KR20230127929A - Biostimulants including sargassum horneri extracts - Google Patents

Abstract

One embodiment of the present invention provides a bioactive agent containing Sargassum horneri extract as an active ingredient. The hoesaeng hatban extract can be extracted by boiling-based extraction method (SBE), immersion-based extraction method (SSE), high-pressure sterilization-based extraction method (SAE), and ethanol extraction method (SEE), among which SBE, SSE, and SAE The extracts extracted with can improve the tolerance to high temperature stress that inhibited the growth of radiation laver ( Neopyropia yezoensis ). According to one embodiment of the present invention, the SBE and SSE extraction method was helpful in increasing the content of phycoviriliprotein, and SBE can increase Superoxide dismutase (SOD) activity.

Description

Physiologically active agent containing hoesaeng japonica extract as an active ingredient {BIOSTIMULANTS INCLUDING SARGASSUM HORNERI EXTRACTS}

The present invention relates to a physiologically active agent containing an extract of genus japonica as an active ingredient. It relates to, and more particularly, to a bioactive agent containing as an active ingredient an extract of spp.

Seaweed is widely cultivated in Asian countries such as China, Indonesia, Korea, and Japan. The seaweed aquaculture industry is growing rapidly in the United States and Europe, and growing interest has increased as applications for food, feed, cosmetics, nutritional supplements and, more recently, biofuels are expanding. In particular, Korea is the third largest producer of seaweed in the world after China and Indonesia. The main aquaculture species is radioactive laver ( Neopyropia spp .; mainly N. yezoensis ), followed by Undaria pinnatifida and Saccharina japonica . has However, the seaweed aquaculture industry is threatened by climate change, particularly global warming, and among many environmental stressors, rising sea surface temperature acts as a major stressor, reducing the production and quality of seaweed. For example, Radiant seaweed ( Neopyropia yezoensis ) is the most economically important red algae cultivated in Korea, China and Japan. Compared to other cultured seaweeds, it has the highest marketability per unit mass. The warming trend of surface seawater has a direct impact on the aquaculture of Neopyropia yezoensis . For example, when the seawater temperature rises, the growth of radiation-patterned seaweed is hindered, reducing aquaculture productivity. Therefore, in order to maintain or increase the current agricultural productivity, it is urgent to develop a new technology for strengthening the heat resistance of the spun patterned seaweed strain.

Biostimulants are known to influence the physiological processes of land plants by providing potential benefits to land plant growth, development, or abiotic stresses. Some examples also highlight that the application of seaweed extracts can increase the resistance of aquacultured seaweed to abiotic and biological stresses. Since 2015, a large group of hoefish ( Sargassum horneri ) has drifted along currents in the East China Sea to Jeju Island and South Jeolla Province. The huge biomass of oxen caps has caused economic damage to tourism and aquaculture. As an approach to utilize some of the biomass accumulated in Korea, attempts have been made to extract useful bioactive compounds from Sargassum horneri (eg, antioxidant, anti-inflammatory, and anti-allergic properties). Although Sargassum horneri has not yet been studied as a source of bioactive agents, some extracts from other Sargassum species have been demonstrated to act as bioactive agents when applied to terrestrial plants. For example, extracts of Sargassum wightti of the genus Sargassum improved root and shoot length when applied to the legume Vigna radiata (green gram), whereas solid and liquid extracts of Sargassum crassifolium improved rice growth and increased yield.

There is no technology using Sargassum horneri as a bioactive agent, and there is a need for a technology that can actively utilize Sargassum horneri in a situation where the damage caused by the temperature rise of seaweed is enormous.

Republic of Korea Patent Publication No. 10-2016-0096995

A technical problem to be achieved by the present invention is to provide a physiologically active agent containing, as an active ingredient, an extract of A.

The technical problem to be achieved by the present invention is not limited to the above-mentioned technical problem, and other technical problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to achieve the above technical problem, one embodiment of the present invention provides a bioactive agent containing Sargassum horneri extract as an active ingredient.

In an embodiment of the present invention, the bioactive agent may exhibit a high-temperature tolerance effect.

In an embodiment of the present invention, the bioactive agent may be applied to radiation patterned seaweed ( Neopyropia yezoensis ).

In an embodiment of the present invention, the hoesaeng capsular extract is a hoesaeng caps boiling extract ( Sargassum horneri boiling extract, SBE), a hoesaengs caps immersion-based extract ( Sargassum horneri soaking extract, SSE) and a hoesaeng caps high-pressure sterilization base extract ( Sargassum horneri autoclaving extract, SAE) may be at least one selected from the group consisting of.

In an embodiment of the present invention, the hoesaengi hatban boiling extract ( Sargassum horneri boiling extract, SBE) can increase SOD activity.

In an embodiment of the present invention, the Sargassum horneri soaking extract (SSE) can increase the content of phycobiliprotein.

In an embodiment of the present invention, the hoesaeng hats and hats boiling-based extract ( Sargassum horneri boiling extract, SBE) or hoesaengs hats and hats soaking-based extract ( Sargassum horneri soaking extract, SSE) can increase the growth rate.

The physiologically active agent according to an embodiment of the present invention contains the extract of the genus chinensis as an active ingredient and may have a high-temperature resistance effect.

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

Figure 1 is a graph showing the specific growth rate (SGR) of radiation patterned laver ( Neopyropia yezoensis ) and four extraction methods using Sargassum horneri as an extraction source at different temperatures (10 ° C, 20 ° C). Different letters in the bars indicate significant differences (p < 0.05). Error bars represent the mean ± standard deviation of three replicates. Control (no extract), hoesaeng capsular boil-based extract (SBE), hoesaengs capsicum ethanol extract (SEE), hoesaengs capsicum immersion-based extract (SSE) and hoesaengs capsicum autoclave extract (SAE).
Figure 2 shows chlorophyll a (a), phycocyanin (b) and phycoerythrin (c) of radioactive laver ( Neopyropia yezoensis ) at different temperatures (10 ° C, 20 ° C), and hoesaeng This is a graph showing four extraction methods using Mojaban as an extraction source. Different letters in the bars indicate significant differences (p < 0.05). Error bars represent the mean ± standard deviation of three replicates. Control (no extract), hoesaeng capsular boil-based extract (SBE), hoesaengs capsicum ethanol extract (SEE), hoesaengs capsicum immersion-based extract (SSE) and hoesaengs capsicum autoclave extract (SAE).
Figure 3 shows superoxide dismutase (SOD; a), catalase (CAT; b), glutathione reductase (GR; c) and ascorbate peroxidase of radiation patterned seaweed ( Neopyropia yezoensis ) at different temperatures (10 ° C, 20 ° C). (APX; d), and a graph showing four extraction methods using hoesaeng caps as an extraction source. Different letters in the bars indicate significant differences (p < 0.05). Error bars represent the mean ± standard deviation of three replicates. Control (no extract), hoesaeng capsular boil-based extract (SBE), hoesaengs capsicum ethanol extract (SEE), hoesaengs capsicum immersion-based extract (SSE) and hoesaengs capsicum autoclave extract (SAE).
Figure 4 shows hydrogen peroxide (H ₂ O ₂ ; a), lipid peroxidation (LPO; b) and reactive oxygen species (ROS; c ) , and hoe This is a graph showing four extraction methods using Saengi Mojaban as an extraction source. Different letters in the bars indicate significant differences (p < 0.05). Error bars represent mean±standard deviation of triplicate. Control (no extract), hoesaeng capsular boil-based extract (SBE), hoesaengs capsicum ethanol extract (SEE), hoesaengs capsicum immersion-based extract (SSE) and hoesaengs capsicum autoclave extract (SAE).
Figure 5 is a graph showing the total protein of radiation patterned seaweed ( Neopyropia yezoensis ) at different temperatures (10 ° C, 20 ° C) and four extraction methods using hoesaeng caps as an extraction source. Different letters in the bars indicate significant differences (p < 0.05). Error bars represent the mean ± standard deviation of three replicates. Control (no extract), hoesaeng capsular boil-based extract (SBE), hoesaengs capsicum ethanol extract (SEE), hoesaengs capsicum immersion-based extract (SSE) and hoesaengs capsicum autoclave extract (SAE).
Figure 6 is a graph showing the total phenol content of radiation patterned seaweed ( Neopyropia yezoensis ) at different temperatures (10 ° C, 20 ° C) and four extraction methods using hoesaeng caps as an extraction source. Different letters in the bars indicate significant differences (p < 0.05). Error bars represent the mean ± standard deviation of three replicates. Control (no extract), hoesaeng capsular boil-based extract (SBE), hoesaengs capsicum ethanol extract (SEE), hoesaengs capsicum immersion-based extract (SSE) and hoesaengs capsicum autoclave extract (SAE).

Hereinafter, the present invention will be described with reference to the accompanying drawings. However, the present invention may be embodied in many different forms and, therefore, is not limited to the embodiments described herein. And in order to clearly explain the present invention in the drawings, parts irrelevant to the description are omitted, and similar reference numerals are attached to similar parts throughout the specification.

Throughout the specification, when a part is said to be "connected (connected, contacted, combined)" with another part, this is not only "directly connected", but also "indirectly connected" with another member in between. "Including cases where In addition, when a part "includes" a certain component, it means that it may further include other components without excluding other components unless otherwise stated.

Terms used in this specification are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "include" or "have" are intended to indicate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.

The bioactive agent according to one aspect of the present invention may include Sargassum horneri extract as an active ingredient.

Effects of bioactive agents and temperature on growth rates

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). Radiated seaweed ( Neopyropia yezoensis ) showed a similar growth rate 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 control (Fig. 1, p<0.004). The growth rate was significantly affected by the interaction of extraction method and temperature (Table 1, p<0.001). These results showed that 20 ℃ has a negative effect than 10 ℃ on the growth of radiation laver ( Neopyropia yezoensis ). In the present invention, it was shown that the extract of S. horneri has a positive effect on the thermal durability of radiation laver ( Neopyropia yezoensis ).

parameter temperature Extraction method Temperature*Extraction method df F p df F p df F p SGR One 248.974 <0.001 4 9.929 <0.001 4 7.622 <0.001 chloroplast a One 153.391 <0.001 4 3.779 <0.05 4 5.325 <0.05 Phycocyanin
(Phycocyanin) One 48.891 <0.001 4 7.961 <0.001 4 5.809 <0.05 Phycoerythrin One 61.736 <0.001 4 13.796 <0.001 4 7.365 <0.001 phenol One 9.765 <0.05 4 2.150 >0.05 4 0.641 >0.05 protein One 8.436 <0.05 4 1.195 >0.05 4 1.681 >0.05 SOD One 31.492 <0.001 4 19.434 <0.001 4 12.138 <0.001 CAT One 49.865 <0.001 4 2.359 >0.05 4 1.312 >0.05 GR One 176.683 <0.001 4 4.529 <0.05 4 3.902 <0.05 APX One 53.845 <0.001 4 9.781 <0.001 4 2.179 >0.05 ROS One 6.859 <0.05 4 8.158 <0.001 4 3.886 <0.05 H ₂ O ₂ One 9.358 <0.05 4 3.741 <0.05 4 3.565 <0.05 LPO One 28.696 <0.001 4 0.706 >0.05 4 2.06 >0.05

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

Effects of bioactive agents and temperature on pigment

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., and a significant difference could only be found in SAE (FIG. 2a). The chlorophyll-a content at 20 °C was significantly lower than that at 10 °C, and no positive effect of the extract from the hoesaeng japonica 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 phycocyanin and phycoerythrin contents were significantly higher than all other conditions (p <0.001) (Fig. 2b and c). Chlorophyll a and phycobiliprotein were significantly affected by the interaction of temperature and extract (Table 1, p <0.05). According to one embodiment of the present invention, the application of seaweed-derived bioactive agents can increase the concentration of phycovirin protein in red algae.

Effects of bioactive agents and temperature on biochemical reactions

According to one embodiment of the present invention, enzymatic (SOD, CAT, GR and APX) and non-enzymatic (total phenolic) antioxidant activities were measured to evaluate oxidative stress. At 10 °C, SOD activity was the highest in SBE, and all other extracts were similar to the control group. SOD in SSE at 20 °C 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 to be the first enzyme controlling the detoxification pathway of ROS. According to an embodiment of the present invention, the SOD activity of Neopyropia yezoensis can be increased by SBE in Sargassum horneri extract (FIG. 3a). According to one embodiment of the present invention, the SOD activity of radiation patterned seaweed ( Neopyropia yezoensis ) was reduced during high temperature treatment. However, SBE can increase the SOD activity of radiation laver ( Neopyropia yezoensis ) at the optimum temperature of 10 ° C. SOD is a metalloprotein and three isoforms can be determined depending on the metal-centered cofactor: According to an embodiment of the present invention, SBE is rich in organic compounds (i.e., polyphenols, organic acids) for chelation. and increase the acceptance of trace metals for SOD synthesis.

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

GR is an enzyme in the Ascorbate-Glutathione (AsA-GSH) cycle and can play an essential role in the defense system against ROS by maintaining the reduced state of glutathione. GR activity was significantly lower at 20 °C than at 10 °C, regardless of the extraction method (Fig. 3c, p < 0.045). According to one embodiment of the present invention, a less increase in lipid peroxidation at 20 ° C. than at 10 ° C. in Neopyropia yezoensis may mean that GR activity increases at 10 ° C.

On the other hand, according to one embodiment of the present invention, APX activity may appear opposite to GR activity (FIG. 3d). APX activity was higher at 20 °C than at 10 °C, and all the extracts of hoesaeng moss slightly increased APX activity compared to the control group at both temperatures (p < 0.036). APX is an important component of the AsA-GSH circuit and can be responsible for converting H ₂ O ₂ to H ₂ O. According to one embodiment of the present invention, the decrease in H ₂ O ₂ at 20° C. may be due to high APX activity ( FIGS. 3D and 4A ). According to one embodiment of the present invention, these results can explain the low LPO at 20 °C (FIG. 4b).

According to one embodiment of the present invention, the ROS value obtained here did not show a significant difference according to temperature (FIG. 4c, p>0.05), showing that antioxidant enzymes can effectively relieve oxidative stress and regulate ROS balance. gave. Balanced ROS may act as important signaling modulators under stress conditions rather than playing a lethal role. Total protein content and total phenol content were only affected by temperature, and neither the extraction method nor the interaction between extract and temperature had a significant effect (Figs. 5 and 6, Table 1, p > 0.05).

According to one embodiment of the present invention, the extract of hoesaengi yezoensis can be a new physiologically active agent source that improves the heat durability of radiation laver ( Neopyropia yezoensis ). According to one embodiment of the present invention, the extraction method for obtaining a 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 radiation patterns. It can be effective in improving the growth and phycovirin protein content of seaweed ( Neopyropia yezoensis ). According to one embodiment of the present invention, regardless of the extraction method used, the expression level of antioxidant enzymes and oxidative stress can be governed by temperature.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Example 1.1. seaweed collection

Radiated seaweed ( Neopyropia yezoensis ) strain (NY-HN-ST1) was originally collected in December 2020 from Haenam radiation seaweed farm (34°57'N, 126°60'E). This strain was propagated through asexual reproduction at Incheon National University's Marine Ecology and Eco-Friendly Aquaculture Laboratory. All objects were cultured in von Stosch enriched solution (VSE) medium, 10 ° C, daylight, 90 ± 10 μmol m ^-2 s ^-1 photosynthetically active radiation (PAR) provided by fluorescent light, 12:12 L: D and salinity 30 psu did

Example 2. Preparation of bioactive agents

Sargassum horneri was collected in Jumunjin, Gangwon-do (37 degrees 90' north latitude, 128 degrees 83' east longitude) in June 2020. The oxen capsular extract (SHE) was prepared based on Zahra, Mehrnaz, Farzaneh, and Kohzad with minor modifications. After drying about 8kg of the hoeer hat half, it was powdered using a mixer mill MM 400 (Letch, Germany). 5 g of the powder was put into 500 ml of distilled water, stirred, and 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 a liquid extract.

The second extract, hereafter referred to as ethanol extract (SEE), was prepared with minor modifications to prior art. 250 g of the powder was put into 2.5 L of distilled water, mixed intermittently for 72 hours at room temperature, and Whatman no. It was filtered with 1 filter paper (11 μm) and concentrated with a rotary vacuum evaporator (R-210, Buchi, Switzerland) at 40°C. As a result, dry powder was obtained and used in the experiment. The soak-based extract and the boil-based extract were prepared according to known techniques with minor modifications. Both extracts were prepared by adding 3 g of powder to 300 mL of distilled water. To obtain the third extract, soak-based extract (SSE), 3 g of the powder was soaked in distilled water at room temperature for 2 days. Finally, the fourth extract, boiling-based extract (SBE), was cooled at room temperature after boiling 3 g of the powder in a water bath for 30 minutes. Both extracts were centrifuged at 3,134 x g for 25 minutes. The supernatant was filtered through Whatman no.1 filter paper (11 μm) to obtain a final liquid extract.

Example 3. Experimental Design

The collected radiation laver ( Neopyropia yezoensis ) was cultured in a 2 L glass cylinder with a density of 1 g L ^-1 . For the SBE, SEE, SSE and SAE treatments, the samples were incubated at 10°C under a cool white fluorescent light and photosynthetically active radiation (PAR) of 90±10 μmol m ^-2 s ^-1 provided by 12:12 L:D. were exposed to 10% of each extract for 10 days. The control group contained only sterilized seawater and VSE medium without extract. Medium was changed every 5 days to avoid nutrient limitations. Salinity was kept constant at 30 psu. After exposure to the extract, samples of Neopyropia yezoensis were washed in sterile seawater to remove residues. Washed radiation laver ( Neopyropia yezoensis ) was grown at different temperatures of 10 or 20 ° C in a 500 mL Erlenmeyer flask with a water density of 1 g L ^-1 using VSE medium supplemented with 0.25% (w / v) germanium dioxide. Incubated at two temperatures. Samples were incubated for 15 days, and medium was changed 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, SOD (superoxide dismutase), CAT (catalase), APX (ascorbate peroxidase), GR (glutathione reductase), ROS (reactive oxygen species), H ₂ O ₂ , LPO (lipid peroxidation), total phenol, total Proteins and pigments (chloroplast a, phycobiliprotein) were analyzed. The specific growth rate (SGR) was calculated using the formula:

W _end and W _initial represent the weight of the frond at T _end and T _initial .

Example 4. Antioxidant enzyme activity assay

Fresh thallus (approximately 100 mg) was ground on ice using a motorized tissue grinder in 1 mL of potassium phosphate buffer (50 mM, pH 7.0) containing 0.25% Triton X-100 and 1% polyvinylpyrrolidone. The homogenates were then centrifuged at 12,000 x g for 10 min at 4 °C. Supernatants were used to measure the activity of SOD, CAT, GR, APX, total protein and ROS. Enzyme activity measurements were performed in triplicate.

The protein content was measured by the known Bradford method. Briefly, 25 μL enzyme extract was mixed with 75 μL distilled water and 2.5 mL Bradford solution (25 mL H ₃ PO ₄ diluted with 0.025 g Coomassie blue dye, 12.5 mL 95% (v:v) ethanol and 250 mL distilled water). added. Reagents were vortexed to mix and allowed to stand for 5 minutes before measuring absorbance at 595 nm. Total protein content was measured based on bovine serum albumin (BSA) and expressed as mg/g.

Superoxide scavenging enzyme (SOD) activity was measured according to a known technique. Briefly, 20 μL of gyth extract was added to 150 μL of carbonate buffer (pH 10.2). The production of adrenochrome due to autoxidation of epinephrine was measured at 480 nm for 3 minutes on a blank, and the activity was expressed as U/mg protein.

Catalase (CAT) activity was measured based on known techniques. Briefly, 100 μL of 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 H ₂ O ₂ (0.1 M). Absorbance was measured at 240 nm (0 and 2 min). Activity was estimated using the molecular extinction coefficient of H ₂ O ₂ (0.043 mM ⁻¹ com ⁻¹ ) and expressed in mmol/mg protein units.

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

Ascorbate peroxidase (APX; EC 1.11.1.11) activity was measured according to known techniques. Briefly, 185 μL reaction buffer (50 mM potassium phosphate buffer, 0.25 mM ascorbic acid) was added to 10 μL enzyme extract. Absorbance was measured at 290 nm for 3 minutes at 25° C. after shaking for 5 seconds to determine non-specific ascorbate degradation. After that, 5 μL H ₂ O ₂ (200 mM) was added, shaken for 5 seconds, and measured at 290 nm for 5 minutes at 25 °C. Enzyme activity was calculated using an extinction coefficient of 2.8 mM ⁻¹ cm ⁻¹ and expressed in units of U/mg protein.

Example 5. Analysis of oxidative stress parameters

Fresh thallus (approximately 100 mg) was ground with 1 mL of 10% (w:v) trichloroacetic acid (TCA) solution on ice using a motorized tissue grinder. The homogenate was centrifuged at 7000 xg for 10 minutes at 4°C. The supernatant was used to measure H ₂ O ₂ and lipid peroxidation (LPO) levels. Oxidative stress parameters were measured in triplicate.

Reactive oxygen species (ROS) concentrations were measured according to known techniques. Briefly, 20 μL extract was added to 180 μL sample buffer and 200 μL 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 1 hour incubation in the dark at 20°C. ROS concentrations were expressed in units of U/mg protein.

H ₂ O ₂ was measured according to a known technique. Briefly, to 50 μL supernatant was added 150 μL potassium phosphate buffer (50 mM, pH 7.0) and 100 μL potassium iodide (1 M). Absorbance was measured at 390 nm compared to the blank. H ₂ O ₂ levels were expressed as nmol/g fresh weight using the H ₂ O ₂ standard curve.

Lipid peroxidation (LPO) levels were detected by measuring malondialdehyde (MDA) content using known techniques. Briefly, 2 mL thiobarbituric acid (TCA) was added to 100 μL supernatant and placed in a water bath at 95 °C for 45 min. Finally, the mixed solution was centrifuged (if necessary) at 4,000 xg, and the absorbance was measured at 532 nm compared to the blank. LPO levels were expressed in μmol MDA/mg protein units using an extinction coefficient of 156 mM ⁻¹ cm ⁻¹ .

Example 6. Total phenol content and pigment analysis

Fresh thallus (approximately 20 mg) was homogenized in 2 mL ice-cold 95% (w:w) methanol using a motor-driven tissue grinder. The homogenate was incubated for 48 hours at room temperature in the dark and then centrifuged at 13,000 x g for 5 minutes. The supernatant was used to determine total phenol content and chlorophyll a. All measurements were repeated three times.

The total phenol content was measured according to known techniques. Briefly, 200 μL of 10% (v:v) Folin-Ciocalteu reagent was added to 100 μL supernatant and thoroughly vortexed. About 800 μL Na ₂ CO ₃ (700 mM) was added, incubated at room temperature for 2 hours, and absorbance was measured at 765 nm. Phenol content was quantified using a gallic acid standard curve and expressed in units of mg GAE/g fresh weight.

Chlorophyll a was estimated according to a known technique. That is, the supernatant was measured at 666 nm and 653 nm, respectively, and expressed in mg/g fresh weight. Phycobiliprotein (phycoerythrin, PE and phycocyanin, PC) was extracted using 2 mL sodium phosphate buffer (50 mM, pH 6.7) according to a known technique. After homogenization using a motorized tissue grinder, samples were centrifuged at 14000 x g for 30 min at 4 °C. The supernatant was measured at 568 nm, 620 nm and 730 nm, respectively, and expressed as mg/g fresh weight.

Example 7. Statistical Analysis

Two-way analyzes of variance (ANOVAs) followed by Tukey's post hoc test (p < 0.05) were performed to explore differences in SGR and antioxidant enzyme activities as a function of temperature, application of bioactive agents, or both. Data are presented as mean±standard deviation. All statistical analyzes were performed using the Social Sciences Statistical Package (SPSS) program version 25 (SPSS Inc., Chicago, IL, USA).

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims (7)

A bioactive agent containing Sargassum horneri extract as an active ingredient.
According to claim 1,
The bioactive agent is characterized in that it exhibits a high temperature resistance effect.
According to claim 1,
The bioactive agent is a bioactive agent, characterized in that applied to radiation seaweed ( Neopyropia yezoensis ).
According to claim 1,
The hoesaeng hatban extract is a hoesaeng hatban boiling extract ( Sargassum horneri boiling extract, SBE), a hoesaeng hatban soaking-based extract ( Sargassum horneri soaking extract, SSE), and a hoesaeng hatban high-pressure sterilization-based extract ( Sargassum horneri autoclaving extract, SAE) ) A bioactive agent, characterized in that at least one selected from the group consisting of.
According to claim 4,
The hoesaengi hatban boiling extract ( Sargassum horneri boiling extract, SBE) is a bioactive agent characterized in that it increases SOD activity.
According to claim 4,
The bioactive agent, characterized in that the Sargassum horneri soaking extract (SSE) increases the content of phycobiliprotein.
According to claim 4,
The bioactive agent, characterized in that the hoesaeng hat half boiling extract ( Sargassum horneri boiling extract, SBE) or hoesaeng hat half soaking base extract ( Sargassum horneri soaking extract, SSE) increases the growth rate.