanol solvent used to elute the column to the combined extraction solution loaded onto the column, the aforementioned conditions of the eluate flowing out of the column when eluting the column with the third ethanol solvent, and / or the conditions when the eluate flowing out of the column when eluting the column with the fourth ethanol solvent. In one embodiment, the ethanol concentration of the fourth ethanol solvent may be, but is not limited to, about 30 to 100 vol%, such as about 40 to 95 vol%, about 45 to 90 vol%, about 50 to 85 vol%, about 60 to 80 vol%, about 70 to 75 vol%, about 30 vol%, about 40 vol%, about 45 vol%, about 50 vol%, about 55 vol%, about 60 vol%, about 65 vol%, about 70 vol%, about 75 vol%, about 80 vol%, about 85 vol%, about 90 vol%, or about 95 vol%. In one embodiment, the ethanol concentration of the fourth ethanol solvent may be about 75%. In another embodiment, the fourth ethanol solvent may be, but is not limited to, an aqueous ethanol solution.

[0065] In yet another embodiment, the method for extracting a water extract of marsh radish described above may further include, but is not limited to, the steps of eluting the column with a fourth ethanol solvent, collecting the eluate flowing out from the column as an initial purified extract solution, subjecting the initial purified extract solution to a second column chromatography, and collecting or combining at least one fraction having the effect of suppressing cellular IL-4 secretion to obtain an active fraction. Furthermore, the active fraction obtained here can be used as the water extract of marsh radish described above.

[0066] In the second column chromatography described above, there is no particular limitation on the solvent that can be used as the mobile phase, as long as it can produce at least one fraction that has the effect of suppressing cellular IL-4 secretion and has no adverse effects on the human or animal body. Solvents that can be used as the mobile phase include, but are not limited to, water, alcohols, esters, alkanes, etc., or any combination thereof in any ratio. Examples of the alcohols described above include, but are not limited to, methanol or ethanol. Examples of the esters described above include, but are not limited to, ethyl acetate. Examples of the alkanes described above include, but are not limited to, n-hexane, cyclohexane, or heptane. In one embodiment, a methanol solvent can be used as the mobile phase in the second column chromatography described above. In another embodiment, a mixed solvent of n-hexane / ethyl acetate can be used as the mobile phase in the second column chromatography described above. The volume ratio of n-hexane to ethyl acetate in the n-hexane / ethyl acetate mixed solvent described above is also not particularly limited, as long as it can produce at least one fraction that has the effect of suppressing cellular IL-4 secretion and has no adverse effects on the human or animal body. For example, the volume ratio of n-hexane to ethyl acetate in the above-mentioned n-hexane / ethyl acetate mixed solvent may be, but is not limited to, about 0.1 to 1:1, such as 0.2 to 0.8:1, 0.3 to 0.7:1, 0.4 to 0.5:1, 0.1:1, 0.2:1, 0.25:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, or 1:1. In a specific embodiment, the volume ratio of n-hexane to ethyl acetate in the above-mentioned n-hexane / ethyl acetate mixed solvent may be about 1:1.

[0067] The type of the second column chromatography is not particularly limited. In one embodiment, the second column chromatography may be silica gel column chromatography. In another embodiment, the second column chromatography may be high-performance liquid chromatography (HPLC).

[0068] In yet another specific embodiment, the method for extracting the water extract of marsh radish described above may further include, but is not limited to, a step of concentrating the active fraction to obtain a concentrated active fraction after the step of subjecting the initial purified extract solution to a second column chromatography to collect, or collect and combine, at least one fraction having the effect of suppressing cellular IL-4 secretion to obtain an active fraction. Furthermore, the concentrated active fraction obtained here can also be used as the water extract of marsh radish described above.

[0069] In addition, in this particular embodiment, the above-mentioned method for extracting a water extract of marsh radish may further include, but is not limited to, the following steps after the step of concentrating the above-mentioned active fraction to obtain a concentrated active fraction:

[0070] First, the concentrated active fraction is subjected to a heating and dissolving step in the fifth ethanol solvent to form an active solution.

[0071] The weight ratio of the fifth ethanol solvent to the concentrated active fraction is not particularly limited and can be adjusted as needed. For example, the weight ratio of the fifth ethanol solvent to the concentrated active fraction can be adjusted depending on the external environment (temperature, humidity, pressure, etc.) during the heat dissolution step and / or the state of the concentrated active fraction (water content, weight, etc.), but is not limited thereto. In one embodiment, the weight ratio of the fifth ethanol solvent to the concentrated active fraction may be about 1 to 10:1, for example, about 1.5 to 8:1, about 2 to 5:1, about 2.5 to 4.5:1, about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 1:10, but is not limited thereto. In a specific embodiment, the weight ratio of the fifth ethanol solvent to the active fraction may be about 4:1.

[0072] The temperature used in the heat dissolution step is not particularly limited, as long as it is higher than room temperature and can dissolve the concentrated active fraction, and can be adjusted as necessary. For example, the temperature used in the heat dissolution step can be adjusted depending on the external environment (e.g., temperature, humidity, pressure, etc.) during the heat dissolution step, the state of the concentrated active fraction (e.g., water content, weight), the weight ratio of the fifth ethanol solvent to the concentrated active fraction, and / or the heating time, but is not limited thereto. In one embodiment, the temperature used in the heat dissolution step may be about 40 to 100°C, for example, about 45 to 95°C, about 50 to 90°C, about 55 to 85°C, about 60 to 80°C, about 65 to 75°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C, or about 100°C, but is not limited thereto. In one particular embodiment, the temperature used in the heat dissolution step may be about 60°C.

[0073] The duration of the heat dissolution step is not particularly limited, and may be adjusted as needed, as long as it is sufficient to dissolve the concentrated active fraction. For example, the duration of the heat dissolution step can be adjusted depending on the external environment (temperature, humidity, pressure, etc.) during the heat dissolution step, the state of the concentrated active fraction (water content, weight, etc.), the weight ratio of the fifth ethanol solvent to the concentrated active fraction, and / or the heating temperature used, but is not limited thereto. In one embodiment, the duration of the heat dissolution step may be about 5 to 60 minutes, for example, about 10 to 55 minutes, about 15 to 50 minutes, about 20 to 45 minutes, about 25 to 40 minutes, about 30 to 35 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes, but is not limited thereto. In a specific embodiment, the duration of the heat dissolution step may be 60 minutes.

[0074] Next, the concentrated active fraction is subjected to a heating and dissolving step in a fifth ethanol solvent to form an active solution, and then the active solution is allowed to stand to form crystals therein.

[0075] The time for which the active solution is allowed to stand is not particularly limited, and may be adjusted as needed, as long as it allows crystals to form in the active solution. For example, the time for which the active solution is allowed to stand can be adjusted depending on the external environment (temperature, humidity, pressure, etc.) during the heating and dissolution process and the state of the active solution (weight, etc.), but is not limited thereto. In one embodiment, the time for which the active solution is allowed to stand may be about 6 to 48 hours, about 9 to 45 hours, about 12 to 42 hours, about 15 to 39 hours, about 18 to 36 hours, about 21 to 33 hours, about 24 to 30 hours, about 6 hours, about 9 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 27 hours, about 30 hours, about 33 hours, about 36 hours, about 39 hours, about 42 hours, about 45 hours, or about 48 hours, but is not limited thereto. In a specific embodiment, the time for which the active solution is allowed to stand may be about 24 hours.

[0076] After the crystals are formed, they are collected and can be used as the water extract of marsh radish.

[0077] Additionally, the collected crystals may be subjected to a drying step.

[0078] The marsh radish extract of the present disclosure obtained by any of the extraction methods described above has the effect of suppressing IL-4 secretion from cells and can at least treat and / or alleviate the symptoms of dermatitis.

[0079] Furthermore, the present disclosure provides the use of a marsh radish extract in the manufacture of a medicament for treating dermatitis.

[0080] In the use of the marsh radish extract in the manufacture of a medicament for treating dermatitis of the present disclosure, the marsh radish extract is not particularly limited, as long as it has the effect of treating and / or alleviating the symptoms of dermatitis. For example, any of the marsh radish extracts described above in the present disclosure can be used as the marsh radish extract in the use of the marsh radish extract in the manufacture of a medicament for treating dermatitis of the present disclosure.

[0081] Additionally, in the use of the marsh radish extract in the manufacture of a medicament for treating dermatitis of the present disclosure, the medicament for treating dermatitis may further comprise, but is not limited to, a pharmaceutically acceptable carrier or salt.

[0082] The present disclosure also provides pharmaceutical compositions for treating dermatitis.

[0083] The pharmaceutical composition for treating dermatitis of the present disclosure may contain, but is not limited to, a marsh radish extract.The marsh radish extract in the pharmaceutical composition for treating dermatitis of the present disclosure is also not particularly limited, as long as it has the effect of treating and / or alleviating the symptoms of dermatitis.For example, the marsh radish extract in the pharmaceutical composition for treating dermatitis of the present disclosure may be any of the marsh radish extracts described above.

[0084] In addition, the pharmaceutical composition for treating dermatitis of the present disclosure may further comprise, but is not limited to, a pharmaceutically acceptable carrier or salt.

[0085] The above-mentioned pharmaceutically acceptable carriers may include, but are not limited to, those suitable for pharmaceutical administration, such as solvents, dispersion media, coatings, antibacterial and antifungal agents, or isotonic and absorption delaying agents, etc. Pharmaceutical compositions can be formulated into dosage forms suitable for different administration routes using conventional methods.

[0086] Furthermore, the above-mentioned pharmaceutically acceptable salts may include, but are not limited to, salts containing inorganic cations, such as alkali metal salts, e.g., sodium salts, potassium salts, or amine salts, alkaline earth metal salts, e.g., magnesium salts or calcium salts, and salts containing divalent or tetravalent cations, e.g., zinc salts, aluminum salts, or zirconium salts. Additionally, the pharmaceutically acceptable salts may be organic salts, such as dicyclohexylamine salts, methyl-D-glucamine salts, and amino acid salts, such as arginine, lysine, histidine, or glutamine salts.

[0087] Additionally, the medicament made in the marsh radish extract of the present disclosure, the pharmaceutical composition for treating dermatitis of the present disclosure, and / or the use of the marsh radish extract in the manufacture of a medicament for treating dermatitis of the present disclosure can be administered to a subject in need thereof, but is not limited thereto.

[0088] The marsh radish extract of the present disclosure, the pharmaceutical composition for treating dermatitis of the present disclosure, and / or the medicament made in the use of the marsh radish extract in the manufacture of a medicament for treating dermatitis of the present disclosure can be administered parenterally, orally, by inhalation spray, or via an implanted reservoir, but is not limited thereto. Parenteral methods may include smearing into the affected area, subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, and intralesional injection, as well as infusion techniques.

[0089] Topical application forms of the smear may include, but are not limited to, ointments, emulsions, solutions, gels, mousses, and the like.

[0090] In addition, the subject to which the marsh radish extract of the present disclosure, the pharmaceutical composition for treating dermatitis of the present disclosure, and / or the medicament produced by the use of marsh radish extract in the manufacture of a medicament for treating dermatitis of the present disclosure may include, but is not limited to, vertebrates.Furthermore, the above-mentioned vertebrates may include, but are not limited to, fish, amphibians, reptiles, birds, or mammals.Examples of mammals may include, but are not limited to, humans, orangutans, monkeys, horses, donkeys, dogs, cats, rabbits, guinea pigs, rats, and mice.In one embodiment, the above-mentioned subject may be human.

[0091] In addition, the present disclosure may also provide a method for treating dermatitis. The method for treating dermatitis of the present disclosure may include, but is not limited to, administering an effective amount of a marsh radish extract or an effective amount of a pharmaceutical composition for treating dermatitis to a subject in need thereof.

[0092] Examples of the dermatitis may include, but are not limited to, atopic dermatitis, contact dermatitis, or any combination thereof. In one embodiment, the dermatitis may be atopic dermatitis. In another embodiment, the dermatitis may be contact dermatitis.

[0093] Furthermore, the marsh radish extract used in the method for treating dermatitis of the present disclosure may be any of the marsh radish extracts described above. Similarly, the pharmaceutical composition for treating dermatitis used in the method for treating dermatitis of the present disclosure may be any of the pharmaceutical compositions for treating dermatitis described above.

[0094] In addition, in the method for treating dermatitis of the present disclosure, the subject who needs the marsh radish extract of the present disclosure or the pharmaceutical composition for treating dermatitis of the present disclosure may include, but is not limited to, vertebrates.Furthermore, the above-mentioned vertebrates may include, but are not limited to, fish, amphibians, reptiles, birds, or mammals.Examples of mammals may include, but are not limited to, humans, orangutans, monkeys, horses, donkeys, dogs, cats, rabbits, guinea pigs, rats, and mice.In one embodiment, the subject may be a human.

[0095] Example

[0096] A. Method

[0097] 1. Evaluation of inhibitory activity against IL-4 secretion by EL4 cells

[0098] EL4 cells were cultured in a 96-well culture plate (1 × 10 5 The cells were seeded in a 96-well plate (100 μL / well). Then, an appropriate concentration of the test sample was added to the cells (10 μL / well) and mixed well. The 96-well culture plate was then cultured in a cell incubator at 37°C and 5% CO for 2 hours. Next, the stimuli A23187 and phorbol 12-myristate-13-acetate (PMA) were added to the cells in the 96-well culture plate. After mixing well, the 96-well culture plate was cultured in a cell incubator for 18 hours.

[0099] Thereafter, the above-mentioned 96-well culture plate was centrifuged at 1,200 rpm for 5 minutes, and the supernatant was collected in a new 96-well culture plate.

[0100] MTT was added to the cells remaining in the original 96-well culture plate, and the original 96-well culture plate was then placed in a cell incubator at 37°C and 5% CO2 for 1 hour. DMSO was then added to the cells to elute the crystal violet. After uniform mixing, the absorbance of each well was measured at 570 nm to calculate the cell viability. The supernatants in the new 96-well plate were then diluted, and the IL-4 content was assayed using a Mouse IL-4 ELISA kit (R&D systems, Mouse IL-4 DuoSet ELISA, Catalog No. 100001). # : DY404).

[0101] For all experimental results, the group to which only A23187 and PMA were added was set as 100%, and the inhibition rate of IL-4 secretion was calculated. Furthermore, the half inhibitory concentration (IC 50 ) and median cytotoxic concentration (CC 50 ) was calculated.

[0102] 2. Experiments on an animal model of 2,4-dinitrochlorobenzene (DNCB)-induced dermatitis

[0103] 2.1 Experimental animals

[0104] Six-week-old BALB / c male mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan). After being transferred to the general mouse experiment area of ​​the Animal Experiment Station of the Industrial Technology Research Institute, the mice were labeled, caged, weighed, and quarantined for one week.

[0105] During the quarantine period, the animals were allowed to acclimate to the environment and their activity was monitored. Once the animals had gained weight normally, they were transferred to the feeding area and prepared for the experiments. The lighting in the breeding area was set to a 12-hour light-dark cycle (7:30 AM to 7:30 PM light cycle) and the room temperature was set at 23 ± 2°C. The animals had free access to food and water. The breeding conditions were in accordance with national laboratory animal standards. There is a wealth of basic reference material and data on this strain of laboratory animal, making it suitable for use in functional evaluation tests of atopic dermatitis and / or contact dermatitis. The experimental methods were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Industrial Technology Research Institute.

[0106] 2.2 Inducing atopic dermatitis in mice with 2,4-dinitrochlorobenzene

[0107] Before the experiment, the hair on the back of each BALB / c male mouse was shaved with an electric shaver. On Day 1 of the experiment, 100 μL of 1% 2,4-dinitrochlorobenzene (prepared in a solution made by mixing acetone and olive oil in a volume ratio of 4:1) was used to shave the hair on the back of the mouse in an area of ​​approximately 2 × 3 cm. 2 The mice were then sensitized / irritated twice a week on the back and ear skin with 100 μL and 20 μL of 0.5% 2,4-dinitrochlorobenzene (prepared in a solution made by mixing acetone and olive oil in a 4:1 volume ratio), respectively, for three weeks (Day 4 to Day 28), causing the mice to develop symptoms of atopic dermatitis.

[0108] Except for the non-sensitized group (Naive), the test sample or control drug was orally administered daily to the other groups of mice for three weeks starting from Day 10 of the experimental period (Day 10 to Day 30). In the animal model experiment of atopic dermatitis induced with 2,4-dinitrochlorobenzene, cyclosporine A (CsA) (Sandimmun, Capsules) was used as a control drug and was orally administered at 30 mg / kg daily.

[0109] During the experiment, photographs were taken periodically to record the swelling and damage (skin lesions) of the mouse skin. On day 31 of the experiment, the animals were sacrificed with CO2 overdose. Skin samples were collected from the backs and ears of the mice and stained with hematoxylin and eosin (H&E) to analyze the epidermal thickness and overall histological score of the mice.

[0110] 2.3 Skin histopathology analysis

[0111] Dorsal skin specimens collected at the time of sacrifice were placed on small pieces of paper and flattened. Their tissue shape and structure, along with those of the ear skin specimens, were fixed in a neutral formalin solution. The fixed specimens were then dehydrated and embedded in paraffin. The completed paraffin specimens were cut into 3-4 μm paraffin sections using a microtome and stained with hematoxylin and eosin. The hematoxylin and eosin-stained sections were measured for epidermal thickness and assessed for skin histopathological severity by a pathologist according to the method described in Shackelford C, Long G, Wolf J, Okerberg C, Herbert R. Qualitative and quantitative analysis of nonneoplastic lesions in toxicology studies. Toxicologic pathology. 2002;30(1):93-6.

[0112] The histopathological grading criteria are as follows: grade 0 = not present, grade 1 = minimal (<1%), grade 2 = slight (1-25%), grade 3 = moderate (26-50%), grade 4 = moderately severe (51-75%), and grade 5 = severe / high (76-100%).

[0113] 2.4 Data analysis

[0114] The data in this experiment are expressed as mean ± standard error of the mean (SEM). All groups were primarily compared with the vehicle-treated group. Depending on the nature of the data, one-way or two-way ANOVA combined with Dunnett's multiple comparison test, Student's t-test, or Mann-Whitney test was used to calculate whether there was a difference. A p value of less than 0.05 indicates a statistically significant difference between groups.

[0115] 3. Experiments on atopic dermatitis model in NC / Nga mice

[0116] 3.1 Experimental animals Six-week-old NC / NgaTndCrlj male mice were purchased from Charles River Japan (Tokyo, Japan). After being brought into the general mouse experiment area of ​​the Animal Experiment Station at the Institute of Industrial Science and Technology, the mice were labeled, caged, weighed, and quarantined for one week.

[0117] During the quarantine period, the animals were allowed to acclimate to the environment and their activity was monitored. Once the animals had gained weight normally, they were transferred to the feeding area and prepared for the experiments. The lighting in the breeding area was set to a 12-hour light-dark cycle (7:30 AM to 7:30 PM light cycle) and the room temperature was set at 23 ± 2°C. The animals had free access to food and water. The breeding conditions were in accordance with national laboratory animal standards. There is a wealth of basic reference material and data on this strain of laboratory animal, making it suitable for use in functional evaluation tests for atopic dermatitis. The experimental methods were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Industrial Technology Research Institute.

[0118] 3.2. Method for inducing atopic dermatitis in NC / Nga mice

[0119] At the start of the experiment, NC / Nga mice were approximately 10 to 12 weeks old and were divided into S-type groups according to body weight, so that the mean body weight and weight distribution trends of each group were similar.

[0120] One day before the start of the experiment, the hair on the back of the experimental animals was shaved. The shaved area was approximately 4 × 2 cm. 2 On the first day of the experiment (Day 0), 150 μL and 20 μL of 4% sodium dodecyl sulfate (SDS) were administered to the back skin and ear skin of the mice by skin smearing, respectively. Three hours later, 100 mg and 10 mg of Dermatophagoides farinae (Dfb) ointment (Biostir AD) (purchased from Biostir, Osaka, Japan) were administered in the same manner to sensitize / irritate the back skin and ear skin of the mice. The frequency of sensitization / irritation was twice a week for six consecutive weeks.

[0121] Test samples or vehicle were administered according to the experimental design at a dose of 10 mL / kg. F4AB was administered orally daily at 30, 100, 200, or 400 mg / kg. The JAK inhibitor upadacitinib (purchased from MedChemExpress, Monmouth Junction, NJ, USA) was used as a control drug at 1, 3, or 10 mg / kg. After sensitization / challenge on Day 0, animals received the test drug. Clinical observations of the dorsal skin were scored on Days 0, 7, 14, 21, 28, 35, and 42. On Day 42, animals were sacrificed, and blood was collected for IgE analysis, and dorsal skin tissue was collected for pathological analysis.

[0122] 3.3 Observation of skin clinical symptoms

[0123] Clinical observations of the skin of NC / Nga mice with atopic dermatitis induced by Dermatophagoides farinae were evaluated for skin damage symptoms, such as erythema / hemorrhage, scarring / dryness, edema, and epidermal peeling / erosion, on the back of the mice. The scaling scale for the above clinical symptoms is as follows: Scale 0 indicates no symptoms, Scale 1 indicates mild, Scale 2 indicates moderate, and Scale 3 indicates severe. The total clinical severity score for dermatitis (skin lesion score) is the sum of the scores for the above clinical symptom scoring items.

[0124] 3.4 Skin histopathology analysis

[0125] Dorsal skin samples collected at the time of sacrifice were placed on small pieces of paper and flattened. Their tissue shape and structure, along with that of ear skin samples, were fixed in a neutral formalin solution. The fixed samples were then dehydrated and embedded in paraffin. The completed paraffin sections were cut into 3-4 μm paraffin sections using a microtome and stained with hematoxylin and eosin. The hematoxylin and eosin-stained sections were measured for epidermal thickness and evaluated for skin histopathological severity, including inflammation, necrosis, hyperplasia, and hyperkeratosis, by a pathologist according to the method described in Shackelford C, Long G, Wolf J, Okerberg C, Herbert R. Qualitative and quantitative analysis of nonneoplastic lesions in toxicology studies. Toxicologic pathology. 2002;30(1):93-6.

[0126] The histopathological grading criteria are as follows: grade 0 = not present, grade 1 = minimal (<1%), grade 2 = slight (1-25%), grade 3 = moderate (26-50%), grade 4 = moderately severe (51-75%), and grade 5 = severe / high (76-100%).

[0127] 3.5 Data analysis

[0128] Data in this experiment are expressed as mean ± standard error of the mean (SEM). All groups were primarily compared with the vehicle-treated group. Depending on the nature of the data, one-way or two-way ANOVA combined with Dunnett's multiple comparison test, Student's t-test, or Mann-Whitney test was used to calculate whether there was a difference. A p-value of less than 0.05 indicated a statistically significant difference between groups.

[0129] B. Preparation of Ragwort Extract

[0130] 1. Preparation of ethanol extract of marsh radish

[0131] 1.1 Preparation of Raphanus sativus extract 0805

[0132] First, dried leaves of Swamp Radish were crushed to obtain Swamp Radish plant material.

[0133] Next, the above-mentioned marsh radish plant material was extracted with 8 times its weight of 95% ethanol at 60°C for 8 hours, and then filtered to obtain the extract solution of the first extraction and a raffinate of the marsh radish plant material.

[0134] Thereafter, the raffinate of the above-mentioned marsh radish plant material was extracted again with 8 times its weight of 95% ethanol at 60°C for 8 hours, and then filtered to obtain the extract solution of the second extraction and the raffinate of the marsh radish plant material.

[0135] The extract solutions from the two extractions were combined to obtain a combined extract solution. Finally, the combined extract solution was concentrated under reduced pressure in a rotary evaporator to remove the solvent to obtain an extract, which was named Swamp Radish Extract 0805.

[0136] 1.2 Preparation of Raphanus sativus extract 0805-F

[0137] 50 g of the above-obtained marsh radish extract 0805 was dissolved in 95% ethanol of 8 times its weight by heating to 50° C. to obtain a mixed solution.

[0138] Five times the weight of silica gel (250 g) was added to the mixed solution to immobilize it, and column chromatography (a 10 cm inner diameter glass column packed with 2 kg of silica gel) was performed using a mixed solvent of n-hexane / ethyl acetate as the mobile phase.

[0139] Six fractions were obtained during column chromatography and named 0805-f1, 0805-f2, 0805-f3, 0805-f4, 0805-f5 and 0805-f6, respectively.

[0140] The IL-4 secretion-suppressing activity of the marsh radish extract 0805 and its six fractions was then evaluated using EL4 cells according to the method described in "1. Evaluation of IL-4 secretion-suppressing activity using EL4 cells" in "A. Methods" above. The results are shown in Table 1.

[0141] [Table 1]

[0142] Table 1 shows that the marsh radish extract 0805 can suppress IL-4 secretion (IC 50 Of the six fractions, 0805-f3 and 0805-f4 had superior IL-4 secretion inhibitory activity, with IC values ​​of 55.4 μg / mL. 50 were 4.6±1.2 μg / mL and 4.4±0.5 μg / mL. Therefore, fraction 0805-f3 and fraction 0805-f4 were combined to obtain a combined fraction.

[0143] The combined fractions were then concentrated under reduced pressure on a rotary evaporator to remove the solvent, yielding a product, which was named Swamp Radish Extract 0805-F.

[0144] 2. Preparation of water extract of marsh radish

[0145] 2.1 Preparation of Raphanus sativus extract W

[0146] First, the leaves of the marsh radish were crushed and then placed in a filter bag to obtain marsh radish plant material.

[0147] Next, the above-mentioned marsh radish plant material was extracted with 30 times its weight of pure water in a pressurized reactor (extraction conditions: 102.97 kPa, 120°C, 20 minutes) to obtain an extract. The extract was then removed from the pressurized reactor, and the above-mentioned marsh radish plant material was repeatedly extracted under the same conditions. A total of three extractions were performed. The extracts obtained from the three extractions were combined to obtain a combined extract, which was named Marsh Radish Extract W.

[0148] 2.2 Preparation of Raphanus sativus extract F4AB

[0149] Column Chromatography I

[0150] The above-mentioned Swamp Radish Extract W was loaded onto a column (12 inches in diameter, containing 100 L of HP20 resin). The column was then eluted with purified water (200 L) and the eluate was discarded. The column was then eluted with 50% aqueous ethanol (200 L) and the eluate was discarded. Next, the column was eluted with 75% aqueous ethanol (200 L), and the eluate was collected and used as the initial purified extract solution.

[0151] The initial purified extract was further concentrated to obtain a concentrated initial purified extract solution.

[0152] Column Chromatography II

[0153] The concentrated initial purified extract solution was immobilized on silica gel in an amount 5 times the solid weight of the solution, and then column chromatography was performed using a silica gel column (a 10 cm diameter column packed with silica gel in an amount 20 times the solid weight of the concentrated initial purified extract solution) with n-hexane / ethyl acetate (1:1) as the mobile phase. Fractions were collected in sections.

[0154] Based on the method described in "1. Evaluation of IL-4 secretion inhibitory activity by EL4 cells" in "A. Methods" above, the IL-4 secretion inhibitory activity of all fractions obtained by column chromatography was evaluated using EL4 cells, and the fractions having IL-4 secretion inhibitory activity were combined to obtain active fractions.

[0155] The active fraction was further concentrated to obtain a concentrated active fraction.

[0156] The concentrated active fraction was dissolved in 95% aqueous ethanol solution of four times its weight by heating at 60° C. to form an active solution.

[0157] The active solution was allowed to stand for 24 hours to allow crystals to form therein. Finally, the crystals were collected and dried. The resulting dried crystals were named Swamp Radish Extract F4AB.

[0158] C. Animal testing of radish extract 0805

[0159] Animal experiments on the marsh radish extract 0805 were conducted based on the method described in "2. Experiments on animal models of 2,4-dinitrochlorobenzene (DNCB)-induced dermatitis" in "A. Methods" above.

[0160] After sensitization with 2,4-dinitrochlorobenzene, the mice were orally administered 300 mg / kg of marsh radish extract 0805 daily for three consecutive weeks (from Day 10 to Day 30) starting from Day 10 of the experiment.

[0161] On day 31 of the experiment, the animals were sacrificed with CO2 overdose, and then the mice were subjected to skin tissue staining and pathological section analysis, epidermal thickness measurement, and a comprehensive skin pathology severity score. The experimental results are shown in Figures 1 to 3.

[0162] Figure 1 shows the results of hematoxylin-eosin staining of dorsal skin tissue sections from mice in the non-sensitized group (Naive), the vehicle-administered group, and the daikon radish extract 0805-administered group. The areas marked with arrows indicate hyperplasia. * The areas marked with a indicate inflammation.

[0163] Figure 2 shows the epidermal thickness of the dorsal skin of mice in the non-sensitized group (Naive), the vehicle-administered group, and the daikon radish extract 0805-administered group, and Figure 3 shows the total histological score of the dorsal skin of mice in the non-sensitized group (Naive), the vehicle-administered group, and the daikon radish extract 0805-administered group. Data are shown as mean ± standard error (Mean ± SEM) (n = 5). Statistical analysis was performed using Student's t-test (for epidermal thickness measurement) or the Mann-Whitney test (for histopathological scoring). Compared to the vehicle-administered group, p < 0.05 indicates a statistically significant difference. * p<0.05, ** p<0.01, *** p<0.001.

[0164] The experimental results shown in Figures 1 to 3 indicate that the marsh radish extract 0805 can significantly reduce the hyperplasia of the dorsal epidermis and the overall pathological damage of the dorsal skin.

[0165] D. Animal experiments with marsh radish extract 0805-F

[0166] Animal experiments were conducted on the marsh radish extract 0805-F based on the method described in "2. Experiments on animal models of 2,4-dinitrochlorobenzene (DNCB)-induced dermatitis" in "A. Methods" above.

[0167] After sensitization with 2,4-dinitrochlorobenzene, mice were orally administered 300 mg / kg of daikon radish extract 0805-F or 30 mg / kg of the control drug, cyclosporin A, daily for three consecutive weeks (from Day 10 to Day 30) starting from Day 10 of the experiment.

[0168] During the experiment, photographs of the mouse ears were taken to record swelling and damage (skin lesions) of the mouse ear skin, and the thickness of the mouse ears was measured. Photographs of the mouse ears on day 30 of the experiment are shown in Figure 4.

[0169] The results of measuring the ear thickness of the mice on day 30 of the experiment are shown in Figure 5. The data are shown as mean ± standard error (Mean ± SEM) (n = 5). In Figure 5, to compare the ear thickness of each group with that of the vehicle-administered group, statistical analysis was performed using a two-way analysis of variance combined with Dunnett's multiple comparison test. p < 0.05 indicates a statistically significant difference. *** p<0.001. In Figure 5, statistical analysis was performed using Student's t-test to compare the ear thickness of the group administered 300 mg / kg of the radish extract 0805-F with that of the group administered cyclosporine A. p<0.05 indicates a statistically significant difference. ### p<0.001.

[0170] On day 31 of the experiment, the animals were sacrificed with CO2 overdose, and then the mouse ear skin tissue sections were stained and analyzed for pathology, and the overall pathological severity of the ear skin was scored. The experimental results are shown in Figures 6 and 7.

[0171] Figure 6 shows the results of hematoxylin-eosin staining of mouse ear skin tissue sections from the non-sensitized group (Naive), the vehicle-treated group, the daikon radish extract 0805-F-treated group, and the cyclosporin A-treated group. The areas marked with arrows indicate hyperplasia. * The areas marked with a indicate inflammation.

[0172] Figure 7 shows the total histological scores of the ear skin of mice in the unsensitized, vehicle-treated, daikon radish extract 0805-F-treated, and cyclosporin A-treated groups. Data are shown as mean ± standard error (Mean ± SEM) (n = 5). Statistical analysis was performed using the Mann-Whitney test to compare the total histological scores of each group with those of the vehicle-treated group. p < 0.05 indicates a statistically significant difference. * p<0.05, **p<0.01. Statistical analysis was performed using the Mann-Whitney test to compare the total histological score of the group administered with 300 mg / kg of daikon radish extract 0805-F with that of the group administered with cyclosporin A. p<0.05 indicates a statistically significant difference. # p<0.05.

[0173] According to the experimental results shown in Figures 4 to 7, the marsh radish extract 0805-F can significantly improve the clinical appearance, pathological symptoms and ear thickness of mice's ear skin, and shows a dose effect, with its efficacy superior to that of the clinical drug cyclosporine A.

[0174] E. Animal experiments with marsh radish extract F4AB

[0175] Animal experiments were conducted on the marsh radish extract F4AB based on the method described in "Experiment on atopic dermatitis model in NC / Nga mice" in "A. Methods" above.

[0176] F4AB was administered orally daily at 30, 100, 200, or 400 mg / kg. The JAK inhibitor upadacitinib was used as a control drug and administered orally daily at 1, 3, or 10 mg / kg. After sensitization / challenge on Day 0, animals were given the test drug. Clinical observations were scored on the dorsal skin on Days 0, 7, 14, 21, 28, 35, and 42. On Day 42, animals were sacrificed, and blood was collected for IgE analysis, and dorsal skin tissue was collected for pathological analysis.

[0177] The results of measuring the serum IgE concentration of mice in each group are shown in Figure 8. In Figure 8, the data are shown as mean ± standard error (Mean ± SEM) (n = 5), and statistical analysis was performed using Student's t-test. When comparing the group administered with the marsh radish extract F4AB with the group administered with the vehicle, p < 0.05 indicates a statistically significant difference.* p<0.05, ** p<0.01. p<0.05 indicates a statistically significant difference between the upadacitinib-treated group and the vehicle-treated group. # p<0.05, ## p<0.01.

[0178] On day 42 of the experiment, before the mice were sacrificed, photographs of the backs were taken and the appearance of skin scaling and damage (skin lesions) was recorded. The results are shown in Figure 9.

[0179] The backs of the mice were given a comprehensive dermatitis clinical severity score (score of skin lesions) every week during the experiment. The results are shown in Figures 10A and 10B. Figure 10A shows the comprehensive dermatitis clinical severity score on the backs of mice administered with the marsh radish extract F4AB. Data are shown as mean ± standard error (Mean ± SEM) (n = 5), and statistical analysis was performed using the Mann-Whitney test. Compared to the vehicle-administered group, p < 0.05 indicates a statistically significant difference. * p<0.05, ** p<0.01. Figure 10B shows the total clinical severity score of dermatitis on the back of mice treated with upadacitinib. Data are presented as mean ± standard error (Mean ± SEM) (n=5), and statistical analysis was performed using the Mann-Whitney test. p<0.05 indicates a statistically significant difference compared to the vehicle-treated group. # p<0.05, ## p<0.01.

[0180] On day 42 of the experiment, before the mice were sacrificed, a comprehensive dermatitis clinical severity score (score of skin lesions) was performed on the backs of the mice. The results are shown in Figure 10C. Data are shown as mean ± standard error (Mean ± SEM) (n = 5), and statistical analysis was performed using the Mann-Whitney test. A p < 0.05 value indicates a statistically significant difference between the group administered with the marsh radish extract F4AB and the group administered with the vehicle. ***p<0.001. p<0.05 indicates a statistically significant difference between the upadacitinib group and the vehicle group. ### p<0.001.

[0181] After the mice were sacrificed, they underwent skin tissue section staining, epidermal thickness measurement, and pathological section analysis, and the results of these experiments are shown in Figures 11, 12, 13, and 14A to 14E.

[0182] Figure 11 shows the results of hematoxylin-eosin staining of dorsal skin tissue sections from mice in the non-sensitized group, the group administered the marsh radish extract F4AB, and the group administered the vehicle. The areas marked with arrows indicate hyperplasia. * The areas marked with a indicate inflammation.

[0183] Figure 12 shows the results of hematoxylin and eosin staining of dorsal skin tissue sections from mice in the unsensitized group, the upadacitinib-treated group, and the vehicle-treated group. * The areas marked with a indicate inflammation.

[0184] Figure 13 shows the epidermal thickness of the dorsal skin of mice in the non-sensitized group, the group administered with the marsh radish vehicle, the group administered with marsh radish extract F4AB, the group administered with the upadacitinib vehicle, and the group administered with upadacitinib. Data are presented as mean ± standard error (Mean ± SEM) (n = 5), and statistical analysis was performed using Student's t-test. A p < 0.05 value indicates a statistically significant difference between the group administered with marsh radish extract F4AB and the group administered with the vehicle. ** p<0.01. p<0.05 indicates a statistically significant difference between the upadacitinib-treated group and the vehicle-treated group. ## p<0.01.

[0185] Figures 14A to 14E show the inflammation score, necrosis score, hyperplasia score, hyperkeratosis score, and skin total histopathological severity score, respectively, in histopathological section analysis of mouse dorsal skin tissue. Data are shown as mean ± standard error (Mean ± SEM) (n = 5), and statistical analysis was performed using the Mann-Whitney test. When comparing the group administered with the marsh radish extract F4AB with the group administered with the vehicle, p < 0.05 indicates a statistically significant difference. * p<0.05, ** p<0.01. p<0.05 indicates a statistically significant difference between the upadacitinib-treated group and the vehicle-treated group. # p<0.05, ## p<0.01.

[0186] According to the above experimental results, compared to the vehicle-administered group, the IgE concentration in the blood of mice in the group administered 30 to 400 mg / kg of the marsh radish extract F4AB was significantly reduced, while the group administered 1 to 10 mg / kg of the control drug upadacitinib had the same effect.

[0187] Oral administration of both marsh radish extract F4AB and upadacitinib significantly improved the clinical appearance and skin damage of the dorsal skin during the experimental period, and this significant improvement and dose effect were maintained until the day of sacrifice. Histopathological analysis showed that oral administration of marsh radish extract F4AB at 30-400 mg / kg and upadacitinib at 1-10 mg / kg significantly improved epidermal thickness in the dorsal skin, while significantly reducing inflammation, hyperkeratosis, hyperplasia, necrosis, and overall pathological damage.

[0188] All the above experiments clearly demonstrate that the marsh radish extract of the present disclosure can be effectively used in the treatment of dermatitis, especially atopic dermatitis and contact dermatitis.

[0189] It will be apparent to those skilled in the art that various modifications and variations can be made to the disclosed embodiments. It is intended that the description and examples be considered as exemplary only, with the true scope of the present disclosure being indicated by the following claims and their equivalents.

Claims

1. Swamp radish extract, a pharmaceutically acceptable carrier or salt; A pharmaceutical composition for treating dermatitis comprising: The marsh radish extract is an ethanol extract of marsh radish or a water extract of marsh radish, The ethanol extract of the marsh radish is obtained by an extraction method for an ethanol extract of the marsh radish, and the extraction method for an ethanol extract of the marsh radish comprises: (a) providing a plant material of marsh radish; (b) subjecting the marsh radish plant material to a hot extraction process followed by a solid-liquid separation process to obtain an extract solution and produce a raffinate of the marsh radish plant material, wherein during the hot extraction process, a first ethanol solvent is used as an extraction solvent, the weight ratio of the first ethanol solvent to the marsh radish plant material is 5-15:1, and the hot extraction process is carried out at 40-80°C for 1-15 hours; (c) repeating step (b) at least once using the raffinate of the marsh radish plant material as the marsh radish plant material and combining all the resulting extract solutions to obtain a combined extract solution; (d) subjecting the combined extraction solution to a drying process to obtain a dried extract; and The water extract of the marsh radish is obtained by an extraction method for extracting a water extract of the marsh radish, and the extraction method for extracting a water extract of the marsh radish comprises the steps of: (a') providing a plant material of marsh radish; (b') subjecting the marsh radish plant material to a pressurized extraction process, followed by a solid-liquid separation process, to obtain an extract solution and produce a raffinate of the marsh radish plant material, wherein water is used as an extraction solvent during the pressurized extraction process, the weight ratio of the water to the marsh radish plant material is 10-50:1, and the pressurized extraction process is carried out at 101.4-140.0 kPa and 100-140°C for 5-60 minutes; (c') repeating step (b) at least once using the raffinate of the marsh radish plant material as the marsh radish plant material and combining all the resulting extract solutions to obtain a combined extract solution; (d') performing a first column chromatography on the combined extraction solution; wherein the first column chromatography comprises: (d1') loading the combined extraction solution into a column packed with resin; (d2') after step (d1'), eluting the column with water and discarding the eluate flowing out of the column; (d3') after step (d2'), eluting the column with a third ethanol solvent and discarding the eluate flowing out of the column; and (d4') after step (d3'), eluting the column with a fourth ethanol solvent, and collecting the eluate flowing out of the column as an initial purified extraction solution; (e') performing a second column chromatography on the initial purified extract solution, and collecting or combining at least one fraction having the effect of suppressing IL-4 secretion from cells to obtain an active fraction, wherein a mixed solvent of n-hexane / ethyl acetate is used as a mobile phase in the second column chromatography. (f') concentrating the active fraction to obtain a concentrated active fraction; (g') performing a heat dissolution process on the concentrated active fraction in a fifth ethanol solvent to form an active solution; (h') allowing the activated solution to stand to form crystals therein; and (i') collecting the crystals; A pharmaceutical composition for treating said dermatitis, comprising:

2. 2. The pharmaceutical composition for treating dermatitis according to claim 1, wherein the marsh radish extract is an ethanol extract of marsh radish.

3. An extraction method for an ethanol extract of marsh radish. (e) subjecting the dry extract to a heating step in a second ethanol solvent until the dry extract dissolves to form a mixture, wherein the weight ratio of the second ethanol solvent to the dry extract is 5-15:1; and (f) subjecting the mixture to column chromatography, and collecting at least one fraction having the effect of suppressing IL-4 secretion from cells, or collecting and combining the collected fractions to obtain an active fraction; wherein a mixed solvent of n-hexane / ethyl acetate is used as a mobile phase in the column chromatography. The pharmaceutical composition for treating dermatitis according to claim 1, further comprising:

4. The pharmaceutical composition for treating dermatitis according to claim 3, wherein the heating step is carried out at 30 to 70°C.

5. 3. The pharmaceutical composition for treating dermatitis according to claim 2, wherein said marsh radish plant material is derived from marsh radish leaves.

6. A pharmaceutical composition for treating dermatitis as described in claim 1, wherein the marsh radish extract is a water extract of marsh radish.

7. 7. The pharmaceutical composition for treating dermatitis according to claim 6, wherein said marsh radish plant material is derived from marsh radish leaves.

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