Cell protection components

Abstract

To inhibit cell death induced by oxidative stress or endoplasmic reticulum stress.SOLUTION: A composition for cell protection includes, as an active ingredient, an extract of an alga belonging to the genus Chlorogonium, the family Haematococcaceae, the order Volvocales, the class Chlorophyceae, and the division Chlorophyta.SELECTED DRAWING: Figure 3

Description

【Technical Field】 【0001】 The present invention relates to a composition for cell protection. 【Background Art】 【0002】 In recent years, various studies have been conducted on the effects of microalgae. For example, Patent Document 1 discloses that components contained in the algal bodies of Chlorogonium, a genus of the Chlorophyceae class, Volvocales order, and Haematococcaceae family of the Chlorophyta phylum, promote the production of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). 【Prior Art Documents】 【Patent Documents】 【0003】 【Patent Document 1】 Japanese Unexamined Patent Application Publication No. 2023-158928 【Summary of the Invention】 【Problems to be Solved by the Invention】 【0004】 The present inventors have newly found that an extract of Chlorogonium capillatum, an algal body of the genus Chlorogonium of the Chlorophyceae class, Volvocales order, and Haematococcaceae family of the Chlorophyta phylum, suppresses cell death caused by oxidative stress and endoplasmic reticulum stress. The present invention has been made based on the above findings. 【Means for Solving the Problems】 【0005】 The composition for cell protection of the invention for solving the above problems is a composition for cell protection that suppresses cell death induced by at least one of oxidative stress and endoplasmic reticulum stress, and contains an extract of Chlorogonium capillatum as an active ingredient. 【0006】 One aspect of the above composition for cell protection is applied as a ferroptosis inhibitor that inhibits ferroptosis induced by oxidative stress. One embodiment of the above-described cell-protective composition is applied as an apoptosis inhibitor that inhibits apoptosis induced by endoplasmic reticulum stress. 【0007】 An apoptosis inhibitor in one embodiment of the above-described cell-protective composition inhibits apoptosis based on the IRE1 pathway. An apoptosis inhibitor in one embodiment of the above-described cell-protective composition inhibits apoptosis based on the PERK pathway. [Effects of the Invention] 【0008】 The cell-protective composition of the present invention can suppress cell death induced by oxidative stress or endoplasmic reticulum stress. [Brief explanation of the drawing] 【0009】 [Figure 1] Figure 1 is an explanatory diagram of the mechanism by which oxidative stress induces ferroptosis. [Figure 2] Figure 2 is an explanatory diagram of the mechanism of apoptosis induction by endoplasmic reticulum stress. [Figure 3] Figures 3(a) and 3(b) are graphs showing the changes in cell viability and cell death rate when oxidative stress is applied. [Figure 4] Figures 4(a) and 4(b) show the results of Western blotting and graphs illustrating the changes in ferritin expression levels when oxidative stress is applied. [Figure 5] Figures 5(a) and 5(b) are graphs showing the changes in cell viability and cell death rate when endoplasmic reticulum stress is applied. [Figure 6] Figure 6 shows the results of Western blotting illustrating the changes in the expression levels of endoplasmic reticulum stress-related proteins when endoplasmic reticulum stress is applied. [Figure 7] Figures 7(a) to 7(e) are graphs showing the changes in the expression levels of endoplasmic reticulum stress-related proteins when endoplasmic reticulum stress is applied. [Modes for carrying out the invention] 【0010】 The following describes one embodiment of the present invention. The composition of this embodiment (hereinafter referred to as "this composition") contains an extract of Chlorogonia capiratum as an active ingredient. 【0011】 [Raw materials] Chlorogonium capillatum is an algal organism belonging to the genus Chlorogonium in the family Haematococcaceae, order Volvoles, class Chlorophyceae, division Chlorophyta. Chlorogonium capillatum (hereinafter sometimes referred to as "specific algal organism") may be a naturally occurring algal organism or an artificially cultured algal organism. However, due to the ability to ensure a stable supply and the ease of maintaining quality, the use of artificially cultured algal organisms is industrially preferable. 【0012】 [Extract] For extracting extracts from specific algae, the extraction solvent can be, for example, water, an organic solvent, or a mixed solvent of an organic solvent and water. Examples of organic solvents include lower alcohols, hexane, ethyl acetate, dimethyl sulfoxide, acetonitrile, acetone, glycerin, and propylene glycol. Examples of lower alcohols include C1-C5 alcohols such as methanol, ethanol, propanol, isopropanol, and butanol. 【0013】 As the organic solvent, a single type may be used, or a mixed solvent of multiple types may be used. When a mixed solvent of water and an organic solvent is used as the extraction solvent, the content of the organic solvent in the mixed solvent is, for example, 1% by volume or more and 99% by volume or less. In addition, the extraction solvent may contain additives. Examples of additives include organic salts, inorganic salts, buffers, and emulsifiers. 【0014】 As an extraction method for extracting an extract from a specific algal body, known extraction methods can be used. Examples of known extraction methods include cooling extraction, normal temperature extraction, and heating extraction. Also, the extraction temperature can be appropriately set according to the type of solvent, extraction efficiency, degradation of components, etc. 【0015】 The extraction operation is performed by immersing the above-mentioned algal body as a raw material in an extraction solvent for a predetermined time. At that time, the concentration of the above-mentioned algal body in the extraction solvent can be appropriately set according to the type of extraction solvent, extraction efficiency, efficiency of the concentration treatment after extraction, etc. In such an extraction operation, in order to enhance the extraction efficiency, treatments such as reflux treatment, stirring treatment, pressurization treatment, and ultrasonic treatment may be further performed as necessary. 【0016】 Then, by performing a solid-liquid separation operation after the extraction operation, the extract as an extract and the residue of the specific algal body are separated. As the method of the solid-liquid separation operation, for example, known separation methods such as filtration and centrifugation can be used. The obtained extract may be subjected to concentration or drying treatment as necessary. 【0017】 The extraction operation and the solid-liquid separation operation may be performed only once on the same specific algal body, or may be performed multiple times. Also, when the extraction operation is performed multiple times, the same extraction operation may be repeated, or different extraction operations may be combined. For example, after performing an extraction operation and a solid-liquid separation operation using an organic solvent, an extraction operation using water may be performed on the residue of the specific algal body on which those operations were performed. When different extraction operations are combined, the extracts obtained by each extraction operation may be mixed and used as one extract, or may be used as separate different extracts. 【0018】 [Actions and Application Fields] [Actions and Application Fields Related to Cell Death Induced by Oxidative Stress] Ingestion of this composition suppresses cell death induced by oxidative stress. Cell death induced by oxidative stress is, for example, ferroptosis. Therefore, this composition can be used as a cell-protective composition aimed at suppressing cell death induced by oxidative stress. Furthermore, this composition can be used as a ferroptosis inhibitor aimed at inhibiting ferroptosis. Examples of the oxidative stress mentioned above include glutamate damage. 【0019】 Ferroptosis is a regulatory form of cell death triggered by iron-dependent lipid oxidation. The mechanism of ferroptosis induction by oxidative stress is described below with reference to Figure 1. 【0020】 As shown in Figure 1, iron is divalent iron (Fe 2+ ) and trivalent iron (Fe 3+ Iron is a transition metal that moves between ferrous and divalent states. Within cells, trivalent iron is stored bound to ferritin, a type of iron-binding protein. Divalent iron acts as a catalyst in the Fenton reaction, in which reactive oxygen species (ROS) are generated from hydrogen peroxide. The reactive oxygen species produced by the Fenton reaction react with membrane lipids, resulting in a chain reaction of lipid oxidation. Ferroptosis occurs when lipid peroxides produced by these iron-dependent lipid oxidation reactions accumulate excessively. 【0021】 Furthermore, cells possess antioxidant mechanisms to suppress the accumulation of lipid peroxides. Cells utilize cystine transporters (xCTs) located in the cell membrane to take in extracellular cystine and release intracellular glutamate to the outside of the cell through exchange transport. The cystine taken into the cell is used to produce reduced glutathione, an antioxidant. Glutathione peroxidase 4 (GPX4) uses reduced glutathione to reduce lipid peroxides produced by the reaction of reactive oxygen species. This suppresses the accumulation of lipid peroxides and inhibits ferroptosis caused by excessive accumulation of lipid peroxides. 【0022】 Ferroptosis is induced by the inhibition of antioxidant mechanisms. For example, when the extracellular glutamate concentration is high, the uptake of cystine into cells via cystine transporters is restricted. In this case, the cells are unable to produce enough reduced glutathione. As a result of the deficiency of reduced glutathione, glutathione peroxidase 4 is unable to reduce lipid peroxides. Consequently, the accumulation of lipid peroxides progresses, and ferroptosis is induced by the excessive accumulation of lipid peroxides. 【0023】 (Mechanisms and application fields of cell death induced by endoplasmic reticulum stress) Ingestion of this composition suppresses cell death induced by endoplasmic reticulum (ER) stress. Examples of such cell death include apoptosis induced by ER stress. Therefore, this composition can be used as a cell-protective composition aimed at suppressing ER stress-induced cell death. Furthermore, this composition can be used as an apoptosis inhibitor aimed at inhibiting apoptosis. 【0024】 Endoplasmic reticulum (ER) stress refers to a state in which proteins accumulate as defective proteins in the ER lumen due to abnormal protein folding, caused by exposure to internal or external environmental changes in cells. Factors that cause ER stress include, for example, nutrient starvation, disruption of intracellular calcium concentration, hypoxia, expression of mutant proteins, and viral infection. These defective proteins are removed by a response mechanism called the ER stress response. When the stress state progresses to a point where the ER stress response can no longer cope, the cell undergoes apoptosis. 【0025】 The mechanism of apoptosis induction by endoplasmic reticulum stress will be described below with reference to Figure 2. Examples of pathways involved in the above apoptosis induction mechanism include the IRE1 pathway, the PERK pathway, and the ATF6 pathway. This composition is preferably applied to inhibit apoptosis based on the IRE1 pathway and the PERK pathway, and more preferably to inhibit apoptosis based on the IRE1 pathway. 【0026】 The IRE1 pathway is a pathway based on the activation of IRE1 (inositol-requiring enzyme-1). IRE1 is a sensor protein possessing a kinase domain and an RNase domain, and is activated by autophosphorylation under endoplasmic reticulum stress. The RNase domain of activated IRE1 splices the mRNA of the transcription factor XBP-1. The protein translated from the spliced ​​XBP-1 mRNA functions as the transcription factor XBP-1. XBP-1 then triggers apoptosis. 【0027】 Furthermore, when IRE1 is activated, the adapter proteins TRAF2 and ASK1 are recruited, and the IRE1 kinase domain phosphorylates them. The phosphorylated ASK1 then phosphorylates JNK (c-Jun N-terminal kinase), and the phosphorylated JNK triggers apoptosis. 【0028】 The PERK pathway is a pathway based on the activation of PERK (PKR-like ER kinase). PERK is activated by autophosphorylation. Activated PERK phosphorylates eIF2α, a subunit of the translation initiation factor. Phosphorylation of eIF2α promotes the translation of the transcription factor ATF4. ATF4 then triggers apoptosis. 【0029】 ATF6 (activating transcription factor 6) is a membrane protein. Under endoplasmic reticulum stress, ATF6 translocates to the Golgi apparatus and undergoes intramembrane cleavage. The cleaved N-terminal fragment (ATF6p50) triggers apoptosis. 【0030】 [effect] Next, the effects of this embodiment will be described. (1) The cell-protective composition contains an extract of Chlorogonia capiratum as an active ingredient. According to the above composition, cell death induced by at least one of oxidative stress and endoplasmic reticulum stress can be suppressed. 【0031】 (2) The above cell-protective composition has the effect of inhibiting ferroptosis induced by oxidative stress. (3) The above cell-protective composition has the effect of inhibiting apoptosis induced by endoplasmic reticulum stress. 【0032】 [Example of changes] This embodiment can be implemented with the following modifications. This embodiment and the following modifications can be combined with each other to the extent that they do not contradict each other technically. 【0033】 This composition may contain other components, provided that they do not impair the intended effects of each component. The amount and duration of intake of this composition are not particularly limited and should be determined as appropriate, taking into consideration the physical condition, age, sex, and other conditions of the person taking the intake. 【0034】 This composition can be applied not only to humans, but also to feed, pharmaceuticals, etc., for livestock and other animals. [Note] Next, we will describe the technical concepts that can be understood from the above embodiments and modified examples. 【0035】 (i) A ferroptosis inhibitor that inhibits ferroptosis induced by oxidative stress, comprising an extract of chloragonium capiratum as an active ingredient. 【0036】 (b) An apoptosis inhibitor that inhibits apoptosis induced by endoplasmic reticulum stress, comprising an extract of chloragonium capiratum as an active ingredient. 【0037】 [Explanation of impossible or impractical circumstances] The description of "extract of chloragonium capiratum" in the claims of this application falls under the category of "circumstances where it is impossible or impractical to directly identify the substance by its structure or properties at the time of filing ('impossible / impractical circumstances')." 【0038】 Chlorogonia capiratum contains countless components that leach into the extraction solvent. Isolating the active ingredient that inhibits specific cell death from among these countless components and analyzing its structure requires an excessive amount of economic expenditure and time. Furthermore, in formulations, it is common practice to include the specific active ingredient in the form of an extract or its crude form, rather than in an isolated and purified state. In particular, biological substances tend to be safer in unpurified or crudely purified states compared to chemically synthesized substances, and their inclusion in unpurified or crudely purified states is actively pursued. 【0039】 Therefore, given the rapid advancements in technology and the intense international competition in patent acquisition, it would be unreasonable to require applicants to isolate active ingredients that suppress specific cell death from chloragonium capiratum extracts and analyze their structures. Accordingly, the description of "chloragonium capiratum extract" in the claims of this application falls under the category of "circumstances where it is impossible or impractical to directly identify the substance by its structure or properties at the time of filing ('impossible / impractical circumstances')." [Examples] 【0040】 The following describes an embodiment that further elaborates on the above embodiment. <Preparation of algal samples> Dimethyl sulfoxide (1 mL) was added to dried Chlorogonia capiratum powder (10.6 mg), and the mixture was stirred in a vortex mixer for 5 minutes, followed by centrifugation (room temperature, 3000 rpm, 5 minutes). The supernatant after centrifugation was filtered and sterilized using a 20 μm filter to obtain the algal sample (10.6 mg / mL). 【0041】 <Test 1: Test on cell death due to oxidative stress> In Experiment 1 and Experiment 2 (described later), glutamate was used as an oxidative stress inducer, and N-acetylcysteine ​​(NAC) was used as a known cell death inhibitor to suppress cell death caused by oxidative stress. 【0042】 Mouse hippocampal cells (HT22 cells) were seeded into a 96-well plate (cell density: 3 × 10⁶). 3 Cells were cultured in a well. After 24 hours, the culture medium was changed and algal samples (final concentrations: 1, 3, 10 μg / mL) were added. One hour later, glutamic acid (final concentration: 5 mM) was added, and the cells were cultured for another 24 hours. Cell viability was then measured by the CCK-8 assay. The results are shown in Figure 3(a). In addition, the rate of dead cells was measured by propidium iodide staining and nuclear staining. The results are shown in Figure 3(b). Each graph in Figure 3 shows the mean and standard error for a sample size of 6. 【0043】 The control groups C1-C3 shown in Figure 3 were the control groups used in this study, and their details are as follows. Control example C1: Test example without the addition of algal tissue samples and glutamic acid. 【0044】 Control example C2: A negative control test example in which dimethyl sulfoxide was added instead of the algal sample. Control example C3: A positive control test example in which NAC (final concentration: 3 mM) was added instead of the algal sample. 【0045】 In Figure 3, "♯♯", "**", and "++" indicate a significant difference from the control group. Details are as follows: "##": There is a statistically significant difference between this case and control example C1, with a p-value of less than 0.01. 【0046】 "**": There is a statistically significant difference between this case and control case C2, with a p-value of less than 0.01. "++": There is a statistically significant difference between this case and control case C2, with a p-value of less than 0.01. As shown in Figures 3(a) and 3(b), control example C2, in which oxidative stress was induced by the addition of glutamic acid, showed a decrease in cell viability and an increase in the rate of dead cells compared to control example C1, in which oxidative stress was not induced. In contrast, the test examples in which glutamic acid and algal samples were added showed a significant increase in cell viability and a significant decrease in the rate of dead cells compared to control example C2. Furthermore, the increase in cell viability and decrease in the rate of dead cells in the above test examples showed a concentration-dependent trend. These results indicate that the addition of algal samples can suppress cell death caused by oxidative stress. 【0047】 <Test 2: Test on the effects on oxidative stress-related proteins> HT22 cells were seeded into a 96-well plate (cell density: 3 × 10⁶). 3 Cells were cultured in a well. After 24 hours, the culture medium was changed and an algal sample (final concentration: 10 μg / mL) was added. One hour later, glutamic acid (final concentration: 5 mM) was added, and the cells were cultured for another 24 hours. The expression of ferritin, one of the oxidative stress-related proteins, was then confirmed by Western blotting. An example of the results is shown in Figure 4(a). Based on the band intensity of the Western blotting, the expression level of ferritin relative to β-actin was quantified. The results are shown in Figure 4(b). The graph in Figure 4(b) shows the mean and standard error for a sample size of 6. 【0048】 The control groups C1, C2, and C4 shown in Figure 4(b) were the control groups used in this study, and their details are as follows. Control example C1: Test example without the addition of algal tissue samples and glutamic acid. 【0049】 Control example C2: A negative control test example in which dimethyl sulfoxide was added instead of the algal sample. Control example C4: Test example without the addition of glutamic acid. In Figure 4(b), "♯♯" and "*" indicate a significant difference from the control group. Details are as follows: 【0050】 "##": There is a statistically significant difference between this case and control example C1, with a p-value of less than 0.01. "**": There is a statistically significant difference between this case and control case C2, with a p-value of less than 0.05. As shown in Figure 4(b), control example C2, in which oxidative stress was induced by the addition of glutamate, showed increased ferritin expression compared to control example C1, in which oxidative stress was not induced. In contrast, the test examples in which glutamate and algal samples were added showed a significant decrease in ferritin expression compared to control example C2. These results indicate that the algal sample is involved in the biological mechanism that induces ferritin expression. Since ferritin is an iron-binding protein involved in ferroptosis, it is thought that the algal sample suppresses ferroptosis caused by oxidative stress. 【0051】 <Test 3: Test on cell death induced by endoplasmic reticulum stress> In Experiment 3 and Experiment 4 (described later), tunicamycin was used as an endoplasmic reticulum stress inducer, and tauroursodeoxycholic acid (TUDCA) was used as a known cell death inhibitor to suppress cell death caused by endoplasmic reticulum stress. 【0052】 HT22 cells were seeded into a 96-well plate (cell density: 3 × 10⁶). 3Cells were cultured in a well. After 24 hours, the culture medium was changed and thallus samples (final concentrations: 1, 3, 10 μg / mL) were added. One hour later, tunicamycin (final concentration: 50 ng / mL) was added, and the cells were cultured for another 24 hours. Cell viability was then measured by the CCK-8 assay. The rate of dead cells was also measured by propidium iodide staining and nuclear staining. The results are shown in Figure 5. Each graph in Figure 5 shows the mean and standard error for a sample size of 6. 【0053】 The control groups C1-C3 shown in Figure 5 were the control groups used in this study, and their details are as follows. Control example C1: Test example without the addition of algal samples and tunicamycin. 【0054】 Control example C2: A negative control test example in which dimethyl sulfoxide was added instead of the algal sample. Control example C3: A positive control test example in which TUDCA (final concentration: 50 ng / mL) was added instead of the algal sample. 【0055】 Furthermore, in Figure 5, "♯♯", "**", "*", "++", and "+" indicate a significant difference from the control group. Details are as follows. "##": There is a statistically significant difference between this case and control example C1, with a p-value of less than 0.01. 【0056】 "**": There is a statistically significant difference between this case and control case C2, with a p-value of less than 0.01. *: There is a statistically significant difference between this case and control case C2, with a p-value of less than 0.05. "++": There is a statistically significant difference between this case and control case C2, with a p-value of less than 0.01. 【0057】 "+": There is a statistically significant difference between this case and control case C2, with a p-value of less than 0.05. As shown in Figures 5(a) and 5(b), control case C2, in which endoplasmic reticulum stress was induced by the addition of tunicamycin, showed a decrease in cell viability and an increase in cell death rate compared to control case C1, in which endoplasmic reticulum stress was not induced. In contrast, the test cases in which tunicamycin and algal samples were added showed a significant increase in cell viability and a significant decrease in cell death rate compared to control case C2. These results indicate that cell death due to endoplasmic reticulum stress can be suppressed by the addition of algal samples. 【0058】 <Test 4: Test on the effects on endoplasmic reticulum stress-related proteins> HT22 cells were seeded into a 96-well plate (cell density: 3 × 10⁶). 3 Cells were cultured in a well. After 24 hours, the culture medium was changed and an algal sample (final concentration: 10 μg / mL) was added. One hour later, tunicamycin (final concentration: 50 ng / mL) was added, and the cells were cultured for another 24 hours. Endoplasmic reticulum stress-related proteins were then detected by Western blotting. An example of the results is shown in Figure 6. The endoplasmic reticulum stress-related proteins targeted for detection in this study were JNK1, JNK2, p-JNK1, p-JNK2, p-PERK, ATF6, and XBP1s. Based on the band intensity of the Western blotting, the phosphorylation ratios of JNK1 and JNK2 (p-JNK1 / JNK1 and p-JNK2 / JNK2) were measured. In addition, based on the band intensity of the Western blotting, the detection amount of p-PERK relative to β-actin, and the expression levels of ATF6 and XBP1s relative to GAPDH were quantified. These results are shown in Figure 7. Each graph in Figure 7 shows the mean and standard error for a sample size of 6. 【0059】 The control groups C1, C2, and C4 shown in Figure 7 were the control groups used in this study, and their details are as follows. Control example C1: Test example without the addition of algal samples and tunicamycin. 【0060】 Control example C2: A negative control test example in which dimethyl sulfoxide was added instead of the algal sample. Control example C4: A test example without tunicamycin. In Figure 7, "♯♯", "♯", and "*" indicate a significant difference from the control group. Details are as follows: 【0061】 "##": There is a statistically significant difference between this case and control example C1, with a p-value of less than 0.01. "#": There is a statistically significant difference between this case and control example C1, with a p-value of less than 0.05. *: There is a statistically significant difference between this case and control case C2, with a p-value of less than 0.05. 【0062】 As shown in Figures 7(a)-(c), in control case C2, where endoplasmic reticulum stress was induced by the addition of tunicamycin, the phosphorylation ratios of JNK1 and JNK2 increased, and the amount of p-PERK detected also increased, compared to control case C1, where endoplasmic reticulum stress was not induced. In contrast, in the test cases to which tunicamycin and algal samples were added, the phosphorylation ratios of JNK1 and JNK2 decreased, and the amount of p-PERK detected also decreased, compared to control case C2. In particular, the phosphorylation ratio of JNK1 decreased significantly. 【0063】 On the other hand, as shown in Figures 7(d) to (e), control case C2, in which endoplasmic reticulum stress was induced by the addition of tunicamycin, showed increased expression levels of ATF6 and XBP1s compared to control case C1, in which endoplasmic reticulum stress was not induced. In contrast, the test cases in which tunicamycin and algal samples were added showed almost no change in the expression levels of ATF6 and XBP1s compared to control case C2. 【0064】 These results indicate that the algal sample is involved in a biological mechanism that phosphorylates endoplasmic reticulum stress-related proteins. JNK is a protein associated with the IRE1 pathway, and p-PERK is a protein associated with the PERK pathway. Therefore, it is thought that the algal sample suppresses apoptosis induced by endoplasmic reticulum stress by acting on the IRE1 pathway and the PERK pathway, particularly the IRE1 pathway.

Claims

[Claim 1] A cell-protective composition that suppresses cell death induced by at least one of oxidative stress and endoplasmic reticulum stress, A cell-protective composition containing an extract of Chlorogonia capiratum as an active ingredient. [Claim 2] The cell-protective composition according to claim 1, which is applied as a ferroptosis inhibitor that inhibits ferroptosis induced by oxidative stress. [Claim 3] The cell-protective composition according to claim 1, which is applied as an apoptosis inhibitor that inhibits apoptosis induced by endoplasmic reticulum stress. [Claim 4] The cell-protective composition according to claim 3, which inhibits apoptosis based on the IRE1 pathway. [Claim 5] The cell-protective composition according to claim 3, which inhibits apoptosis based on the PERK pathway.

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