KR102524067B1 - Antioxidant composition comprising loliolide as an active ingredient - Google Patents

Abstract

The present invention relates to an antioxidant composition containing loriolide as an active ingredient, wherein (-)-loliolide isolated/extracted from Sargassum horneri can be used for lipid peroxidation in cells and zebrafish animal models. , By confirming that cell death and reducing ROS levels, (-)-loliolide is not only provided as an antioxidant agent, but also a treatment for diseases caused by excessive accumulation of active oxygen, health functional food, and functional It is provided as a cosmetic composition.

Description

Antioxidant composition comprising loliolide as an active ingredient

The present invention relates to an antioxidant composition comprising loriolide as an active ingredient.

Free radicals and reactive oxygen species (ROS) are generated during aerobic metabolism in vivo and are the main cause of cell damage with various side effects that cause DNA, protein, and cell membrane degradation. Free radicals and reactive oxygen species generated during normal cell metabolism play a role in killing pathogens invading from the outside and are regulated at the cellular level by clearance mechanisms. However, when free radicals and reactive oxygen species accumulate in cells without being removed by the scavenging mechanism, they cause cell damage and, in extreme cases, induce cell death, resulting in inflammation including cancer, anemia, myocardial infarction, cerebral palsy, arteriosclerosis, diabetes, It has been reported to cause various diseases such as rheumatoid arthritis, Parkinson's disease, skin damage, and autoimmune diseases, and to be a major cause of aging.

Antioxidant enzymes such as peroxidase and catalase exist as mechanisms to scavenge free radicals and reactive oxygen species, and antioxidant substances such as vitamin E and vitamin C can also scavenge free radicals and reactive oxygen species. However, when free radicals and reactive oxygen species are present in excess, these scavenging mechanisms fail to remove all reactive oxygen species, resulting in damage due to oxidative stress.

In order to reduce oxidative stress caused by excessive amounts of free radicals and active oxygen, research on safe and efficient antioxidants is being conducted, and functional products are being developed using them. Therefore, the demand and preference for new natural antioxidants that can be used in various fields such as medicine and health functional foods, are safe, and have less side effects in vivo are gradually increasing.

Meanwhile, products using seaweed as a natural source of physiologically active compounds are increasing in various fields such as functional foods, cosmetics, and pharmaceuticals. It has been reported that most seaweeds contain high levels of abundant physiologically active secondary metabolites with structural diversity. The inventors of the present invention discovered a substance exhibiting antioxidant activity from the seaweed Sargassum horneri and tried to provide it as a pharmaceutical composition, health functional food, and cosmetic composition for diseases caused by excessive accumulation of active oxygen.

Korean Registered Patent Publication No. 10-1481758

The present invention relates to an antioxidant composition containing loriolide as an active ingredient, wherein (-)-loliolide isolated/extracted from Sargassum horneri can be used for lipid peroxidation in cells and zebrafish animal models. , By confirming that cell death and reducing ROS levels, (-)-loliolide is not only provided as an antioxidant agent, but also a treatment for diseases caused by excessive accumulation of active oxygen, health functional food, and functional It is provided as a cosmetic composition.

The present invention provides an antioxidant composition comprising loliolide or a salt thereof as an active ingredient.

In addition, the present invention provides a method for preparing an antioxidant composition comprising the step of extracting loliolide from Sargassum horneri .

In addition, the present invention provides a pharmaceutical composition for preventing or treating diseases caused by excessive accumulation of active oxygen, comprising the antioxidant composition as an active ingredient.

In addition, the present invention provides a health functional food composition comprising the antioxidant composition as an active ingredient.

In addition, the present invention provides a cosmetic composition comprising the antioxidant composition as an active ingredient.

According to the present invention, by confirming that (-)-loliolide isolated/extracted from Sargassum horneri reduces lipid peroxidation, cell death and ROS levels in cells and zebrafish animal models, In addition to providing (-)-loliolide as an antioxidant agent, it can be provided as a treatment for diseases caused by excessive accumulation of active oxygen, a health functional food, and a functional cosmetic composition.

1 is a schematic diagram showing a method for separating/extracting (-)-loliolide from Sargassum horneri .
FIG. 2 is a result of evaluating the effects of (-)-loliolide on oxidative stress (A) and production (B) of reactive oxygen species (ROS) in a cell model.
Figure 3 is a result of evaluating the effect of (-) -loliolide on survival rate (A) and heart rate (B) in a zebrafish model.
Figure 4 evaluates the effects of (-)-loliolide on production (A), cell death (B), and lipid peroxidation (C) of reactive oxygen species (ROS) in a zebrafish model is a result

The terms used in this specification have been selected from general terms that are currently widely used as much as possible while considering the functions in the present invention, but these may vary depending on the intention of a person skilled in the art, precedent, or the emergence of new technologies. In addition, in a specific case, there is also a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, not simply the name of the term.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in this application, it should not be interpreted in an ideal or excessively formal meaning. don't

Numerical ranges are inclusive of the values defined therein. Every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written. Every numerical limitation given throughout this specification will include every better numerical range within the broader numerical range, as if the narrower numerical limitations were expressly written.

Hereinafter, the present invention will be described in more detail.

The present invention provides an antioxidant composition comprising loliolide or a salt thereof as an active ingredient.

The loliolide is derived from Sargassum horneri and is (-)-loliolide, specifically (6S,7aR)-6-hydroxy- 4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one ((6S,7aR)-6-hydroxy-4,4,7a-trimethyl-5,6, 7,7a-tetrahydrobenzofuran-2(4H)-one).

[Formula 1]

Since the loliolide has been demonstrated to have activity to reduce lipid peroxidation, cell death, and ROS levels in cells and animal models, it can be used as an active ingredient in an antioxidant composition.

In addition, the present invention provides a method for preparing an antioxidant composition comprising the step of extracting loliolide from Sargassum horneri .

The loliolide may be obtained by extracting Sargassum horneri with methanol to prepare a methanol extract, and fractional extraction of the prepared methanol extract with chloroform.

In addition, the present invention provides a pharmaceutical composition for preventing or treating diseases caused by excessive accumulation of active oxygen, comprising the antioxidant composition as an active ingredient.

Diseases caused by excessive accumulation of active oxygen may be at least one selected from the group consisting of cancer, inflammation, anemia, myocardial infarction, cerebral palsy, arteriosclerosis, diabetes, rheumatoid arthritis, Parkinson's disease, and autoimmune diseases, It is not limited thereto.

The pharmaceutical composition may be formulated in the form of one or more external agents selected from the group consisting of creams, gels, patches, sprays, ointments, warning agents, lotions, liniment agents, pasta agents, and cataplasma agents.

The pharmaceutical composition of the present invention may further contain pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include excipients such as starch, sugar and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose, gelatin, alginates, and polyvinyl fibres. binders such as rolidone, etc., lubricants such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone, crospovidone, surfactants such as polysorbates, cetyl alcohol, and glycerol; don't The pharmaceutically acceptable carrier and diluent may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents and/or dispersion media.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the desired method, and is preferably administered orally. In addition, the dosage of the pharmaceutical composition of the present invention varies depending on the subject's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease, but is not limited thereto.

In addition, the present invention provides a health functional food composition comprising the antioxidant composition as an active ingredient.

The present invention can be generally used as a commonly used food.

The food composition of the present invention can be used as a health functional food. The term "health functional food" refers to food manufactured and processed using raw materials or ingredients having useful functionalities for the human body in accordance with the Health Functional Food Act, and "functional" refers to food that is not related to the structure and function of the human body. It refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological functions.

The food composition of the present invention may include conventional food additives, and the suitability as the "food additive" is determined according to the general rules of the food additive code approved by the Ministry of Food and Drug Safety and general test methods, etc., unless otherwise specified. It is judged according to the specifications and standards for the item.

Items listed in the "Food Additive Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as dark pigment, licorice extract, crystalline cellulose, goyang pigment, guar gum, and mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations.

The food composition of the present invention can be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like.

For example, among the health functional foods in the form of capsules, hard capsules can be prepared by mixing and filling the composition according to the present invention with additives such as excipients in conventional hard capsules, and soft capsules can be prepared by filling the composition according to the present invention. It can be prepared by mixing with additives such as excipients and filling in a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.

Definitions of terms for the excipients, binders, disintegrants, lubricants, corrigents, flavoring agents, etc. are described in literature known in the art, and include those having the same or similar functions. There is no particular limitation on the type of food, and it includes all health functional foods in the usual sense.

In addition, the present invention provides a cosmetic composition comprising the antioxidant composition as an active ingredient.

The cosmetic composition includes softening lotion, astringent lotion, nutrient lotion, nutrient cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil, body It may be a formulation selected from the group consisting of essence, makeup base, foundation, hair dye, shampoo, rinse and body cleanser.

It can be prepared in various forms according to conventional cosmetic preparation methods using extracts or fractions, and may include conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments, and fragrances commonly used in the field of cosmetic compositions. can

Hereinafter, experimental examples and examples will be described in detail to aid understanding of the present invention. However, the following experimental examples and examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following experimental examples and examples. Experimental examples and examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Experimental Example> Experimental Materials and Methods

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Samples and Reagents

RPMI-1640 medium used in this experiment, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent, trypsin-EDTA, FBS (fetal bovine serum), and penicillin-strepto Penicillin-streptomycin (P/S) was purchased from Gibco-BRL (Grand Island, NY, USA). 2,2'-Aazobis-(2-amidinopropane) dihydrochloride (AAPH), acridine orange, dimethyl sulfoxide (DMSO), 1,3-Bis(diphenylphosphino)propane (DPPP), and DCfh-DA ( 2',7'-dichlorofluorescein diacetate) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

2. Preparation and analysis of loliolide

The (-)-loliolide used in this experiment was prepared by extraction/purification from Sargassum horneri. Specifically, as shown in FIG. 1, Sargassum horneri was extracted with 80% methanol and then fractionated with chloroform. The chloroform fraction was separated using high-performance centrifugal partition chromatography (HPCPC) by applying a solvent system in which the n-hexane:ethyl acetate:methanol:DW ratio was 5:5:5:5 (v/v).

The isolated (-)-loliolide was analyzed for single compounds using a high-performance liquid chromatography (HPLC) system (Waters Corporation, Milford, MA, USA) equipped with a PDA detector. The structure of (-)-loliolide was confirmed by ¹ H, ¹³ C, DEPT, COSY, and HMBC NMR spectra on a JEOL DELTA-400 (400 MHz) spectrometer (Peabody, MA, USA).

3. Vero cell model

Monkey kidney fibroblast Vero cells (KCLB, Seoul, Korea) were cultured in RPMI-1640 medium containing 1% P/S and 10% FBS. Vero cells were subcultured every 3 days and seeded at a concentration of 5 X 10 ⁴ cells/well in 24 well plates for subsequent experiments.

4. Intracellular AAPH clearance assay using DCFH-DA

After the Vero cells were inoculated and cultured for 16 hours, they were treated with (-)-loliolide for 30 minutes to confirm changes in intracellular ROS levels. Thereafter, 10 mM AAPH was treated for 30 minutes, followed by 500 μg/mL DCFH-DA. Finally, ROS levels in these cells were measured by DCFDA (2',7'-dichlorofluorescein diacetate) assay.

5. Assessment of cell viability

After culturing the cells in a humidified condition at 37° C. for 16 hours, they were treated with various concentrations of (-)-loliolide and cultured for 30 minutes. Thereafter, 10 μL of AAPH (10 mM) was added and cultured at 37° C. for 24 hours. Thereafter, MTT assay was performed on the cells for 4 hours, and absorbance was measured at a wavelength of 540 nm.

6. Assessment of apoptotic body formation by Hoechst 33342 staining

The level of apoptotic body formation in Vero cells stimulated with AAPH (2,2'-Aazobis-(2-amidinopropane) dihydrochloride) was evaluated based on cell nuclei morphology observed using the cell-permeable DNA dye Hoechst 33342. Following treatment and stimulation, cells were cultured for 1 hour prior to exposure to AAPH at a concentration of 10 mM. After 24 hours, the cells were stained with 1.5 μL of Hoechst 33342 at a concentration of 10 mg/ml and incubated for 10 minutes. The morphology of cell nuclei was observed using a fluorescence microscope (Olympus Life Science, Tokyo, Japan).

7. Effect of loliolide and AAPH in zebrafish embryo model

The effect of (-)-loliolide on AAPH-induced oxidative stress in a zebrafish embryo model was evaluated. Embryos 7-9 hours post fertilization (hpf) were transferred to each well of a 12 well plate. 900 μL of embryo medium mixed with (-)-loliolide and AAPH was added. Embryos were incubated for 1 hour in various concentrations of (-)-loliolide before exposure to 15 mM AAPH. After AAPH treatment, embryos were cultured for 24 hours and replaced with fresh embryo medium. Zebrafish experiments were approved by the Animal Care Committee of Jeju National University (approval number 2019-O-0074).

8. Assessment of survival rate and heart rate pattern

Survival (mortality) of AAPH-treated zebrafish embryos was evaluated at 72 hours post fertilization (72 hpf). To evaluate the toxicity of the samples, atrial and ventricular heart rates were evaluated at 48 hours post fertilization (48 hpf).

9. Evaluation of intracellular ROS production, lipid peroxidation and apoptosis in zebrafish embryos

Intracellular ROS levels, lipid peroxidation and cell death were measured with oxidation-sensitive fluorescent dyes such as DCFDA, DPPP, and acridine orange. Embryos 2 days after fertilization (2 dpf) were transferred to a 96-well plate and treated with 20 μg/mL DCFDA-DA for 1 hour, 25 μg/mL DPPP for 1 hour, and 7 μg/mL embryos at 28.5 ° C in the dark. Acridine orange was treated with each fluorescent dye in the well for 30 minutes. The treated fluorescent dye is used to measure intracellular lipid peroxidation, apoptosis and ROS levels.

10. Data and statistical analysis

Data are presented as mean ± standard deviation (SD). All statistical analyzes were performed using SPSS® software. Values were evaluated using one-way analysis of variance (ANOVA) followed by Duncan's multiple range tests.

Example 1. Separation of (-)-loliolide

The (-)-loliolide used in this experiment was extracted from Sargassum horneri, fractionated with chloroform, and further separated by HPCPC to obtain 1D and 2D structures of the separated compound from deuterated methanol. ¹ H, ¹³ C, HSQC, COSY, DEPT, and HMBC spectra were confirmed using NMR. Based on the correlation of two-dimensional nuclear magnetic resonance spectroscopy (NOESY), the isolated compound was identified as (-)-loliolide corresponding to the (6S, 7aR)-configuration. .

As shown in FIG. 1, the conventional method for separating (-)-loliolide is performed in 5 steps, but in this experiment, it is performed in 3 steps, which is simple and high-yield (-)-loliolide. (loliolide) was confirmed to be separated.

Example 2. Cell protection and antioxidant effect of (-)-loliolide

The effect of (-)-loliolide on AAPH-induced cell viability reduction in Vero cells was evaluated by MTT assay.

As shown in Figure 2, it was found that the survival rate of Vero cells increased according to the concentration treated with (-)-loliolide compared to the control group treated with AAPH alone. ROS analysis using DCFH-DA showed that the ROS level of cells treated with (-)-loliolide gradually decreased.

In addition, the level of apoptosis in AAPH-stimulated Vero cells was assessed by chromatin condensation and nuclear fragmentation observed with Hoechst 33342 staining. As shown in FIG. 2C , apoptosis due to AAPH treatment showed apoptotic bodies, but the number of apoptotic bodies decreased as (-)-loliolide was treated. AAPH-stimulated cells showed higher amounts of chromatin condensation and nuclear fragmentation (bright spots). At the same time, as the concentration of (-)-loliolide treatment increased, the frequency of chromatin condensation and nuclear fragmentation decreased, and the number of apoptotic bodies decreased.

Overall, these results demonstrate that (-)-loliolide has an antioxidant effect that protects cells from AAPH-induced ROS.

Example 3. Effects of (-)-loliolide on AAPH toxicity and cell death in animal models

To evaluate the effect of (-)-loliolide in an animal model, the survival rate and heart rate change of zebrafish larvae were evaluated by AAPH-induced toxicity.

As shown in FIG. 3, when AAPH was treated with zebrafish larvae, the survival rate was reduced to 50%. It has been shown that the survival rate of zebrafish increases as the treatment dose of (-)-loliolide increases, suggesting that (-)-loliolide protects zebrafish from AAPH-induced toxicity. prove that it protects In addition, AAPH-treated zebrafish larvae showed an increase in heart rate compared to the control group, but a gradual decrease in heart rate as (-)-loliolide was treated.

Example 4. Effects of (-)-loliolide on ROS generation, apoptosis, and lipid peroxidation in animal models

DCFDA staining with a fluorescent probe is used as a method to evaluate intracellular ROS levels in zebrafish embryos. The fluorescence intensity of DCF (2',7'-dichlorofluorescein), an oxide of DCFDA, is used as a measure of intracellular ROS concentration.

As shown in FIG. 4a, when AAPH was treated in zebrafish embryos, the ROS concentration represented by green fluorescence was increased by 250% compared to the control group. ROS levels were shown to decrease with increasing treatment doses of (-)-loliolide.

As shown in Figure 4b, acridine orange is used to evaluate apoptotic cells, and when zebrafish embryos are treated with AAPH, the fluorescence intensity of acridine orange increases by 210% compared to the control group. appear. The fluorescence intensity of acridine orange was found to decrease with increasing treatment dose of (-)-loliolide.

As shown in Figure 4c, oxidized DPPP (diphenyl-1-pyrenylphosphine) is used to measure lipid peroxidation, and when zebrafish embryos were treated with AAPH, DPPP fluorescence was 161% higher than that of the control group. DPPP fluorescence was shown to decrease with increasing treatment dose of (-)-loliolide.

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (11)

delete delete delete delete delete A method for producing an antioxidant composition comprising the step of extracting (-)-loliolide from Sargassum horneri ,
The step of extracting the (-)-loliolide,
(a) extracting Sargassum horneri with 80% methanol; and
(b) fractionally extracting the extract according to step (a) with chloroform in a solvent having a volume ratio of n-hexane, ethyl acetate, methanol and water of 5:5:5:5; Antioxidant composition comprising Manufacturing method of. delete delete delete delete delete

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