KR101121883B1 - A skin external composition containing callus extract drived from saint john's wart - Google Patents

Abstract

PURPOSE: An external use skin composition containing callus extract derived from Saint John's Wart is provided to improve physiological activity. CONSTITUTION: A method for preparing callus extract derived from Saint John's Wart with enhanced secondary metabolic products comprises: a step of culturing seeds of Saint John's Wart to induce germination; a step of inoculating germinated stems and leaves into a medium for inducing callus; a step of treating an elicitor to the proliferated callus and culturing to produce secondary metabolic products; and a step of extracting the callus with solvent. The elicitor includes light/darkness, UV-A, shikimic acid, or jasmonic acid. An external use skin composition for enhancing anti-oxidation or collagen synthesis contains 1.0-10.0 volume% of the extract.

Description

A Skin External Composition Containing Callus Extract Drived from Saint John's Wart}

The present invention relates to a composition for external application for skin containing St. John's wort callus extract as an active ingredient, more specifically, St. John's wort Callus induced by plant tissue culture technology, contrast, UV-A, Sikkim acid and Jasmonic acid The present invention relates to an external preparation composition for skin, which is treated with an elicitor such as to improve physiological activity.

Callus is an amorphous tissue or cell mass that has lost its ability to cause normal organogenesis or tissue differentiation. Cultured tissues from the plant are cultured in auxin or cytokine-containing media, wounded or injured. It is produced when the upper mouth is treated with auxin. In addition, callus can be derived from any part of the plant, such as leaves, stems, roots, rhizomes and fruits through plant cell culture, in particular tissue culture, it can be continuously produced by subculture.

It has been found that useful components showing various physiological activities are contained in plants, and these useful components are secondary metabolites that are produced secondary to normal metabolism of cells and accumulate in special places in cells. From the point of view, there is a growing interest in protecting the plant itself from microorganisms or external animals or in terms of physiological activity. Secondary metabolites produced by plants include alkaloids, flavonoids, carotenoids, glycosides and terpenoids. It is known to show the effects of soothing and converging, sun protection, whitening, exfoliation, skin aging prevention, antioxidant, skin wrinkle improvement, antibacterial, anti-inflammatory, anti-cancer.

Physiologically active substances in plants are produced in order to defend themselves from microorganisms or animals, or to gain an advantage in food competition between plants or to secure habitat. Some plants accumulate only at certain times or under stress conditions. Murthy et al ., J. Biotechnol . , 24: 711-716, 2008). In general, the production of physiologically active substances by using cell or organ cultures allows the production of bioactive substances of uniform quality throughout the year. This has a low disadvantage (Buitelaar et al ., J. Biotechnol . , 23: 111-141, 1992). When the plant is invaded by microorganisms, the content of physiologically active substances in the plant increases, which generally increases the content of phytoalexin, which acts like an antibiotic. Therefore, when culturing cells, organs, etc., in order to overcome the disadvantage of low bioactive substance content in the in-flight culture, by artificially treating the pathogen-derived compound inducing factor (elicitor), phyto-Alec in defense culture by invasives such as microorganisms Elicitation, which increases the amount of God and induces measurement of the final bioactive substance, is widely used (Ahn et al. al . , J. Kor . For . Soc ., 95: 27-32, 2006; Hu et al ., Process Biochem ., 43: 113-118, 2008).

St. John's Wort (Hypericum perforatum) is a globally distributed plant called the "grass of the holy". This plant is a perennial plant that blooms from June to September. From ancient Greek times to the Middle Ages, this plant was believed to stop witches and evil spirits and defeat demons. Nervous diseases, depression, neuralgia, wounds and burns, It has been used to treat many kinds of diseases, such as kidney disease, and has been used by people for centuries because of its antibacterial and anti-inflammatory properties. In recent years, the plant has received considerable attention as it contains as a major component hyperhydrin and similar hypericin, which have been shown to inhibit the AIDS virus. The buds of St. John's wort contain a large number of structurally different compounds that act directly or indirectly on the central nervous system (CNS), which are active compounds and dimers such as hypericin and hyperporin. Flavones are included, which are known to have antidepressant and anxiolytic effects in animals and humans. In view of its mechanism of action, hypericin has been demonstrated antidepressant action in the presence of dimers of procyanidines contained in extracts of St. John's wort (Regensburg, Germany, V. Butterwecke et . Al ., Annual Congress of the Society for Medicinal Plant Research , 1997), hyperporin has been reported to elevate 5-HT (Serotonine) levels in the hypothalamus and hippocampus of mice, suggesting that the antidepressant effects of hyperporin are associated with the serotonin system ( J Pharm Pharmacol , 53 (5): 583-600, 2001; Pharmacopsychiatry , 33 (2): 60-65, 2000). However, about 20% of the depressed patients are not treated with conventional antidepressants, and the side effects of recently developed antidepressants such as SSRI are relatively less than the existing antidepressants, but still cannot be ignored.

In recent years, there has been a trend to recognize and study the effects of natural herbal extracts in the West, and researches using extracts of hypericum perforatum , mainly called St. John's plants, have been actively conducted in relation to depression ( Neuropharmacology , 21 (2): 247-257, 1999; Cochrane Database Syst Rev , (2): CD000448, 2000,; Drugs Aging , 16 (3): 189-197, 2000).

Accordingly, the present inventors have made intensive efforts to prepare a topical composition for skin having excellent physiological activity using St. John's wort callus, and as a result, the St. John's wort callus extract having an increased content of secondary metabolites has antioxidant, cell regeneration, etc. functions. It was confirmed that the present invention was completed.

The present invention provides an external preparation composition for skin containing the callus extract of St. John's wort having functions such as antioxidant and cell regeneration, and a method of preparing St. John's wort callus extract with an increased content of secondary metabolites.

In order to achieve the above object, the present invention provides an external composition for skin containing the callus extract of St. John's wort as an active ingredient.

The present invention also comprises the steps of (a) incubating seeds of St. John's wort to induce germination; (b) inoculating and incubating the germinated stems and leaves with a callus induction medium to induce and propagate callus; (c) treating the proliferated callus with an attractant and culturing to induce the production of secondary metabolites; And (d) provides a method for producing a St. John's wort callus extract with increased content of the secondary metabolite comprising the step of extracting the secondary metabolite derived callus with a solvent.

St. John's wort callus of the present invention is treated with attractants such as light and shade, UV-A, shikimic acid and jasmonic acid to provide an external composition for skin having antioxidant, cell regeneration.

1 is a diagram showing the observation of the callus of St. John's wort under an electron microscope (SEM).
Figure 2 shows the results of HPLC analysis after treatment with the concentration of shikimic acid in callus extract derived from St. John's wort.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention relates to an external composition for skin containing the callus extract of St. John's wort as an active ingredient.

In the present invention, the callus of St. John's wort may be characterized in that it is treated with an attractant selected from the group consisting of light and dark, UV-A, shikimic acid and jasmonic acid.

In another aspect, the present invention comprises the steps of (a) inducing germination by culturing St. John's wort; (b) inoculating and incubating the germinated stems and leaves with a callus induction medium to induce and propagate callus; (c) treating the proliferated callus with an attractant and culturing to induce the production of secondary metabolites; And (d) relates to a method for producing a St. John's wort callus extract with increased content of the secondary metabolite comprising the step of extracting the secondary metabolite derived callus with a solvent.

In the present invention, the extract may be characterized in that extracted with a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, and mixtures thereof.

In the present invention, the attractant may be selected from the group consisting of light and dark, UV-A, shikimic acid and jasmonic acid.

In the present invention, the callus induction medium may be characterized in that it contains IAA (indole-3-acetic acid), zeatine, sucrose and gelatin.

"Callus (callus)" refers to the tissue formed on the wound site of the plant, but is an amorphous cell mass formed by tissue culture of sections, organs, tissues, etc., refers to the oily tissue, oily body, and generally auxin cut tissue from the plant It refers to a special tissue or cell mass produced when cultured in a medium containing, wounding a certain kind of plant, or treating the epidermis with auxin. Normally, callus is an amorphous tissue or cell mass that has lost its ability to cause normal organogenesis or tissue differentiation, and is mostly composed of flow cells. Callus and tumor tissue are distinguished in the following ways: In case of tissue culture, callus needs to give auxin to its growth from the outside, whereas tumor tissue does not need auxin, so callus is itself a synthesis of auxin Although not capable, tumor tissue is believed to be able to synthesize auxin. By culturing tissues, callus can be produced from any part of the leaves, stems, roots, etc., and subcultured to produce callus continuously. The growth of completely undifferentiated cells is repeated, and the amount of plant hormone in the medium is repeated. By controlling the morphology, irregular catheter and phloem differentiation, denudation, and the formation of the abscess, and also the formation of a complete individual in callus, callus is a powerful material for the study of morphogenesis.

"Shimkimic acid" acts as a precursor to the synthesis of phenolic compounds in plants, phenylalanine (Aromatic Amino acid), tryptophan and gallic acid or pyrogallol (tyrosine or polyphenol) It is known to be involved in biosynthesis such as).

"Jasmonic acid" is a plant signaling molecule derived from linolenic acid present in the membrane lipids. Jasmonate and methyl jasmonate, which are signaling agents involved in the expression of plant defense mechanisms, increase the contents of bioactive substances in plants. In addition, it is a substance involved in plant growth, development, aging of leaves and defense mechanisms of the whole plant.

"Phytoalexin" generally refers to a generic term for low molecular weight antimicrobial compounds that are synthesized and accumulated by plants when they come in contact with microorganisms, but are produced by mechanical injury or chemicals of any species. Involved in the plant's disease resistance response. It does not show any specificity for pathogens and inhibits the development of many other pathogens. The type of phytoalexin is determined by the host plant regardless of the type of pathogen.

An "elicitor" is a substance that induces a plant's defense response and its related reactions, such as the accumulation of phytoalexin. In general, a substance produced by plant pathogens (biological outflower) is used here. Secreting components include -β glucan, peptides, glycoproteins and lipids such as arachidonic acid. In addition, heavy metal salts, surfactants, and ultraviolet light are also called abiotic attractants because of their activity of inducing phytoalexin. In addition, the attractant responds to the specificity of the disease between the pathogen and the host plant, and induces a defense response regardless of the specific attractant and plant variety, which induces a defense response only to plant varieties that have a resistance gene against the pathogen producing the attractant. Distinguish by inducing nonspecific attractant.

Since each bioactive substance is finally synthesized through different biosynthetic pathways, it is necessary to select an appropriate attractant type and concentration that can increase the amount of bioactive substance to be produced while minimizing a decrease in growth of the culture.

In the present invention, the "antioxidative action" refers to inhibiting or incapacitating the action of representative components of the oxidizing substance causing aging or disease in the human body or skin. Oxidizing materials include oxygen radicals in which stable oxygen molecules are destabilized, lipid peroxides in oxidation forms of lipid components, and hydroxyl radicals, and various free radicals. Antioxidants refer to components having an effect of removing them.

Total phenolic content refers to the total amount of phenolic compounds and is deeply related to antioxidant activity. Generally, phenolic compounds include compounds such as flavonoids, phenolic acids, and lignans, and in general, the higher the total phenolic content, the higher the antioxidant activity.

In one embodiment of the present invention, the seed of St. John's wort sterilizes the plant, induces and proliferates the callus from the plant, and uses a physical and chemical elicitor in the grown callus, a polyphenol compound that is a secondary metabolite. Promotes the content of (polyphenolic compounds) to provide an external skin composition with antioxidant properties.

In one embodiment of the present invention, in order to purchase seeds of St. John's wort (St. John's wort) and sterilize the surface of the seeds, the stem and leaf portions of the plant of St. John's wort are immersed in ethanol, shaken with sodium chlorite, After sterilization, washing with sterile water. After washing, sterile seeds were germinated in the medium, and growth regulators of indole-3-acetic acid (ZAA), zeatin (Zeatin), sucrose and gellite were obtained from the stems and leaves of germinated St. John's wort. After inoculation into MS medium containing the culture to induce callus, and cultured and propagated. The grown callus was harvested to remove moisture, and dried. The dried St. John's wort callus powder was placed in a container, purified water was added thereto, and the reaction extract was filtered to prepare St. John's Callus extract.

In another embodiment of the present invention to determine the content of carotenoids, flavonoids and phenols having antioxidant activity from the callus secondary metabolites, and to increase the content of the callus of St. John's wort (Culture grass), photoperiod, UV-A By treating with Sikkim acid and Jasmonic acid, the content of the active ingredient was improved.

In another embodiment of the present invention, when treated with a photoperiod from 0h / 24h to 12h / 12h photoperiod from 0h / 24h, it was confirmed that the content of carotenoids, flavonoids and phenolic compounds slightly reduced when UV-A is treated for a long time. In addition, as the concentration of Shikimic acid was increased in the St. John's wort callus, the contents of carotenoids, flavonoids and phenolic compounds of the callus secondary metabolite increased, but the concentration of Sikkim acid was added to the medium at a concentration of 600 μM. It was confirmed that the content of carotenoids, flavonoids and phenolic compounds slightly reduced. In addition, when 100 and 250 μM of jasmonic acid was added to the St. John's wort callus, the contents of carotenoids, flavonoids and phenolic compounds of the secondary metabolite of callus were increased, but the concentration of jasmonic acid was increased to 500 μM. When added to it was confirmed that the content of carotenoids, flavonoids and phenolic compounds decreased.

In another embodiment of the present invention, an experiment was performed to compare the antimicrobial activity, collagen synthesis enhancement effect, iNOS activity inhibition and antioxidant activity of the callus extract derived from St. John's wort. As a result of measuring antimicrobial activity, Pseudomonas aeruginosa or Escherichia coli contained 10% of St. John's wort callus extract, similar to ampicillin, which had positive antibacterial activity, and St. John's wort callus extract treated with attractant was attractant. It was confirmed that the superior antimicrobial activity appeared compared to the untreated group. In addition, as a result of measuring the amount of collagen synthesis, as the content of St. John's Callus extract increased, collagen synthesis increased, and St. John's Callus extract treated with attractant was much more than the untreated group without attractant. Excellent collagen synthesis was shown, and among them, the highest collagen synthesis was shown by the treatment with jasmonic acid. In addition, as a result of measuring the activity of iNOS, the activity of iNOS was inhibited according to the concentration of St. John's wort callus extract, and it was confirmed that the anti-cancer effect was excellent when treated with the extract of shikimic acid and jasmonic acid. In addition, the antioxidant activity of St. John's Callus extract was measured, and the antioxidant activity of St. John's Callus extract without the attractant was higher than that of St. John's Callus extract with the attractant. On the other hand, by treatment with Jasmonic acid it was confirmed that showed an excellent antioxidant effect compared to the St. John's wort callus extract not treated with attractant.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

From St. John's Wort Callus  Judo

Example  1-1: Of St. John's Wort  Seed germination of grass

Seeds of St. John's wort (St. John's wort) were purchased from the Korean Wild Plant Seed Bank and germinated seedlings were used. To sterilize the surface of the seed, cut the stems and leaves of the plant of St. John's wort (St. John's wort) into appropriate sizes, soak for 30 seconds in 70% ethanol, and 10-15 in 0.3% sodium hypochlorite. After sterilization by shaking with a minute, it was washed with sterile water. After washing, sterile seeds were grown in MS medium for 10 days to induce germination.

Example  1-2: germinated From St. John's Wort Callus  Judo

Stem and leaves of germinated St. John's wort (grown grass) were grown with IAA (indole-3-acetic acid) 0.5 mg / L, zeatin 0.2 mg / L growth regulator, sucrose 30 g / L, gellite ( Gelite) inoculated in MS (Murashinge and Skoog, 1962) medium containing 2.0g / L and incubated for 4 weeks in a culture room at 23 ~ 27 ℃ to induce callus. Induced callus was proliferated by subculture at 4 week intervals.

The propagated callus was harvested and sufficiently dehydrated with a clean tissue and then dried in a drier at 60 ° C. for 2 days. 100 g of dried St. John's wort callus powder was placed in a container, 1 L of purified water was added thereto, followed by stirring at 65 ° C. for 2 hours, and the reaction extract was filtered to prepare 1 L of St. John's wort callus extract.

Callus  Determination of Carotenoids, Flavonoids and Phenols as Secondary Metabolites

Example  2-1: Determination of Content of Carotenoids

The total carotenoid content was extracted by adding an acetone: nucleic acid (3: 7, v / v) mixture to the freeze-dried St. John's wort (Critic grass) callus sample by AOAC method, and then extracting the filtrate and washing with distilled water. : A chromatograph column was prepared from a mixture of diatomaceous earth (1: 9, v / v), and the extract was injected and aspirated. The absorbance was measured at 436 nm with acetone: nucleic acid (1: 9, v / v) mixture.

Example  2-2: Determination of Flavonoid Content

The total flavonoid content was measured according to the method based on the colorimetric method in which the coloring material was added according to the component properties and the content was compared with the standard solution according to the degree of color development. 1.25 ml of distilled water was added to 0.25 ml of St. John's wort callus extract and standard solution, followed by 0.075 ml of 5% (w / v) NaNO ₂ solution. After 6 minutes, 0.15 ml of a 10% (w / v) AlCl ₃ solution was added to the mixed solution, which was allowed to stand for 5 minutes and 0.5 ml of 1N NaOH solution was added thereto. Distilled water was added so that the final volume of the mixed solution was 2.5 ml and the absorbance was measured at 510 nm. (+/-) catechin standard solution was used as a standard.

Example  2-3: Content of Phenol

The total phenol content was determined by using a method based on the Folin-Ciocalteu method, in which the Foline-Ciocalteu reagent was reduced by the total phenolic substance of St. John's wort (Cultivars) callus extract, and developed in molybdenum blue. 2.55 ml of distilled water was added to 0.05 ml of the extract and the standard solution, and then 0.1 ml of 2N Folin-Ciocalteu reagent solution (10 time dilution; Sigma Chemical Co., St. Louis, Mo., USA) was added. After 6 minutes, 0.5 ml of a 20% (w / v) Na ₂ CO ₃ solution was added to the mixed solution, which was left in the dark for 30 minutes, and then absorbance was measured at 760 nm. Gallic acid was used as a standard.

Callus Secondary metabolites  Enhancement and Effect

The callus of St. John's wort (St. John's wort) prepared in Example 1 was treated with photoperiod, UV-A, shikimic acid and jasmonic acid to improve the content of the active ingredient.

Example  3-1: According to the photoperiod Callus Secondary metabolites  Content enhancement

Callus of St. John's wort (St. John's wort) prepared in Example 1 was added to MS medium by adding growth regulators of 0.5 mg / L IAA (indole-3-acetic acid) and 0.2 mg / L zetin (Zeatin). Incubation temperature and 30 μmol / m ² s Treated photoperiod with 0h / 24h, 3h / 12h, 6h / 18h, 12h / 12h on light-controlled growth and incubated for 8 weeks, and the carotenoids, flavonoids and phenols of St. John's wort callus extract by the method of Example 2 The content of compound mule was measured. As a result, when the photoperiod treatment from 0h / 24h to 12h / 12h, it was confirmed that the content of carotenoids, flavonoids and phenolic compounds of the secondary metabolite of callus increased.

Callus secondary metabolite content according to the change of photoperiod Light / dark Carotenoids (mg / g) Flavonoids (mg / g) Phenolic Compound (mg / g) 0h / 24h 34 26 35 3h / 12h 45 43 67 6h / 18h 47 42 61 12h / 12h 50 37 73

Example  3-2: UV According to -A Callus Secondary metabolites  Content enhancement

Callus of St. John's wort (St. John's wort) prepared in Example 1 was added to MS basic medium by adding growth regulators of 0.5 mg / L IAA (indole-3-acetic acid) and 0.2 mg / L zetin (Zeatin). After incubation for 5 weeks at the incubation temperature, and after 6 weeks of treatment with UV-A at 0h, 0.5h, 1h, 4h and 8h daily using a UV-A fluorescent lamp (20W, Sankyo Denki, Japan), The content of carotenoids, flavonoids and phenol compound of St. John's Callus extract was measured by the method of Example 2. As a result, it was confirmed that the content of carotenoids, flavonoids and phenolic compounds of the secondary metabolite of callus increased.

Callus secondary metabolite content according to UV-A change UV-A Carotenoids (mg / g) Flavonoids (mg / g) Phenolic Compound (mg / g) 0h 34 32 45 0.5h 37 37 63 2h 43 36 72 4h 46 37 72 8h 54 38 79

Example  3-3: Sikkim  According Callus Secondary metabolites  Content enhancement

Callus of St. John's wort (St. John's wort) prepared in Example 1 was added to MS basic medium by adding growth regulators of 0.5 mg / L IAA (indole-3-acetic acid) and 0.2 mg / L zetin (Zeatin). Incubated for 5 weeks at the incubation temperature. Shikimic acid was added to the culture medium at the concentrations of 200, 400 and 600 μM at 6 weeks, and the contents of carotenoids, flavonoids and phenolic compounds of St. John's wort callus extract were measured by the method of Example 2 (FIG. 2). ). As a result, the content of carotenoids, flavonoids and phenolic compounds of callus secondary metabolites increased as the concentration of shikimic acid was increased.However, when the concentration of shikimic acid was added to the medium at a concentration of 600 μM, carotenoids and flavonoids were added. And it was confirmed that the content of the phenol compound is somewhat reduced.

Callus secondary metabolite content according to the change of shikimic acid concentration Shikimic acid concentration (μM) Carotenoids (mg / g) Flavonoids (mg / g) Phenolic Compound (mg / g) 0 43 37 57 100 42 39 67 200 45 36 80 400 46 35 89 600 42 35 62

Example  3-4: On Mount Jasmon  According Callus Secondary metabolites  Content enhancement

Callus of St. John's wort prepared in Example 1 was added with growth regulators of 0.5 mg / L IAA (indole-3-acetic acid) and 0.2 mg / L zeatin (Zeatin) to MS medium at 25 ° C. incubation temperature. After culturing weekly, Jasmonic acid was added to the culture medium at concentrations of 100, 250 and 500 μM at 6 weeks of culture, respectively, and the contents of carotenoids, flavonoids and phenolic compounds of St. John's wort callus extract were measured by the method of Example 2. When the concentration of jasmonic acid was added to 100 and 250 μM, the contents of carotenoids, flavonoids and phenolic compounds of callus secondary metabolites were increased, but when the concentration of jasmonic acid was added to the medium at a concentration of 500 μM, carotenoid, It was confirmed that the content of flavonoids and phenolic compounds was reduced.

Callus Secondary Metabolites Containing Different Levels of Jasmonic Acid Jasmonic Acid Concentration (μM) Carotenoids (mg / g) Flavonoids (mg / g) Phenolic Compound (mg / g) 0 43 37 57 100 100 80 130 250 130 160 250 500 80 76 150

Experimental Example 1

Antimicrobial Activity Test

Experimental strains of Staphylococcus aureus (Staphylococcus aureus subsp . aureus, KCTC 1927), Pseudomonas aeruginosa , KCTC 2513) and E. coli (Escherichia coli, KCTC 2571) were transferred to liquid medium (beef extract 3.0 g, peptone 5.0 g, agar 15.0 g, distilled water 1.0 L, pH6.8) for 8-24 hours at 37 ° C. After culturing and activating, the bacterial suspension was diluted by adjusting to a constant concentration (optical density 0.5 at 620 nm), inoculated in petri dishes by 0.1 ml, and then injected with 10 ml of sterilized medium to form a medium.

St. John's wort callus extract prepared in Example 1 was diluted by concentration and incubated for 30 hours in a sterile paper disk adsorbed 30ul, the antimicrobial activity was measured by the size of the growth inhibitory ring around the disk. As a positive control, ampicillin (20 µg) used as an antibiotic of the strain was used.

As a result of measuring the antimicrobial activity, the inhibition zones of the respective strains by the St. John's wort callus extract diluted with concentration and attractant treated St. John's wort callus extract are shown in Table 5. In the case of Pseudomonas aeruginosa or Escherichia coli, the antimicrobial activity of 10% St. John's wort callus extract was found to be similar to that of ampicillin, a positive control group, and the concentration-dependent activity was confirmed to increase. In addition, it was confirmed that St. John's wort callus extract treated with attractants such as photoperiod, UV-A, shikimic acid and jasmonic acid showed superior antimicrobial activity compared to the untreated group without the attractant.

Growth Inhibition of Strains by St. John's Wort Callus Extract Diluted by Concentration and St. John's Wort Callus Extract Treated with Attractant
Attractant Treatment
extract
Treatment concentration (%)
Growth inhibition size (mm) P. aeruginosa
(KCTC 2513) E. coli
(KCTC 2571) S. aureus
(KCTC 1927) Untreated Extract 0.5% ND ND ND Extract 1.0% ND ND 3 Extract 5.0% ND ND 12 Extract 10.0% 4 4 13 Gwangju period Extract 0.5% ND ND ND Extract 1.0% ND ND ND Extract 5.0% ND ND ND Extract 10.0% 6 5 13 UV-A Extract 0.5% ND ND ND Extract 1.0% 3 2 5 Extract 5.0% 7 3 8 Extract 10.0% 9 5 12 Sikkim Mountain Extract 0.5% ND ND ND Extract 1.0% ND ND ND Extract 5.0% ND ND ND Extract 10.0% 5 6 10 Jasmon Mountain Extract 0.5% ND ND ND Extract 1.0% ND ND ND Extract 5.0% ND ND ND Extract 10.0% 3 One 5 Ampicillin 20µg 3 2 17 Purified water ND ND ND

Experimental Example 2

Collagen Synthesis Enhancement Test

DMEM containing 10% FBS, penicillin (50U / ml) and streptomycin (50µg / ml) in human skin fibroblasts at 37 ° C. in a 5% CO ₂ incubator with constant humidity (Dulbecco's Modified Eagle's Medium, Gibco, USA), 500 μl was dispensed in 24 well plates at 1 × 10 ⁵ / ml and then incubated for 24 hours. A control medium (DMEM medium) and a medium containing St. John's wort callus extract at various concentrations of 0.1 to 10% were added and cultured in a 37 ° C., 5% CO ₂ incubator for 48 hours. After 48 hours, 20 μl of each supernatant of the medium was newly synthesized by measuring using a Procollagen Type I C-Peptide EIA Kit (PICP, Takara, Cat No. MK101). The amount of collagen was measured. The PICP amount was converted into ng / 1 × 10 ⁵ cells. As a result, it was found that the synthesis of collagen increased as the content of St. John's Callus extract increased. In addition, St. John's wort callus extract treated with attractants such as photoperiod, UV-A, shikimic acid and jasmonic acid showed much better collagen synthesis effect than untreated group without attractant. Showed the highest collagen synthesis effect (Table 6).

Collagen synthesis according to the concentration of St. John's wort callus extract and St. John's wort callus extract treated with attractant Attractant Treatment Extract Treatment Concentration (%) Collagen Synthesis Amount (ng / 1 × 10 ⁵ cells)) Control group 105 Untreated Extract 0.1% 103 Extract 0.5% 110 Extract 1.0% 123 Extract 5.0% 139 Extract 10.0% 157 Gwangju period Extract 0.1% 101 Extract 0.5% 109 Extract 1.0% 135 Extract 5.0% 149 Extract 10.0% 160 UV-A Extract 0.1% 112 Extract 0.5% 120 Extract 1.0% 139 Extract 5.0% 154 Extract 10.0% 171 Sikkim Mountain Extract 0.1% 109 Extract 0.5% 109 Extract 1.0% 139 Extract 5.0% 145 Extract 10.0% 151 Jasmon Mountain Extract 0.1% 101 Extract 0.5% 112 Extract 1.0% 158 Extract 5.0% 172 Extract 10.0% 197

[Experimental Example 3]

iNOS  Activity inhibition test

To investigate the inhibitory effect of artificially induced inflammation using macrophage Raw264.7 cells, 10% FBS-DMEM (Fetal Bovine Serum, Fetal Bovine Serum, Dulbecco's Modified Eagle Medium, GIBCO) containing no iNOS G) 1 × 10 ⁵ cells were suspended in the medium and inoculated in 24 well plates. One day later, the St. John's wort callus extract prepared in Example 1 was treated by concentration, and cultured for 18 hours, followed by 1 µg / ml lipid polysaccharide (Lipopolysaccharide (Sigma, USA)) and cultured for 24 hours. Thereafter, the supernatant was collected, 100 µl each was added to a 96 well plate, and the same amount of grease reagent (Griess reagent (Sigma, USA)) was added thereto, and the mixture was gently shaken at room temperature for 10 minutes to measure absorbance at 570 nm. . A calibration curve was prepared using sodium nitrite as a standard product, and the amount of nitric oxide produced in each culture was determined for each sample. The yield of each sample was measured using the amount of nitrogen oxide produced in the group treated with the lipid polysaccharide as 100%. As a result, as shown in Table 7, it was confirmed that the activity of iNOS was inhibited according to the concentration of St. John's wort callus extract, and it was confirmed that the antioxidant effect was excellent when treated with the extract of shikimic acid and jasmonic acid.

Measurement of Yield by Treatment of Lipid Polysaccharides in St. John's Callus Extract and St. John's Callus Extract Treated with Attractant Treatment condition Yield (%) Attractant Treatment LPS processing Extract Treatment Concentration (%) 100 Untreated - Control (DMEM Medium) 8.9 Untreated

+

Extract 0.1% 86.13 Extract 0.5% 77.36 Extract 1.0% 46.02 Extract 5.0% 30.24 Extract 10.0% 21.4 Gwangju period

+

Extract 0.1% 90.2 Extract 0.5% 85.9 Extract 1.0% 71.1 Extract 5.0% 69.8 Extract 10.0% 58.4 UV-A

+

Extract 0.1% 78.8 Extract 0.5% 62.1 Extract 1.0% 53.9 Extract 5.0% 42.8 Extract 10.0% 35.1 Sikkim Mountain

+

Extract 0.1% 80.1 Extract 0.5% 72.9 Extract 1.0% 30.0 Extract 5.0% 12.1 Extract 10.0% 5.2 Jasmon Mountain

+

Extract 0.1% 76.2 Extract 0.5% 32.5 Extract 1.0% 25.2 Extract 5.0% 20.5 Extract 10.0% 10.1

[Experimental Example 4]

Antioxidant Activity Comparison

Experimental Example  4-1: DPPH Radical  Scavenging action

The antioxidant activity of St. John's wort is the most widely used for screening the antioxidant activity of natural products by using the principle of reducing the relatively stable DPPH radical by donating electrons to free radicals.

DPPH (2,2-diphenyl-1-picrylhydrazyl, Sigma, Keyuk), a nitrogen-based radical present in a dark purple and stable state in the absence of radicals, reacts with the radicals of the extract to discolor (Blois, 1958). DPPH radical scavenging activity of St. John's wort callus extract was measured using the 0.2 ml of St. John's wort callus extract and 0.8 ml of 200 μM DPPH solution dissolved in 100% (w / v) ethanol were mixed in the dark, and left for 5 minutes, and then absorbance was measured at 517 nm. The antioxidative activity of St. John's wort callus extract was higher than that of St. John's wort callus extract treated with photoperiod, UV-A and shikimic acid, while it was treated with jasmonic acid. It showed an excellent antioxidant effect compared to the St. John's wort callus extract was not treated (Table 8).

※ DPPH radical scavenging activity

= [(Absorbance of control)-(absorbance of sample addition) / (absorbance of control)] X100

DPPH Radical Scavenging Activity According to the Concentration of St. John's Callus Extract and Attractant Treated St. John's Callus Extract Attractant Treatment Extract Treatment Concentration (%) DPPH radical scavenging activity (%) (antioxidant effect) Untreated Extract 0.1% 8 Extract 0.5% 21 Extract 1.0% 36 Extract 5.0% 67 Extract 10.0% 77 Gwangju period Extract 0.1% 2 Extract 0.5% 5 Extract 1.0% 15 Extract 5.0% 25 Extract 10.0% 30 UV-A Extract 0.1% 9 Extract 0.5% 27 Extract 1.0% 31 Extract 5.0% 40 Extract 10.0% 55 Sikkim Mountain Extract 0.1% 5 Extract 0.5% 7 Extract 1.0% 12 Extract 5.0% 23 Extract 10.0% 35 Jasmon Mountain



Extract 0.1% 20 Extract 0.5% 41 Extract 1.0% 50 Extract 5.0% 82 Extract 10.0% 90 Positive control 0.1% Vitamin C 85

Experimental Example  4-2: ABTS Radical Scatters

Determination of antioxidant activity using ABTS radicals was carried out by the method in which the ABTS free radicals produced by the reaction with potassium persulfate were removed by the antioxidants in the extract to decolorize the blue color of the radicals. It was measured by.

1.0 mM AAPH (2,2'-azo-bis 2-amidinopropanedeihydrochloride) was mixed with 2.5 mM ABTS (2,2'-azino-bis 3-ethylbenzenothiazolin-6-sulfonic acid) dissolved in 100 mM PBS buffer and 68 ° C. The reaction was continued for 12 minutes in a thermostat. 20 μl of St. John's wort callus extract diluted by concentration and 980 μl of ABTS solution were measured in a 37 ° C. water bath for 10 minutes to measure the degree of absorbance at 734 nm. The positive control group used Trolox ^? 0.1%. As a result, it was confirmed that the antioxidant activity of St. John's wort callus extract without the attractant was higher than that of St. John's wort callus treated with the attractant of photoperiod, UV-A and Sikkim acid, while Jasmon The acid treatment showed an excellent antioxidant effect compared to the St. John's wort callus extract that was not treated with attractant (Table 9).

ABTS radical scavenging ability was calculated according to the following equation.

※ ABTS radical scavenging activity (%)

= [(Blank OD-Sample OD) x 100] / Blank OD

ABTS radical scavenging activity according to the concentration of St. John's Callus extract and St. John's Callus extract treated with attractant Attractant Treatment Extract Treatment Concentration (%) ABTS radical scavenging activity (%) (antioxidant effect) Untreated Extract 0.1% 8 Extract 0.5% 21 Extract 1.0% 34 Extract 5.0% 67 Extract 10.0% 81 Gwangju period Extract 0.1% 5 Extract 0.5% 10 Extract 1.0% 13 Extract 5.0% 19 Extract 10.0% 25 UV-A Extract 0.1% 2 Extract 0.5% 8 Extract 1.0% 15 Extract 5.0% 19 Extract 10.0% 35 Sikkim Mountain Extract 0.1% 4 Extract 0.5% 17 Extract 1.0% 34 Extract 5.0% 39 Extract 10.0% 42 Jasmon Mountain Extract 0.1% 5 Extract 0.5% 19 Extract 1.0% 45 Extract 5.0% 71 Extract 10.0% 95 Positive control 0.1% Trolox 84

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

delete delete delete delete A composition for external application for skin to enhance antioxidant or collagen synthesis, which contains 1.0 to 10.0% by volume of St. John's wort callus extract cultured by treating with Jasmonic acid.


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