Use of Kogomemizu Extract for Preparing Anti-inflammatory Preparations

Abstract

To provide a natural preparation that improves skin conditions by exerting an anti-inflammatory effect.SOLUTION: The present invention provides use of Pilea microphylla extract for manufacturing anti-inflammatory preparations. The Pilea microphylla extract is prepared using a phosphate-buffered saline solution as the extraction solvent and extracting a Pilea microphylla sample at a pressure in the range of 1-1.2 Kgf / cm2 and a temperature in the range of 121-135°C for 20-30 minutes.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the use of a Zucchini extract, in particular to the use of a Zucchini extract for preparing an anti-inflammatory preparation. [Background technology]

[0002] Skin is the outermost organ of the human body, yet it is extremely fragile. When human skin cells are stimulated by excessive free radicals, the body's immune system triggers an inflammatory response, further leading to skin cell aging. Research has shown that the human body's inflammatory response is related to nitric oxide (NO). Nitric oxide plays a major role in blood vessels, the heart, nerve conduction, and other functions, and is an essential compound for protecting the human body. However, excessively high levels of nitric oxide in the human body can easily trigger an inflammatory response.

[0003] In the human body, nitric oxide production is primarily controlled by nitric oxide synthase (NOS), which uses L-arginine as a starting material and generates nitric oxide through a series of catalytic reactions. Nitric oxide synthases can be further classified into neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS). Neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) are constitutive nitric oxide synthases that generally function only under specific physiological conditions. The former is responsible for generating nitric oxide in the central and peripheral nervous systems, whereas the latter is responsible for generating nitric oxide in blood vessels. Inducible nitric oxide synthase (iNOS) is normally present at very low concentrations in the human body, but when the human body is exposed to stimuli (e.g., free radicals), it is induced to proliferate and produce large amounts of nitric oxide, which leads to harmful phenomena in the human body, such as inflammatory responses. Therefore, since the inflammatory responses in the human body are partly related to the induced expression of inducible nitric oxide synthase (iNOS), if appropriate means can be found to reduce the expression of inducible nitric oxide synthase (iNOS) and convert the excess nitric oxide already produced into other substances, the inflammatory responses in the human body can be effectively improved.

[0004] Currently, many skincare product manufacturers on the market achieve the above effects by adding chemical additives to their skincare products, but today's consumers place great importance on the naturalness of skincare product ingredients, and excessive chemical additives may place unnecessary strain on the skin.

[0005] In view of this, there is certainly a need to develop natural formulations that improve skin conditions by providing anti-inflammatory effects. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Sajad Papi et al., Immunopathologia Persa(2019) 5(1):e08 Summary of the Invention

[0007] In order to solve the above problems, the object of the present invention is to provide a use of a Zucchini extract for preparing an anti-inflammatory preparation.

[0008] For the parts and elements described in the entire text of the present invention, the counter words "one" or "one" are used merely for ease of description and to indicate the general meaning of the scope of the present invention, and should be interpreted as including one or at least one in the present invention, and the singular concept also includes the plural case unless clearly indicated otherwise.

[0009] Use of the extract of the present invention for the preparation of an anti-inflammatory preparation, wherein the extract of the present invention is extracted using a phosphate buffered saline solution as an extraction solvent at a concentration of 1 to 1.2 kgf / cm 2 The present invention relates to the use of the rice grain extract of the present invention, which is obtained by extracting a rice grain sample at a pressure in the range of 100 to 120°C and a temperature in the range of 121 to 135°C for 20 to 30 minutes.

[0010] According to this, the use of the Zizania kogomemizu extract of the present invention is such that, when the Zizania kogomemizu extract is administered to a desired individual, the active ingredients contained in the Zizania kogomemizu extract produce effects in the epidermis or body of the desired individual, effectively suppressing and / or alleviating inflammatory responses by scavenging free radicals and nitric oxide in the epidermis or body, and further providing an anti-inflammatory effect. Furthermore, since the Zizania kogomemizu extract has low toxicity to cells and can therefore promote cell growth, the Zizania kogomemizu extract can be used to prepare an anti-inflammatory preparation, which is the effect of the present invention.

[0011] The phosphate buffered saline solution is a use of the Siberian ginseng extract of the present invention, which contains sodium chloride at a concentration ranging from 0.12 to 0.14 M and sodium dihydrogen phosphate at a concentration ranging from 0.008 to 0.012 M. When phosphate buffered saline containing these components is used as an extraction solvent, the active ingredients abundantly contained in the Siberian ginseng sample can be completely extracted, and the anti-inflammatory effect of the Siberian ginseng extract can be enhanced.

[0012] 1~1.2Kgf / cm 2 The present invention also provides a method for extracting the Z. chinensis extract of the present invention, which comprises extracting the Z. chinensis sample at a pressure in the range of 1000 to 15000 kJ / min, thereby further enhancing the anti-inflammatory effect of the Z. chinensis extract, and further enhancing the effective ingredients contained in the Z. chinensis sample.

[0013] The Kogomemizu extract is 100 to 300 μg / cm 2 The present invention relates to the use of the extract of Z. kogomemizu of the present invention, which is administered to a desired individual at a dose of 100 mg / kg / day. Thus, the dose of the extract of Z. kogomemizu of the present invention can effectively reduce and / or suppress the inflammatory reaction in the affected area, thereby achieving a high anti-inflammatory effect.

[0014] The extract of the present invention is administered to a desired individual by topical application to suppress inflammation in the desired individual. In this way, the user can easily obtain the active ingredients of the extract of the extract of the present invention and reduce and / or suppress the inflammatory reaction in the affected area.

[0015] The extract of the present invention is administered to the patient in an amount of 100-300 μg per square centimeter of skin, 2-3 times a day. This amount of the extract of the present invention can effectively reduce and / or suppress the inflammatory response in the affected area, thereby achieving a high anti-inflammatory effect.

[0016] The anti-inflammatory preparation is the use of the extract of the present invention in the form of a lotion, cream, liquid or gel for use as a cosmetic or for use as a skin dressing, thereby allowing the user to reduce and / or suppress the inflammatory reaction in the affected area by applying it. [Brief explanation of the drawings]

[0017] [Figure 1] 1 is a histogram of cell viability of test samples from groups A0 to A7 in Test (A). [Figure 2] 10 is a histogram of cell viability of test samples of groups B0 to B7 in Test (B). [Figure 3] 1 is a histogram of the DPPH scavenging rates of test samples of groups C0 to C10 in Test (C). [Figure 4] 1 is a histogram of the nitric oxide scavenging rates of test samples from groups D0 to D9 in test (D). [Figure 5] (Drawing substitute photograph) This shows the results of immunofluorescence staining of the keratinocyte cell line HaCat in test (E). [Figure 6] (Photograph substitute for drawing) This shows the results of immunofluorescence staining of mouse macrophages RAW264.7 in test (F). DETAILED DESCRIPTION OF THE INVENTION

[0018] In order to make the above and other objects, features and advantages of the present invention more clearly comprehensible, preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings.

[0019] The term "preparation" as used herein means a substance in which active ingredients are processed and mixed to form a specific dosage form and which contains a certain amount of medicine or cosmetic product, whereas the term "anti-inflammatory preparation" as used herein means a substance that has an activity such as improving inflammatory responses in users, which is understandable to those skilled in the art and will not be described in detail here.

[0020] "Pilea microphylla" as used in this invention refers to a plant of the genus Pilea in the family Urticaceae, which is currently distributed throughout the mid-altitude areas of Taiwan. Its stems are green-white and transparent, and have the property of transmitting light under sunlight, so it is also known as "transparent grass" and is also known by other common names such as small water hemp, small-numbered pearl grass, small-leaf cold water flower, or small-leaf cold water grass.

[0021] The Zizania kojomei extract of the present invention can scavenge free radicals and nitric oxide in the epidermis or body, effectively suppressing and / or reducing inflammatory responses, and further providing anti-inflammatory effects. Furthermore, since the Zizania kojomei extract has low toxicity to cells and can promote cell growth, the Zizania kojomei extract can be used to prepare anti-inflammatory preparations. The Zizania kojomei extract may constitute the anti-inflammatory preparation together with at least one pharmaceutically acceptable carrier, or other active ingredients having anti-inflammatory effects may be added to the anti-inflammatory preparation and administered together to a desired individual (for example, 100 to 300 μg / cm). 2 The carrier is an excipient or additive, and is preferably one that can be used in combination to form a preparation in various forms such as a liquid, emulsion, cream, or gel, and that can be easily administered to the skin of the desired individual.

[0022] The term "Zogomeji water extract" as used herein may refer to any extract obtained by extracting Zogomeji water sample, and the Zogomeji water extract may be prepared by the steps of preparing Zogomeji water sample, using phosphate buffer saline (abbreviated as PBS) as an extraction solvent, and extracting Zogomeji water at a concentration of 1 to 1.2 kgf / cm. 2 It is preferable that the extract can be prepared by a method comprising the steps of extracting the Zostera japonica sample at a pressure in the range of 100 to 120°C and a temperature in the range of 121 to 135°C to obtain a Zostera japonica crude extract, and concentrating the Zostera japonica crude extract to obtain the Zostera japonica extract.

[0023] Specifically, the sample of Rhizome may be the whole plant of Rhizome. After obtaining the sample of Rhizome, it may be directly extracted with the phosphate buffered saline solution, or the sample may be pre-dried before extraction, for example, by drying to reduce the water content of the sample to 3-10%. Furthermore, to increase the contact surface area when extracted with the phosphate buffered saline solution, the sample of Rhizome may be pre-pulverized, for example, to a particle size of 5-20 nm, thereby improving the efficiency of subsequent extraction.

[0024] The phosphate buffered saline solution may contain sodium chloride (NaCl) and sodium dihydrogen phosphate (NaH2PO4), and has a pH value of approximately neutral. For example, the phosphate buffered saline solution may contain sodium chloride at a concentration of 0.12 to 0.14 M and sodium dihydrogen phosphate at a concentration of 0.008 to 0.012 M, but preferably contains sodium chloride at a concentration of 0.13 M and sodium dihydrogen phosphate at a concentration of 0.01 M, and the phosphate buffered saline solution has a pH value of 7.0.

[0025] In one embodiment, 100 grams of the water sample can be mixed with 200 milliliters of the extraction solvent to obtain a mixture, which can then be extracted with 1 to 1.2 kgf / cm 2 range (e.g., 1.033Kgf / cm 2The extraction is carried out at a pressure of 1000 kJ / cm 2 (at a temperature in the range of 121 to 135°C (e.g., 127°C) for a period of 20 to 30 minutes (e.g., 20 minutes), allowing the active ingredients abundantly contained in the Zongzi water sample to be completely dissolved in the extraction solvent, thereby obtaining the Zongzi water crude extract. In one embodiment, microorganisms are further filtered through a filter hole with a pore size of 0.22 μm. The extraction step is preferably carried out in a sterilizer to reduce the content of bacteria and / or microorganisms in the extract and to prevent the effects of the active ingredients contained in the Zongzi water from being affected by bacteria and / or microorganisms.

[0026] After obtaining the crude extract of Porphyra sieboldii, the crude extract of Porphyra sieboldii can be freeze-dried to obtain the Porphyra sieboldii extract. This process allows the active ingredients of the Porphyra sieboldii extract to be more concentrated, so that only a small amount of the Porphyra sieboldii extract can exert its optimal effect. In one embodiment, the Porphyra sieboldii crude extract is centrifuged at 12,000 rpm for 20 minutes using a centrifuge to remove residues from the Porphyra sieboldii extract, and then freeze-dried at a temperature in the range of -50 to -80°C to obtain a dry powder, thereby obtaining the Porphyra sieboldii extract. [Example]

[0027] In order to demonstrate that the Kogomemizu extract of the present invention is not toxic to human skin cells and has the activity of promoting the regeneration of skin cells, the following tests (A) and (B) are carried out.

[0028] (A) Effect of Kogomemizu extract on cell viability of keratinocyte cell lines

[0029] Referring to Table 1, in this test, the extract of the present invention was added to the keratinocyte line HaCat, and after culturing for 24 hours, the cell viability of each model cell line was calculated by MTT analysis. The absorbance value of the control group A0 (i.e., the group with the extract of the present invention at a concentration of 0 μg / mL) was set at 100%, and the absorbance values of the remaining groups were calculated, which allowed the relative cell viability of the test samples in each group to be calculated. The results are shown in Figure 1.

[0030] Table 1 shows the test samples for each group in this study. [Table 1]

[0031] As shown in Figure 1, the relative cell viability of groups A1 to A4 was higher than that of group A0, indicating that the addition of medium to low concentrations (20 μg / mL to 80 μg / mL) of Kogomemizu extract can promote the survival of the keratinocyte cell line HaCat. With increasing concentrations of Kogomemizu extract, groups A5 to A7 showed a gradual increase in relative cell viability, indicating that the addition of medium to high concentrations (100 μg / mL to 200 μg / mL) of Kogomemizu extract was not cytotoxic to the keratinocyte cell line HaCat.

[0032] (B) Effect of Kogomemizu extract on cell viability of fibroblast cell lines

[0033] Referring to Table 2, in this test, the extract of the present invention was added to the fibroblast cell line HS68, and after culturing for 24 hours, the cell viability of each model cell line was calculated by MTT analysis. The absorbance value of the control group B0 (i.e., the group with the extract of the extract at a concentration of 0 μg / mL) was set at 100%, and the absorbance values of the remaining groups were calculated, which allowed the relative cell viability of the test samples in each group to be calculated. The results are shown in Figure 2.

[0034] Table 2 shows the test samples for each group in this study. [Table 2]

[0035] As shown in Figure 2, similar to the results obtained in Test (A), the relative cell viability of groups B1 to B4 was higher than that of group B0, indicating that the addition of medium to low concentrations (20 μg / mL to 80 μg / mL) of Z. sieboldii extract could promote the survival of the fibroblast cell line HS68. With increasing concentrations of Z. sieboldii extract, groups B5 to B7 showed a gradual increase in relative cell viability, indicating that the addition of medium to high concentrations (100 μg / mL to 200 μg / mL) of Z. sieboldii extract was not cytotoxic to the fibroblast cell line HS68.

[0036] Next, the following test (C) is carried out to demonstrate that the Kogomemizu extract of the present invention can indeed be used to scavenge free radicals and further inhibit the expression of inducible nitric oxide synthase (iNOS).

[0037] (C) DPPH free radical scavenging effect test

[0038] This test measures the antioxidant capacity of test samples using the DPPH free radical scavenging assay. DPPH (α,α-diphenyl-β-pricrylhydrazyl) is a compound that can generate stable free radicals and is often used as an indicator of antioxidant capacity. DPPH exhibits a blue-purple color in alcoholic solutions. When the free radicals generated by DPPH are scavenged by the test sample, the solution changes color from blue-purple to yellow. Therefore, the greater the degree of decolorization of the DPPH solution, the greater the antioxidant capacity of the test sample. Referring to Table 3, this test involves taking 50 μL of each test solution, adding 1 mL of DPPH solution and 0.95 mL of Tris-HCl buffer solution, sequentially mixing (shaking), and allowing to stand at room temperature for 30 minutes. The absorbance at 517 nm is then measured using a spectrophotometer. The DPPH scavenging rate of each test sample can then be calculated by substituting the absorbance values for each group into the following equation: In this study, Group C1 was the negative control group, containing only 75% ethanol solution and no kogomemizu extract, while Group C2 was the positive control group, containing 40 μg / mL vitamin C but no kogomemizu extract. DPPH solution has a high absorbance at a wavelength of 517 nm, so the lower the absorbance measured, the higher the DPPH scavenging rate (antioxidant power) of the test sample.

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[0039] Table 3 shows the test samples for each group in this study. [Table 3]

[0040] As shown in Figure 3, the DPPH scavenging rate of the Kogomemizu extract of the present invention was positively correlated with the concentration of the Kogomemizu extract, and when the concentration was 200 μg / mL or higher (i.e., groups C8 to C10), the DPPH scavenging rate reached 50% or higher, indicating that the Kogomemizu extract of the present invention can indeed effectively scavenge free radicals and further suppress the expression of inducible nitric oxide synthase (iNOS).

[0041] Next, the following test (D) is carried out to demonstrate that the Kogomemizu extract of the present invention can indeed be used to scavenge already produced nitric oxide and further suppress / reduce inflammatory responses.

[0042] (D) Nitric oxide scavenging effect test

[0043] This test measured the nitric oxide scavenging effect of test samples using a sodium nitroprusside (SNP) scavenging assay. Sodium nitroprusside (SNP) is a substance that can stably generate nitric oxide in solution and is often used as an indicator of nitric oxide scavenging effect. Referring to Table 4, this test was performed by taking 0.12 mL of test solution from each group, adding 0.08 mL of SNP solution (12.5 mM, dissolved in PBS), and incubating at room temperature for 30 minutes. Subsequently, 0.02 mL of sulfanilamide solution (1% by weight, dissolved in 5% phosphoric acid aqueous solution) and 0.02 mL of N-(naphthyl)ethylenediamine dihydrochloride aqueous solution (1% by weight) were added, followed by shaking for 5 minutes. The absorbance at 550 nm was then measured. The absorbance values of each group were then substituted into the following formula to obtain the nitric oxide scavenging rate of the test sample for each group, with a lower absorbance value indicating a higher nitric oxide scavenging ability of the test sample. Group D1 of this study was the negative control group, to which only phosphate-buffered saline (PBS) solution was added, without any extract of the water hyacinth.

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[0044] Table 4 shows the test samples for each group in this study. [Table 4]

[0045] As shown in Figure 4, the nitric oxide scavenging rate of the inventive Zucchini extract was positively correlated with the extract concentration, and when the extract concentration was 400 μg / mL or higher (i.e., groups D8 to D9), the nitric oxide scavenging rate reached 50% or higher, demonstrating that the inventive Zucchini extract can indeed effectively scavenge already produced nitric oxide and further eliminate and / or reduce inflammatory responses in the human body.

[0046] Next, the following test (E) is carried out to demonstrate that the extract of the present invention can promote the anti-aging expression of skin cells.

[0047] (E) Effect of Kogomemizu extract on the expression of the anti-aging protein Sirt6

[0048] In this test, 0 μg / mL, 100 μg / mL, and 200 μg / mL of the Kogomemizu extract were added to the keratinocyte cell line HaCat and cultured for 24 hours. After that, to observe the effect of the Kogomemizu extract of the present invention on the expression of the anti-aging protein Sirt6, the anti-aging protein Sirt6 was stained by immunofluorescence staining, where DAPI is a cell nuclear stain that can clearly mark the position of the cell nucleus.

[0049] As shown in Figure 5, the control group did not receive the extract of the present invention, and the anti-aging protein Sirt6 was hardly expressed. With increasing concentrations of the extract of the present invention, the expression of the anti-aging protein Sirt6 was significantly enhanced, indicating that the extract of the present invention can indeed enhance the expression of the anti-aging protein Sirt6 and provide further anti-aging effects.

[0050] Subsequently, the following test (F) is carried out to demonstrate that the Kogomemizu extract of the present invention can provide an anti-inflammatory effect.

[0051] (F) Effect of Kogomemizu extract on the expression of transcription factor NF-κB

[0052] The transcription factor NF-κB is a representative factor that can regulate inflammatory responses. Its functions include regulating pro-inflammatory genes in innate immune cells and controlling the activation and differentiation of inflammatory T cells. In this study, mouse macrophages RAW264.7 were used as a model cell line. After inducing a cellular immune response with lipopolysaccharide (LPS), 100 μg / mL and 200 μg / mL of the Scutellaria baicalensis extract were added to the immune-responsive mouse macrophages RAW264.7 and cultured for 6 hours. To observe the effect of the Scutellaria baicalensis extract of the present invention on the expression of the transcription factor NF-κB, immunofluorescence staining was used to stain the transcription factor NF-κB. DAPI is a nuclear stain that can clearly identify the location of cell nuclei.

[0053] As shown in Figure 6, the control group was a group that did not receive the inventive extract of P. cerevisiae and did not induce a cellular immune response with lipopolysaccharide (LPS). The expression of the transcription factor NF-κB was very weak and localized in the cytoplasm. After inducing a cellular immune response with LPS, the fluorescence intensity of the transcription factor NF-κB was significantly increased, and most of the fluorescence was observed to be concentrated in the cell nucleus, indicating that the mouse macrophages RAW264.7 had indeed undergone an immune response in response to LPS. Subsequently, when different concentrations (i.e., 100 μg / mL and 200 μg / mL) of P. cerevisiae extract were treated with mouse macrophages RAW264.7 that had already undergone an immune response, the fluorescence intensity of the transcription factor NF-κB was significantly reduced, and the fluorescence dispersed away from the cell nucleus and into the cytoplasm, indicating that the inventive extract of P. cerevisiae can indeed effectively inhibit and / or alleviate inflammatory responses and further provide anti-inflammatory effects.

[0054] In summary, the use of the Zucchini extract of the present invention is such that, when the Zucchini extract is administered to a desired individual, the active ingredients contained in the Zucchini extract produce effects in the epidermis or body of the desired individual, effectively suppressing and / or reducing inflammatory responses by scavenging free radicals and nitric oxide in the epidermis or body, and further providing an anti-inflammatory effect. Furthermore, since the Zucchini extract has low toxicity to cells and can promote cell growth, the Zucchini extract can be used to prepare anti-inflammatory preparations, which is the effect of the present invention.

[0055] Although the present invention has been disclosed by the above preferred embodiments, it is not intended to limit the present invention, and those skilled in the art will be able to make various changes and modifications to the above embodiments without departing from the spirit and scope of the present invention, which will still fall within the scope of protection of the present invention. The scope of protection of the present invention should include the contents of the appended claims and all modifications within their equivalent scope.

Claims

1. Use of a Zizania kogomemizu extract for the preparation of an anti-inflammatory preparation, wherein the Zizania kogomemizu extract is obtained by extracting a Zizania kogomemizu sample using a phosphate buffered saline solution as an extraction solvent at a temperature in the range of 121 to 135°C for 20 to 30 minutes.

2. The use of the extract of claim 1, wherein the phosphate buffered saline solution contains sodium chloride in a concentration ranging from 0.12 to 0.14 M and sodium dihydrogen phosphate in a concentration ranging from 0.008 to 0.012 M.

3. 1~1.2Kgf / cm ２ 2. The use of the extract of claim 1, wherein the extract of the sample of the water hyacinth is extracted at a pressure in the range of 0.1 to 1.0 MPa.

4. The Kogomemizu extract is 100 to 300 μg / cm ２ 2. Use of the extract of claim 1, wherein the extract is administered to a desired individual at a dose of

5. The use of the extract of Z. kogomemizu according to any one of claims 1 to 3, wherein the extract of Z. kogomemizu is administered to a desired individual by topical application to suppress inflammatory phenomena in the desired individual.

6. The use of the Kogomemizu extract according to claim 5, wherein the Kogomemizu extract is administered to the desired individual in an amount of 100 to 300 μg per square centimeter of skin two to three times a day.

7. 6. The use of the extract of claim 5, wherein the anti-inflammatory preparation is used as a cosmetic in the form of a milk, cream, liquid or gel, or as a skin dressing.

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