Composition for cell protection

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 protecting cells. [Background technology]

[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 algae of the genus Chlorogonia, family Haematococcaceae, order Volvocales, class Chlorophyceae, division Chlorophyta, promote the production of brain-derived trophic factor (BDNF) and nerve growth factor (NGF). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 2023-158928 Summary of the Invention [Problem to be solved by the invention]

[0004] The present researchers have newly discovered that extracts from Chlorogonium capillatum, a member of the Chlorogonium genus, Haematococcaceae family, Volvocales order, Chlorophyceae, Division of Chlorophyta, can suppress cell death caused by oxidative stress and endoplasmic reticulum stress. The present invention was made based on these findings. [Means for solving the problem]

[0005] The cell protective composition of the invention that solves the above problems is a cell protective composition that suppresses cell death induced by at least one of oxidative stress and endoplasmic reticulum stress, and contains an extract of Chlorogonia capillatum as an active ingredient.

[0006] One embodiment of the cell protection composition is applied as a ferroptosis inhibitor that inhibits ferroptosis induced by oxidative stress. One embodiment of the cell protection composition is applied as an apoptosis inhibitor that inhibits apoptosis induced by endoplasmic reticulum stress.

[0007] In one embodiment of the above-mentioned cell protection composition, the apoptosis inhibitor inhibits the apoptosis based on the IRE1 pathway. In one embodiment of the above-mentioned cell protection composition, the apoptosis inhibitor inhibits the apoptosis based on the PERK pathway. [Effects of the Invention]

[0008] The cell protection composition of the present invention can suppress cell death induced by oxidative stress or endoplasmic reticulum stress. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 is an explanatory diagram of the mechanism by which ferroptosis is induced by oxidative stress. [Figure 2] FIG. 2 is an explanatory diagram of the mechanism of apoptosis induction by endoplasmic reticulum stress. [Figure 3] 3(a) and 3(b) are graphs showing the changes in cell viability and cell death rate when oxidative stress is applied. [Figure 4] 4(a) and 4(b) are the results of Western blotting and a graph showing the change in the expression level of ferritin when oxidative stress is applied. [Figure 5] 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] FIG. 6 shows the results of Western blotting showing changes in the expression levels of endoplasmic reticulum stress-related proteins when endoplasmic reticulum stress is applied. [Figure 7] 7(a) to 7(e) are graphs showing changes in the expression levels of endoplasmic reticulum stress-related proteins when endoplasmic reticulum stress is applied. DETAILED DESCRIPTION OF THE INVENTION

[0010] An embodiment of the present invention will be described below. The composition of this embodiment (hereinafter referred to as the present composition) contains an extract of Chlorogonium capratum as an active ingredient.

[0011] [Raw materials] Chlorogonium capillatum is an alga of the Chlorogonium genus, Volvocales order, Haematococcaceae family, Chlorophyta division. Chlorogonium capillatum (hereinafter sometimes referred to as "specific alga") may be a naturally occurring alga or an artificially cultivated alga. However, the use of artificially cultivated alga is industrially preferable due to the stable supply and ease of maintaining quality.

[0012] [Extract] Examples of extraction solvents that can be used to extract an extract from specific algae include water, organic solvents, and mixed solvents of organic solvents and water. Examples of the organic solvents include lower alcohols, hexane, ethyl acetate, dimethyl sulfoxide, acetonitrile, acetone, glycerin, and propylene glycol. Examples of lower alcohols include alcohols with 1 to 5 carbon atoms, such as methanol, ethanol, propanol, isopropanol, and butanol.

[0013] The organic solvent may be a single type or a mixed solvent of multiple types. 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. The extraction solvent may also contain additives. Examples of additives include organic salts, inorganic salts, buffers, and emulsifiers.

[0014] A known extraction method can be used to extract an extract from a specific alga. Examples of known extraction methods include cold extraction, room temperature extraction, and heated extraction. The extraction temperature can be appropriately set depending on the type of solvent, extraction efficiency, deterioration of components, etc.

[0015] The extraction operation is carried out by immersing the raw algae cells in the extraction solvent for a predetermined period of time. The concentration of the algae cells in the extraction solvent can be appropriately set depending on the type of extraction solvent, extraction efficiency, efficiency of the concentration treatment after extraction, etc. In this extraction operation, further treatments such as refluxing, stirring, pressurization, and ultrasonic treatment may be carried out as necessary to increase the extraction efficiency.

[0016] After the extraction, a solid-liquid separation operation is performed to separate the extract from the residue of the specific algae. Known separation methods such as filtration and centrifugation can be used for the solid-liquid separation operation. The obtained extract may be concentrated or dried as needed.

[0017] The extraction operation and solid-liquid separation operation may be performed once or multiple times on the same specific algae. When performing the extraction operation multiple times, the same extraction operation may be performed repeatedly, or different extraction operations may be performed in combination. For example, after performing the extraction operation using an organic solvent and the solid-liquid separation operation, the residue of the specific algae that has undergone these operations may be subjected to an extraction operation using water. When different extraction operations are performed in combination, the extracts obtained by each extraction operation may be mixed and used as a single extract, or may be used as separate, different extracts.

[0018] [Action and application field] (Actions and application areas related to cell death induced by oxidative stress) By ingesting this composition, cell death induced by oxidative stress is suppressed. Cell death induced by oxidative stress is, for example, ferroptosis. Therefore, this composition can be used as a cell protection composition for suppressing cell death induced by oxidative stress. Furthermore, this composition can be used as a ferroptosis inhibitor for inhibiting ferroptosis. In addition, examples of the above-mentioned oxidative stress include glutamate damage.

[0019] Ferroptosis is a regulated cell death induced by iron-dependent lipid oxidation. The mechanism of ferroptosis induction by oxidative stress is explained below with reference to Figure 1.

[0020] As shown in Figure 1, iron is present in the form of divalent iron (Fe 2+ ) and iron(III) 3+ ) is a transition metal that travels between the iron-binding and iron-sulfate complexes. Within cells, trivalent iron is stored bound to ferritin, a type of iron-binding protein. Divalent iron catalyzes the Fenton reaction, which generates reactive oxygen species (ROS) from hydrogen peroxide. The reactive oxygen species generated by the Fenton reaction react with membrane lipids, causing a chain reaction of lipid oxidation. Ferroptosis occurs when the lipid peroxides generated by these iron-dependent lipid oxidation reactions accumulate excessively.

[0021] Cells also possess antioxidant mechanisms to suppress the accumulation of lipid peroxides. Cells use the cystine transporter (xCT) present in the cell membrane to transport extracellular cystine into the cell and release intracellular glutamate out of the cell. Once cystine is taken up into the cell, it is used to produce reduced glutathione, an antioxidant. Glutathione peroxidase 4 (GPX4) uses reduced glutathione to reduce lipid peroxides produced by reaction with reactive oxygen species. This suppresses the accumulation of lipid peroxides and also suppresses ferroptosis, which is 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, reduced glutathione cannot be produced sufficiently within the cell. Furthermore, the lack of reduced glutathione prevents glutathione peroxidase 4 from reducing lipid peroxides. As a result, lipid peroxides accumulate, and excessive accumulation of lipid peroxides induces ferroptosis.

[0023] (Effects and applications of endoplasmic reticulum stress-induced cell death) Ingestion of this composition suppresses cell death induced by endoplasmic reticulum stress. Examples of such cell death include apoptosis induced by endoplasmic reticulum stress. Therefore, this composition can be used as a cell protection composition for suppressing cell death induced by endoplasmic reticulum stress. Furthermore, this composition can be used as an apoptosis inhibitor for inhibiting apoptosis.

[0024] Endoplasmic reticulum stress refers to a state in which proteins do not fold normally in the lumen of the endoplasmic reticulum due to exposure of cells to internal or external environmental changes, and accumulate as defective proteins. Factors that cause endoplasmic reticulum stress include, for example, nutrient starvation, disturbances in intracellular calcium concentration, hypoxia, expression of mutant proteins, and viral infection. The defective proteins are removed by a response mechanism known as the endoplasmic reticulum stress response. When the stress state progresses to a point where the endoplasmic reticulum stress response cannot cope with it, the cell undergoes apoptosis.

[0025] The mechanism of apoptosis induction by endoplasmic reticulum stress will be described below with reference to Figure 2. Pathways involved in the apoptosis induction mechanism include, for example, the IRE1 pathway, the PERK pathway, and the ATF6 pathway. The present composition is preferably used to inhibit apoptosis via the IRE1 pathway and the PERK pathway, and more preferably used to inhibit apoptosis via the IRE1 pathway.

[0026] The IRE1 pathway is based on the activation of IRE1 (inositol-requiring enzyme-1). IRE1 is a sensor protein with a kinase domain and an RNase domain, and is activated by autophosphorylation upon endoplasmic reticulum stress. The RNase domain of activated IRE1 splices the mRNA for the transcription factor XBP-1. The protein translated by the spliced ​​XBP-1 mRNA acts as the transcription factor XBP-1. XBP-1 induces apoptosis.

[0027] Activation of IRE1 also recruits the adaptor proteins TRAF2 and ASK1, which are then phosphorylated by the IRE1 kinase domain. The phosphorylated ASK1 then phosphorylates JNK (c-Jun N-terminal kinase), which then induces apoptosis.

[0028] The PERK pathway is 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 induces 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) induces apoptosis.

[0030] [effect] Next, the effects of this embodiment will be described. (1) A cell-protecting composition containing an extract of Chlorogonium capillatum as an active ingredient can suppress cell death induced by at least one of oxidative stress and endoplasmic reticulum stress.

[0031] (2) The above-mentioned cell protecting composition has the effect of inhibiting ferroptosis induced by oxidative stress. (3) The above-mentioned cell protective composition has the effect of inhibiting apoptosis induced by endoplasmic reticulum stress.

[0032] [Example of change] This embodiment can be modified as follows: This embodiment and the following modifications can be combined and implemented within the scope of technical compatibility.

[0033] This composition may contain other ingredients to the extent that the intended effects of each ingredient are not impaired. The intake amount and intake period of this composition are not particularly limited and may be determined appropriately taking into consideration the state of the user's physical function, age, sex, and other conditions.

[0034] The present composition can be applied not only to humans but also to livestock and other domestic animals as feed, medicine, etc. [Note] Next, the technical ideas that can be understood from the above-described embodiment and modified examples will be described.

[0035] (i) A ferroptosis inhibitor that inhibits ferroptosis induced by oxidative stress, comprising an extract of Chlorogonia capillatum as an active ingredient.

[0036] (b) An apoptosis inhibitor that inhibits apoptosis induced by endoplasmic reticulum stress, the apoptosis inhibitor comprising an extract of Chlorogonia capillatum as an active ingredient.

[0037] [Explanation of impossible / impractical circumstances] The description of "extract of Chlorogonia capratum" in the claims of this application falls under the case where "circumstances exist where it is impossible or practical to directly identify the substance by its structure or properties at the time of filing ('impossible or impractical circumstances')."

[0038] Chlorogonia capillatum contains numerous components that dissolve in the extraction solvent. Isolating the active ingredient that inhibits specific cell death from these numerous components and analyzing its structure requires significant financial and time expenditures. Furthermore, formulations typically contain specific active ingredients in the form of extracts or crudely purified products rather than in their isolated and purified form. In particular, biologically derived substances tend to be safer in their unpurified or crudely purified state compared to chemically synthesized substances, and therefore are actively used in these forms.

[0039] Therefore, in the context of rapid technological advances and fierce international competition in the patent acquisition arena, it is unreasonable to require an applicant to isolate the active ingredient that inhibits specific cell death from an extract of Chloragonium capiratum and analyze its structure. Therefore, the description of "an extract of Chloragonium capiratum" in the claims of this patent application falls under the case where "at the time of filing, there exist circumstances that make it impossible or impractical to directly identify the product by its structure or properties ('impossible or impractical circumstances')." [Example]

[0040] Hereinafter, a more specific example of the above embodiment will be described. <Preparation of algal samples> Dimethyl sulfoxide (1 mL) was added to dried powder of Chlorogonia capillatum (10.6 mg), stirred for 5 minutes using a vortex mixer, and then centrifuged (room temperature, 3000 rpm, 5 minutes). The supernatant was filtered and sterilized using a 20 μm filter to prepare the algae sample (10.6 mg / mL).

[0041] <Test 1: Test on cell death due to oxidative stress> In Test 1 and Test 2 described below, glutamic acid (Glutamate) was used as an oxidative stress inducer, and N-acetylcysteine ​​(NAC) was used as a known cell death inhibitor that inhibits cell death due to oxidative stress.

[0042] Mouse hippocampal-derived cells (HT22 cells) were seeded in a 96-well plate (cell density: 3 × 10 3 The cells were cultured in a 1000-well culture medium (1, 3, or 10 μg / mL). After 24 hours, the medium was replaced and algal samples (final concentrations: 1, 3, or 10 μg / mL) were added. One hour later, glutamic acid (final concentration: 5 mM) was added, and the culture was continued for another 24 hours. The cell viability was measured using a CCK-8 assay. The results are shown in Figure 3(a). The cell death rate was also measured using 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 six samples.

[0043] Control examples C1 to C3 shown in FIG. 3 are the control groups used in this test, and the details thereof are as follows. Control Example C1: A test example in which no algae sample or glutamic acid was added.

[0044] Control Example C2: A negative control test example in which dimethyl sulfoxide was added instead of the algae sample. Control Example C3: A positive control test example in which NAC (final concentration: 3 mM) was added instead of the algae sample.

[0045] In Figure 3, "##", "**" and "++" indicate significant differences from the control group. The details are as follows: "##": There is a significant difference between the control example C1 with a p-value of less than 0.01.

[0046] "**": There is a significant difference with the control example C2, with a p-value of less than 0.01. "++": There is a significant difference with the control example 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 cell death compared to control example C1, in which oxidative stress was not induced. In contrast, test examples in which glutamic acid and algal samples were added showed a significant increase in cell viability and a significant decrease in cell death compared to control example C2. Furthermore, the increase in cell viability and decrease in cell death in the above test examples showed a concentration-dependent tendency. These results demonstrate that the addition of algal samples can suppress cell death due to oxidative stress.

[0047] <Test 2: Test on effects on oxidative stress-related proteins> HT22 cells were seeded in a 96-well plate (cell density: 3 × 10 3 The cells were cultured in a 1000-well culture medium (3000 cells / well). After 24 hours, the medium was replaced and algal samples (final concentration: 10 μg / mL) were added. One hour later, glutamic acid (final concentration: 5 mM) was added, and the culture was continued for another 24 hours. The expression of ferritin, an oxidative stress-related protein, was confirmed by Western blotting. An example of the results is shown in Figure 4(a). The expression level of ferritin relative to β-actin was quantified based on the band intensity in the Western blotting. The results are shown in Figure 4(b). The graph in Figure 4(b) shows the mean and standard error for six samples.

[0048] Control examples C1, C2, and C4 shown in FIG. 4(b) are the control groups used in this test, and their details are as follows. Control Example C1: A test example in which no algae sample or glutamic acid was added.

[0049] Control Example C2: A negative control test example in which dimethyl sulfoxide was added instead of the algae sample. Control Example C4: A test example in which glutamic acid was not added. In Figure 4(b), "##" and "*" indicate significant differences from the control group. The details are as follows:

[0050] "##": There is a significant difference between the control example C1 with a p-value of less than 0.01. "**": There is a significant difference with the control example C2, with a p-value of less than 0.05. As shown in Figure 4(b), the control example C2, in which oxidative stress was induced by the addition of glutamic acid, showed an increased level of ferritin expression compared to the control example C1, in which oxidative stress was not induced. In contrast, the test example in which glutamic acid and the algal cell sample were added showed a significant decrease in ferritin expression compared to the control example C2. These results indicate that the algal cell sample is involved in the biological mechanism that expresses ferritin. Since ferritin is an iron-binding protein involved in ferroptosis, it is thought that the algal cell sample suppresses ferritosis caused by oxidative stress.

[0051] <Test 3: Test on cell death caused by endoplasmic reticulum stress> In Test 3 and Test 4 described below, tunicamycin was used as an endoplasmic reticulum stress inducer, and tauroursodeoxycholic acid (TUDCA) was used as a known cell death inhibitor that suppresses cell death due to endoplasmic reticulum stress.

[0052] HT22 cells were seeded in a 96-well plate (cell density: 3 × 10 3The cells were cultured in a 1000-well culture medium (300 μg / well). After 24 hours, the medium was replaced and algal samples (final concentrations: 1, 3, and 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. The cell viability was measured by CCK-8 assay. The cell death rate 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 six samples.

[0053] Control examples C1 to C3 shown in FIG. 5 are the control groups used in this test, and the details thereof are as follows. Control Example C1: A test example in which no algal sample or tunicamycin was added.

[0054] Control Example C2: A negative control test example in which dimethyl sulfoxide was added instead of the algae sample. Control Example C3: A positive control test example in which TUDCA (final concentration: 50 ng / mL) was added instead of the algae sample.

[0055] In Figure 5, "##", "**", "*", "++", and "+" indicate significant differences from the control group. The details are as follows: "##": There is a significant difference between the control example C1 with a p-value of less than 0.01.

[0056] "**": There is a significant difference with the control example C2, with a p-value of less than 0.01. "*": There is a significant difference with the control example C2, with a p-value of less than 0.05. "++": There is a significant difference with the control example C2, with a p-value of less than 0.01.

[0057] "+": There is a significant difference with the control example C2, with a p-value of less than 0.05. As shown in Figures 5(a) and 5(b), control example C2, in which endoplasmic reticulum stress was induced by the addition of tunicamycin, showed a decreased cell viability and an increased cell death rate compared to control example C1, in which endoplasmic reticulum stress was not induced. In contrast, test examples in which tunicamycin and the algal sample were added showed a significant increase in cell viability and a significant decrease in cell death compared to control example C2. These results demonstrate that the addition of the algal sample can suppress cell death due to endoplasmic reticulum stress.

[0058] <Test 4: Test on effects on endoplasmic reticulum stress-related proteins> HT22 cells were seeded in a 96-well plate (cell density: 3 × 10 3 The cells / well were cultured. After 24 hours, the medium was replaced and the algal sample (final concentration: 10 μg / mL) was added. One hour later, tunicamycin (final concentration: 50 ng / mL) was added. After further culture for 24 hours, endoplasmic reticulum stress-related proteins were detected by Western blotting. An example of the results is shown in Figure 6. The endoplasmic reticulum stress-related proteins detected in this study were JNK1, JNK2, p-JNK1, p-JNK2, p-PERK, ATF6, and XBP1s. The phosphorylation ratios of JNK1 and JNK2 (p-JNK1 / JNK1 and p-JNK2 / JNK2) were measured based on the band intensity of Western blotting. Furthermore, the amount of p-PERK detected relative to β-actin, and the expression levels of ATF6 and XBP1s relative to GAPDH were quantified based on the band intensity of Western blotting. The results are shown in Figure 7. Each graph in FIG. 7 shows the average value and standard error for six samples.

[0059] Control examples C1, C2, and C4 shown in FIG. 7 are the control groups used in this test, and their details are as follows. Control Example C1: A test example in which no algal sample or tunicamycin was added.

[0060] Control Example C2: A negative control test example in which dimethyl sulfoxide was added instead of the algae sample. Control Example C4: Test example in which tunicamycin was not added. In Figure 7, "##", "#" and "*" indicate significant differences from the control group. The details are as follows:

[0061] "##": There is a significant difference between the control example C1 with a p-value of less than 0.01. "#": There is a significant difference with the control example C1, with a p-value of less than 0.05. "*": There is a significant difference with the control example C2, with a p-value of less than 0.05.

[0062] As shown in Figures 7(a) to 7(c), in Control Example C2, in which endoplasmic reticulum stress was induced by the addition of tunicamycin, the phosphorylation rates of JNK1 and JNK2 increased, and the detected amount of p-PERK increased, compared to Control Example C1, in which endoplasmic reticulum stress was not induced. In contrast, in the test example in which tunicamycin and an algal sample were added, the phosphorylation rates of JNK1 and JNK2 decreased, and the detected amount of p-PERK decreased, compared to Control Example C2. In particular, the phosphorylation rate of JNK1 decreased significantly.

[0063] 7(d)-(e), the expression levels of ATF6 and XBP1s in control C2, in which endoplasmic reticulum stress was induced by the addition of tunicamycin, were increased compared to control C1, in which endoplasmic reticulum stress was not induced. In contrast, the expression levels of ATF6 and XBP1s in the test examples in which tunicamycin and algal samples were added were almost unchanged compared to control C2.

[0064] These results indicate that the algal cell sample is involved in the biological mechanism of phosphorylating ER stress-related proteins. JNK is a protein related to the IRE1 pathway, and p-PERK is a protein related to the PERK pathway. Therefore, the algal cell sample is thought to suppress apoptosis caused by ER stress by acting on the IRE1 and PERK pathways, especially the IRE1 pathway.

Claims

1. A cytoprotective composition that suppresses cell death induced by at least one of oxidative stress and endoplasmic reticulum stress, comprising: A cell-protective composition containing an extract of Chlorogonium capiratum as an active ingredient.

2. The cell protecting composition according to claim 1, which is applied as a ferroptosis inhibitor that inhibits ferroptosis induced by oxidative stress.

3. The cell protecting composition according to claim 1, which is used as an apoptosis inhibitor that inhibits apoptosis induced by endoplasmic reticulum stress.

4. The cell-protecting composition according to claim 3, which inhibits the apoptosis based on the IRE1 pathway.

5. The cell protective composition according to claim 3, which inhibits the apoptosis based on the PERK pathway.

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