Agent for increasing cardiolipin molecular species as mitochondrial component

Abstract

[Problem] The purpose of the present invention is to provide an increasing agent which contains 3, 5-dihydroxy-4-methoxybenzyl alcohol (3, 5-dihydroxy-4-methyloxyl alcohol) as an active ingredient, and which has a useful effect such as an effect of increasing the number of cardiolipin molecular species, which is a component of the mitochondrial inner membrane. [Solution] The present invention is characterized by having 3, 5-dihydroxy-4-methoxybenzyl alcohol (3, 5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient, and by having an effect of increasing the number of cardiolipin molecular species, which is a component of the mitochondrial inner membrane.

Description

Technical Field

[0001] The present invention relates to various agents for increasing cardiolipin molecular species (Japanese: molecular species, English: molecular species) which is a component of the inner membrane of mitochondria in human renal proximal tubular cells (HK-2) under oxidative stress stimulation. Background Art

[0002] Oxidative stress refers to a state in which the balance of oxidation and antioxidation in cells is disrupted, and excessive reactive oxygen species accumulate in cells. Oxidative stress is the cause of various diseases such as aging and kidney disease.

[0003] On the other hand, when ATP, which is an energy source, is produced in mitochondria, reactive oxygen species are produced as byproducts. Reports have shown that when oxidative stress is enhanced, the composition and total amount of cardiolipin, a phospholipid in the inner membrane of mitochondria, change, leading to decreased function of intracellular proteins and cell death.

[0004] However, there are many mitochondria in human renal proximal tubular cells in the kidney. The reason for this is believed to be that mitochondria need to produce a large amount of adenosine triphosphate when reabsorbing nutrients, etc.

[0005] Therefore, it is believed that the cardiolipin composition of mitochondria plays an important role in human renal proximal tubular cells in the kidney. This is because, as mentioned above, it has been reported that cardiolipin, a phospholipid in the mitochondrial inner membrane, changes in composition and decreases in total amount under conditions of enhanced oxidative stress.

[0006] Oysters (マガキ, Pacific oysters) are bivalves belonging to the Japanese Mytilidae family, and their habitat is headed by Japan and spread throughout East Asia. In addition to glycogen and protein, oysters also contain a large amount of minerals such as calcium and zinc, and are a food with high nutritional value. The inventors explored and studied physiologically active substances from oysters, and by exploring substances with antioxidant ability from oyster extracts, the antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (hereinafter referred to as DHMBA) was discovered. So far, the free radical scavenging ability of DHMBA and its role in activating the Keap1-Nrf2 pathway and inducing the expression of the antioxidant gene group (HO-1 and NQO1, etc.) associated with it have been confirmed.

[0007] The former is called direct antioxidant capacity, and the latter is called indirect antioxidant capacity. It has been proven that DHMBA is a substance that has both functions. Moreover, studies have shown that DHMBA has lower cytotoxicity than existing antioxidant substances. Therefore, it is further verified that DHMBA is useful in kidney protection.

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: Japanese Patent Application Publication No. 2017-132753 Summary of the invention

[0011] Problem that the invention aims to solve

[0012] The object of the present invention is to further develop the inventions of various useful agents having 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient that the inventors have already obtained, and to provide various agents such as an agent for increasing cardiolipin molecular species as a component of the mitochondrial inner membrane, which has 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and has useful effects such as an effect of increasing cardiolipin molecular species as a component of the mitochondrial inner membrane.

[0013] Solutions for solving problems

[0014] The invention is characterized in that 3,5-dihydroxy-4-methoxybenzyl alcohol is used as an effective ingredient and has the effect of increasing the molecular species of cardiolipin, which is a component of the mitochondrial inner membrane;

[0015] Alternatively, it is characterized in that

[0016] It contains 3,5-dihydroxy-4-methoxybenzyl alcohol as its active ingredient and has the effect of increasing the cardiolipin molecular species, which is a component of the mitochondrial inner membrane, in human renal proximal tubular cells (HK-2) under oxidative stress.

[0017] Alternatively, it is characterized in that

[0018] It contains 3,5-dihydroxy-4-methoxybenzyl alcohol as its active ingredient and has the effect of increasing the amount of total cardiolipin, a component of the mitochondrial inner membrane.

[0019] Alternatively, it is characterized in that

[0020] It contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and has the effect of increasing the amount of total cardiolipin, a component of the mitochondrial inner membrane, in human renal proximal tubular cells (HK-2) under oxidative stress.

[0021] Alternatively, it is characterized in that

[0022] It contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and has the effect of increasing the number of unsaturated fatty acid chains by changing the acyl groups of cardiolipin, a component of the inner membrane of mitochondria.

[0023] Alternatively, it is characterized in that

[0024] It contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and has the effect of increasing unsaturated fatty acid chains by changing the acyl groups of cardiolipin, a component of the inner membrane of mitochondria, in human renal proximal tubular cells (HK-2) under oxidative stress.

[0025] Alternatively, it is characterized in that

[0026] It contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and has the function of activating the biosynthesis, reconstruction and degradation of cardiolipin, a component of the mitochondrial inner membrane.

[0027] Alternatively, it is characterized in that

[0028] It contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and has the effect of activating the biosynthesis, reconstruction and degradation of cardiolipin, a component of the mitochondrial inner membrane, in human renal proximal tubular cells (HK-2) under oxidative stress.

[0029] Effects of the Invention

[0030] According to the present invention, there is provided an increasing agent having 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and having useful effects such as increasing cardiolipin molecular species as a component of the mitochondrial inner membrane. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is an explanatory diagram for explaining the strategy for exploring the effect of increasing cardiolipin (CL).

[0032] Figure 2This is an explanatory diagram for explaining the effect of DHMBA on the increase of CL molecular species under oxidative stress conditions. HK-2 cells were cultured with 100 μM BSO, 250 μM, and 500 μM DHMBA, and then recovered and CL was measured by LC-MS / MS. Mean ± SD (n = 4).

[0033] Figure 3 This is an explanatory diagram for explaining the effect of DHMBA on increasing the total CL amount under oxidative stress conditions. HK-2 cells were cultured with 100 μM BSO, 250 μM, and 500 μM DHMBA, and then recovered and CL was measured by LC-MS / MS. Mean ± SD (n = 4).

[0034] Figure 4 This is an explanatory diagram for explaining the change of acyl groups in CL caused by the addition of DHMBA under oxidative stress conditions. HK-2 cells were cultured with 100 μM BSO, 250 μM, and 500 μM DHMBA, and then recovered and CL was measured by LC-MS / MS. Mean ± SD (n = 4).

[0035] Figure 5 This is an explanatory diagram for explaining the changes in the expression levels of genes related to the biosynthesis of CL caused by DHMBA under oxidative stress conditions. HK-2 cells were cultured with 100 μM BSO, 250 μM, and 500 μM DHMBA, and the expression levels of genes related to the biosynthesis of CL were measured by qPCR. Mean ± SD (n = 4).

[0036] Figure 6 This is an explanatory diagram to illustrate the changes in the expression levels of genes related to CL reconstruction and degradation caused by DHMBA under oxidative stress conditions. HK-2 cells were cultured with 100 μM BSO, 250 μM, and 500 μM DHMBA, and the expression levels of genes related to mitochondrial biogenesis were measured by qPCR. Mean ± SD (n = 4).

[0037] Figure 7 is an explanatory diagram for explanation by Table 1 showing the primer sequences used in the present invention; DETAILED DESCRIPTION

[0038] (Example)

[0039] Hereinafter, the present invention will be described based on one embodiment shown in the drawings.

[0040] The present invention focuses on mitochondria and verifies the strategy of using human renal proximal tubule cells (HK-2) to explore the effect of DHMBA on increasing cardiolipin. Figure 1 ).

[0041] like Figure 1 As shown, when human renal proximal tubular cells (HK-2) are subjected to oxidative stress by, for example, an oxidative stress inducer, the amount of glutathione decreases.

[0042] Here, oxidative stress is generated when free radicals or reactive oxygen species are present in excess in a living body. Studies have shown that the accumulation of excessive reactive oxygen species is associated with cell damage such as damage to DNA, proteins, and lipid membranes. In addition, it is suggested that cell damage caused by reactive oxygen species is associated with the onset of various pathological conditions such as aging, asthma, arthritis, diabetes, cancer, inflammation, cardiovascular disease, atherosclerosis, Down syndrome, and neurodegenerative diseases.

[0043] Glutathione is an important tripeptide thiol in cells, composed of glutamic acid, cysteine ​​and glycine.

[0044] In addition, glutathione acts as an antioxidant, thereby protecting cells from free radical damage. Glutathione in cells exists in the form of reduced glutathione (GSH) and oxidized glutathione (GSSG). In normal cells and tissues, reduced glutathione (GSH) accounts for 90% of the total glutathione, while oxidized glutathione (GSSG) as a disulfide type is less than 10%.

[0045] The reason why the reduced glutathione (GSH) ratio is high is that the enzyme that converts the oxidized form (GSSG), glutathione reductase (GSH reductase), is always active and induced by oxidative stress, and an increase in the GSSG / GSH ratio is also considered an indicator of oxidative stress.

[0046] like Figure 1 As shown in the literature, if the amount of glutathione decreases, endogenous reactive oxygen species will increase, resulting in damage to mitochondria. In addition, changes in the amount of cardiolipin will also occur, leading to abnormal cell function or cell death.

[0047] Therefore, the inventors focused on mitochondria and used human renal proximal tubule cells (HK-2) to verify the following situations: the effect of DHMBA on increasing cardiolipin, that is, the effect of increasing the total amount of cardiolipin when DHMBA is added under oxidative stress stimulation, the effect of increasing the molecular weight of cardiolipin when DHMBA is added under oxidative stress stimulation, the effect of changing the fatty acid chain of cardiolipin when DHMBA is added under oxidative stress stimulation, and further, the mechanism of cardiolipin change: gene expression level, that is, the change in the expression level of genes related to the biosynthesis of cardiolipin when DHMBA is added under oxidative stress stimulation, and the change in the expression level of genes related to the reconstruction and decomposition of cardiolipin when DHMBA is added under oxidative stress stimulation.

[0048] "Verification experiment for exploring the effect of increasing cardiolipin, etc."

[0049] (Materials and methods)

[0050] Cell culture

[0051] Human renal proximal tubule cells (HK-2) were subcultured in a 37°C, 5% CO2 incubator using a culture medium of Dulbecco's Modified Eagle's Medium (DMEM, Nakalai Tesque) containing 10% fetal bovine serum and 1% penicillin-streptomycin. In order to confirm the effect of DHMBA on the increase of cardiolipin under oxidative stress, the following experiment was conducted using an oxidative stress inducer.

[0052] "Verification of total intracellular cardiolipin content"

[0053] HK-2 cells were seeded into 6-well plates at a density of 1.0 × 10 5 The cells were cultured for 24 hours. Then, the cells were divided into 4 groups with different stimulation conditions, and 2 mL of each was added to each well.

[0054] The four groups with different stimulation conditions are as follows.

[0055] Control group (culture medium); oxidative stress group (final concentration of 100 μM oxidative stress); oxidative stress + DHMBA 250 group (final concentration of 100 μM oxidative stress + final concentration of 250 μM DHMBA); oxidative stress + DHMBA 500 group (final concentration of 100 μM oxidative stress + final concentration of 500 μM DHMBA).

[0056] After 24 hours, the cells were washed twice with phosphate buffered saline (PBS), 1 mL of PBS was added to each well, and the cells were recovered with a scraper. 100 μL of the cell suspension was used for protein quantification by the bicinchoninic acid (BCA) method. The protein concentration was used to correct the cardiolipin concentration. The remaining cell suspension was centrifuged at 1200 rpm for 5 minutes, the supernatant was removed, and the precipitated cells were stored at -80°C.

[0057] For cardiolipin determination, total intracellular lipids were extracted as reported in the past (Chen Z, et al. Metabolomics, 16:115, 2020).

[0058] Briefly, lipids in the test body were extracted with hexane / isopropanol 3:2 (v / v, containing 0.05% butylated hydroxytoluene) and dried and solidified with an evaporator. They were then dissolved with methanol (MeOH) and centrifuged at 15,000 rpm for 15 minutes at 4°C to remove insoluble matter. The extracted total lipids were stored at -80°C. Cardiolipin was determined by LC-MS / MS in the same order as before.

[0059] "Expression levels of genes related to the biosynthesis, remodeling, and degradation of cardiolipin"

[0060] For example, it has been reported that oxidative stress stimulation caused by oxidative stress inducers induces changes in the composition of cardiolipin molecular species and a decrease in the total amount of cardiolipin, while also inducing a decrease in the function of mitochondrial membrane proteins containing the electron transport chain and a form of cell death, apoptosis (Paradies G, et al. FEBS Lett, 466: 323-326, 2000; Paradies G, et al. Cells, 8: 728, 2019).

[0061] Therefore, in the present invention, the expression levels of genes related to the biosynthesis, reconstruction, and degradation of cardiolipin under oxidative stress stimulation were analyzed and verified.

[0062] HK-2 cells were seeded into 6-well plates at a density of 5.0 × 10 4 cells / well and cultured for 24 hours.

[0063] Then, 2 mL was added to each well under the same stimulation conditions as above. After 24 hours, total RNA was recovered using NucleoSpin (registered trademark) RNA kit (MACHEREY-NAGEL). The concentration of purified RNA was quantified using Nanodrop (Invitrogen). cDNA was synthesized from 1.0 μg of total RNA according to the operating procedures of ReverTra Ace (registered trademark) qPCR RTMaster Mix with gDNA Remover (TOYOBO).

[0064] The gene expression level was measured according to the protocol of THUNDERBIRD (registered trademark) SYBR (registered trademark) qPCR Mix (TOYOBO). TM Real-time PCR analysis system (BioRad) was used for the determination. -(ΔΔCT) method to conduct analysis.

[0065] The gene expression level of the control group was set to 1 for comparison. The mean ± SD is shown in the figure (n = 4 per group). Each expression level was corrected by the expression level of β-actin as a housekeeping gene. The primer sequences used are as follows Figure 7 As shown in the table shown.

[0066] "Statistical Processing"

[0067] The data obtained were expressed as mean ± standard deviation (SD) and statistically analyzed using JMP Pro 16 (SAS Institute Inc.). Dunnett's test was used for multiple comparisons between groups, and P < 0.05 was considered statistically significant.

[0068] “Results and Investigations”

[0069] (Changes in cardiolipin molecular species)

[0070] Results to date suggest that DHMBA has a mitochondrial protective effect under conditions of oxidative stress. Here, we investigated changes in cardiolipin, an important component of the inner mitochondrial membrane ( Figure 2 ).

[0071] In the group that added the oxidative stress inducer, no significant fluctuations were found in the various cardiolipin molecular species compared to the control group; however, in the group that added the oxidative stress inducer and DHMBA, almost all cardiolipin molecular species increased significantly. Cardiolipin molecular species are related to mitochondrial function and cell function. Based on this result, it can be considered that the function of mitochondria is improved by adding DHMBA.

[0072] (Changes in total cardiolipin content)

[0073] Since almost all cardiolipin molecular species increased after the addition of DHMBA, the changes in total cardiolipin were also studied.

[0074] like Figure 3 As shown, in HK-2 cells to which 100 μM oxidative stress was added (100 μM oxidative stress + 0 μM DHMBA group), no decrease in the amount of total cardiolipin was observed.

[0075] On the other hand, the total amount of cardiolipin increased to about 1.5 times by co-treatment with 100 μM oxidative stress and 250 μM or 500 μM DHMBA.

[0076] This result indicates that oxidative stress stimulation did not change the total amount of cardiolipin, but DHMBA increased the total amount of cardiolipin. Figure 2That is, oxidative stress stimulation did not change the cardiolipin molecular species and the total amount of cardiolipin, but DHMBA increased both the cardiolipin molecular species and the total amount of cardiolipin. Therefore, it can be considered that the function of mitochondria has been improved by adding DHMBA.

[0077] (Changes in the cardiophosphatidyl group)

[0078] In addition, there are reports showing that the acyl composition of cardiolipin is related to mitochondrial function (Chicco AJ and Sparagna GC. Am J Physiol Cell Physiol, 292: C33-C44, 2007). In the verification of the present invention, the distribution of cardiolipin acyl groups was also investigated (see Figure 4 ).

[0079] After adding oxidative stress, the saturated fatty acid chain FA16:0 increased, while after adding DHMBA, the saturated fatty acid chain FA16:0 decreased, while the unsaturated fatty acid chains FA18:1 and FA18:2 increased.

[0080] It is believed that the increase in unsaturated fatty acid chains improves the fluidity of the mitochondrial inner membrane and enhances the function of membrane proteins (Fouret G, et al. Biochim Biophys Acta Bioenerg, 1847: 1025-1035, 2015).

[0081] Therefore, it is believed that the changes in the cardiolipin profile caused by DHMBA can explain the protective effect of DHMBA on mitochondria.

[0082] In addition, regarding the results of CL acyl changes, if we explain it in advance, fatty acids can be divided into two categories based on their structural differences: "saturated fatty acids" and "unsaturated fatty acids." Among them, fatty acids that do not have double bonds between carbon atoms are called saturated fatty acids, and fatty acids that contain double bonds are called unsaturated fatty acids.

[0083] The results mentioned that "after adding oxidative stress, saturated fatty acid chains FA16:1 and FA16:0 increased, while after adding DHMBA, saturated fatty acid chain FA16:0 decreased, while unsaturated fatty acid chains FA18:1 and FA18:2 increased". As we know, the more phospholipids with a large number of double bonds (i.e., high unsaturation), the higher the fluidity of the lipid bilayer. The reason is that since it is an unsaturated fatty acid, the fatty acid will bend and form a gap between adjacent phospholipids, so the membrane will become a soft membrane with fluidity.

[0084] Therefore, by adding DHMBA, "it is believed that due to the increase in unsaturated fatty acid chains, the fluidity of the mitochondrial inner membrane is improved and the function of membrane proteins is also enhanced" (Fouret G, et al. Biochim Biophys Acta Bioenerg, 1847: 1025-1035, 2015).

[0085] (Expression levels of genes related to the biosynthesis, reconstruction, and degradation of cardiolipin)

[0086] In order to study the mechanism by which DHMBA causes an increase in cardiolipin, the expression of genes related to the biosynthesis, remodeling, and degradation of cardiolipin was verified.

[0087] First, if Figure 5 As shown, in the oxidative stress group, the gene expression level of CDS2 related to cardiolipin biosynthesis was increased, but by adding DHMBA, its gene expression level was reduced to the same extent as that of the control group.

[0088] The CDS2 mentioned here is one of the synthases that functions in the CL biosynthesis pathway. Under the action of CDS, CDP-DAG is synthesized from phosphatidic acid (PA) and cytidine triphosphate (CTP). Its activity is known to exist in the endoplasmic reticulum membrane and the inner membrane of mitochondria. As CDS, two genes, CDS1 and CDS2, have been identified.

[0089] In addition, the gene expression level of PNPLA8, which is related to cardiolipin remodeling and degradation, was also increased in the oxidative stress group, but its gene expression level was reduced to the same level as the control group by adding DHMBA (see Figure 6 ).

[0090] The PNPLA8 mentioned here refers to the enzyme that cuts off the second position among phospholipase A (PLA) that can cut off fatty acids. It is an enzyme located in mitochondria and peroxisomes, involved in the metabolism of oxidized cardiolipin, and is very important for maintaining mitochondrial function. For this reason, the absence or mutation of PNPLA8 is associated with abnormal lipid metabolism and mitochondrial damage, and will produce phenotypes such as muscle weakness, atrophy of adipose tissue, hypothermia, and neurodegeneration, centered on skeletal muscle, myocardium, adipose tissue, and the brain and nervous system with active energy metabolism.

[0091] This study on the biosynthesis, remodeling and decomposition activation of cardiolipin under oxidative stress conditions shows that HK-2 cells damaged by oxidative stress may try to maintain mitochondrial function by enhancing mitochondrial synthesis and remodeling. Therefore, it may be for this reason that no changes in the total amount and composition of cardiolipin were observed after the addition of oxidative stress.

[0092] On the other hand, the addition of DHMBA reduced CDS2, which is related to cardiolipin biosynthesis, and decreased and increased the expression levels of PNPLA8 and HADHB, which are related to cardiolipin remodeling and degradation, respectively. Although the correlation between changes in gene expression levels related to cardiolipin biosynthesis, remodeling, and degradation and changes in cardiolipin profiles has not yet been clarified, it can be explained that DHMBA reduces oxidative stress, eliminating the need for HK-2 cells to compensate for oxidative stress (changes in gene expression levels).

[0093] Here is an explanation of HADH (3-hydroxyacyl-CoA dehydrogenase) as follows: long-chain fatty acids taken up into cells are metabolized in the mitochondria in sequence by various dehydrogenases corresponding to the carbon chain lengths of the fatty acids. With each step, the carbon chain is shortened by two carbon atoms, eventually generating acetyl-CoA, which contributes to energy production.

[0094] 3-Hydroxyacyl-CoA dehydrogenase (HADH) is an enzyme present in the inner membrane of mitochondria. It works synergistically with other β-oxidases and is responsible for the metabolism of medium- and short-chain acyl-CoA.

[0095] Judging from the results this time, after adding DHMBA, the function of mitochondria was maintained and there were signs of improvement thanks to the numerous cardiolipins whose functions were maintained.

[0096] (Regarding the manufacturing method of the present invention)

[0097] The following is an explanation of the methods for producing the following types of preparations with 3,5-dihydroxy-4-methoxybenzylalcohol as an active ingredient, including: an agent for increasing the molecular species of cardiolipin as a component of the inner mitochondrial membrane, an agent for increasing the total amount of cardiolipin as a component of the inner mitochondrial membrane, an agent for increasing the total amount of cardiolipin as a component of the inner mitochondrial membrane, an agent for increasing the unsaturated fatty acid chain based on the acyl change of cardiolipin as a component of the inner mitochondrial membrane, an active agent for the biosynthesis, reconstruction, and decomposition of cardiolipin as a component of the inner mitochondrial membrane, and the like.

[0098] As mentioned above, 3,5-dihydroxy-4-methoxybenzyl alcohol can be obtained from oysters, and the inventors of the present invention have obtained numerous patents for methods for efficiently extracting and producing 3,5-dihydroxy-4-methoxybenzyl alcohol from oysters. In addition, 3,5-dihydroxy-4-methoxybenzyl alcohol can also be produced by synthesis, and the inventors of the present invention have also obtained relevant patents for the synthesis method.

[0099] Furthermore, in the present invention, it is presumed that 3,5-dihydroxy-4-methoxybenzyl alcohol has many protective usefulnesses on various organs of the human body, and experiments were conducted to verify this, thereby completing the present invention.

[0100] Therefore, the useful agent of the present invention is produced after performing these verifications.

Claims

1. An agent for increasing the cardiolipin molecular species as a component of the mitochondrial inner membrane, characterized in that It contains 3,5-dihydroxy-4-methoxybenzyl alcohol as its active ingredient and has the effect of increasing the number of cardiolipin molecules, which is a component of the mitochondrial inner membrane.

2. An agent for increasing the molecular species of cardiolipin, which is a component of the inner membrane of mitochondria, characterized in that It contains 3,5-dihydroxy-4-methoxybenzyl alcohol as its active ingredient and has the effect of increasing the molecular species of cardiolipin, a component of the mitochondrial inner membrane, in human renal proximal tubular cells (HK-2) under oxidative stress.

3. An agent for increasing the amount of total cardiolipin as a component of the mitochondrial inner membrane, characterized in that It contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and has the effect of increasing the total amount of cardiolipin, a component of the mitochondrial inner membrane.

4. An agent for increasing the amount of total cardiolipin as a component of the mitochondrial inner membrane, characterized in that It contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and has the effect of increasing the amount of total cardiolipin, a component of the mitochondrial inner membrane, in human renal proximal tubular cells (HK-2) under oxidative stress.

5. An unsaturated fatty acid chain increasing agent based on the acyl change of cardiolipin which is a component of the mitochondrial inner membrane, characterized in that It contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and has the effect of increasing the number of unsaturated fatty acid chains by changing the acyl groups of cardiolipin, a component of the inner membrane of mitochondria.

6. An unsaturated fatty acid chain increasing agent based on the acyl group change of cardiolipin which is a component of the mitochondrial inner membrane, characterized in that It contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and has the effect of increasing unsaturated fatty acid chains by changing the acyl groups of cardiolipin, a component of the inner membrane of mitochondria, in human renal proximal tubular cells (HK-2) under oxidative stress.

7. An active agent for the biosynthesis, reconstruction and degradation of cardiolipin, a component of the inner membrane of mitochondria, characterized in that It contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and has the effect of activating the biosynthesis, reconstruction and degradation of cardiolipin, a component of the mitochondrial inner membrane.

8. An active agent for the biosynthesis, reconstruction and degradation of cardiolipin, a component of the inner membrane of mitochondria, characterized in that It contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and has the effect of activating the biosynthesis, reconstruction and degradation of cardiolipin, a component of the mitochondrial inner membrane, in human renal proximal tubular cells (HK-2) under oxidative stress.

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