Thiol-modified botryococcene or squalene with antioxidant activity

Abstract

To provide a novel compound derived from botryococcene or squalene having antioxidant activity, a method for producing the same, and an antioxidative composition comprising such a novel compound as an active ingredient.SOLUTION: This invention has been completed with the finding that: a thiol acetate derivative having thioacetic acid added to botryococcene or squalene and a Py-S-S-Py modification thereof have a potent antioxidant activity.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to novel compounds derived from botryococcene or squalene that have antioxidant activity, methods for producing them, and antioxidant compositions containing these novel compounds as active ingredients. [Background technology]

[0002] Reactive oxygen species (ROS), generated during various metabolic processes in the body, are known to be involved in the onset and progression of various symptoms and diseases (e.g., inflammation, allergic reactions, joint damage, rheumatoid arthritis, osteoporosis, cardiovascular disease, and cancer). For example, when lipid molecules are oxidized by ROS, peroxidation of low-density lipoproteins can occur, leading to the formation of plaque in arteries, which can lead to cardiovascular disease and atherosclerosis. Furthermore, oxidative modification of protein molecules by ROS can cause changes in tissue structure and immune abnormalities, leading to rheumatoid arthritis and connective tissue damage, and altering the appearance and function of the skin. Oxidative stress is also known to contribute not only to physical stress such as fatigue, but also to mental disorders.

[0003] The gastrointestinal tract is known to be the largest ROS-generating tissue in the body. ROS are responsible for diseases such as gastric ulcers, ulcerative colitis, pancreatitis, and fatty liver. Nonsteroidal anti-inflammatory drugs (NSAIDs), widely used to prevent infarction, induce a decrease in gastric mucus and ROS production in the gastrointestinal tract as side effects. When tissue is exposed to gastric juice due to the decrease in gastric mucus, the tissue is damaged, inducing ROS production. Furthermore, ROS production is induced by a decrease in mitochondrial membrane potential. This ROS production results in gastrointestinal mucosal damage and gastrointestinal bleeding. Because excessive bleeding can be fatal, NSAIDs are often used in combination with stomach medications. These stomach medications suppress ROS by promoting the production of reduced mucus. Furthermore, some anticoagulants have been reported to cause esophageal injury, suggesting that ROS may also be involved in this injury.

[0004] Superoxide dismutase (SOD), a representative ROS-scavenging enzyme, catalyzes the conversion of superoxide, a reactive oxygen species, into oxygen and hydrogen peroxide. The resulting hydrogen peroxide, which is also harmful, is then eliminated by catalase and glutathione peroxidase (GPx).

[0005] Various small molecules, such as various vitamins, polyphenols, catechins, glutathione, ascorbate, tocopherol, ubiquinone, bilirubin, uric acid, and sulfur compounds, can function as exogenous antioxidants, either in cooperation with the body's own antioxidant mechanisms or independently. For example, vitamin B2 acts as a coenzyme for GPx, and polyphenols have SOD-like activity. Carotenoids also function as antioxidants and may exert protective effects against oxidative stress and related chronic diseases. For example, Non-Patent Document 1 summarizes reports on the relationship between carotenoids and various chronic diseases, such as coronary heart disease, cataracts, and cancer.

[0006] Therefore, pharmaceuticals containing such substances with antioxidant activity as active ingredients can be preferably used for the treatment or prevention of diseases or symptoms related to the action of ROS. In fact, it has been reported that taking antioxidants can prevent gastrointestinal damage (Non-Patent Document 2) and improve the intestinal flora (Non-Patent Document 3).

[0007] The discovery of new compounds with good antioxidant activity, or the discovery of existing compounds with good antioxidant activity, is greatly welcomed as it will broaden the options for antioxidants, which are in high demand in the fields of medicine, food, and cosmetics.

[0008] For example, Patent Document 1 discloses the synthesis of luteolin 6-C-arabinoside as a flavonoid compound exhibiting excellent antioxidant activity, and discloses the compound, a method for producing the compound, and an antioxidant containing the compound as an active ingredient. Patent Document 2 discloses that epigallocatechin dimers and trimers have the activity of scavenging superoxide anion radicals. Patent Document 3 discloses novel nitrogen-containing heterocyclic compounds or salts thereof, and therapeutic agents containing them for oxidative stress-related diseases. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] : JP 2006-265249 [Patent Document 2] :Patent Publication No. 2010-280716 [Patent Document 3] : JP2007-016011 [Patent Document 4] :Retable 2012 / 077801 [Patent Document 5] :Retable 2012 / 077799 [Patent Document 6] : JP2013 / 035791 [Non-patent literature]

[0010] [Non-Patent Document 1] :Canfield, et al. (1992), Proc. Soc. Exp. Biol. Med. 200:260 [Non-patent document 2] :Sha S. et al. (2013), Biomaterials, Nov; Vol34 (33), pp8393-8400) [Non-patent document 3] :Long BV. Et al. (2014), J. Gastroenterol, May; Vol49(5), pp806-813) [Non-patent document 4] :BioScience, Biotechnology, and Biochemistry 75, 2246-2248 [Non-Patent Document 5] :Science Advances 01 Jan 2020: Vol. 6, no. 1, eaax8358 [Non-patent document 6] :Kawashima et al., Algal Research 46 (2020) 101724 Summary of the Invention [Problem to be solved by the invention]

[0011] An object of the present invention is to provide novel compounds derived from botryococcene or squalene that have antioxidant activity, methods for producing them, and antioxidant compositions containing these novel compounds as active ingredients. [Means for solving the problem]

[0012] Among sulfur compounds known as antioxidants, persulfur (>S=S) compounds such as oxidized glutathione are the only compounds that have been shown to mitigate environmental stress and be effective against various diseases, not only due to their antioxidant function but also due to sulfur metabolism through sulfur respiration, a process recently discovered in mammals. Therefore, compared to other antioxidant compounds, they have great potential for future development. However, the total content of the well-known oxidized (S=S) and reduced (S-) glutathione is generally around 0.3% in the body, and even in highly accumulating yeast, it is around 4-5%. Furthermore, the oxidized form only exists at about one-tenth of that (Imahori, 2007). This has resulted in extremely high market prices, which has been a major obstacle to practical application and widespread use.

[0013] The excellent antioxidant function of glutathione is ensured by the action of its thiol group (RSH). This is due to the high nucleophilicity of the thiol group. It was expected that compounds modified with thiols would also have good antioxidant function, but there have been few reports of thiol-modified compounds, and their antioxidant function has not been investigated.

[0014] Kawashima et al. have reported the unsaturated hydrocarbon botryococcene: [ka] reported that a thiol was introduced into the double bond of botryococcene using a thiol-ene reaction, resulting in the synthesis of botryococcene thiol derivatives, botryococcene pentathiolates 2a, 2b, and 2c (Non-Patent Document 6). Focusing on this research, the inventors prepared various thiol derivatives of botryococcene using the thiol-ene reaction and evaluated their antioxidant activity. The antioxidant effects of the derivatives varied depending on the type of thiol introduced. For example, a botryococcene ol derivative (BOT-SH) in which a thiol was polymerized onto the double bond in the side chain of botryococcene: [ka] On the other hand, botryococcene ol acetate derivative (Bot-SAc), in which thiol acetate is polymerized to the double bond of the side chain of botryococcene, showed no antioxidant effect. [ka] Particularly strong antioxidant activity was observed in

[0015] Also, squalene, which has a similar structure to botryococcene: [ka] Squarenethiol acetate derivative (SQ-SAc) in which thiol acetate is polymerized to the double bond of the main chain: [ka] was a known compound, but it was found to have antioxidant activity similar to botryococcinol acetate derivatives.

[0016] Furthermore, the Py-SS-Py modified product of the above BOT-SAc (BOT-SSPy): [ka] was found to have stronger antioxidant activity than the original BOT-SAc.

[0017] ROS are known to be involved in various diseases or symptoms in the body, and the administration of antioxidants is useful for preventing or suppressing such diseases or symptoms. As shown in the examples below, the antioxidant composition of the present invention potently suppressed ROS production induced by exposure of normal rat gastric mucosal cells to the nonsteroidal anti-inflammatory drug indomethacin or the anticoagulant dabigatran, as well as the cell damage and apoptosis associated with the effects of these drugs. These cytoprotective effects shown in the examples indicate that the antioxidant ability of the pharmaceutical composition of the present invention extends to improving cellular conditions involving ROS.

[0018] There are established methods for producing botryococcene and squalene on an industrial scale by culturing the microalgae that produce them (Patent Document 4, Non-Patent Document 4, and Patent Document 5). Starting from these, the present invention makes it possible to obtain compounds with high antioxidant activity using a simple thiol-ene reaction. As a new antioxidant biological material that is inexpensive and suitable for mass production, it will greatly satisfy the high demand in the pharmaceutical, food, feed, and health care markets.

[0019] Therefore, the present application provides the following inventions.

[0020] 1. Formula (I): [ka] A compound represented by the formula: 2. Formula (II): [ka] A compound represented by the formula: 3. A compound according to any one of items 1 or 2, and a compound of formula (III): [ka] An antioxidant composition having antioxidant activity, comprising, as an active ingredient, a compound selected from the group consisting of compounds represented by the formula: 4. The antioxidant composition according to item 3 for treating, preventing and / or alleviating a disease or symptom or a cosmetic problem associated with reactive oxygen species (ROS) in a living body. 5. The antioxidant composition according to either item 3 or 4, which is a nutritional supplement composition or a feed composition for supporting, improving or enhancing antioxidant function in the body. 6. The antioxidant composition according to any one of items 3 to 5, which is in the form of a lozenge, pill, capsule, liquid medicine or tablet for oral administration. 7. A method for producing the compound according to item 1, comprising: i) mixing botryococcene, 2,2-dimethoxy-2-phenylacetophenone and THF; ii) adding thioacetic acid to the mixture obtained in step i); iii) stirring the mixture obtained in step ii) under UV irradiation; and iv) concentrating the reaction product of step iii) and purifying the compound described in item 1; A manufacturing method comprising: 8. A method for producing the compound according to item 2, comprising: i) mixing the compound according to item 1, LiOH, EtOH and distilled water; ii) removing EtOH by fractional distillation from the reaction product obtained in step i), and adding saturated saline and dichloromethane; iii) recovering the dichloromethane phase from the mixture obtained in step ii); iv) adding chloroform and 2,2'-dipyridyl disulfide to the crude product obtained by concentrating the dichloromethane phase of step iii) and stirring the mixture; and v) concentrating the reaction product of step iv) and purifying the compound described in item 2; A manufacturing method comprising: [Brief explanation of the drawings]

[0021] [Figure 1] The antioxidant effects of BOT-SH and BOT-SAc were evaluated using ESR. BOT-SH was not found to have the ability to scavenge singlet oxygen or hydroxyl radicals. On the other hand, BOT-SAc was found to have the ability to scavenge these radicals.

[0022] [Figure 2] The antioxidant activity of BOT-SAc and SQ-SAc was evaluated using ESR. Both compounds were confirmed to have significant scavenging abilities for singlet oxygen, superoxide anion, and hydroxyl radical.

[0023] [Figure 3] The antioxidant activity of BOT-SAc, assessed using ESR, is significant compared to the control.

[0024] [Figure 4] The antioxidant activity of BOT-SSPy evaluated using ESR is significant compared to the control.

[0025] [Figure 5] The results of evaluating the antioxidant effects of BOT-SAc and BOT-SSPy on normal rat gastric mucosal cells, RGM1, are shown. The intracellular fluorescence was weaker when BOT-SAc or BOT-SSPy was added than when it was not added, demonstrating that indomethacin-induced ROS production was significantly suppressed by BOT-SAc or BOT-SSPy.

[0026] [Figure 6]The antioxidant effect of BOT-SSPy on normal rat gastric mucosal cells, RGM1, was evaluated. Intracellular fluorescence was weaker with the addition of BOT-SSPy than without, demonstrating that BOT-SSPy significantly suppresses tabigatran-induced ROS production.

[0027] [Figure 7] The results of evaluating the effect of BOT-SSPy in reducing indomethacin-induced cell damage are shown below. Exposure to indomethacin reduced cell viability to 50% of the group without BOT-SSPy compared to the group without indomethacin, but with 100 μM BOT-SSPy, cell damage was significantly reduced to 86%.

[0028] [Figure 8] The results of evaluating the effect of BOT-SSPy in reducing dabigatran-induced cell injury are shown below. Exposure to dabigatran reduced cell viability to 33% compared to the group without BOT-SSPy, but with 100 μM BOT-SSPy, cell injury was significantly reduced to 54%.

[0029] [Figure 9] The results of evaluating the inhibitory effect of BOT-SSPy on indomethacin-induced apoptosis are shown below. Compared to samples not exposed to indomethacin, BAX expression was significantly increased in indomethacin-exposed samples, whereas BAX expression was significantly decreased in the BOT-SSPy-containing condition. DETAILED DESCRIPTION OF THE INVENTION

[0030] Botryococcene is produced by some strains of Botryococcus braunii and has the following structure: [ka] Botryococcus braunii race-B, which produces botryococcene, is useful for mass production of botryococcene because it accumulates high-purity botryococcene in large quantities and secretes the produced botryococcene extracellularly (Patent Document 4).

[0031] Botryococcene itself can be used directly as diesel oil in ships and agricultural vehicles, but it can also be converted into diesel, naphtha, kerosene, and gasoline using conventional catalytic cracking. Furthermore, because botryococcene has excellent moisturizing properties, it has also been found to be used as a moisturizing ingredient in cosmetics (Patent Document 6). Thus, botryococcene is a valuable material that is expected to be used in a variety of industrial applications. The BOT-SAc of the present invention proposes a further promising use for botryococcene: as a material for a novel compound with excellent antioxidant activity.

[0032] Furthermore, based on the finding that the SS structure provides strong antioxidant activity (Non-Patent Document 5), a Py-SS-Py modified product of BOT-SAc was produced: [ka] was found to have stronger antioxidant activity than the original BOT-SAc.

[0033] Squalene has the following structure: [ka] Squalene is a type of triterpene hydrocarbon with a hydroxyl group. As a precursor to sterols, squalene is expected to be effective in preventing heart disease and suppressing tumor formation. Hydrogenation can also produce squalane, a cosmetic material with excellent lubricating and moisturizing properties. Until now, squalene has been obtained exclusively from deep-sea shark liver oil, but overfishing has led to a decline in the population of deep-sea sharks, making it highly likely that a sustainable supply of squalene will become difficult in the future. Meanwhile, the Aurantiochytrium algae strain tsukuba-3, which produces squalene with extremely high efficiency, has been discovered, and attempts are being made to use it as an alternative squalene source to deep-sea sharks (Non-Patent Document 4).

[0034] Thiolacetate derivatives of squalene: [ka] was already known, but its functionality was unknown. In the present invention, since it was shown that the thiol acetate derivative of botryococcene, which has a molecular structure similar to squalene, has strong antioxidant activity, the antioxidant activity of SQ-SAc was also tested and it was newly found to have similar antioxidant activity.

[0035] In the present invention, thiol acetate derivatives are produced by adding thioacetic acid to botryococcene. This addition reaction, known as the thiol-ene reaction, is considered click chemistry due to its simplicity, high reactivity, and wide range of available reagents. Botryococcene has five double bonds in its hydrocarbon side chain and one in its main chain. Using this thiol-ene reaction, thioacetic acid is added to all five double bonds in the side chain. Depending on the reaction conditions, there may be a small number of compounds in which thioacetic acid is not attached to all of the side chains or in which thioacetic acid is attached to double bonds in the main chain. However, these compounds are also included in the BOT-SAc of the present invention.

[0036] Squalene differs from botryococcene in that all six double bonds are located in the hydrocarbon backbone. Thioacetate addition via the thiol-ene reaction results in the addition of thioacetate to all six double bonds. As discussed below, BOT-SAc and SQ-SAc have significant differences in antioxidant activity, which are thought to be related to the positional relationship between the thioacetate group and the hydrocarbon backbone.

[0037] Py-SS-Py reacts with thiols to form RSS-Py, whereas RSS-Py and R'-SH readily react to form RSS-R'.

[0038] The thiol acetate derivatives and their modified products have high antioxidant activity. In the present invention, "antioxidant activity" refers to the ability to remove reactive oxygen species (ROS) such as singlet oxygen (1O2), hydroxyl radicals (OH-), and superoxide anions (O2-), or the ability to prevent or suppress cell or tissue damage caused by ROS in vivo, ROS-related diseases or symptoms, or cosmetic problems.

[0039] Preferably, antioxidant activity may be evaluated using any appropriate method, and examples of available evaluation methods include measurement of reactive oxygen species scavenging activity used in the Examples, measurement of DDPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity, and ORAC (Oxygen Radical Absorbance Capacity) evaluation. The evaluation results can be used to optimize the production process of the thiol acetate derivatives and their modified products according to the present invention, or to estimate the dosage required to produce the desired antioxidant activity.

[0040] The thiol acetate derivatives and their modified products can be prepared as antioxidant compositions containing them as active ingredients. Various additives may be added to the antioxidant compositions to prevent denaturation during the preparation process, enhance antioxidant activity, improve storage stability, change physicochemical properties, or impart other nutritional properties. Examples of such additives include lipids, carbohydrates, organic acids, vitamins, minerals, antibiotics, flavorings, colorings, preservatives, excipients, bulking agents, thickeners, adhesives, hydrating agents, disintegrants, emulsifiers, pH adjusters, and any other additives acceptable in the field of the final product.

[0041] The antioxidant composition can be prepared as an antioxidant pharmaceutical composition or antioxidant cosmetic composition for treating, preventing, and / or alleviating various diseases or symptoms associated with ROS in the body. Alternatively, the antioxidant composition can be prepared as a nutritional supplement or feed composition with antioxidant activity. The various conditions of the composition for each application, such as form, composition, physicochemical properties, dosage, and method of administration, are design matters that can be appropriately selected by those skilled in the art according to the specific application.

[0042] In a further aspect of the present invention, the antioxidant composition may be used alone or in combination with other substances that also have antioxidant activity or other biological activities.

[0043] Preferably, the antioxidant composition can be prepared as an oral formulation, which can further contain one or more optional excipients that are generally accepted as pharmaceuticals.

[0044] Preferably, the antioxidant composition is formulated as a troche, pill, capsule, liquid medicine, tablet, or the like.

[0045] The oral formulation may preferably be taken as a nutritional supplement for the treatment or prevention of diseases or physical disorders associated with oxidative stress.

[0046] The oral formulation may be taken in combination with one or more additional substances, compounds, drugs or compositions useful in achieving the desired effect. [Example]

[0047] Example 1 Synthesis of BOT-SAc [ka] Under an argon atmosphere, botryococcene (1.20 g, 2.57 mmol), 2,2-dimethoxy-2-phenylacetophenone (33.3 mg, 0.130 mmol, 5 mol%), and THF (2 mL) were added to a quartz test tube and mixed at room temperature. The mixture was cooled to 0 °C, and thioacetic acid (1.32 mL, 18.5 mmol, 7.2 mole equiv.) was added. The mixture was then stirred at room temperature for 1.5 hours under UV irradiation (365 nm, using a handy UV lamp, one 4W UV lamp). After completion of the reaction, the reaction mixture was concentrated and purified by silica gel column chromatography (hexane:ethyl acetate 6:1) to obtain the desired thiol acetate derivative BOT-SAc (1.72 g, 2.03 mmol, 79%). 1 H-NMR (400 MHz, CDCl3)δ = 0.76-1.01 (m, 24 H, CH3 x 8), 1.02-2.02 (m, 27 H, CH2x 9 + CH x 9), 2.28-2.36 (m, 15 H, CH3COS - x 5), 2.63-3.05 (m, 10 H, AcS-CH2x 5), 5.09-5.27 (m, 2 H, C=CH x 2) ppm

[0048] Example 2 Synthesis of SQ-SAc [ka] Under an argon atmosphere, squalene (1.03 g, 2.51 mmol), 2,2-dimethoxy-2-phenylacetophenone (33.6 mg, 0.131 mmol, 5 mol%), and THF (2 mL) were added to a quartz test tube and mixed at room temperature. The mixture was cooled to 0 °C, and thioacetic acid (1.29 mL, 18.1 mmol, 7.2 mole equiv.) was added. The mixture was then stirred at room temperature for 1.5 hours under UV irradiation (365 nm, using a handheld UV lamp, one 4W UV lamp). After completion of the reaction, the reaction mixture was concentrated and purified by silica gel column chromatography (hexane:ethyl acetate 6:1) to obtain the desired thiolate derivative SQ-SAc (1.89 g, 2.18 mmol, 87%). 1 H-NMR (400 MHz, CDCl3)δ = 0.78-1.04 (m, 24 H, CH3x 8), 1.06-1.82 (m, 24 H, CH2x 10 + CH x 4), 1.85-2.02 (m, 2 H, CH x 2), 2.31-2.36 (m, 18 H, CH3COS - x 6), 3.37-3.62 (m, 6 H, AcS - CH - x 6) ppm

[0049] Example 3 Synthesis of BOT-SSPy [ka] A 30 mL recovery flask was charged with BOT-SAc (550 mg, 0.649 mmol), LiOH (164 mg, 6.86 mmol, 10 eq.), EtOH (12 mL), and distilled water (1 mL) obtained in Example 1 and stirred at room temperature for 4 hours. After confirming the progress of the reaction in the reaction mixture by TLC, the ethanol was removed once using an evaporator. Saturated saline and dichloromethane were added and the mixture was transferred to a separatory funnel. The pH was adjusted to approximately 1-2 using 1N hydrochloric acid, and then extraction was performed five times with dichloromethane. The dichloromethane layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated using an evaporator to obtain a crude product. Chloroform (3 mL) and 2,2'-dipyridyl disulfide (718 mg, 3.26 mmol) were added to the 30 mL recovery flask containing the crude product. The internal gas was replaced with argon using an argon balloon, and the mixture was stirred at room temperature for 12 hours. After checking the progress of the reaction by TLC, the mixture was concentrated using an evaporator, and the resulting concentrate was purified by column chromatography (silica gel, hexane:ethyl acetate = 4:1 → 1:1 → ethyl acetate only). Because numerous spots were observed without interruption, all spots except for Py-SS-Py and a Py-SS-Py modified product (isolated and analyzed by NMR, but the structure has not yet been determined) at the origin with hexamethyl acetic acid (EA) = 4:1 were combined and concentrated to obtain 414 mg of the desired BOT-SSPy.

[0050] Example 4 Antioxidant effects of BOT-SAc, SQ-SAc, and BOT-SSPy The antioxidant activity of BOT-SAc, SQ-SAc, and BOT-SSPy obtained in Examples 1 to 3 was evaluated using electron spin resonance (ESR). BOT-SAc and SQ-SAc were dissolved in ethanol, and BOT-SSPy was dissolved in DMSO to prepare 10,000 μM test samples.

[0051] Singlet oxygen (1O2) measurement method Acid red and TEMPO were dissolved in MilliQ water to final concentrations of 2 mM and 100 mM, respectively. 100 μL of Acid red, 100 μL of TEMPO, and 30 μL of the test sample were mixed with 70 μL of MilliQ water and irradiated with 540 nm light for 30 seconds. After irradiation, the solution was loaded into a Pasteur pipette and measured using an electron spin resonance spectrometer (JEOL-TE, JEOL Ltd., Tokyo). The measurement conditions were as follows: sweep width: 7.5 mT, gain: 50-500, modulation width: 0.2 mT, time contrast: 0.1 s, center field: 335.5 mT, sweep time: 0.5 min.

[0052] Hydroxy radical (OH-) measurement method The spin trap CYPMPO was dissolved in MilliQ water to a final concentration of 100 mM. 16 μL of hydrogen peroxide, 20 μL of CYPMPO, and 20 μL of the test sample were mixed with 144 μL of MilliQ water and irradiated with 405 nm light for 10 seconds. The ESR of the sample solution after irradiation was measured in the same manner as for singlet oxygen.

[0053] Superoxide anion (O2-) measurement method The spin trap CYPMPO was dissolved in MilliQ water to a final concentration of 100 mM. Hypoxanthine was dissolved in NaOH to a final concentration of 20 mM. A mixture of 20 μL hypoxanthine and 10 μL CYPMPO was designated Solution A. B mixture of 1.6 μL xanthine oxidase (25 U / mL) and 10 μL CYPMPO was designated Solution B. C mixture of 20 μL of the test sample and 138.4 μL MilliQ water was used. The ESR of the sample solutions prepared by mixing Solutions A, B, and C was measured in the same manner as for singlet oxygen measurement.

[0054] Measurement results SQ-SAc and BOT-SAc were confirmed to have the ability to scavenge singlet oxygen, superoxide anion, and hydroxyl radical (Fig. 2). Furthermore, the ability of BOT-SAc (Fig. 3) and BOT-SSPy (Fig. 4) to scavenge these ROS was shown to be significant compared to the control. On the other hand, BOT-SH did not exhibit significant ROS scavenging ability (Fig. 1).

[0055] Example 5 Antioxidant effect of BOT-SAc and BOT-SSPy on cells The antioxidant effects of BOT-SAc and BOT-SSPy on normal rat gastric mucosal cells (RGM1) were evaluated by inhibiting ROS production induced by exposure to the nonsteroidal anti-inflammatory drug indomethacin and the anticoagulant drug dabigatran.

[0056] RGM1 cells were seeded in 24-well plates and cultured overnight. After incubation, the cells were incubated for 1 hour in medium containing 10 or 100 μM BOT-SAc or BOT-SSPy and 1 mM indomethacin. After incubation, the supernatant was removed, and 5 μM hydroxyphenylfluorescein (HPF) was added and incubated for 15 minutes. HPF is a fluorescent reagent for detecting reactive oxygen species. The supernatant was replaced with buffer, and intracellular fluorescence was observed under a fluorescence microscope. Fluorescence was observed in the cells without BOT-SAc or BOT-SSPy, indicating that indomethacin increased intracellular ROS production. In contrast, intracellular fluorescence was weaker in the presence of BOT-SAc or BOT-SSPy than in the absence of BOT-SAc or BOT-SSPy, demonstrating that indomethacin-induced ROS production was significantly suppressed by BOT-SAc or BOT-SSPy (Figure 5).

[0057] The inhibitory effect of BOT-SSPy on ROS production induced by dabigatran exposure was also evaluated. RGM1 cells were seeded in 24-well plates and cultured overnight. After incubation, the cells were cultured for 6 hours in medium supplemented with 10 or 100 μM BOT-SSPy and 25 μM dabigatran. After incubation, the supernatant was removed, and 5 μM HPF was added and cultured for 15 minutes. The supernatant was replaced with buffer, and intracellular fluorescence was observed under a fluorescence microscope. Fluorescence was observed in cells without BOT-SSPy, indicating that dabigatran increased intracellular ROS production. In contrast, the intracellular fluorescence was weaker in cells with BOT-SSPy than in the absence of BOT-SSPy, demonstrating that BOT-SSPy significantly suppressed tabigatran-induced ROS production (Figure 6).

[0058] Example 6: Cell-protective effect of BOT-SSPy against drug-induced cytotoxicity ROS production induced by indomethacin and dabigatran induces cell injury. Such cell injury can be reduced by antioxidants. In this study, we investigated whether BOT-SSPy can reduce the cell injury caused by these drugs.

[0059] RGM1 cells were seeded in 96-well plates and cultured overnight. After incubation, the cells were incubated for 24 hours in medium supplemented with 10 or 100 μM BOT-SSPy and 500 μM indomethacin or 50 μM dabigatran. The supernatant was removed, and the cells were incubated with 10% CCK8-containing medium. The absorbance at 450 nm was measured until it reached 1. The viability of cells not exposed to indomethacin was defined as 100%. Exposure to indomethacin reduced cell viability to 50% without BOT-SSPy, whereas exposure to 100 μM BOT-SSPy significantly reduced cell damage (86%) (Figure 7). Exposure to dabigatran reduced cell viability to 33% without BOT-SSPy, whereas exposure to 100 μM BOT-SSPy significantly reduced cell damage (54%) (Figure 8).

[0060] Example 7: Cell-protective effect of BOT-SSPy against indomethacin-induced apoptosis Indomethacin-induced ROS production induces apoptosis. In this study, we investigated whether BOT-SSPy can suppress indomethacin-induced apoptosis.

[0061] BAX is a protein that interacts with mitochondrial voltage-dependent anion channels, increasing their opening, leading to loss of membrane potential and release of cytochrome c. Increased BAX expression is an indicator of the induction of apoptosis.

[0062] RGM1 cells were seeded in 60-mm dishes and cultured overnight. After incubation, they were cultured for 24 hours in medium supplemented with 10 or 100 μM BOT-SSPY and 500 μM indomethacin. After incubation, the cells were washed three times with PBS, harvested with RIPA buffer, and sonicated to prepare the suspension. The BAX levels in the test samples were measured by Western blotting. BAX expression was significantly increased in the indomethacin-exposed samples compared with the unexposed samples. On the other hand, BAX expression was significantly decreased in the BOT-SSPy-containing conditions, indicating that BOT-SSPy suppressed indomethacin-induced apoptosis (Figure 9).

Claims

1. Formula (I): 【Chemistry 1】 A compound represented by the formula:

2. Formula (II): 【Chemistry 2】 A compound represented by the formula:

3. A compound according to any one of claims 1 or 2 and a compound of formula (III): 【Transformation 3】 An antioxidant composition having antioxidant activity, comprising, as an active ingredient, a compound selected from the group consisting of compounds represented by the formula:

4. The antioxidant composition according to claim 3 for the treatment and prevention and / or alleviation of diseases or symptoms or cosmetic problems associated with reactive oxygen species (ROS) in the body.

5. 5. The antioxidant composition according to claim 3, which is a nutritional supplement or a feed composition for supporting, improving or enhancing antioxidant function in the body.

6. The antioxidant composition according to any one of claims 3 to 5, which is in the form of a troche, pill, capsule, liquid medicine or tablet for oral administration.

7. 10. A method for preparing the compound of claim 1, comprising: i) mixing botryococcene, 2,2-dimethoxy-2-phenylacetophenone and THF; ii) adding thioacetic acid to the mixture obtained in step i); iii) stirring the mixture obtained in step ii) under UV irradiation; and iv) concentrating the reaction product of step iii) and purifying the compound of claim 1; A manufacturing method comprising:

8. A method for preparing the compound of claim 2, comprising: i) mixing the compound of claim 1, LiOH, EtOH and distilled water; ii) removing EtOH by fractional distillation from the reaction product obtained in step i), and adding saturated saline and dichloromethane; iii) recovering the dichloromethane phase from the mixture obtained in step ii); iv) adding chloroform and 2,2'-dipyridyl disulfide to the crude product obtained by concentrating the dichloromethane phase of step iii) and stirring the mixture; and v) concentrating the reaction product of step iv) and purifying the compound of claim 2; A manufacturing method comprising:

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