KR20110072153A - Composition comprising white rosa rugosa flower extract as active ingredient - Google Patents

Abstract

PURPOSE: An antioxidative and antibacterial composition containing Rosa rugosa var. alba flower extract is provided to remove free radical and to suppress DNA fragmentation and apoptosis. CONSTITUTION: An antioxidative composition contains Rosa rugosa var. alba flower extract as an active ingredient. The extract is isolated using water, anhydrous or hydrous alcohol of 1-6 carbon atoms, hexane, acetone, ethyl acetate, butyl acetate, 1,3-butylene glycol, or mixture solvent thereof. The extract contains cyclododecane or dodecyl acrylate. The composition is a cosmetic composition or functional food composition.

Description

Composition Comprising White Rosa rugosa Flower Extract as Active Ingredient}

The present invention relates to a composition comprising white rose rugosa flower extract as an active ingredient. More specifically, the present invention relates to an antioxidant or antimicrobial composition comprising white rose flower extract as an active ingredient.

Plants have been provided as a very useful resource for drug development, and research in many countries continues to identify plants that are effective for health. In the past decades, many scientists have been interested in finding plant chemicals, not chemical synthetic compounds, from plants for use in pharmaceuticals, therapeutic aids, or foods in finding synthetic or semisynthetic inducers. Plants can produce a wide variety of low molecular weight natural substances known as secondary metabolites, such as ascorbic acid, glutathione, uric acid, tocopherols, carotenoids, polyphenols, etc., and these compounds have a protective activity against active oxygen species. Have These substances, which accumulate in stressed plants that contain various stimulants or signal molecules, can perform a variety of functions in the plant, but their ecological function is not only provided as a unique medicinal resource, but also a beneficial medicinal feature for humans. (Zhao et al. 2005).

Of the few low molecular weight substances present in plants, polyphenols (hydrolyzable tannins, phenylpropanoids, etc.) are the most antioxidants in our diet and are clinically responsible for preventing some chronic diseases. It has been known. Polyphenol antioxidants such as resveratrol have been reported to inhibit the development and growth of mammalian cancer cells, and various polyphenolic substances derived from plants have been reported to have significant homeostasis and anti-inflammatory activity. The potential of therapy is known to be very high. In recent studies, antioxidant properties in plants are associated with the defense against oxidative stress, and several diseases in humans are known to be associated with aging. When the production of reactive oxygen species exceeds the cell's antioxidant capacity, modification of the cell's proteins, lipids and DNA (Deoxyriboneucleic acid) may result, leading to cell death or promoting aging and diseases associated with aging. Thus, this natural resource antioxidant overall understanding of the potential reactive oxygen species / active nitrogen to check for the feature species (O ₂ ^{and -,} HO ^and and ONOO ^-), extensive research on is required (Prior et al 2005.) .

White Rosa rugosa is a rose species in a wide area of East Asia. This plant has been used to make raw materials such as tea and rose oil and jams, and its roots have been used for diabetes, pain and chronic inflammatory diseases as orthodox therapy in Korea. However, despite these pharmacological characteristics, there are not many studies on antioxidant and antimicrobial activity of white rose flower extract or solvent fractions.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made an effort to search for and develop a physiologically active ingredient having antioxidant ability from natural substances derived from plants. As a result, the extracts from the flowers of white Rosa rugosa were found to be DPPH (2,2-diphenyl-1-picrylhydrazyl hydrate) and ABTS (2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)). In the evaluation experiments, such as antioxidant activity and nitric oxide scavenging activity was confirmed to have a very strong antioxidant activity, and at the same time experimentally confirmed that it also has an antimicrobial activity that inhibits the growth of the bacteria was completed.

Accordingly, it is an object of the present invention to provide a composition for antioxidant use comprising white rose flower extract as an active ingredient.

In addition, another object of the present invention is to provide a composition for antimicrobial use comprising a white rose flower extract as an active ingredient.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention is to provide an antioxidant composition comprising a white rose (Rose, Rosa rugosa var. Alba) flower extract as an active ingredient.

According to another aspect of the present invention, the present invention provides an antimicrobial composition comprising a white rose flower extract as an active ingredient.

The present inventors have made an effort to search for and develop a physiologically active ingredient having antioxidant ability from natural substances derived from plants. As a result, it was confirmed that the extract obtained from the flowers of white rose rosa (Rosa rugosa, Rosa rugosa var. Alba) has very strong antioxidant activity in the evaluation experiments such as antioxidant activity and nitric oxide scavenging activity using DPPH and ABTS. The present invention was completed by experimentally confirming that it also has antibacterial activity that inhibits the growth of bacteria.

The white rose flower extract of the present invention can be separated according to conventional methods known in the art for extracting extracts from natural products, that is, using conventional solvents under the conditions of conventional temperature, pressure.

As an extraction solvent for extracting the white rose flower extract of the present invention, a solvent that can be generally used in the extraction process may be used, and preferably, water, anhydrous or hydrous alcohol having 1 to 6 carbon atoms, hexane, acetone , Ethyl acetate, butyl acetate, 1,3-butylene glycol or a combination thereof can be used.

According to a preferred embodiment of the present invention, the white rose flower extract of the present invention is a fraction extract extracted using water, anhydrous or hydrous alcohol having 1 to 6 carbon atoms, hexane or a combination thereof as a solvent, more preferably water, methanol, Solvent fraction extract of butanol, hexane or a combination thereof.

The white rose flower extract of the present invention contains various polyphenol compounds, volatile compounds, and gallic acid. In particular, the extract of the present invention contains a large amount of dodecyl acrylate (dodecyl accrylate) and cyclododecane (cyclododecane) components.

According to a preferred embodiment of the present invention, the white rose flower extract of the active ingredient of the present invention is free radical scavenging activity, lipid peroxidation inhibitory activity, nitric oxide scavenging and inhibitory activity, DNA fragmentation induced by nitric oxide and It has the effect of suppressing cell death.

White rose flower extract of the present invention can also be used as an active ingredient of the cosmetic composition for antioxidant use.

Cosmetic compositions of the present invention can be prepared in any formulations conventionally prepared in the art and include, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing It may be formulated as cleansing, oil, powder foundation, emulsion foundation, wax foundation, spray, and the like, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be.

In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

In addition to the active ingredient and the carrier component, the components included in the cosmetic composition of the present invention include components conventionally used in cosmetic compositions, for example, conventional auxiliary agents such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings. It may include.

White rose flower extract of the present invention can also be used as an active ingredient of functional food composition for antioxidant use.

Functional food compositions of the present invention include ingredients that are commonly added in the manufacture of food, and include, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when prepared with a drink, flavoring agents or natural carbohydrates may be included as additional ingredients in addition to the active ingredient. For example, natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.); Disaccharides (eg maltose, sucrose, etc.); oligosaccharide; Polysaccharides (eg, dextrins, cyclodextrins, etc.); And sugar alcohols (eg, xylitol, sorbitol, erythritol, and the like). As the flavoring agent, natural flavoring agents (e.g., taumartin, stevia extract, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) can be used.

The present invention can be used as an active ingredient of antimicrobial composition because white rose flower extract has antimicrobial activity.

According to a preferred embodiment of the present invention, the white rose flower extract is an active ingredient of the antimicrobial composition is water, anhydrous or hydrous alcohol having 1 to 6 carbon atoms, hexane, acetone, ethyl acetate, butyl acetate, 1,3-butylene glycol Or a fraction extract of a solvent of a combination thereof, more preferably a fraction extract of a solvent of water, methanol, butanol, hexane or a combination thereof.

The features and advantages of the present invention are summarized as follows:

(i) White Rosa rugosa (Rosa rugosa var. alba) flower extract of the active ingredient of the present invention is free radical (free radical), lipid peroxidation inhibitory activity, nitric oxide scavenging and inhibitory activity, by nitric oxide Effects of inhibiting induced DNA fragmentation and cell death.

(Ii) The white rose flower extract, which is the active ingredient of the present invention, also has the growth inhibitory activity of bacteria, and thus retains antibacterial activity.

(Iii) Therefore, the white rose flower extract of the present invention can be developed as an active ingredient of antioxidant composition, cosmetic composition for antioxidant use, functional food composition, antimicrobial composition.

The present invention relates to an antioxidant composition and an antimicrobial composition comprising white rose flower extract as an active ingredient. The white rose flower extract of the present invention has free radical scavenging activity, peroxidation inhibitory activity of lipids, nitric oxide scavenging and inhibitory activity, nitrogen fragmentation induced by nitric oxide, and inhibits cell death. It can be very usefully used as an active ingredient in the composition. In addition, the white rose flower extract of the present invention also has a growth inhibitory activity against bacteria, it can be developed as an active ingredient of the antimicrobial composition.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Methods

1. Plant Materials and Chemicals

In May 2006, fresh white rose (Rosa rugosa var. Alba) flower was obtained from Jincheon, Chungbuk, Korea and completely dried under sunlight. The completely dried petals were ground in a rotor speed mill (Laval Lab Inc., Laval, Que), powdered and sterilized using a 70% ethanol spray, dried at 80 ° C. and stored at 4 ° C. until use. .

2. Reagents and Compounds

L-ascorbic acid, dimethyl sulfoxide (DMSO), H ₂ O ₂ (hydroperoxide), SNAP (S-nitroso-N-acetylpenicillamine), DPPH (2,2-diphenyl-1-picrylhydrazyl hydrate), ABTS (2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)) , gallic acid, BHA (butylated hydroxyanisole), BSA (bovine serum albumin) and Folin-Ciocalteu's phenol reagent were purchased from Sigma (St. Louis, Mo.). Peroxynitrite (ONOO ^-) was purchased from Calbiochem Co. (San Diego, Calif.).

3. Extract Separation

The finely ground flower powder was extracted with methanol for 24 hours at room temperature. The ratio of the flower powder and the solvent was 1:20 (w / v). The extracted suspension was filtered through Whatman No. 1 filter paper. This process was repeated twice for the residue, and all the filtrates were collected. All filtrates obtained in the above procedure were collected, concentrated under vacuum at 50 ° C. and freeze dried. The freeze-dried methanol extract (1 g) was dissolved again in methanol (1.5 mL) and 20 mL of water was added (methanol-water, 1.5: 20, v / v). The mixture was partitioned into hexane and butanol sequentially in the same solvent ratio as water. Three fractions (H ₂ O, butanol, hexane layer) were obtained and evaporated to dry under vacuum conditions and used directly for antioxidant experiments. Each fraction was taken up in 10% DMSO and a high concentration of stock solution was prepared. The DMSO concentration did not exceed 0.1% during all experiments.

3. Experimental Animal

Male Spraque-Dawley rats weighing between 300-350 g were purchased from Kyung Gi, Korea and allowed to acclimate for at least one week prior to experimental use. Animals were reared individually to allow for food and water intake, and maintained a normal contrast cycle. Animal experiments were approved by the Animal Care and Use Committee of the Experimental Animal Research Center, Chungbuk National University, Korea.

4. Cell Culture

RAW 264.7 (mouse leukaemic monocyte macrophage cell line) cells were treated with 10% fetal bovine serum (FBS), 4 mmol / L L-glutamate (Invitrogen, Calif.), 100 U / ml penicillin and 100 μg / ml streptomycin (Invitrogen). Supplemented with Dulbecco's modified Eagle's medium (Hyclone, Utah). The culture was carried out in a tissue culture flask while maintaining 5% CO ₂ 37 ° C. All experiments were grown to at least 90% confluency conditions and subcultures were not exceeded 20 times.

5. Determination of Total Phenolic Compounds

The amount of total phenolic compounds in each fraction was measured and expressed in milligrams of the corresponding gallic acid per gram of fraction using a 96-well microplate reader. That is, 10 μL of each fraction is transferred to a 96-well microplate containing 160 μL of distilled water and 10 μL of 1 mol / L Folin-Ciocalteu's phenol reagent (Sigma), followed by a 20% aqueous sodium carbonate solution. 20 μL was added. After the mixture was stored at room temperature for 20 minutes, the color absorbance was measured at 735 nm using a spectrophotometer. The concentration of the phenolic compound was calculated using a measurement curve set as a gallic standard solution in the range of 25-800 mg / ml.

6. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

Volatile compounds in the hexane fraction of the white rose flower extract were measured using an Agilent 6890 gas chromatograph / 5973 mass selective detector (Palo Alto, Calif.) And HP-FFAP (HP-free fatty acid phase) capillary column (30 m length × 0.25 mm inner diameter, 0.25 μm film thickness). Helium was used as a carrier gas at a constant rate of 1 ml / min. The oven temperature was set at 50 ° C. for 5 minutes, and then increased to 230 ° C. at a rate of 4 ° C./min. Hold for a minute. The temperature of the detector and injector was kept at 250 ° C. The ionization energy of the mass selective probe was 70 eV, and the mass range of m / z 35-500 was scanned. Most peaks were identified using a computer library (Wiley 275L program). Chromatographic peaks were checked for their homogeneity with the aid of mass chromatograms for characteristic fragment ions.

7. High Performance Liquid Chromatography (HPLC) Analysis

The hexane fraction of the white rose flower extract was analyzed by HPLC using the Younglin-ACME 900 system (Younglin, Anyang, South Korea) and the YL9160 PDA detector. Spectrum data were recorded from 190nm to 900nm. Mightysil RP-18 GP column (4.6 × 250 nm, 5 μm, Kanto Chemical, Japan) was used for separation, and the flow rate of the solvent was kept constant at 0.6 mL / min. The mobile phase for separation consisted of solution A (acetonitrile, acetonitrile) and solution B (10 mmol / L phosphoric acid, phosphoric acid, pH 2.5). The concentration gradient elution process was carried out as follows: 0 min 10% solution A, 0-10 min 5% solution A, 10-40 min 50% solution A, 40-41 min 100% solution A, 41-51 min 100 % Solution A, 51-61 minutes 10% Solution A. The injection volume for analysis was 20 μL and gallic acid standards were prepared in HPLC-grade methanol. The concentration of gallic acid was measured by a standard curve obtained by injecting different concentrations of gallic acid standards. Gallic acid was identified and quantified by comparing the UV-visible spectral data with the retention time known above.

8. Determination of DPPH and ABTS radical scavenging activity

The DPPH (2,2-diphenyl-1-picrylhydrazyl hydrate) radical is one of the most unstable organonitrogen radicals in dark purple. To assess free radical scavenging activity, fractions were reacted with DPPH solution. Each lyophilized fraction was dissolved in DMSO at the corresponding concentration from the stock solution (100 mg / mL), and each fraction was reacted with 0.3 mmol / L of DPPH dissolved in methanol. Each fraction (1-32 μg / mL) at various concentrations was reacted with DPPH radical solution for 30 minutes at room temperature and then absorbance was measured at 517 nm. DPPH free radical scavenging activity was calculated using the following equation [1].

 [One]

In the formula [1] Ac is the absorbance of the control DPPH solution, A is the absorbance of the sample measured with the DPPH solution, As is the absorbance of the sample alone.

ABTS (2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) forms relatively stable free radicals, which are colorless in non-free form. 2 mmol / L ABTS dissolved in H ₂ O and 2.5 Prepare ABTS ^{• +} by mixing mmol / L potassium persulfate (K ₂ S ₂ O ₈ ) and storing in dark at room temperature for 4 hours, and react 100 μL of the prepared ABTS ^{● +} solution with each fraction in 96-well microplate. ABTS ⁺ left after a predetermined time was quantified by absorbance at 734 nm.

9. Measurement of Lipid Peroxidation

Lipid peroxidation was measured by measuring the formation of malondialdehyde (MDA) based on the presence of thibarbituric acid reactive substance (TBARS) in the brain. That is, the rat brain was perfused and homogenized in a 10-fold volume of buffer solution. Centrifuge at 4000 r / min to obtain a homogeneous suspension supernatant. To induce lipid peroxidation, the brain homogenate (450 μL) was incubated with 50 μmol / L iron chloride (25 μL) for 30 minutes at 37 ° C., with or without fractions (25 μL). The reaction was terminated by the addition of 1 mL of sodium dodecyl sulphate (500 μL of 8.1% w / v solution) and 20% acetic acid (adjusted to pH 3.5). The clear supernatant (500 μL) was mixed with an equal volume of TBA (thiobarbituric acid) solution (0.8% w / v), placed in a glass tube, covered with aluminum foil, and heated at 95 ° C. for 30 minutes. Samples were cooled on ice and 100 μL of each sample was transferred to a 96-well plate and absorbance was measured at 532 nm. Butylated hydroxyanisole (BHA) was used as an antioxidant control.

10. Nitric oxide scavenging assay

A calibrated non-cell nitric oxide scavenging assay (SNAP assay) was performed to confirm that the hexane layer fraction had nitric oxide scavenging activity. That is, a solution containing 100 μmol / L SNAP (S-nitroso-N-acetylpenicillamine), 10% DMSO, and various concentrations of hexane layer fractions (0.1-100 μg / mL) was prepared. The final volume of the reaction was 150 μL and incubated at 37 ° C. for 3 hours. After completion of the reaction, each sample (50 μL) was mixed with Greases reagent according to the manual and absorbance was measured at 540 nm.

11. Measurement of Nitric Oxide Production in RAW264.7 Cells

To obtain more in-depth results on the antioxidant effect of the hexane layer fraction, the mammalian cell line RAW264.7 was used. RAW264.7 cells (1 × 10 ⁶ cells / ml) were dispensed into 24-well tissue culture plates and pre-cultured at 37 ° C. for 12 hours to ensure stable attachment of cells. The cells are then washed with phosphate buffered saline (PBS) and replenished with 1% FBS DMEM containing samples (lipopolysaccharide (LPS), ascorbic acid (Asc), hexane layer fraction (0.1-25 μg / ml)) and Incubated for 24 hours. The production of nitric oxide was measured by measuring the amount of nitrate in the medium using a grease reagent (Promega).

12. hydroxyl-mediated oxidation analysis

In this experiment, hydroxyl-mediated oxidation experiments were performed using metal catalysis. The target protein, BSA, was dissolved in 150 mmol / L phosphate buffer (pH 7.3) to give a final concentration of 0.5 mmol / L. BSA solution was treated with a fraction or alone, and cultured in the conditions other than or along with 100 μmol / L copper (Cu ^{+ 2)} and 2.5 mmol / L hydrogen peroxide. 50 μmol / L ascorbic acid was used as an antioxidant control, which was dissolved directly in PBS. The tube was reacted in an open state in a 37 ° C. vibrating water tank. After the reaction was completed, each mixture was separated using 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and stained with 0.1% Coomassie blue solution.

13. Observation of DNA fragmentation and apoptosis

RAW 264.7 cells were cultured in a 60 cm ² dish and then starved in DMEM medium without FBS for 6 hours. Cells were harvested and washed twice with cold PBS after treatment as indicated and in lysis buffer (10 mmol / L Tris-HCl, pH 8.0), 10 mmol / L EDTA, and 0.2% Triton X-100). Put on ice for 30 minutes. Cells were centrifuged at 13500 r / min at 4 ° C. for 10 minutes and the supernatant transferred to a new tube. To remove RNA contamination, 10 μg / mL of RNase A was added and incubated at 37 ° C. for 30 minutes. Soluble DNA was isolated by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. DNA lumps dried in air were dissolved in tertiary distilled water and subjected to periodic electrophoresis on 1.6% agarose gel for 1 hour before analysis. DNA fragments were observed by staining with ethidium bromide. To confirm the presence of apoptosis, the HaCaT (human keratinocyte line) was treated with 0.5 mmol / L SNAP (nitric oxide (NO) donor) in the presence or absence of a hexane layer fraction (0.1-50 μg / mL). And incubated for 6 hours. The prepared cells were washed three times with cold PBS, fixed with 4% paraformaldehyde for 15 minutes, stained with ice for 1 hour in dark with Annexin V-FITC and propiodium iodide (1: 2.5 ratio). It was. Cell death progress was confirmed using an Olympus® 71 inverted microscope (Olympus, Tokyo, Japan). Morphological changes were compared by combining photographs taken of the same part.

14. Escherichia coli E. coli Of antioxidant effect

Cells were prestressed with hexane layer fractions of various concentrations (1-1000 μg / mL) and directly stressed with 1 mmol / L hydrogen peroxide. It was then incubated at 250 ° C. at 37 ° C. for 2 hours in liquid Luria-Bertani (LB) medium. One drop was taken from each culture tube and inoculated in two LB agar plates and incubated overnight at 37 ° C. Cell growth was compared with the group exposed to stress without ascorbic acid (0.5 mmol / L) or fraction treatment.

15. Statistical Analysis

The obtained results were analyzed by one-way ANOVA (SPSS 12.0 version) and analyzed by each group by Tukey method. The obtained information was expressed as mean ± SD and was considered statistically significant when p value was less than 0.05.

Experiment result

1. Measurement of Phenolic Compounds

The extract was prepared by fractionation from white rose flower extract with two solvents (hexane and butanol) and water. The total amount of polyphenols present in these three fractions is shown in FIG. 1A. The hexane layer fraction contained the most total polyphenols (330.3 ± 20.1 mg), which was about 2 times the butanol fraction (177.3 ± 7.1 mg) and about 2.6 times the water layer fraction (127.1 ± 10.8 mg). Amount. From this result, it was found that the hexane layer fraction would have a stronger antioxidant effect, and thus this fraction was selected as a promising fraction for further analysis.

2. GC-MS and HPLC Analysis

In the GC-MS analysis, the inventors found a volatile component having the antioxidant potency included in the hexane layer fraction. The major aromatic components of the hexane layer fraction of white rose flower were dodecyl acrylate (41.17%) and cyclododecane (23.83%) (see Table 1). Furthermore, large amounts of gallic acid (> 10%) were identified in the hexane layer fractions by HPLC (FIG. 1B). The presence of gallic acid was confirmed by UV spectral analysis of the hexane layer fraction and gallic acid as standard.

Table 1 Volatile Components of White Rose Flower Hexane Fractions Verified by GC-MS

3. DPPH and ABTS free radical scavenging activity

DPPH is a commonly used reagent to evaluate the free radical scavenging activity of various antioxidants. DPPH is a stable free radical and accepts electrons or hydrogen groups to become stable diamagnetic molecules. ABTS also forms relatively stable free radicals, which lose color in the non-radical state. Thus, this method was primarily used to evaluate the antioxidant potential of the fractions. In DPPH analysis, the hexane layer fraction reached the highest level at 2 μg / ml, while the butanol layer fraction reached the highest level at 4 μg / ml. However, the water layer fraction did not remove free radicals effectively as compared to the hexane and butanol layer fractions (FIG. 2A). Moreover, the hexane layer fraction removed free radicals faster and more strongly than the butanol layer fraction (FIG. 2B). Also in the ABTS radical scavenging activity analysis, the hexane layer fraction was observed to have stronger antioxidant activity than the butanol or water layer fraction (FIG. 2C).

4. Inhibition of lipid peroxide

Lipid peroxide assay using thiobarbituric acid reacting substances (TBARS) is a sensitive method widely adopted to evaluate antioxidant activity. The potential to inhibit MDA (malondialdehyde) formation of two solvent fractions (hexane and butanol) obtained from white rose flowers was analyzed at 532 nm (FIG. 3). The addition of butylated hydroxyanisole (BHA) and hexane layer fractions was found to decrease the absorbance (form TBARS) in a concentration-dependent manner. Surprisingly, the hexane layer fraction showed a similar inhibitory effect to the positive control BHA at 31.6 μg / ml. However, butanol layer fractions were not very effective in inhibiting lipid peroxidation. These results suggest that the hexane layer fraction can have similar pharmacological effects as chemical antioxidants.

5. Nitric oxide scavenging and inhibitory activity

As a more comprehensive method, the removal of nitric oxide (NO) in non-cells and cells using the grease reagent method to evaluate the properties of antioxidant, scavenging and inhibition of hexane layer fractions. The analysis was performed. In the noncellular nitric oxide scavenging assay, 100 μmol / L SNAP was mixed with the hexane layer fraction and absorbance was measured for up to 3 hours by time and dose. In this experiment, the hexane layer fraction showed prominent nitric oxide scavenging properties at all test concentrations (0.1-100 μg / ml) (FIG. 4A), and the scavenging pattern during the reaction time (0-3 hours) showed a pattern of ascorbic acid. Followed. The IC ₅₀ of the hexane layer fraction for nitric oxide was 0.9 μg / ml. Inhibition of nitric oxide production by the fraction was measured in RAW264.7 cell line. To stimulate nitric oxide production, 5 μg / mL of LPS was added and the hexane layer fractions (0.1-25 μg / mL) of various concentrations were treated with the cells for up to 36 hours. As shown in FIG. 4C, the hexane layer fraction significantly inhibited the production of nitric oxide at 10 μg / ml, which was almost similar to 50 μmol / L ascorbic acid. More interestingly, the hexane layer fraction was more effective than ascorbic acid when incubated for longer periods (36 hours) (FIG. 4D). These two results suggest that the hexane layer fraction can regulate the concentration of nitric oxide at the cellular level by removing nitric oxide or inhibiting the production of nitric oxide.

6. hydroxyl-mediated oxidation analysis

The hexane layer fraction was observed the decomposition (degradation) of the BSA by the Cu ^{+ 2} and H ₂ O ₂ in the presence of a hexane layer fraction of 0.1-10μg / ㎖ to see if the antioxidant activity at the protein level. As shown in Figure 5, the hexane layer fractional fire, such as 0.5 mmol / L ascorbic acid inhibited the damage of BSA. In other words, the hexane layer fraction effectively prevented the decomposition of BSA by free radicals, and showed the greatest effect at 10 μg / ml.

7. Cell Damage and Growth Inhibition Analysis

Nuclear DNA fragmentation tests were performed, which is a biochemical feature of apoptosis in RAW264.7 cells treated with hexane layer fraction and peroxide. As shown in FIG. 6A, DNA ladders were observed in the DNA extracted from the cells treated with the low concentration of the hexane layer fraction. However, treatment with high concentrations of hexane layer fractions significantly reduced DNA fragmentation. These results suggest that the hexane layer fraction can effectively remove nitrate peroxide, which can occur when high concentrations of nitric oxide are present, thus protecting cells from DNA damage. 6B also shows that the hexane layer fraction effectively protects cells from nitric oxide-induced cell death at high concentrations (> 50 μg / mL). This scavenging ability was confirmed by the growth of cells when E. coli was incubated with 1 mmol / L hydrogen peroxide. Interestingly, in the cell growth assay, it was observed that the hexane layer fraction effectively removed hydrogen peroxide at low concentrations (~ 1 μg / ml) but had a bactericidal effect at high concentrations (> 100 μg / ml) (FIG. 6C). This suggests that the hexane layer fraction can act as an scavenger or bactericidal factor for reactive oxygen species in a dose selective manner.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

1a and 1b show the total amount of phenolic compounds and chromatogram analysis results. 1A shows the total amount of phenolic compounds (milligrams of gallic acid equivalent) was measured in water, butanol and hexane fractions. Data were expressed as mean ± standard deviation of three replicates. **, p <0.01 vs. Water fraction, †††, p <0.001 vs. Butanol fraction.

1B is a chromatogram of hexane fractions. Peak 4 (arrow) shows gallic acid present at a high rate.

2a to 2c show radical scavenging activity according to time and concentration. Figure 2a shows the DPPH free radical scavenging activity (time fixed, 20 minutes) by concentration of water fraction, butanol fraction, hexane fraction, Figure 2b shows the DPPH free radical scavenging activity (concentration fixed, 2) of butanol fraction, hexane fraction μg / mL), and FIG. 2C shows ABTS radical scavenging activity (fixed time, 30 minutes) by concentration of water fraction, butanol fraction and hexane fraction. Data are expressed as mean ± standard deviation.

3 shows the measurement results of lipid peroxide. TBARS inhibitory activity formed in brain homogenates in rats with butanol and hexane fractions was measured at absorbance 532 nm. In this experiment, BHA was used as a positive control and no treatment was used as a normal control.

4A-4D show the results of nitric oxide scavenging and inhibition experiments. Two separate experiments were performed on splenocytes (nitric oxide scavenging) and cells (nitric oxide suppression).

In FIG. 4A, nitric oxide scavenging was measured using SNAP at various concentrations (0.1-100 μg / ml) of the hexane fraction. Ascorbic acid (50 μM) was used as a positive control and 10% DMSO was used as a solvent control to confirm the noise.

4b was measured within 0-3 hours to determine the time dependence of nitric oxide scavenging by hexane fraction (10 μg / ml) or ascorbic acid (50 μM) in the presence of SNAP.

4C and 4D, RAW264.7 cell lines were dispensed into 96-well culture plates for nitric oxide production analysis followed by treatment with hexane fractions (0.1-25 μg / mL) and incubated for 24 or 36 hours. Ascorbic acid (50 μM) was used as a positive control and LPS (5 μg / mL) was used as a cell stimulator. The presence of nitric oxide was measured at absorbance 540 with a grease reagent, and the results were expressed as the mean standard deviation of three replicates. *, p <0.05, **, p <0.01, ***, p <0.001 vs. LPS.

5 shows the BSA proteolytic PAGE profile by Cu ²⁺ / H ₂ O ₂ in the presence of hexane fractions. Gel band of the figure shows the efficacy of hexane fractions (0.1-10 μg / ㎖) to effectively inhibit the BSA protein attack by the oxide material produced during Cu ²⁺ / H ₂ O ₂ treatment. Ascorbic acid (0.5 mM) was used as a positive control and 10% DMSO as a solvent control. Finally it was analyzed in 10% SDS-PAGE after 2 hours reaction.

6A-6C show the results of cytoprotective and cell growth assays.

Figure 6a shows agarose gel electrophoresis results of DNA extracted from RAW264.7 cells. RAW264.7 cells were treated with various concentrations of hexane fractions (0.1-50 μg / ml) and peroxide (ONOO ⁻ ) followed by DNA fragmentation. Ascorbic acid (0.1 mM) was used as a positive control. M, 100 bp DNA marker.

6B shows various captured photographs of cells undergoing apoptosis. HaCaT cells were treated with hexane fractions (0.1 and 50 μg / ml) and incubated for 6 hours in the presence of 0.5 mM SNAP, and cell death progress was observed by adding Annexin V-FITC + PI (Propidium iodide). PI-stained condensed cells (black red) show the cells being killed.

Figure 6c shows the results of observing the growth inhibitory effect of E. coli in the presence of hexane fractions of various concentrations (1-1000 μg / ㎖) and 1 mM hydrogen peroxide. 10% DMSO was used as the solvent control and ascorbic acid (0.5 mM) as the positive control. Disappearing circles indicate the inhibition of fungal cell growth of hexane fractions.

Claims (8)

Antioxidant composition comprising white rose (white Rugosa rose, Rosa rugosa var. Alba) flower extract as an active ingredient. The method of claim 1, wherein the white rose flower extract is selected from the group consisting of water, anhydrous or hydrous alcohol having 1 to 6 carbon atoms, hexane, acetone, ethyl acetate, butyl acetate, 1,3-butylene glycol or a combination thereof Composition characterized in that the fraction extract of the solvent. The composition of claim 1 or 2, wherein the white rose flower extract comprises cyclododecane or dodecyl acrylate. According to claim 1 or 2, The white rose flower extract has free radical scavenging activity, lipid peroxidation inhibitory activity, nitric oxide scavenging and inhibitory activity, or DNA fragmentation and cell death inhibitory activity induced by nitric oxide Composition. The composition of claim 1 wherein the composition is a cosmetic composition. The composition of claim 1 wherein the composition is a functional food composition. Antimicrobial composition comprising white rose (Rose rugosa var. Alba) flower extract as an active ingredient. The method of claim 7, wherein the white rose flower extract is selected from the group consisting of water, anhydrous or hydrous alcohol having 1 to 6 carbon atoms, hexane, acetone, ethyl acetate, butyl acetate, 1,3-butylene glycol and combinations thereof. Composition characterized in that the fraction extract of the solvent.

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