WO2023085873A1 - Anti-oxidant and anti-inflammatory composition containing, as active ingredient, oil extracted of xylem from pinus densiflora siebold & zucc. - Google Patents

Abstract

The present invention relates to an anti-oxidant and anti-inflammatory composition containing, as an active ingredient, oil of the xylem of Pinus densiflora Siebold & Zucc. Oil of the xylem (truck and stem) of Pinus densiflora Siebold & Zucc., extracted through steam distillation, has excellent anti-oxidant and anti-inflammatory effects, and thus a composition containing the oil as an active ingredient can be effectively used in anti-oxidant and anti-inflammatory functional cosmetic products, foods, medical products and the like.

Description

Antioxidant and anti-inflammatory composition containing oil extracted from pine xylem as an active ingredient

The present invention relates to an antioxidant and anti-inflammatory composition comprising Pinus densiflora Siebold & Zucc. xylem oil as an active ingredient. Pine xylem (body and stem) oil extracted by steam distillation has excellent antioxidant and anti-inflammatory effects, so a composition containing this oil as an active ingredient can be usefully used for antioxidant and anti-inflammatory functional cosmetics, food, and pharmaceuticals.

The human body generates necessary energy by supplying oxygen in the air to the body through breathing. However, reactive oxygen species (ROS) generated in this process accelerate aging by oxidizing various organelles including DNA and cell membranes with high oxidizing power. In addition, when the human body is continuously exposed to oxidative stress from harmful environments such as air pollution, exposure to ultraviolet rays, stress or disease, increased radicals in the body stimulate collagen and elastin, which are connective tissues of the dermis. , hyaluronic acid (Hyaluronic acid), etc. can cause subsidence (wrinkles) in certain parts of the skin, and by oxidizing the lipid part of the cell membrane to cause cell destruction, it can cause diseases such as dermatitis, acne or skin cancer.

Therefore, antioxidants such as substances capable of scavenging radicals or substances inhibiting lipid peroxidation are expected as inhibitors for preventing various diseases or aging caused by active oxygen, or as inhibitors of inflammation.

On the other hand, the inflammatory response is a defense mechanism of the human body against external stimuli in order to maintain homeostasis, and is caused by external stimulants such as microbial-derived LPS (lipopolysaccharide) and internal stimulants such as arachidonic acid. do. LPS is a substance present in the outermost layer of the cell wall of Gram-negative bacteria and is known to play various roles in interactions between pathogenic bacteria and eukaryotic organisms. In particular, LPS is recognized as a foreign substance in the human body and activates the cell defense system, thereby activating one of the immune defense systems, the inflammatory pathway.

When cells are stimulated by external stimulants such as LPS, the expression of iNOS (inducible NO synthas) and COX-2 are induced, resulting in nitric oxide (nitric oxide), an intracellular signaling substance that mediates the inflammatory response. , NO) and prostaglandin E2 (PGE2) are released. Inflammatory reactions occur in the human body and tissues due to the excessive release of NO and PGE2.

In general, NO is a regulator that regulates normal physiological activities, such as regulating blood pressure, which is a physiological phenomenon, and acting as a neurotransmitter mediator, and plays a pivotal role in immune responses. However, NO overexpressed by iNOS causes an inflammatory response and causes abnormalities in the immune system.

Regarding the prior art literature, Korean Patent Registration No. 10-1817505 discloses 'an anti-inflammatory composition containing an essential oil extract derived from a pine tree trunk and a method for preparing the extract'. Referring to claim 1, it is described that the essential oil extract contains longipollen as an active ingredient. In addition, looking at claims 4 to 6, it can be confirmed that longipollen inhibits cytokines and inflammatory mediating enzymes. In addition, under the identification item [0097], the chemical composition of the essential oil is shown, and the ratio of longipolene is 9.85%, and in the identification item [0118], it is described that the essential oil was fractionated to obtain a fraction with a high content of longipolene. . That is, among the components of pine essential oil, the experiment was conducted focusing on the anti-inflammatory effect of longipolene, and it can be seen that the anti-inflammatory effect is based on the inhibition test of cytokines and inflammatory mediating enzymes.

Republic of Korea Patent Registration No. 10-2125895 discloses 'Natural plant extract-derived NO activity inhibitor, anti-inflammatory agent containing the same, cosmetics containing the same, and a manufacturing method thereof'. Looking at the claims, it can be seen that NO activity inhibitors based on the NO production inhibition rate of natural plant extracts, anti-inflammatory agents and cosmetics using the same have been registered.

Looking at the thesis 'Essential oil of pine leaves and branches and anti-oxidation and anti-aging of their components', it is described that DPPH scavenging ability was tested after obtaining essential oil of leaves and stem by hot-water extraction of pine leaves and stems, respectively. .(p.216 right column, p.219 right column)

The problem to be solved is to provide an antioxidant and anti-inflammatory composition containing, as an active ingredient, an oil obtained by distillation and extraction of pine xylem by steam distillation.

The solution is an antioxidant and anti-inflammatory composition containing pine xylem oil obtained by steam distillation of pine ( Pinus densiflora Siebold & Zucc.) xylem as an active ingredient.

In the above, the pine tree oil is characterized by having an ABST radical scavenging ability of 25 to 78% at 0.01 to 0.1 v / v%.

In the above, the pine tree oil is characterized by having a DPPH radical scavenging ability of 14 to 56% at 0.01 to 0.1 v / v%.

*In the above, pine xylem oil is characterized in that it inhibits NO generation in LPS-treated RAW264.7 cells.

The solution is an antioxidant and anti-inflammatory food composition containing pine xylem oil obtained by steam distillation of pine ( Pinus densiflora Siebold & Zucc.) xylem as an active ingredient.

The solution is an antioxidant and anti-inflammatory cosmetic composition containing pine xylem oil obtained by steam distillation of pine ( Pinus densiflora Siebold & Zucc.) xylem as an active ingredient.

The present invention secures productivity and economy because all pine wood is utilized. According to the present invention, pine xylem oil extracted by steam distillation has ABST radical scavenging activity and DPPH radical scavenging activity. According to the present invention, pine xylem oil extracted by steam distillation has an effect of inhibiting nitric oxide production. Since the composition containing pine xylem oil according to the present invention has antioxidant and anti-inflammatory functions, it can be used in antioxidant and anti-inflammatory functional foods, cosmetics and pharmaceuticals.

1 is a graph showing the ABTS radical scavenging ability of pine xylem oil. Figure 2 is a graph showing the DPPH radical scavenging ability of pine xylem oil. Figure 3 is a graph showing the cell viability and inhibition of nitric oxide (NO) production of pine xylem oil.

The terms or words used in this specification and claims should not be construed as being limited to their ordinary or dictionary meanings, and the inventors can properly define the concept of terms in order to best explain their invention. Based on the principle, it should be interpreted as a meaning and concept consistent with the technical idea of the present invention.

Therefore, the matters described in the embodiments, reference examples, and drawings described in this specification are only the most preferred examples of the present invention and do not represent all the technical ideas of the present invention, so that they can be replaced at the time of the present application. It should be understood that there may be many equivalents and variations.

The pine ( Pinus densiflora Siebold & Zucc.) xylem oil used in the following experimental examples is oil extracted and collected from the trunk and trunk of pine trees by steam distillation.

'Xylem' is one of the components of the vascular bundle and refers to a complex tissue composed of conduits, tracheids, xylem fibers, and xylem tissue. It serves as a passageway for water and nutrients and also serves as a mechanical support for trees.

Steam distillation is a method of collecting volatile components by distillation through steam, and is a distillation method for obtaining a distillate repeatedly.

Experimental Example 1. Measurement of ABTS radical scavenging ability of pine tree oil

1 is a graph showing the ABTS radical scavenging ability of pine xylem oil (**p < 0.001 compared to control.).

Antioxidant activity was measured with ABTS radicals by ABTS ^+· cation decolourisation assay. ABTS + Radical cation scavenging action was measured by dissolving 7.4 mM 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid in distilled water, mixing 1:1, and then reacting in the dark at room temperature for more than 15 hours to obtain ABTS ⁺ to form a radical. The absorbance of the ABTS ⁺ solution was 0.70 (±0.01) diluted with distilled water before reaction with pine xylem oil at 720 nm (SpectraMax iD3 Molecular Devices, Austria).

180 μl of ABTS ⁺ solution was added to 20 μl of pine xylem oil, reacted for 5 minutes at room temperature in a dark room, and absorbance was measured at 720 nm. ABTS radical scavenging activity was calculated by the following equation. All experimental results in this study were repeated three times, and the experimental results were expressed as mean ± standard deviation. Statistical significance for the average difference between each group was verified at p-value < 0.05.

ABTS radical scavenging activity (%) = [1 - (Aa / Ab)] × 100

Aa: absorbance of sample-added area, Ab: absorbance of untreated area

As a result of the experiment, as shown in FIG. 1, the ABTS radical scavenging ability of pine xylem oil was 26.72±1.17% and 41.38±, respectively, at concentrations of 0.01 v/v%, 0.02 v/v%, 0.04 v/v%, and 0.1 v/v%. It was confirmed to have ABTS radical scavenging ability of 1.92%, 56.47±1.99%, and 75.75±0.80%.

Experimental Example 2. Measurement of DPPH radical scavenging ability of pine tree oil

Figure 2 is a graph showing the DPPH radical scavenging ability of pine xylem oil (**p < 0.001 compared to control.).

Free radical scavenging activity by reducing power was measured using a free group called DPPH (2,2-diphenyl-1-picrylhydrazyl). The degree of decrease in absorbance due to reduction of DPPH by the test substance was compared with the absorbance of the blank test solution to measure the free radical scavenging rate at a wavelength of 517 m.

To measure the free radical scavenging activity, 180 μl of 150 μM DPPH dissolved in methanol was added to 20 μl of pine tree oil, and allowed to stand at room temperature in the dark for 15 minutes, and then absorbance was measured at 517 nm. DPPH radical scavenging activity (%) was calculated by the following equation. All experimental results in this study were repeated three times, and the experimental results were expressed as mean ± standard deviation. Statistical significance for the average difference between each group was verified at p-value < 0.05.

DPPH radical scavenging activity (%) = [1 - (Aa / Ab)] × 100

Aa: absorbance of sample-added area, Ab: absorbance of untreated area

As a result of the experiment, as shown in FIG. 2, the DPPH radical scavenging ability of pine tree oil was 15.15 ± 0.54% and 26.32 ± 0.01 v / v%, 0.02 v / v%, 0.04 v / v%, and 0.1 v / v%, respectively. It was confirmed to have DPPH radical scavenging activity of 0.53%, 48.87±0.56%, and 54.90±0.73%.

Experimental Example 3. Inhibition of Cell Viability and Nitric Oxide (NO) Production by Pine Tree Oil

3 is a graph showing cell viability and inhibition of nitric oxide (NO) production by pine xylem oil (#p < 0.05 vs. the control group; **p < 0.001 vs. the LPS-treated group.).

[Cell culture]

The RAW 264.7 cell line, a murine macrophage cell line, was distributed from the Korea Cell Line Bank (KCLB, Seoul, Korea) and used in the experiment. As a medium for cell growth, Dulbeco's modified Eagle's medium high glucose (DMEM) medium supplemented with 10% FBS (fetal bovine serum) and 1% antibiotics (penicillin/streptomycin) was used, and 5% CO ₂ was maintained in a 37°C incubator. cultured while maintaining. FBS and antibiotics were purchased from Gibco (Rockville, MD, USA) and media from Hyclone (Logan, UT, USA). The cultured cells were used for experiments while exchanging the medium every 2-3 days, and when the cells grew more than 80%, they were washed with phosphate buffered saline (PBS, Gibco, Rockville, MD, USA) and subcultured. .

[Measurement of cell viability]

In order to confirm the degree of cell viability, it was measured by the WST-1 test method. RAW 264.7 cells were dispensed in a 96 well plate at a concentration of 1×10 ⁴ cells/ml and cultured in a 37°C, 5% CO ₂ incubator for 24 hours. The cultured cells were treated with pine xylem oil at different concentrations, reacted for 24 hours, and then 10 μl of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium ( WST-1) solution (Premix WST-1 Cell Proliferation Assay System, Takara, Japan) was treated and incubated for 30 minutes.

Cell viability was calculated by measuring absorbance at a wavelength of 440 nm using a micro reader (SpectraMax iD3, Molecular Devices, Sunnyvale, CA). At this time, WST-1 solution is based on the conversion of tetrazolium salt (WST-1) to formazan pigment by mitochondrial dehydrogenase in living cells, and the tetrazolium salt added to the medium is It is converted to formazan pigment by succinate-tetrazolium-reductase (EC 1.3.99.1), which is present in the respiratory chain and is active only in viable cells.

As the number of living cells increases, the activity of the entire mitochondrial dehydrogenase enzyme in the sample increases, and the increase in this enzyme activity leads to an increase in the production of formazan pigment. Therefore, the formazan pigment and the number of cells with metabolic activity in the medium have a linear correlation. will indicate The viability of the cells pre-treated with the extract was expressed as a percentage of the relative cell viability to the average value of the viability of the sample-free group.

As a result of the experiment, as shown in FIG. 3, it was confirmed that the pine xylem oil of the present invention did not exhibit cytotoxicity in the range of 0.0001 v / v% or less.

* [Inhibition of Nitric oxide (NO) generation]

NO is one of the representative free radicals and acts as an important cell signaling substance in vivo. It has a property that can easily pass through cell membranes, so it has various actions in vivo. cause cell damage.

Nitric oxide (II) content measurement in the culture medium was carried out using the Griess reaction using nitrite as an indicator of nitric oxide (II) production. RAW 264.7 cells were dispensed in a 24 well plate at a concentration of 1×10 ⁵ cells/ml and cultured for 24 hours in a 37°C, 5% CO ₂ incubator.

The cultured cells were treated with pine xylem oil at different concentrations, cultured for 2 hours, and then treated with lipopolysaccharides (LPS) to a final concentration of 1 μg/ml, stimulated for 24 hours, and then cultured supernatant was taken and added 100 μl each. After transferring to a new 96-well plate, 100 μl of Griess reagent (0.1% naphthylethylenediamine and 1% sulfanilamidein 5% H ₃ PO ₄ solution) was mixed, reacted at room temperature for 15 minutes, and absorbance was measured at 540 nm. A standard calibration curve was prepared using sodium nitrite and used for content calculation.

All reagents used in the experiment were obtained from Sigma-Aldrich (St. Louis, MO, USA). All experimental results in this study were repeated three times, and the experimental results were expressed as mean ± standard deviation. Statistical significance for the average difference between each group was verified at p-value < 0.05.

In this experiment, RAW 264.7 cells were treated with various concentrations of pine xylem oil (0.000001 v/v%, 0.00001 v/v%, 0.0001 v/v%) before LPS treatment, and the pine xylem oil treatment concentration of the present invention The degree of NO production according to the method was measured. As a result, as shown in FIG. 3, it was confirmed that NO production was reduced according to the pine xylem oil treatment, and a statistically significant reduction was confirmed (p<0.001).

Example 1. Antioxidant and anti-inflammatory composition containing oil extracted from pine xylem as an active ingredient

The composition according to the present invention contains pine xylem oil obtained by extracting and collecting pine ( Pinus densiflora Siebold & Zucc.) xylem - trunk and stem - by steam distillation as an active ingredient.

Based on the results of the above experimental example, pine xylem oil was included in an amount of 0.01 to 0.1 v/v% to exert an antioxidant effect and contained in an amount of 0.000001 to 0.0001 v/v% to exhibit an anti-inflammatory effect based on nitric oxide production inhibitory function. done

The present invention may provide an antioxidant, NO activity inhibitor, and anti-inflammatory agent formulated in a pharmaceutical unit dosage form by adding the pine xylem oil as an active ingredient and adding a pharmaceutically acceptable carrier, excipient, or diluent. Here, the carrier, excipient, and diluent include toze, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, undecided quality cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the pharmaceutical dosage form may be used in the form of a pharmaceutically acceptable salt, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set. In addition, when formulating the active ingredient, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. In addition, the pharmaceutical dosage form is formulated according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions. can

The solid preparation for oral administration may be prepared by mixing the extract with at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used as the non-aqueous solvent or suspending agent.

As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

The preferred dosage of the composition of the present invention varies depending on the condition and weight of the patient, the severity of the disease, age, sex, drug type, administration route and period, but can be appropriately selected by those skilled in the art. However, for a desirable effect, the extract of the present invention is preferably administered at 0.001 to 300 mg/kg, and administration may be administered once a day or divided several times. The dosage is not intended to limit the scope of the present invention in any way.

The extract of the present invention can be administered to mammals such as rats, mice, livestock, and humans through various routes. All modes of administration are contemplated, eg oral, rectal or intravenous, intramuscular or subcutaneous administration.

Antioxidant foods, NO activity inhibiting foods, and anti-inflammatory foods can be provided by adding food additives to the active ingredients of the present invention.

Foods to which the active ingredient can be added include, for example, various foods, beverages, chewing gum, tea, vitamin complexes, health functional foods, and the like.

The amount of the active ingredient in the food or beverage may be added in an amount of 0.01 to 20% by weight of the total weight of the food or beverage, and the health drink composition may be added in an amount of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml. there is.

The health functional beverage composition of the present invention is not particularly limited in components other than containing pine xylem oil, and may contain various flavoring agents or natural carbohydrates as additional components like conventional beverages. Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose; disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. In addition to the above, natural flavors (thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavoring agents. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, organic acids, protection It may contain a colloidal thickener, a pH adjusting agent, a stabilizer, a preservative, glycerin, alcohol, a carbonating agent used in carbonated beverages, and the like.

In addition, the composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages. These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

So far, the present invention has been mainly looked at with respect to preferred embodiments.

The embodiments described in this specification and the configurations shown in the drawings relate to one of the most preferred embodiments of the present invention, and do not represent all of the technical spirit of the present invention, so various equivalents and modifications that can replace them It should be understood that there may be examples.

Therefore, the present invention is not limited to the presented embodiments, and within the equivalent scope of the technical idea of the present invention and the technical idea described in the claims to be described below by those skilled in the art to which the present invention belongs There may be embodiments in which various modifications and changes are possible.

Claims (6)

1. An antioxidant and anti-inflammatory composition containing pine xylem oil obtained by steam distillation of pine ( Pinus densiflora Siebold & Zucc.) xylem as an active ingredient.
2. The method of claim 1,

   The pine tree oil is an antioxidant and anti-inflammatory composition, characterized in that it has an ABST radical scavenging ability of 25 to 78% at 0.01 to 0.1 v / v%.
3. The method of claim 1,

   The pine tree oil is an antioxidant and anti-inflammatory composition, characterized in that it has a DPPH radical scavenging ability of 14 to 56% at 0.01 to 0.1 v / v%.
4. The method of claim 1,

   The pine tree oil is an antioxidant and anti-inflammatory composition, characterized in that for inhibiting NO generation in LPS-treated RAW264.7 cells.
5. An antioxidant and anti-inflammatory food composition comprising pine xylem oil obtained by steam distillation of pine ( Pinus densiflora Siebold & Zucc.) xylem as an active ingredient.
6. An antioxidant and anti-inflammatory cosmetic composition containing pine xylem oil obtained by steam distillation of pine ( Pinus densiflora Siebold & Zucc.) xylem as an active ingredient.

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