Composition comprising extract of cruciferous plant sprout powder and method for preparing same

Abstract

The present invention relates to a cruciferous plant sprout extract and, more specifically, to an extract of cruciferous plant sprout powder with increased isothiocyanate including sulforaphane, and a method for preparing same.

Description

Composition containing extract of cruciferous plant sprout powder and method for preparing same

[0001] The present invention relates to a method for producing an extract of cruciferous plant sprout powder having an increased content of isothiocyanates and indole, an extract of cruciferous plant sprout powder having an increased content of isothiocyanates and indole obtained through the method, and a pharmaceutical composition and a food composition comprising the extract of the cruciferous plant sprout powder.

[0002] Cruciferous plants, including Brassica crops, contain significant amounts of isothiocyanates and indole compounds, particularly glucosinolates and phytoalexins. Glucosinolates are sulfur-containing glycosides commonly found in various plant species. These glucosinolates are secondary plant metabolites believed to play a key role in protecting cruciferous plants from microorganisms, fungi, and insects.

[0003] Isothiocyanates (ITCs) and indoles, derived from naturally occurring glucosinolate precursors, are attracting significant attention as chemopreventive agents. Representative examples of isothiocyanates include 4-methylsulfinylbutyl isothiocyanate and phenethyl isothiocyanate (PEITC), also known as sulforaphane (SFN), while representative examples of indoles include indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM).

[0004] Sulforaphane, an isothiocyanate compound, is a metabolite of glucoraphanin, which is abundant in cruciferous vegetables such as broccoli, Brussels sprouts, and cabbage. It exhibits antioxidant, anti-inflammatory, and detoxifying properties. Furthermore, high intake of cruciferous vegetables is known to reduce the risk of cardiovascular disease.

[0005] Sulforaphane plays a crucial role in DNA transcription and proinflammatory cytokine production by regulating the nuclear factor-kappa B (NFkB) pathway, and is produced by the interaction of myrosinase and glucoraphanin, which is formed naturally by the hydrolytic action of an endogenous thioglucosidase enzyme known as myrosinase, which acts on stored precursor glutathione upon physical damage to cells.

[0006] Sulforaphane chemically contains an isothiocynate functional group (- N = C = S) and a methylsulfonyl side chain (R-(SO)-R), has an electrophilic structure, and does not have an aromatic group, so it is water-soluble and enhances pharmacological activity in the neutral pH environment of the intestine.

[0007] Sulforaphane has the ability to activate Nrf2, a protective protein in the body, which helps relieve cellular stress associated with various diseases, including pain, cardiovascular disease, cancer, and Alzheimer's.

[0008] Sulforaphane exhibits potent activation of the cellular antioxidant pathway Keep1 / Nrf2 / ARE and other anti-inflammatory mechanisms by inhibiting the NFkB production pathway. This promotes the expression of antioxidant genes, increases the activity of phase II detoxification enzymes, and increases intracellular glutathione levels via the Nrf2 / ARE pathway, thereby protecting cells from harmful substances. Furthermore, the neuroprotective potential of sulforaphane in neurodegenerative diseases is likely due to activation of the Nrf2 / ARE pathway. Furthermore, sulforaphane exhibits anti-inflammatory actions by binding to toll-like receptor-4 (TLR4) and blocking NFkB, a downstream transcription factor that regulates inflammatory cytokines.

[0009] Sulforaphane can be administered orally or, more commonly, by intraperitoneal injection, but it can also be consumed daily through the diet to achieve similar benefits. The recommended daily intake of sulforaphane is 2–6 mg / day for an adult weighing 60 kg, with doses exceeding 150 mg / kg body weight potentially causing toxicity. While cruciferous vegetables contain some sulforaphane, it is nearly impossible to obtain the required amount from a normal diet. To address this issue, a naturally occurring, concentrated form of sulforaphane that can be taken orally as a nutritional supplement is needed.

[0010] Meanwhile, naturally occurring sulforaphane is highly unstable, making it difficult to extract high concentrations from raw materials. While efforts are being made to apply eco-friendly, non-traditional extraction methods to extract sulforaphane, traditional solvent extraction methods such as aqueous and high-pressure process (HPP), high-voltage electric discharge (HVED), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), supercritical fluid extraction (SFE), and pressurized fluid extraction (PEE) are still widely used to ensure sulforaphane quality and stable extraction yield. However, sulforaphane extracted using these traditional solvent extraction methods has low bioavailability and is uneconomical due to the complex process.

[0011] Furthermore, due to the instability of sulforaphane, efforts have been made to stabilize sulforaphane, including α-cyclodextrin-encapsulated sulforaphane and a stabilized version of pure plant-derived sulforaphane known as Prostaphan®, but these efforts involve the hassle of separate chemical treatments and have uncertainties about their performance.

[0012] Information on prior art documents related to this: ① Patent Publication No. 10-1965391 (published on April 3, 2019) “Broccoli sprout extract with increased sulforaphane content and method for producing the same”, ② U.S. Patent Publication No. US8491944 (published on January 31, 2013) “Process for extracting glucosinolate from broccoli seeds”, ③ U.S. Patent Publication No. US9017666 (published on December 5, 2013) “Broccoli-based nutritional supplement”, ④ Patent Publication No. 10-1729913 (published on April 25, 2017) “Method for producing broccoli with increased sulforaphane content and method for using broccoli produced by the method”, ⑤ U.S. Patent Publication US9433654 (publication date: June 19, 2014) “Composition for treating or preventing prostate cancer comprising broccoli seeds”, ⑥ Patent Publication No. 10-2467837 (publication date: November 21, 2022) “Composition comprising radish sprout extract with increased isothiacyanate and preparation method thereof”, ⑦ European Patent Publication EP2854862 (publication date: August 1, 2018) “Separation and purification of sulforaphane”, ⑧ Japanese Patent Publication JP4712346 (publication date: June 9, 2011) “Method for making ice and ice made by making ice”, and ⑨ Japanese Patent Publication JP6677688 (publication date: April 8, 2020) “Stabilized sulforaphane”

[0013] ① “Broccoli sprout extract with increased sulforaphane and its preparation method” is to supply a pozzolan mixture to broccoli seeds to obtain broccoli sprouts and to obtain the obtained broccoli sprout extract. However, the process is complicated, including the step of preparing the pozzolan mixture to be supplied to the broccoli seeds and the vacuum-reduced pressure drying step to obtain the broccoli sprout extract, and thus has an uneconomical aspect. ② “Process of extracting glucosinolate from broccoli seeds” is to extract glucosinolate, a precursor of sulforaphane, from broccoli seeds, etc., and sulforaphane extracted in this way using an ethanol extraction method has an aspect of low absorption rate in the body. ③ “Broccoli-based nutritional supplement” is a nutritional supplement based on broccoli sprout extract, but in order to increase the sulforaphane content, broccoli seeds are additionally used in addition to broccoli sprouts, and the process is complicated and the equipment costs are high using a supercritical fluid extraction method. Furthermore, high temperature treatment was performed to inhibit epithiospecifier protein (ESP), a protein that acts competitively with myrosinase, but it is difficult to expect a high sulforaphane content in an extract obtained by such high temperature treatment. ④ “Method for producing broccoli with increased sulforaphane content and method for using broccoli produced by the method” uses a traditional solvent extraction method using high-voltage pulse electric field treatment, so it has the problems of the traditional solvent extraction method described above. ⑤ “Composition for treating or preventing prostate cancer comprising broccoli seeds” discloses a method for extracting sulforaphane, but it is limited to a specific broccoli variety, and the process is complicated and uneconomical when considering that broccoli seeds are cold-pressed, acetone and ethylacetone are added, and sulforaphane is mixed with another substance and encapsulated.⑥ “Composition containing radish sprout extract with increased isothiocyanate content and preparation method thereof” includes a method for preparing a radish sprout extract with increased isothiocyanate content or a step of cultivating radish sprouts from radish seeds, but does not present specific conditions such as content ratio, temperature, and time for increasing the content of isothiocyanate containing sulforaphane. Furthermore, “sulforaphane isolation and purification,” “ice making method and ice made by making ice,” and “stabilized sulforaphane,” corresponding to ⑦ to ⑨ above, have the inconvenience of separate chemical treatment and uncertainty about performance in that they use dextrin or cyclodextrin to stabilize sulforaphane.

[0014] The present invention is intended to solve the above problems, and provides a method for producing an extract of cruciferous plant sprout powder having a high content of isothiocyanates and indole including sulforaphane in the extract, and provides a method for producing an extract of cruciferous plant sprout powder for improving the absorption rate of the above substances in the body.

[0015] The present invention provides an extract of cruciferous plant sprout powder having a high extractable content of isothiocyanates and indoles including sulforaphane by the above method.

[0016] The present invention provides a pharmaceutical composition comprising an extract of the cruciferous plant sprout powder capable of preventing or treating pain, cardiovascular disease, cancer, dementia or Alzheimer's.

[0017] The present invention provides a food composition comprising an extract of the cruciferous plant sprout powder and capable of preventing or improving pain, cardiovascular disease, cancer, dementia or Alzheimer's disease, which comprises the extract of the cruciferous plant sprout powder and can be administered orally.

[0018] The above and other objects and advantages of the present invention will become apparent from the following description of preferred embodiments.

[0019] In order to solve the above problem, the present invention includes a method for producing a cruciferous plant extract, comprising: a culturing step of stirring a mixture containing cruciferous plant sprout powder and a solvent; a cooling step of cooling the cultured mixture; an extract producing step of filtering the cooled mixture to remove sprout powder and obtain a filtered extract; a concentrating step of concentrating the extract; and a powdering step of drying and powdering the concentrate.

[0020] Hereinafter, the present invention will be described in more detail.

[0021] Unless otherwise defined, all technical terms used in this invention have the same meanings as those commonly understood by those skilled in the art in the relevant fields of the present invention. Furthermore, while preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention. The contents of all publications cited as references herein are incorporated herein by reference in their entirety.

[0022] Within this specification, isothiocyanates (ITCs) may include sulforaphane (SFN), phenethyl isothiocyanate (PEITC) and other components.

[0023] Within this specification, indole may include indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM) and other components.

[0024] The above cruciferous plant may be one selected from the group consisting of broccoli sprouts, cabbage or watercress sprouts.

[0025] The mixing ratio of the above cruciferous plant and water or distilled water may be 1:20 to 1:40.

[0026] The above solution is characterized by using a hot water extraction method using water or distilled water.

[0027] In the step of preparing the above mixture, the step of storing it in a constant temperature water bath may include a step of reacting so that the myrosinase enzyme present in cruciferous plants can convert glucosinolates into isothiocyanates. The constant temperature water bath may be maintained at, for example, a temperature of 35°C to 70°C, 40°C to 70°C, 45°C to 70°C, 45°C to 65°C, 55°C ± 1°C, or 55°C.

[0028] The above cooling step is characterized in that it is carried out at room temperature or low temperature for 1.5 to 3.5 hours.

[0029] The above extraction may be a stirring extraction of cruciferous plants, and the stirring is characterized in that it is performed for less than 60 minutes at a speed of, for example, 50 to 100 rpm.

[0030]

[0031] As an example, a cruciferous plant extract having an increased content of isothiocyanates manufactured by the above method is provided.

[0032] The above extract may contain, for example, at least 150 ppm of isothiocyanates including sulforaphane based on 1 g of the total weight of the extract. In particular, in one embodiment, an extract manufacturing method capable of increasing the content of isothiocyanates including sulforaphane compared to a conventional extraction method may be used, so that the extract may contain 150 ppm to 500 ppm of sulforaphane based on 1 g of the total weight of the extract.

[0033] The above cruciferous plant extract may be manufactured into a powder form through additional processes such as filtration, concentration, or drying, and may be stored at low temperature or in a frozen state. In the case of such cruciferous plant sprout extract powder, the content of isothiacyanates including sulforaphane may be 4 mg to 15 mg based on 1 g of the total sprout weight.

[0034] In this specification, the term "extract" broadly includes the meaning of a fraction obtained by further fractionating an extract. That is, the extract of cruciferous plant sprout powder includes not only that obtained using the above-described extraction solvent, but also that obtained by additionally applying a purification process thereto.

[0035] As an example, a pharmaceutical composition for preventing or treating pain, cardiovascular disease, cancer, dementia or Alzheimer's disease is provided, comprising an extract of the cruciferous plant sprout powder.

[0036] Another example provides a food composition for preventing or improving pain, cardiovascular disease, cancer, dementia or Alzheimer's disease, comprising an extract of the cruciferous plant sprout powder.

[0037] The above composition may be a pharmaceutical composition, a food composition or an over-the-counter drug composition.

[0038] According to the present invention, a stabilized extract containing a large amount of isothiocyanates and indoles, including sulforaphane, and having a high absorption rate in the body can be produced from a cruciferous plant through an optimal manufacturing method and manufacturing process.

[0039] According to the present invention, the relatively low temperature extraction method can be used to achieve effects such as activation of myrosinase in cruciferous plants, removal of other enzymes in plants, and sterilization of extracts.

[0040] Accordingly, the extract not only has an increased content of isothiocyanates and indoles, including sulforaphane, compared to conventional extraction methods, but also has a high absorption rate in the body and is stabilized, so it can be widely used as a pharmaceutical or food composition for preventing, improving, or treating pain, cardiovascular disease, cancer, dementia, or Alzheimer's disease.

[0041] However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0042] FIG. 1 is a schematic diagram illustrating the anti-inflammatory, chemoprotective, and epigenetic modulatory activities of sulforaphane according to one embodiment of the present invention.

[0043] Figure 2 is a graph showing the results of measuring the SFN content (ppm) compared to the initial detection over time in the mixture preparation stage of Experimental Example 1.

[0044] Figure 3 is a graph showing the results of measuring the I3C content (ppm) compared to the initial detection over time in the mixture preparation stage of Experimental Example 1.

[0045] Figure 4 is a graph showing the results of measuring the PEITC content (ppm) compared to the initial detection over time in the mixture preparation stage of Experimental Example 1.

[0046] Figure 5 is a graph showing the results of measuring the SFN content (ppm) according to temperature in the mixture manufacturing step of Experimental Example 2.

[0047] Figure 6 is a graph showing the results of measuring the I3C content (ppm) according to temperature in the mixture manufacturing step of Experimental Example 2.

[0048] Figure 7 is a graph showing the results of measuring the PEITC content (ppm) according to temperature in the mixture manufacturing step of Experimental Example 2.

[0049] Figure 8 is a graph showing the results of measuring the SFN content (ppm) over time in the cooling stage of Experimental Example 3.

[0050] Figure 9 is a graph showing the results of measuring the I3C content (ppm) over time in the cooling stage of Experimental Example 3.

[0051] Figure 10 is a graph showing the results of measuring the PEITC content (ppm) over time in the cooling stage of Experimental Example 3.

[0052] Figure 11 is a graph showing the fold change in gene expression of TNFα according to the in vitro anti-inflammatory assay of Experimental Example 5.

[0053] Figure 12 is a graph showing the fold change in gene expression of IL-6 according to the in vitro anti-inflammatory assay of Experimental Example 5.

[0054] Figure 13 is a graph showing the fold change in gene expression of COX2 according to the in vitro anti-inflammatory assay of Experimental Example 5.

[0055] Hereinafter, the present invention will be described in detail with reference to examples and drawings. These examples are provided solely as examples to more specifically illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0056] Additionally, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and in case of conflict, the description in this specification, including definitions, shall prevail.

[0057] To clearly explain the invention proposed in the drawings, irrelevant parts have been omitted, and similar parts have been designated with similar drawing reference numerals throughout the specification. Furthermore, when a part is said to "include" a component, this does not mean that other components are excluded, but rather that other components may be included, unless otherwise specifically stated. Furthermore, the term "part" described in the specification refers to a single unit or block that performs a specific function.

[0058] The identifiers (1, 2, etc.) for each step are used for convenience of explanation and do not describe the order of each step. Each step may be performed in a different order than stated unless the context clearly indicates a specific order. In other words, each step may be performed in the same order as stated, may be performed substantially simultaneously, or may be performed in the opposite order.

[0059] Unless otherwise defined, all technical terms used in this invention have the same meanings as those commonly understood by those skilled in the art in the relevant fields of the present invention. Furthermore, while preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention. The contents of all publications cited as references herein are incorporated herein by reference in their entirety.

[0060]

[0061] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. However, the present invention may not be limited to these embodiments and drawings.

[0062]

[0063] One aspect of the present invention provides a method for producing an extract of cruciferous plant sprout powder, comprising: a culturing step of stirring a mixture containing cruciferous plant sprout powder and a solvent; a cooling step of cooling the cultured mixture; an extract producing step of filtering the cooled mixture to remove sprout powder and obtain a filtered extract; a concentrating step of concentrating the extract; and a powdering step of drying and powdering the concentrate.

[0064]

[0065] Within this specification, isothiocyanates (ITCs) may include sulforaphane (SFN), phenethyl isothiocyanate (PEITC) and other components.

[0066] Within this specification, indole may include indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM) and other components.

[0067] In this specification, the term "extract" broadly refers to an extract, and further includes fractions obtained by fractionation. That is, the extract of cruciferous plant sprout powder includes not only those obtained using the above-described extraction solvent, but also those obtained by additionally applying a purification process thereto.

[0068]

[0069] The above cruciferous plant may be one selected from the group consisting of broccoli sprouts, cabbage or watercress sprouts.

[0070] The mixing ratio of the above cruciferous plant sprout powder and water or distilled water may be 1:20 to 1:40.

[0071] The solvent may be water or distilled water.

[0072] The above culturing step may include a process of stirring the mixture in a constant temperature water bath.

[0073] The above culturing step may include a step of reacting so that the myrosinase enzyme present in cruciferous plants can convert glucosinolates into isothiocyanates.

[0074] The above-mentioned constant temperature water bath may be maintained at a temperature of, for example, 35°C to 70°C, 40°C to 70°C, 45°C to 70°C, 45°C to 65°C, 55°C ± 1°C or 55°C.

[0075] The above cooling step may be performed at room temperature or a low temperature, for example, 19 to 26°C, specifically, 20 to 25°C, for 1.5 to 3.5 hours.

[0076] The above extract production step may include a hot water extraction method that is performed including the above-mentioned culturing step and cooling step, and more specifically, a stirring process in a constant temperature water bath.

[0077] The above stirring is characterized in that it is performed for less than 60 minutes at a speed of, for example, 50 to 100 rpm.

[0078]

[0079] An extract of cruciferous plant sprout powder having an increased content of isothiocyanates and / or indoles produced by the above method can be provided.

[0080] The above extract may contain, for example, at least 150 ppm of isothiocyanates including sulforaphane based on 1 g of the total weight of the extract. In particular, in one embodiment, an extract manufacturing method capable of increasing the content of isothiocyanates including sulforaphane compared to a conventional extraction method may be used, so that the extract may contain 150 ppm to 500 ppm of sulforaphane based on 1 g of the total weight of the extract.

[0081] The extract of the above cruciferous plant sprout powder can be manufactured into a powder form by an additional process such as filtration, concentration or drying, and can be stored at a low temperature or in a frozen state.

[0082]

[0083] The extract of the cruciferous plant sprout powder prepared according to one embodiment of the present invention can be specifically included in a pharmaceutical composition for preventing or treating pain, cardiovascular disease, cancer, dementia or Alzheimer's.

[0084] Another example may provide a food composition for preventing or improving pain, cardiovascular disease, cancer, dementia or Alzheimer's disease, comprising the cruciferous plant extract.

[0085] The above composition may be a pharmaceutical composition, a food composition or an over-the-counter drug composition.

[0086]

[0087] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0088]

[0089] Example 1: Preparation of extract from broccoli sprout powder

[0090] 1 g of broccoli sprout powder was mixed with water at 50 to 60°C, and stirred at 60 rpm for 30 minutes. The temperature of the water was maintained at 50 to 60°C during stirring. After stirring was completed, the mixture was cooled to room temperature (20 to 25°C) for 2 hours to prepare an extract of broccoli sprout powder. The prepared extract was filtered through a 5 μm filter to remove the sprout particles, and the obtained filtrate was concentrated and dried to prepare a powder form. The prepared broccoli sprout extract powder was stored at a low temperature or frozen (-20°C to +4°C).

[0091]

[0092] Experimental Example 1. Determination of isothiocyanate and indole contents in cruciferous plants according to culture time.

[0093] To determine the content of isothiocyanates in cruciferous plants according to the culture time, the content of isothiocyanates was determined from broccoli sprout powder, purple cabbage sprout powder, and watercress sprout powder.

[0094] Broccoli sprout powder, purple cabbage sprout powder, and watercress sprout powder, 1 g each, were added to 50 ml tubes containing distilled water and gently stirred for proper mixing. After maintaining each tube in a constant temperature water bath set to 65℃, they were incubated for 30 minutes, 1 hour, 2 hours, and 3 hours, and samples were extracted from the culture medium at each designated time point. The extracted samples were cooled as is to obtain extracts. The extracts were filtered using Whatman filter paper, and the filtrate was further filtered using a 0.45 μm syringe filter. Then, 20 μl of the volume was injected into an HPLC column equilibrated with the mobile phase according to each detection condition. Each sample was injected twice and the average value was calculated to measure the content of sulforaphane (SFN) in the broccoli sprout powder sample, indole-3-carbinol (I3C) in the purple cabbage sprout powder sample, and phenethyl isothiocyanate (PEITC) in the watercress sprout powder sample, which are shown in Figures 2 to 4, respectively.

[0095] As confirmed in FIGS. 2 to 4, the sulforaphane (SFN) content of each sample was approximately 300 ppm at the time of incubation for 30 minutes in a water bath set at 65°C, and indole-3-carbinol (I3C) and phenethyl isothiocyanate (PEITC) showed approximately 150 ppm and 120 ppm, respectively, and it was confirmed that the contents of sulforaphane, isocyanate, and indole were the highest. However, after 30 minutes, it was found that these contents decreased significantly as the incubation time increased, decreasing at a significant rate compared to the initial value.

[0096]

[0097] Experimental Example 2. Determination of isothiocyanate and indole contents in cruciferous plants according to culture temperature.

[0098] Broccoli sprout powder, purple cabbage sprout powder, and watercress sprout powder, 1 g each, were added to a 50 ml tube containing distilled water and gently stirred to ensure proper mixing. The tubes were then exposed to various incubation temperatures of 35°C, 45°C, 55°C, 65°C, and 75°C, and incubated at each temperature for 30 and 60 minutes, respectively. Samples were extracted and the extracted samples were then cooled to obtain extracts. The extracts were filtered using Whatman filter paper, and the filtrates were further filtered using a 0.45 μm syringe filter. 20 μl of the resulting volume was injected into an HPLC column equilibrated with the mobile phase according to each detection condition. Each sample was injected twice, and the average value was calculated to determine the contents of the selected isothiacyanates and indoles in the samples, which are shown in Figures 5 to 7.

[0099] As confirmed in Figures 5 to 7, the isothiocyanate content of the extract extracted after culturing for 30 minutes was measured, and the sulforaphane (SFN) content of the broccoli sprout extract was the highest at 460.75 ± 10.27 ppm at 65°C, while at 75°C, it was measured to be 405.41 ± 67.98 ppm, which was lower than that at 65°C.

[0100] Meanwhile, indole-3-carbinol (I3C) from purple cabbage sprout extract and phenethyl isothiocyanate (PEITC) from watercress sprout extract were also detected in the highest yield in the sample cultured at 65°C.

[0101] Furthermore, all samples showed a tendency for the content of isothiocyanates and indoles to decrease rapidly when extracted at temperatures above 70℃.

[0102] Meanwhile, the extracts extracted after culturing for 1 hour were measured at three temperatures (35℃, 45℃, and 55℃). The sulforaphane (SFN) content of the broccoli sprout extract was the highest at 55℃ (435.24 ± 1.09 ppm), whereas the sulforaphane (SFN) content decreased rapidly at 65℃, in contrast to the extracts extracted after culturing for 30 minutes. On the other hand, indole-3-carbinol (I3C) of the red cabbage extract extracted after culturing for 1 hour and phenethyl isothiocyanate (PEITC) of the watercress sprout extract were the highest at 65℃.

[0103]

[0104] Experimental Example 3. Determination of isothiocyanate and indole contents in cruciferous plants according to cooling time.

[0105] Broccoli sprout powder, purple cabbage sprout powder, and watercress sprout powder, 1 g each, were added to 50 ml tubes containing distilled water and gently stirred to ensure proper mixing. The tubes were then exposed to the optimal incubation temperature (65°C) and incubated for 30 minutes before extracting the samples. The extracted samples were cooled to room temperature (21–24°C) at 1-hour intervals for up to 4 hours to obtain extracts. The filtrates were further filtered using a 0.45 μm syringe filter and then injected in a volume of 20 μl onto an HPLC column equilibrated with the mobile phase according to each detection condition. Each sample was injected twice and the average value was calculated to measure the content of sulforaphane (SFN) in the broccoli sprout powder sample, indole-3-carbinol (I3C) in the purple cabbage sprout powder sample, and phenethyl isothiocyanate (PEITC) in the watercress sprout powder sample, which are shown in Figures 8 to 10.

[0106] As confirmed in Figures 8 to 10, it was confirmed that the extractive contents of sulforaphane (SFN), indole-3-carbinol (I3C), and phenethyl isothiocyanate (PEITC) were the highest when the room temperature incubation time was 1.5 to 3.5 hours.

[0107]

[0108] Experimental Example 4. Antioxidant Capacity Analysis

[0109] An analytical experiment was conducted to measure the antioxidant capacity of the broccoli sprout extract powder prepared in Example 1. The analytical experiment was conducted using the Zen-Bio Oxygen Radical Absorbance Capacity (ORAC) antioxidant assay kit, which is a useful tool used to measure the total antioxidant capacity of naturally occurring or synthetic compounds. The principle of the assay kit is to monitor the loss of fluorescein fluorescence over time, which is caused by the formation of peroxyl radicals due to the decomposition of 2,2'-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH).

[0110] To assess antioxidant activity, the breakdown of fluorescein was assayed against the positive control, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water-soluble vitamin E analogue. Trolox demonstrated a dose-dependent inhibition of fluorescein breakdown, allowing for a meaningful comparison.

[0111] The ORAC assay is a kinetic assay that measures antioxidant protection and fluorescein breakdown over a specific period of time, quantifying the antioxidant activity of a test sample and expressing it in equivalent Trolox units, thus allowing a measure of the complex antioxidant activity present in a sample.

[0112] Specifically, at 37°C, 2,2'-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) decomposes to generate peroxyl radicals (ROO-), which oxidize fluorescein (3',6'-dihydroxy-spiro[isobenzofuran-1[3H], 9'[9H]-xanthene]-3-one) to form nonfluorescent products. Antioxidants inhibit this reaction via a hydrogen atom transfer mechanism, protecting the fluorescein signal from oxidative degradation. The assay measures fluorescence intensity over 30 minutes using excitation at 485 nm, emission at 538 nm, and cutoff at 530 nm. The antioxidant concentration in the test sample is determined by comparing the net area under the curve with that of Trolox, a known antioxidant.

[0113] For ORAC analysis, first, the plate reader incubation chamber was equilibrated to 37°C, and the plate reader was set to perform kinetic readings at 1-minute intervals for a total of 30 minutes. Fluorescence measurements used an excitation wavelength of 485 nm and an emission range of 528–538 nm (with a cutoff at 530 nm, if necessary). The plate reader was configured for bottom readings. To prepare the fluorescein working solution, 11.8 mL of AOX Assay Buffer was combined with 0.2 mL of stock fluorescein solution in an empty tube, and the mixture was protected from light. Next, the contents of a 1.5 mM Trolox standard tube were thawed, briefly spun, and 280 μL of AOX Assay Buffer was added to the tube and mixed well by vortexing to prepare the Trolox standard. This step produced a diluted stock Trolox standard with a concentration of 100 μM.

[0114] Next, 150 μL of AOX assay buffer was pipetted into four tubes and a dilution series was created using freshly prepared diluted stock Trolox solution as described in the instructions. Each new dilution was thoroughly mixed before proceeding to the next step. The 100 μM stock dilution was used as the top standard, and the assay buffer was used as the zero standard for calibration purposes. 150 μL of working fluorescein solution was added to each of the inner 60 wells of the provided assay plate.

[0115] Next, 25 μL of sample or Trolox standard was added to individual wells, and 25 μL of assay buffer was used as a negative control. The plate was incubated at 37°C for at least 10 minutes. To prepare the AAPH working solution, 2.0 mL of AOX assay buffer was added to the provided AAPH tube, gently inverted, and placed on ice until needed. Once ready, 25 μL of AAPH working solution was added to each well containing the standards and samples from step 5. Finally, the assay plate was inserted into the plate reader, and kinetic fluorescence readings were initiated.

[0116] Table 1 shows the area under the curve (AUC), net AUC, and ORAC values ​​of broccoli sprout extract powder solution (BSEP) samples and sulforaphane (SFN), and as shown in Table 2, the antioxidant activity of broccoli sprout extract powder solution (BSEP) at the same concentration level (1 mM) is about 4 times that of net sulforaphane (SFN) and about 7.8 times that of Trolox. This may be due to the combined effects of other isothiocyanates and glucoraphanin in the extract.

[0117] ParameterBSEPSFNAUC17.9715.27Net AUC6.016.35ORAC Value15.6018.61

[0118] Oxygen Radical Absorbance Capacity (ORAC) Analysis

[0119] Sl NoSample NameFinal ORAC11% Broccoli sprout extract powder (BSEP)78002Sulforaphane (SFN)1918.353Trolox (Vit. E analogue)1000

[0120] <Oxygen radical absorbance of 1% broccoli sprout extract powder solution (BSEP) sample and other common antioxidants>

[0121]

[0122] Experimental Example 5. Expression Profiles of Anti-Inflammatory and Pro-Inflammatory Genes

[0123] Mouse macrophage cell lines (RAW 264.7) were purchased from ATCC, and passage 3 cells were used in the experiment. The split ratio was 1:4, and cells were passaged after reaching 60-70% confluence. Cells were cultured in T75 flasks (Corning). The cell culture medium used was 1x DMEM (4.5 g / L glucose, sodium pyruvate, L-glutamine, Corning) supplemented with 10% fetal bovine serum (Corning) and 1x antibiotic-antimycotic solution (penicillin 10,000 U, streptomycin 10 mg, amphotericin B 25 μg). The medium was changed every 3 days for the first 2 days, and then every 2 days until the required concentration was reached.

[0124] RAW 264.7 cells from passage 3 were seeded at 1 x 10 in each well of a 6-well cell culture plate. 5Cells were seeded at 100 μg / mL. Cells were cultured until 70–80% confluent, and the medium was changed every two days. Upon confluence, the existing medium was removed, and the cells were pretreated with 1 mL of serum-free medium for 10–15 minutes in an incubator. All controls, samples, and dilutions were prepared in serum-free medium. The serum-free medium was removed, and LPS (1 μg / mL) was added to the serum-free medium for 24 hours. The following day, the LPS-containing medium was removed, washed once with 1x PBS, and the cells were treated with SFN solution for 1 hour. The treatment solution was removed, and the cells were washed twice with 1x PBS, pH 7.4. The cells were dissociated and collected by sedimentation (1500 rpm, 6 minutes).

[0125] Total RNA content was extracted using the TRIzol method according to the manufacturer's protocol. mRNA abundance was quantified using one-step reverse transcription real-time quantitative PCR using the QuantiTect SYBR Green RT-PCR kit (Qiagen, Germany) performed on a Rotor-Gene Q Real-Time system (Qiagen, Germany).

[0126] Gene primers were used in the study of Wang et al. (2008) (IL6, TNFα, COX2, and ACTb). Each reaction contained 100 ng of total RNA, and for gene expression, 0.5 pmol of each primer, 5 μl of the one-step reaction mixture, and 0.1 μl of Quantiscript reverse transcriptase were added to a 10 μl reaction system, and the reaction was performed at 50°C for 30 min, 95°C for 15 min, and 40 cycles of 94°C for 15 s and 52-58°C for 30 s. Each sample analysis was performed in triplicate to determine the average threshold cycle (Ct) value.

[0127] Target gene expression was normalized to the constitutively expressed ACTb gene, and the relative amount of target gene mRNA was calculated using the relative comparison threshold cycle method described above. - Ct expressed as (Voge et al., 2004; Lagaly et al., 2008; Grado-Ahuir et al., 2011). Non-template and non-reverse transcriptase controls were performed for each PCR run to ensure that there was no genomic DNA contamination in the samples or master mix. Tumor necrosis factor alpha (TNFα) and IL-6 are major inflammatory cytokines. COX-2 is regulated by IL-1. It is induced in cells when stimulated by and plays an important role in inflammation. The experimental results are shown in Figs. 11 to 13 and Tables 3 to 5, respectively.

[0128] Table 3 shows the fold change in gene expression of TNFα according to the in vitro anti-inflammatory assay, Table 4 shows the fold change in gene expression of IL-6 according to the in vitro anti-inflammatory assay, and Table 5 shows the fold change in gene expression of COX2 according to the in vitro anti-inflammatory assay.

[0129] Sl No.Treatment% Fold expression (TNF )1Control100.00 0.012SFN 210 ppm50.00 0.1331% BSEP36.35 0.18

[0130] Sl No.Treatment% Fold expression(IL6)1Control100.00 0.212SFN 210 ppm18.05 0.2631% BSEP0.55 0.33

[0131] Sl No.Treatment% Fold expression(COX2)1Control100.00 0.052SFN 210 ppm1.11 0.0931% BSEP1.12 0.08

[0132] As shown in Figures 11 to 13 and Tables 3 to 5, the experimental results confirmed a downregulation of TNFα in the 210 ppm SFN standard solution and the 1% BSEP diluted solution, which appears to be influenced by cell viability. This suggests that SFN in broccoli sprout extract exerts anti-inflammatory effects. A similar trend was also observed in IL-6 and COX2 gene expression.

[0133]

[0134] Although this specification describes only a few examples among various experimental examples performed by the inventors, the technical idea of ​​the present invention is not limited or restricted thereto, and can be modified and implemented in various ways by those skilled in the art.

Claims

1. A culturing step of stirring a mixture containing cruciferous plant sprout powder and a solvent; A cooling step for cooling the cultured mixture; An extract preparation step of filtering the cooled mixture to remove the sprout powder and obtain a filtered extract; A concentration step for concentrating the above extract; and, A method for producing an extract of cruciferous plant sprout powder, comprising a powdering step of drying and powdering the above concentrate.

2. In paragraph 1, A method for producing an extract of cruciferous plant sprout powder, characterized in that the solvent is water or distilled water.

3. In paragraph 1, A method for producing an extract of cruciferous plant sprout powder, characterized in that the cruciferous plant is broccoli sprout, cabbage or watercress sprout.

4. In paragraph 1, A method for producing an extract of cruciferous plant sprout powder, characterized in that the stirring is performed at a speed of 50 to 100 rpm for less than 60 minutes.

5. In paragraph 1, A method for producing an extract of cruciferous plant sprout powder, characterized in that in the above-mentioned cultivating step, the temperature during stirring is maintained at 45°C to 70°C.

6. In paragraph 1, A method for producing an extract of cruciferous plant sprout powder, characterized in that the cooling step is performed at a temperature of 20°C to 25°C for 1.5 to 3.5 hours.

7. In paragraph 1, A method for producing an extract of cruciferous plant sprout powder, characterized in that it further comprises a step of storing the powdered extract at a low temperature or in a frozen state.

8. An extract of cruciferous plant sprout powder having an increased content of isothiocyanates, manufactured by any one of the methods of claims 1 to 7.

9. An extract of cruciferous plant sprout powder having an increased indole content, manufactured by any one of the methods of claims 1 to 7.

10. A pharmaceutical composition for preventing or treating pain, cardiovascular disease, cancer, dementia, or Alzheimer's disease, comprising an extract of cruciferous plant sprout powder according to claim 8 or 9.

11. A food composition for preventing or improving pain, cardiovascular disease, cancer, dementia, or Alzheimer's disease, comprising an extract of cruciferous plant sprout powder of claim 8 or 9.

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