Biostimulants including sargassum horneri extracts

Abstract

One embodiment of the present invention provides a bioactive agent comprising Sargassum hornerie extract as an active ingredient. The Sargassum hornerie extract can be extracted by a boiling-based extraction method (SBE), an immersion-based extraction method (SSE), a high-pressure sterilization-based extraction method (SAE), or an ethanol extraction method (SEE). Among these, extracts obtained by SBE, SSE, and SAE can improve resistance to high-temperature stress that inhibits the growth of Neopyropia yezoensis. According to one embodiment of the present invention, the SBE and SSE extraction methods helped to increase the content of picoviriprotein, and SBE can increase Superoxide dismutase (SOD) activity.

Description

Technology Field

[0001] The present invention relates to a physiologically active agent containing Sargassum horneri extract as an active ingredient. More specifically, it relates to a physiologically active agent containing Sargassum horneri extract as an active ingredient having a high temperature resistance effect. Background Technology

[0002] Seaweed is widely cultivated in Asian countries such as China, Indonesia, Korea, and Japan. The seaweed farming industry is growing rapidly in the United States and Europe, and interest in cultivation has increased as applications expand to include food, animal feed, cosmetics, nutritional supplements, and recently, biofuels. In particular, Korea is the world's third-largest producer of seaweed, following China and Indonesia. The main cultivated species is *Pteridium aquilinum* ( Neopyropia spp .; mostly N . yezoensis ) and next is Undaria pinnatifida and Saccharina japonica There is, and the amount of seaweed produced in Korea exceeds 500,000 tons, which has an economic value of $600 million. However, the seaweed farming industry is under threat due to climate change, particularly global warming, and among many environmental stressors, rising sea surface temperatures act as a major stressor, reducing seaweed production volume and quality. For example, *Pteridium aquilinum* ( Neopyropia yezoensis ) is the most economically important red algae cultivated in Korea, China, and Japan. It has the highest marketability per unit mass compared to other farmed seaweeds. The warming trend of surface seawater is ( Neopyropia yezoensis It is having a direct impact on aquaculture. For example, when seawater temperatures rise, it hinders the growth of pyrethrum, thereby lowering aquaculture productivity. Therefore, in order to maintain or increase current agricultural productivity, it is urgent to develop new technologies to enhance the heat resistance of pyrethrum strains.

[0003] Biostimulants are known to influence the physiological processes of terrestrial plants by providing potential benefits to their growth, development, or abiotic stress. Several examples also highlight that the application of seaweed extracts can increase the resistance of cultured seaweed to abiotic and biotic stress. Since 2015, large-scale Sargassum horneri ( Sargassum horneri ) drifted from the East China Sea to Jeju Island and South Jeolla Province along ocean currents. The massive biomass of Sargassum horneri caused economic damage to the tourism and aquaculture industries. As an approach to utilize a portion of the domestically accumulated biomass, Sargassum horneri ( Sargassum horneri There have been attempts to extract useful bioactive compounds from )(e.g., antioxidant, anti-inflammatory, anti-allergic properties). Sargassum horneri ( Sargassum horneri Although ) has not yet been studied as a raw material for bioactive agents, other Sargassum species ( Sargassum Some extracts derived from species have been proven to act as bioactive agents when applied to terrestrial plants. For example, among the Sargassum species Sargassum wightti The extract is from mung beans, a legume ( Vigna radiata While it improved root and shoot length when applied to )(green gram), Sargassum crassifolium The solid and liquid extracts of increased the growth and yield of rice.

[0004] Sargassum horneri as a bioactive agent ( Sargassum horneri With no technology utilizing ) available and massive damage caused by rising temperatures to seaweed, Sargassum horneri ( Sargassum horneri There is a need for technology that can actively utilize ). Prior art literature

[0005] Republic of Korea Published Patent No. 10-2016-0096995 The problem to be solved

[0006] The technical problem that the present invention aims to solve is to provide a bioactive agent containing Sargassum horneri extract as an active ingredient that has a high temperature resistance effect.

[0007] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below. means of solving the problem

[0008] To achieve the above technical problem, one embodiment of the present invention is a Sargassum extract ( Sargassum horneri A bioactive agent containing extract) as an active ingredient is provided.

[0009] In an embodiment of the present invention, the bioactive agent may exhibit a high temperature resistance effect.

[0010] In an embodiment of the present invention, the bioactive agent is radial seaweed ( Neopyropia yezoensis It can be applied to ).

[0011] In an embodiment of the present invention, the Sargassum extract is a Sargassum boiling-based extract ( Sargassum horneri boiling extract, SBE), Sargassum horneri immersion-based extract ( Sargassum horneri Soaking extract (SSE) and Sargassum horneri high-pressure sterilization-based extract ( Sargassum horneri It may be at least one selected from a group consisting of autoclaving extracts (SAE).

[0012] In an embodiment of the present invention, the Sargassum boiling-based extract ( Sargassum horneri Boiling extract (SBE) can increase SOD activity.

[0013] In an embodiment of the present invention, the Sargassum immersion-based extract ( Sargassum horneriSoaking extract (SSE) can increase the content of phycobiliprotein.

[0014] In an embodiment of the present invention, the Sargassum boiling-based extract ( Sargassum horneri boiling extract, SBE) or Sargassum horneri immersion-based extract ( Sargassum horneri Soaking extract (SSE) can increase the growth rate. Effects of the invention

[0015] The bioactive agent according to an embodiment of the present invention contains Sargassum horneri extract as an active ingredient and may have a high temperature resistance effect.

[0016] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description of the invention or the claims. Brief explanation of the drawing

[0017] Fig. 1 shows radial seaweed (at different temperatures (10℃, 20℃) Neopyropia yezoensis Specific Growth Rate (SGR) of ) and Sargassum horneri ( Sargassum horneri This is a graph showing four extraction methods using ) as the extraction source. Different letters on the bars indicate a significant difference (p < 0.05). Error bars represent the mean ± standard deviation of three replicates. The samples are the control group (no extract), Sargassum boiling-based extract (SBE), Sargassum ethanol extract (SEE), Sargassum immersion-based extract (SSE), and Sargassum autoclave extract (SAE). Fig. 2 shows radial seaweed at different temperatures (10℃, 20℃) Neopyropia yezoensisThis is a graph showing chlorophyll a(a), phycocyanin(b) and phycoerythrin(c) of Sargassum horneri, and four extraction methods using Sargassum horneri as the extraction source. Different letters on the bars indicate significant differences (p < 0.05). Error bars represent the mean ± standard deviation of three replicates. The samples are the control group (no extract), Sargassum horneri boiling-based extract (SBE), Sargassum horneri ethanol extract (SEE), Sargassum horneri immersion-based extract (SSE), and Sargassum horneri autoclave extract (SAE). Fig. 3 shows radial seaweed at different temperatures (10℃, 20℃) Neopyropia yezoensis This is a graph showing the superoxide dismutase (SOD; a), catalase (CAT; b), glutathione reductase (GR; c), and ascorbate peroxidase (APX; d) of Sargassum horneri, and four extraction methods using Sargassum horneri as the extraction source. Different letters in the bars indicate significant differences (p < 0.05). Error bars represent the mean ± standard deviation of three replicates. The samples are the control group (no extract), Sargassum horneri boiling-based extract (SBE), Sargassum horneri ethanol extract (SEE), Sargassum horneri immersion-based extract (SSE), and Sargassum horneri autoclave extract (SAE). Fig. 4 shows radial seaweed at different temperatures (10℃, 20℃) Neopyropia yezoensis This is a graph showing hydrogen peroxide (H2O2; a), lipid peroxidation (LPO; b), and reactive oxygen species (ROS; c) of Sargassum horneri, and four extraction methods using Sargassum horneri as the extraction source. Different letters on the bars indicate significant differences (p < 0.05). Error bars represent the mean ± standard deviation of three replicates. The samples are the control group (no extract), Sargassum horneri boiling-based extract (SBE), Sargassum horneri ethanol extract (SEE), Sargassum horneri immersion-based extract (SSE), and Sargassum horneri autoclave extract (SAE). Fig. 5 shows radial seaweed at different temperatures (10℃, 20℃) Neopyropia yezoensis This is a graph showing the total protein of ) and four extraction methods using Sargassum horneri as the extraction source. Different letters on the bars indicate a significant difference (p < 0.05). Error bars represent the mean ± standard deviation of three replicates. The samples are the control group (no extract), Sargassum horneri boiling-based extract (SBE), Sargassum horneri ethanol extract (SEE), Sargassum horneri immersion-based extract (SSE), and Sargassum horneri autoclave extract (SAE). Fig. 6 shows radial seaweed at different temperatures (10℃, 20℃) Neopyropia yezoensis This is a graph showing the total phenolic content of Sargassum horneri and four extraction methods using Sargassum horneri as the extraction source. Different letters on the bars indicate significant differences (p < 0.05). Error bars represent the mean ± standard deviation of three replicates. The samples are the control group (no extract), Sargassum horneri boiling-based extract (SBE), Sargassum horneri ethanol extract (SEE), Sargassum horneri immersion-based extract (SSE), and Sargassum horneri autoclave extract (SAE). Specific details for implementing the invention

[0018] The present invention will be described below with reference to the attached drawings. However, the present invention may be implemented in various different forms and is therefore not limited to the embodiments described herein. Furthermore, in order to clearly explain the present invention in the drawings, parts unrelated to the explanation have been omitted, and similar parts throughout the specification have been given similar reference numerals.

[0019] Throughout the specification, when it is stated that a part is "connected (connected, in contact, combined)" with another part, this includes not only cases where they are "directly connected," but also cases where they are "indirectly connected" with other members interposed between them. Furthermore, when it is stated that a part "includes" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but rather allows for the inclusion of additional components.

[0020] The terms used herein are merely for describing specific embodiments and are not intended to limit the invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as “comprising” or “having” are intended to indicate the presence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0022] A bioactive agent according to one aspect of the present invention is a Sargassum extract ( Sargassum horneri It may include extract) as an active ingredient.

[0024] Effects of bioactive agents and temperature on growth rate

[0025] According to one embodiment of the present invention, the growth rate at 10°C may be higher than the growth rate at 20°C (Fig. 1). Radial patterned laver ( Neopyropia yezoensis) showed similar growth rates at 10°C regardless of the extraction method. However, at 20°C, SBE and SAE showed the highest growth rates, followed by SSE, SEE, and the control group (Fig. 1, p<0.004). Growth rates were significantly affected by the interaction between extraction method and temperature (Table 1, p<0.001). These results suggest that 20°C is [specifically related to] *Pterygos japonicus* ( Neopyropia yezoensis It was shown that it has a more negative effect than 10℃ on the growth of ). In the present invention, Sargassum horneri extract is used for radial laver ( Neopyropia yezoensis It was shown that it has a positive effect on the thermal durability of ).

[0026] parameters temperature extraction method Temperature * Extraction Method df F p df F p df F p SGR 1 248.974 <0.001 4 9.929 <0.001 4 7.622 <0.001 Chloroplast a 1 153.391 <0.001 4 3.779 <0.05 4 5.325 <0.05 Phycocyanin 1 48.891 <0.001 4 7.961 <0.001 4 5.809 <0.05 Phycoerythrin 1 61.736 <0.001 4 13.796 <0.001 4 7.365 <0.001 phenol 1 9.765 <0.05 4 2.150 >0.05 4 0.641 >0.05 protein 1 8.436 <0.05 4 1.195 >0.05 4 1.681 >0.05 SOD 1 31.492 <0.001 4 19.434 <0.001 4 12.138 <0.001 CAT 1 49.865 <0.001 4 2.359 >0.05 4 1.312 >0.05 GR 1 176.683 <0.001 4 4.529 <0.05 4 3.902 <0.05 APX 1 53.845 <0.001 4 9.781 <0.001 4 2.179 >0.05 ROS 1 6.859 <0.05 4 8.158 <0.001 4 3.886 <0.05 H2O2 1 9.358 <0.05 4 3.741 <0.05 4 3.565 <0.05 LPO 1 28.696 <0.001 4 0.706 >0.05 4 2.06 >0.05

[0027] It was found that the extraction method according to one embodiment of the present invention exhibits an effect of improving thermal durability. In particular, SBE and SAE are radial patterned seaweed ( Neopyropia yezoensis It was the most effective extraction method for improving the heat resistance of ). Radiating seaweed ( Neopyropia yezoensis In terms of production, in one embodiment of the present invention, the daily production of the control group at 10°C was 0.054g, and the control group at 20°C showed a production yield 717.64% lower than that of the control group at 10°C. However, compared to the control group at 20°C, the SBE according to one embodiment of the present invention (radial patterned laver) Neopyropia yezoensis It can increase the production of ) by 284.47%. Boiling extracts contain important inorganic nutrients (N, P, S, and B) than soaking extracts and may contain higher levels of polyphenols. In addition, brown seaweed Sargassum sp.Boiled extracts and auto-cooked extracts may contain higher levels of polyphenols than green and red seaweed. These polyphenols are known as effective antioxidants. Organic compounds contained in brown seaweed, such as organic acids, methionine, polyamines, polyphenols, and mannitol, can chelate available nutrients to increase nutrient absorption and enable the effective use of nutrients.

[0029] Effects of bioactive agents and temperature on pigments

[0030] According to one embodiment of the present invention, the chlorophyll a measurement results showed similar results in SBE, SEE, SSE, and the control group at 10°C, with a significant difference appearing only in SAE (Fig. 2a). At 20°C, the chlorophyll a content was significantly lower than at 10°C, and no positive effect of the Sargassum horneri extract was observed (Fig. 2a, p>0.05). According to one embodiment of the present invention, the chlorophyll a content was found to decrease at high temperatures. In SBE and SSE at 10°C, the content of phycocyanin and phycoerythrin was significantly higher than in all other conditions (p <0.001) (Figs. 2b and c). Chlorophyll a and phycobiliprotein were significantly affected by the interaction between temperature and the extract (Table 1, p <0.05). According to one embodiment of the present invention, the application of a bioactive agent derived from seaweed can increase the concentration of phycobirin protein in red algae.

[0032] Effects of bioactive agents and temperature on biochemical reactions

[0033] According to one embodiment of the present invention, enzymatic (SOD, CAT, GR, and APX) and non-enzymatic (total phenol) antioxidant activities were measured to evaluate oxidative stress. At 10°C, SOD activity was highest in SBE, while all other extracts were similar to the control group. At 20°C, SOD in SSE was similar to the control group, and all other extraction methods showed lower SOD than the control group (Fig. 3a). Temperature stress can induce oxidative stress. SOD is known as the first enzyme controlling the detoxification pathway of ROS. According to one embodiment of the present invention, in Sargassum horneri extract, radial patterned seaweed ( Neopyropia yezoensis The SOD activity of ) can be increased (Fig. 3a). According to one embodiment of the present invention, when treated at a high temperature, radial seaweed ( Neopyropia yezoensis The SOD activity of ) decreased. However, at the optimal temperature of 10℃, SBE ( Neopyropia yezoensis It can increase the SOD activity of ). SOD is a metalloprotein, and three isoforms can be determined depending on the metalloproteinase: According to one embodiment of the present invention, SBE is abundant in organic compounds for chelation (i.e., polyphenols, organic acids) and can increase the acceptance of trace metals for SOD synthesis.

[0034] CAT is an enzyme directly involved in photosynthetic reactions. It is a temperature-sensitive enzyme, and its activity decreases as the temperature rises. One embodiment of the present invention showed a significant effect of temperature on CAT activity (Fig. 3b, p < 0.001), while the extraction method did not have a significant effect and no interaction between temperature and the extraction method was detected (Fig. 3b and Table 1, p > 0.05).

[0035] GR is an enzyme in the Ascorbate-Glutathione (AsA-GSH) cycle that maintains the reduced state of glutathione, thereby performing an essential function in the defense system against ROS. GR activity was significantly lower at 20°C compared to 10°C, regardless of the extraction method (Fig. 3c, p < 0.045). According to one embodiment of the present invention, *Pteridium aquilinum* ( Neopyropia yezoensis The fact that lipid peroxidation increased less at 20°C than at 10°C may mean that GR activity increased at 10°C.

[0036] On the other hand, according to one embodiment of the present invention, APX activity may appear inversely to GR activity (Fig. 3d). APX activity was higher at 20°C than at 10°C, and all Sargassum extracts showed a slight increase in APX activity compared to the control group at both temperatures (p < 0.036). APX is an important component of the AsA-GSH cycle and may be responsible for converting H2O2 to H2O. According to one embodiment of the present invention, the decrease in H2O2 at 20°C may be due to high APX activity (Figs. 3d and 4a). According to one embodiment of the present invention, these results may explain the low LPO at 20°C (Fig. 4b).

[0037] According to one embodiment of the present invention, the ROS values ​​obtained therefrom did not show a significant difference depending on the temperature (Fig. 4c, p > 0.05), demonstrating that antioxidant enzymes can effectively alleviate oxidative stress and regulate ROS balance. Balanced ROS can act as important signaling regulators under stress conditions rather than playing a lethal role. Total protein content and total phenolic content were affected only by temperature, and neither the extraction method nor the interaction between the extract and temperature had a significant effect (Figs. 5 and 6, Table 1, p > 0.05).

[0039] According to one embodiment of the present invention, Sargassum horneri extract is radial Sargassum ( Neopyropia yezoensis It can serve as a new source of bioactive agents that improves the thermal durability of ). According to one embodiment of the present invention, the extraction method for obtaining the bioactive agent can play an important role in the effect of the extract, and among the extraction methods, the boiling-based extraction method (SBE) and the immersion-based extraction method (SSE) are used for radial-patterned seaweed ( Neopyropia yezoensis It may be effective for the growth and improvement of phycovirine protein content. According to one embodiment of the present invention, regardless of the extraction method used, the expression level of antioxidant enzymes and oxidative stress may be governed by temperature.

[0041] Embodiments of the present invention will be described in detail below with reference to the attached drawings.

[0043] Example 1.1. Collection of Seaweed

[0044] Radial pattern seaweed Neopyropia yezoensis The strain (NY-HN-ST1) was originally collected in December 2020 from the Haenam Bangsamunui Pteris farm (34°57'N, 126°60'E). This strain was propagated asexually by the Marine Ecology and Eco-friendly Aquaculture Laboratory at Incheon National University. All individuals were 90±10 μmol m⁻¹ provided by fluorescent light, von Stosch enriched solution (VSE) medium, 10°C, daylight color. -2 s -1 It was cultured in photosynthetically active radiation (PAR), 12:12 L:D, and a salinity of 30 psu.

[0046] Example 2. Preparation of a bioactive agent

[0047] Sargassum horneri Sargassum horneriThe sample was collected in June 2020 in Jumunjin, Gangwon-do (37°90'N, 128°83'E). The Sargassum extract (SHE) was prepared based on Zahra, Mehrnaz, Farzaneh, and Kohzad with minor modifications. Approximately 8 kg of Sargassum was dried and then powdered using a mixer mill MM 400 (German Lech). 5 g of the powder was added to 500 ml of distilled water and stirred, then autoclaved at 121 °C for 15 minutes. The extract was cooled at room temperature for 3 hours and then centrifuged at 2,220 g for 10 minutes. The resulting supernatant was obtained as the liquid extract.

[0048] The second extract, hereinafter referred to as the ethanol extract (SEE), was prepared by slightly modifying the known technology. 250 g of powder was placed in 2.5 L of distilled water and mixed intermittently at room temperature for 72 hours, then filtered through Whatman No. 1 filter paper (11 μm) and concentrated using a rotary vacuum evaporator (R-210, Buchi, Switzerland) at 40°C. The resulting dried powder was used in the experiment. The immersion-based extract and the boiling-based extract were prepared by slightly modifying the known technology. Both extracts were prepared by placing 3 g of powder in 300 mL of distilled water. To obtain the third extract, the immersion-based extract (SSE), 3 g of powder was soaked in distilled water at room temperature for 2 days. Finally, for the fourth extract, the boiling-based extract (SBE), 3 g of powder was boiled in a water bath for 30 minutes and then cooled to room temperature. Both extracts were centrifuged at 3,134 xg for 25 minutes. The supernatant was filtered through Whatman No. 1 filter paper (11 μm) to obtain the final liquid extract.

[0050] Example 3. Experimental Design

[0051] Collected radial seaweed ( Neopyropia yezoensis ) has a density of 1 g L -1They were cultured in 2 L glass cylinders. For SBE, SEE, SSE, and SAE treatments, samples were exposed to a cool white fluorescent lamp and 90 ± 10 μmol m⁻² provided at a 12:12 L:D ratio. -2 s -1 Under photosynthetically active radiation (PAR), it was exposed to 10% of each extract for 10 days at 10°C. The control group consisted only of sterilized seawater without extract and VSE medium. To avoid nutrient limitations, the medium was replaced every 5 days. Salinity was maintained constant at 30 psu. After exposure to the extract, *Pterygota var. radiata* ( Neopyropia yezoensis The ) samples were washed in sterilized seawater to remove residues. Washed radial laver ( Neopyropia yezoensis ) is used with a storage density of 1 g L using VSE medium supplemented with 0.25% (w / v) germanium dioxide. -1 The samples were cultured in 500 mL Erlenmeyer flasks at two different temperatures, 10 or 20°C. The samples were cultured for 15 days, and the medium was replaced every 5 days. On the same day, the fresh weight of the thalli was measured to calculate the specific growth rate. At the end of the experiment, superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), reactive oxygen species (ROS), H2O2, lipid peroxidation (LPO), total phenols, total protein, and pigments (chloroplast a, phycobiliprotein) were analyzed. The specific growth rate (SGR) was calculated using the following formula.

[0052]

[0053] W end and Winitial Eun T end and T initial It represents the weight of the thallus.

[0055] Example 4. Antioxidant Enzyme Activity Analysis

[0056] Fresh thallus (approx. 100 mg) was ground on ice using a motor-driven tissue grinder in 1 mL of potassium phosphate buffer (50 mM, pH 7.0) containing 0.25% Triton X-100 and 1% polyvinylpyrrolidone. The homogenate was centrifuged at 12,000 xg for 10 minutes at 4 °C. The supernatant was used to measure the activity of SOD, CAT, GR, APX, total protein, and ROS. Enzyme activity was measured three times.

[0057] Protein content was measured using the known Bradford method. In summary, 75 μL of distilled water and 2.5 mL of Bradford solution (0.025 g Coomassie blue dye, 12.5 mL of 95% (v:v) ethanol, and 25 mL of H3PO4 diluted with 250 mL of distilled water) were added to 25 μL of enzyme extract. The reagents were vortexed for mixing and left to stand for 5 minutes before measuring absorbance at 595 nm. Total protein content was measured relative to bovine serum albumin (BSA) and expressed in mg / g.

[0058] Superoxide dismutase (SOD) activity was measured according to known technology. In summary, 20 μL of gyth extract was added to 150 μL of carbonate buffer (pH 10.2). The production of adrenochrome due to the auto-oxidation of epinephrine was measured at 480 nm for 3 minutes against a blank, and activity was expressed in U / mg protein.

[0059] Catalase (CAT) activity was measured based on known technology. In summary, 100 μL of the enzyme extract was added to 750 μL of potassium phosphate buffer (50 mM, pH 7.0), 500 μL of distilled water, and 150 μL of H2O2 (0.1 M). Absorbance was measured at 240 nm (0 and 2 min). Activity was measured in H2O2 (0.043 mM -1 com -1 It was estimated using the molecular absorption coefficient of ) and expressed in mmol / mg protein units.

[0060] The activity of glutathione reductase (GR; EC 1.8.1.7) was measured according to known techniques. Briefly, 600 μL of Tris-HCl buffer (100 mM, pH 7.8) containing 100 μM NADPH, 1 mM EDTA, and 0.5 mM oxidized glutathione was added to 15 μL of enzyme extract. Absorbance was measured against a blank at 340 nm for 3 minutes. Enzyme activity was calculated using the extinction coefficient of NADPH (6.2 mM / cm) and expressed in U / mg protein units.

[0061] Ascorbate peroxidase (APX; EC 1.11.1.11) activity was measured according to the known art. In summary, 185 μL of reaction buffer (50 mM potassium phosphate buffer, 0.25 mM ascorbic acid) was added to 10 μL of enzyme extract. To determine non-specific ascorbate degradation, the mixture was shaken for 5 seconds, and then absorbance was measured at 290 nm for 3 minutes at 25 °C. Subsequently, 5 μL of H2O2 (200 mM) was added, the mixture was shaken for 5 seconds, and absorbance was measured at 290 nm for 5 minutes at 25 °C. Enzyme activity was 2.8 mM -1 cm -1 It was calculated using the absorption coefficient and expressed in U / mg protein units.

[0063] Example 5. Analysis of Oxidative Stress Parameters

[0064] Fresh thallus (approx. 100 mg) was ground using a motor-driven tissue grinder with 1 mL of 10% (w:v) trichloroacetic acid (TCA) solution on ice. The homogenized mixture was centrifuged at 7000 xg for 10 minutes at 4°C. The supernatant was used to measure H2O2 and lipid peroxidation (LPO) levels. Oxidative stress parameters were measured in three repetitions.

[0065] Reactive oxygen species (ROS) concentrations were measured according to known technology. In summary, 20 μL of the extract was added to 180 μL of sample buffer and 200 μL of dye solution (1 mM DCFDA, 0.01 N NaOH, and 25 mM, pH 7.2 sodium phosphate buffer). Fluorescence absorption (λex = 485 nm and λem = 535 nm) was measured after incubation in the dark at 20°C for 1 hour. ROS concentrations were expressed in U / mg protein units.

[0066] H2O2 was measured according to known technology. In summary, 150 μL of potassium phosphate buffer (50 mM, pH 7.0) and 100 μL of potassium iodide (1 M) were added to 50 μL of the supernatant. Absorbance was measured at 390 nm relative to the blank. The H2O2 level was expressed as nmol / g fresh weight using an H2O2 standard curve.

[0067] Lipid peroxidation (LPO) levels were detected by measuring malondialdehyde (MDA) content using known technology. In summary, 2 mL of thiobarbituric acid (TCA) was added to 100 μL of the supernatant, and the mixture was incubated in a water bath at 95 °C for 45 minutes. Finally, the mixture was centrifuged at 4,000 xg (if necessary), and the absorbance was measured at 532 nm relative to the blank. The LPO level was 156 mM. -1 cm-1 It was expressed in μmol MDA / mg protein units using the absorption coefficient.

[0069] Example 6. Total phenolic content and pigment analysis

[0070] Fresh thallus (approx. 20 mg) was homogenized with 2 mL of ice-cold 95% (w:w) methanol using a motor-driven tissue grinder. The homogenate was incubated at dark room temperature for 48 hours, followed by centrifugation at 13,000 xg for 5 minutes. The supernatant was used to measure total phenolic content and chlorophyll a. All measurements were repeated three times.

[0071] Total phenol content was measured according to known technology. Briefly, 200 μL of 10% (v:v) Folin-Ciocalteu reagent was added to 100 μL of the supernatant and thoroughly vortexed. Approximately 800 μL of Na2CO3 (700 mM) was added, and the mixture was incubated at room temperature for 2 hours, after which the absorbance was measured at 765 nm. Phenol content was quantified using a gallic acid standard curve and expressed in mg GAE / g fresh weight.

[0072] Chlorophyll a was estimated according to known technology. Specifically, the supernatant was measured at 666 nm and 653 nm, respectively, and expressed in mg / g fresh weight. Phycobiliproteins (phycoerythrin, PE and phycocyanin, PC) were extracted using 2 mL of sodium phosphate buffer (50 mM, pH 6.7) according to known technology. After homogenization using a motor-driven tissue grinder, the samples were centrifuged at 14,000 xg for 30 minutes at 4 °C. The supernatant was measured at 568 nm, 620 nm, and 730 nm, respectively, and expressed in mg / g fresh weight.

[0074] Example 7. Statistical Analysis

[0075] Two-way analyses of variance (ANOVAs) were performed according to Tukey's post-hoc test (p < 0.05) to explore differences in SGR and antioxidant enzyme activity according to temperature function, application of bioactive agents, or both. Data are expressed as mean ± standard deviation. All statistical analyses were performed using the Social Science Statistical Package (SPSS) program version 25 (SPSS Inc., Chicago, Illinois, USA).

[0077] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0078] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

Claim 1 Sargassum extract ( Sargassum horneri Radial patterned seaweed containing extract) as an active ingredient ( Neopyropia yezoensis As a bioactive agent for ), the above Sargassum extract is characterized by increasing the APX activity of Pteris versicolor, and the above Sargassum extract is a Sargassum boiling-based extract ( Sargassum horneri boiling extract, SBE), Sargassum horneri immersion-based extract ( Sargassum horneri Soaking extract (SSE) and Sargassum horneri high-pressure sterilization-based extract ( Sargassum horneri A bioactive agent characterized by being at least one selected from the group consisting of autoclaving extract (SAE), wherein the above-mentioned boiled Sargassum extract is characterized by increasing the production yield of Sargassum at 20°C compared to 10°C, wherein the above-mentioned boiled Sargassum extract is characterized by increasing the SOD activity of Sargassum, and wherein the above-mentioned soaking extract is characterized by increasing the content of phycobiliprotein of Sargassum. Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete

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