Composition for improving skin comprising extract of lavandula angustifolia stem-derived callus as active ingredient and method for preparing same

Abstract

The present invention relates to a composition for improving skin, comprising an extract of Lavandula angustifolia stem-derived callus or a fraction thereof as an active ingredient and a method for preparing same, the method comprising the steps of: (a) inducing callus from Lavandula angustifolia stem tissue; (b) culturing the Lavandula angustifolia stem-derived callus induced in step (a) in the presence of methyl jasmonate; and (c) obtaining an extract of the Lavandula angustifolia stem-derived callus cultured in step (b).

Description

Composition for improving skin comprising callus extract derived from lavender stem as an effective ingredient and method for producing the same

[0001] The present invention relates to a composition for improving skin, comprising an extract of lavender stem-derived callus or a fraction thereof as an effective ingredient, and a method for producing the same.

[0002]

[0003] Lavender (Lavandula angustifolia) is a perennial plant in the Lamiaceae family, commonly used in herbal teas, aromatherapy, and as an ornamental. It is known to have pain-relieving, mentally calming, insect-repelling, and sterilizing effects. The genus name, Lavandula, is derived from the Latin word for "to wash." This is because it is effective in calming the nerves, mainly aiding anemia, insomnia, headaches, and neuralgia, and the Romans enjoyed using lavender in water as a perfume for bathing or laundry.

[0004] Callus, a mass of dedifferentiated plant cells, is created using plant tissue culture techniques. By cultivating meristematic cells in a nutrient medium containing appropriate macronutrients and micronutrients, and adjusting the concentrations of the plant hormones auxin and cytokinin, plant tissue-derived callus can be formed.

[0005] Meanwhile, although the industrial use of lavender oil through research using lavender plants as raw materials is common, the industrial use of callus extracts obtained through callus cell culture derived from lavender plants, such as in functional cosmetics, has not yet been reported.

[0006]

[0007] [Prior Art Literature]

[0008] [Patent Document]

[0009] KR 10-2328353 B1

[0010]

[0011] The present inventors completed the present invention by confirming the excellent anti-oxidation, wrinkle improvement, whitening, etc. effects of callus extract and its fractions through treatment with an elicitor and mass cultivation of callus suspension culture derived from lavender stem tissue.

[0012]

[0013] One object of the present invention is to provide a method for producing a cosmetic composition comprising an extract of lavender stem-derived callus or a fraction thereof as an active ingredient.

[0014] Another object of the present invention is to provide a method for producing a pharmaceutical composition comprising an extract of lavender stem-derived callus or a fraction thereof as an active ingredient.

[0015] Another object of the present invention is to provide a composition for improving skin, which comprises an extract of lavender stem-derived callus or a fraction thereof as an effective ingredient.

[0016]

[0017] A composition comprising a lavender stem-derived callus extract or a fraction thereof prepared according to the method of the present invention as an active ingredient can be usefully used for improving skin.

[0018]

[0019] Figure 1 shows stem tissue of a lavender plant (A), callus derived from lavender stem tissue (B), and callus derived from lavender stem tissue proliferating in the culture medium at two-week intervals (C).

[0020] Figure 2 shows the growth curve of callus derived from lavender stem tissue (A) and the liquid culture appearance of callus derived from lavender stem tissue (B).

[0021] Figure 3 shows the results of antioxidant activity analysis according to the treatment of various activation inducers in callus suspension cultures derived from lavender stem tissue. In Figure 3, CON: untreated group; MJ: methyl jasmonate treatment group; ET: ethephon treatment group; ABA: abscisic acid treatment group; SA: salicylic acid treatment group; Fla: flagellin 22 treatment group; Chi: chitosan treatment group; AsA: L-ascorbic acid.

[0022] Figure 4 shows the appearance of mass cultivation of lavender stem tissue-derived callus using a 1-ton tank bioreactor (A), the tank bioreactor-cultured callus harvest (B), and the hot-air-dried callus sample (C).

[0023] Figure 5 is a schematic diagram illustrating a method for preparing an extract of a mass culture sample of callus derived from lavender stem tissue.

[0024] Figure 6 shows the results of a cytotoxicity test of a mass culture extract of lavender stem tissue-derived callus, where LA: lavender plant extract; CON: untreated group with lavender stem tissue-derived callus; MJ: MJ treated group with lavender stem tissue-derived callus.

[0025] Figure 7 shows the results of an analysis of antioxidant activity according to MJ treatment in mass culture of callus derived from lavender stem tissue, where LA: lavender plant extract; CON: untreated callus extract; MJ: MJ treated callus extract; AsA: L-ascorbic acid.

[0026] Figure 8 shows the results of a collagen production test for wrinkle improvement of a mass culture extract of lavender stem tissue-derived callus. Control: serum-free medium; Positive control: L-ascorbic acid (200 μM); MJ: extract of lavender stem tissue-derived callus MJ treatment group.

[0027] Figure 9 shows the results of a melanin production inhibition test for whitening activity of a mass culture extract of lavender stem tissue-derived callus, with negative control: α-MSH (200 nM); positive control: Arbutin (100 μg / mL); LA: lavender plant extract; CON: untreated group with lavender stem tissue-derived callus; MJ: MJ treated group with lavender stem tissue-derived callus.

[0028]

[0029] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0030] Furthermore, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Furthermore, such equivalents are intended to be encompassed by the present invention.

[0031] Additionally, numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the present invention.

[0032]

[0033] One aspect of the present invention is a method for producing a composition comprising an extract of lavender stem-derived callus or a fraction thereof as an active ingredient.

[0034] In one specific example, the production method comprises culturing lavender stem-derived callus in the presence of methyl jasmonate.

[0035] In another specific embodiment, the production method comprises the steps of (a) inducing callus from lavender stem tissue; (b) culturing the lavender stem-derived callus induced in step (a) in the presence of methyl jasmonate; and (c) obtaining an extract of the lavender stem-derived callus cultured in step (b).

[0036] A production method according to any one of the above-described specific examples, wherein the production method comprises the step of (d) obtaining a fraction of the callus extract obtained in step (c).

[0037] A production method according to any one of the above-described specific examples, wherein the composition is a pharmaceutical or cosmetic composition.

[0038] A production method according to any one of the above-described specific examples, wherein the concentration of methyl jasmonate is greater than 0 and less than 200 uM.

[0039] As a production method according to any one of the above-described specific examples, the cosmetic composition is a composition for improving skin.

[0040] A production method according to any one of the aforementioned specific examples, wherein the skin improvement has at least one characteristic selected from among antioxidation, antiaging, skin whitening, and wrinkle improvement. For example, the skin improvement may have at least one characteristic selected from among skin whitening and wrinkle improvement.

[0041] A production method according to any one of the above-described specific examples, characterized in that step (b) comprises culturing in a medium to which B5 vitamins (Duchefa), Sucrose, MES, 2,4-Dichlorophenoxyacetic acid, α-Naphthaleneacetic Acid, Kinetin, and Indole-3-Acetic Acid are added to Murashige & Skoog (MS) basal medium.

[0042] As a production method according to any one of the above-described specific examples, the lavender stem-derived callus is characterized in that it has been deposited as a patent resource (deposit number KCTC 15497BP) at the Korea Research Institute of Bioscience and Biotechnology (KCTC), a domestic patent depository for biological resources.

[0043]

[0044] For example, in the present invention, the lavender stem-derived callus can be obtained by cutting a lavender stem tissue section, or can be used by receiving and depositing a callus derived from lavender stem tissue.

[0045] As an example, the callus induction medium and / or proliferation medium for lavender stem tissue explants may be Murashige & Skoog (MS) basal medium supplemented with B5 vitamins (Duchefa), Sucrose, MES, 2,4-Dichlorophenoxyacetic acid, α-Naphthaleneacetic Acid, Kinetin, and Indole-3-Acetic Acid. If the medium is solid, the medium may additionally contain Gelrite or Agar.

[0046] In one embodiment of the present invention, a callus induction medium was used with a composition of Murashige & Skoog (MS) basal medium, B5 vitamins (Duchefa) 4.4 g / L, Sucrose 20 g / L, MES 0.5 g / L, 2,4-Dichlorophenoxyacetic acid 1.5 mg / L, α-Naphthaleneacetic Acid 0.1 mg / L, Kinetin 0.25 mg / L, Indole-3-Acetic Acid 0.1 mg / L, and Gelrite 4 g / L.

[0047] For example, a callus proliferation medium for culturing callus obtained from a callus induction medium may include Murashige&Skoog (MS) basic medium, B5 vitamins (Duchefa), sucrose, MES, 2,4-Dichlorophenoxyacetic acid, α-Naphthaleneacetic Acid, Kinetin, Indole-3-Acetic Acid, and methyl jasmonate. The methyl jasmonate may be added after a certain period of time has passed since the start of culturing.

[0048] In one embodiment of the present invention, a callus proliferation medium was used with a composition of B5 vitamins (Duchefa) 4.4 g / L, Sucrose 20 g / L, MES 0.5 g / L, 2,4-Dichlorophenoxyacetic acid 1.5 mg / L, α-Naphthaleneacetic Acid 0.1 mg / L, Kinetin 0.25 mg / L, and Indole-3-Acetic Acid 0.1 mg / L, and liquid suspension culture was performed by adding methyl jasmonate thereto.

[0049] In the present invention, "methyl jasmonate" is also abbreviated as MeJA, and may also be referred to as "MJ" in the present invention. Methyl jasmonate is a type of volatile organic compound, and it is known that some plants can synthesize it through their defense system, and as a type of activation elicitor, it also has an activation effect of the biological defense mechanism, and its role and effect in plants are known to differ depending on the type of plant. In one embodiment of the present invention, as a result of confirming the antioxidant activity using various types of activation elicitors, it was confirmed that methyl jasmonate has a particularly excellent effect in enhancing the antioxidant activity of a callus extract derived from lavender stems and its fractions.

[0050]

[0051] The extract of the present invention refers to the resultant substance, such as a liquid component obtained by immersing a target substance in various solvents and then extracting it at room temperature or a heated state for a certain period of time, or a solid component obtained by removing the solvent from the liquid component. In addition, it can be comprehensively interpreted to include, in addition to the resultant substance, a dilution of the resultant substance, a concentrate thereof, a controlled substance thereof, a purified substance thereof, etc.

[0052] Accordingly, the callus extract provided in the present invention can be interpreted as including the extract itself and all formulations that can be formed using the extract, such as an extract obtained by extracting callus, a diluted or concentrated extract, a dried product obtained by drying the extract, a controlled or purified product of the extract, or a mixture thereof.

[0053] In the present invention, the type of solvent used for the extraction is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water, alcohol, or a mixture thereof, and these may be used alone or in combination of one or more. When alcohol is used as the solvent, an alcohol having 1 to 4 carbon atoms may be used, such as methanol or ethanol.

[0054] The “fraction” of the present invention means a result obtained by performing fractionation to separate a specific component or a specific group of components from a mixture containing various components.

[0055] In the present invention, the method for obtaining the fraction may be, for example, a solvent fractionation method performed by treating various solvents, an ultrafiltration fractionation method performed by passing the fraction through an ultrafiltration membrane having a certain molecular weight cut-off value, a chromatographic fractionation method performed by performing various chromatographies (designed for separation according to size, charge, hydrophobicity, or affinity), and combinations thereof.

[0056] Examples of fractionation solvents of the present invention include polar solvents such as water, distilled water, and alcohol; and non-polar solvents such as hexane, ethyl acetate, chloroform, and dichloromethane. These solvents may be used alone or in combination of two or more. For example, ethyl acetate may be used as the fractionation solvent.

[0057] In one embodiment, the callus fraction derived from lavender stems of the present invention may be an ethyl acetate fraction of an extract extracted with an alcohol solvent. For example, the alcohol solvent may be ethanol.

[0058] In one embodiment of the present invention, a callus culture derived from lavender stems was dried with hot air, and extracted three times with 50% ethanol for two hours each by reflux and cooling, and then concentrated under reduced pressure and frozen to prepare an extract powder. The dried extract powder was suspended in distilled water, and non-polar components such as lipids and chlorophyll were removed using hexane, and then repeatedly treated with ethyl acetate three times to obtain a fraction. The fraction was concentrated under reduced pressure to completely remove the organic solvent, and the ethyl acetate fraction was used to confirm the free radical scavenging, collagen synthesis promotion, and melanin production inhibition abilities.

[0059]

[0060] The composition of the present invention may be a composition for improving skin.

[0061] The term "improvement" of the present invention means any action that at least reduces the degree of symptoms, for example, a parameter related to alleviation or treatment of a condition, and may be at least one selected from among anti-oxidation, anti-aging, skin whitening, and wrinkle improvement.

[0062] For example, the skin improvement may be achieved through free radical scavenging, collagen synthesis promotion, and melanin production inhibition.

[0063] In one embodiment of the present invention, it was confirmed that the lavender stem-derived callus extract and its fractions of the present invention have excellent free radical scavenging ability, collagen synthesis promoting ability, and melanin production inhibitory ability, and thus can be usefully used in a composition for improving skin.

[0064]

[0065] Since the “cosmetic composition” of the present invention is basically applied to the skin, it can be manufactured in any formulation that is commonly manufactured with reference to cosmetic compositions in the art. For example, it can be manufactured in a formulation selected from the group consisting of solutions, external ointments, creams, foams, nourishing toners, emollients, packs, emulsions, makeup bases, foundations, essences, soaps, liquid cleansers, bath agents, sunscreens, sun oils, suspensions, gels, lotions, powders, surfactant-containing cleansers, patches, and sprays, but is not limited thereto.

[0066] In addition to the lavender stem-derived callus extract or a fraction thereof, the above cosmetic composition may further include conventional auxiliary ingredients and carriers, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, fragrances, etc., which are commonly used in cosmetic compositions. For example, the above cosmetic composition may further include auxiliary ingredients such as glycerin, butylene glycol, polyoxyethylene hydrogenated castor oil, tocopheryl acetate, citric acid, panthenol, squalane, sodium citrate, allantoin, etc., but is not limited thereto.

[0067] The above cosmetic composition may be formulated as a softening toner, astringent toner, nourishing toner, nourishing cream, massage cream, essence, pack, skin adhesive patch, skin adhesive gel, powder, ointment, paste, gel, suspension, emulsion, spray, cosmetic solution or capsule, but is not particularly limited thereto.

[0068] The cosmetic composition of the present invention may additionally include one or more cosmetically acceptable carriers that are incorporated into general skin cosmetics, and may appropriately incorporate conventional ingredients such as oil, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, etc., but is not limited thereto. The cosmetically acceptable carriers included in the cosmetic composition of the present invention vary depending on the formulation.

[0069] In the present invention, "quasi-drug composition" refers to products used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing human or animal diseases, and products with a milder effect than pharmaceuticals. For example, quasi-drugs under the Pharmaceutical Affairs Act exclude products used for pharmaceutical purposes, and include products used for the treatment or prevention of human / animal diseases, as well as products with a mild or no direct effect on the human body.

[0070] Specifically, the above-mentioned over-the-counter drugs may include external skin preparations and personal hygiene products. More specifically, they may include, but are not limited to, disinfectants, shower foams, mouthwashes, wet wipes, detergent soaps, hand washes, or ointments.

[0071] When the composition according to the present invention is used as an over-the-counter drug additive, the composition may be added as is or used together with other over-the-counter drugs or over-the-counter drug ingredients, and may be used appropriately according to conventional methods. The amount of active ingredient mixed may be appropriately determined depending on the intended use.

[0072]

[0073] Another aspect of the present invention is a composition for improving skin, comprising a callus extract derived from lavender stems or a fraction thereof as an effective ingredient.

[0074] In one specific example, the composition is characterized in that it comprises, as an active ingredient, a lavender stem-derived callus extract prepared by the steps of: (a) inducing callus from lavender stem tissue; (b) culturing the lavender stem-derived callus induced in step (a) in the presence of methyl jasmonate; and (c) obtaining an extract of the lavender stem-derived callus cultured in step (b).

[0075] In another specific embodiment, the composition is characterized in that it comprises, as an active ingredient, a fraction of a lavender stem-derived callus extract prepared by the steps of (a) inducing callus from lavender stem tissue; (b) culturing the lavender stem-derived callus induced in step (a) in the presence of methyl jasmonate; (c) obtaining an extract of the lavender stem-derived callus cultured in step (b); and (d) obtaining a fraction of the extract obtained in step (c).

[0076] A composition according to any one of the above-described specific examples, characterized in that the composition is a pharmaceutical or cosmetic composition.

[0077] The lavender stem tissue, callus, methyl jasmonate, culture and extract, pharmaceutical and cosmetic compositions are as described above.

[0078]

[0079] Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, these examples and experimental examples are intended to exemplify the present invention, and the scope of the present invention is not limited to these examples and experimental examples.

[0080]

[0081] Example 1: Induction and development of callus from lavender stem tissue.

[0082] Callus derived from lavender stem tissue was induced through the following three steps.

[0083] 1. Sterilizing and disinfecting the stem tissue of lavender plants

[0084] 2. Step of cutting the sterile tissue provided in step 1 into an appropriate size and inoculating it on a callus induction medium containing various plant hormones.

[0085] 3. Step of proliferating and culturing the callus induced in step 2 in the same medium as the induction medium.

[0086] In this example, a portion of the tissue of a lavender (Lavandula angustifolia) plant grown at the lavender farm ‘Herbwon (188-29 Guryang 1-gil, Jeongeup-si, Jeollabuk-do)’ in Jeongeup was used.

[0087] In step 1 of the above method, stem tissues of plants grown in a farm were separated and sterilized to develop sterile dedifferentiated cells (callus). This was done by washing with running water for less than 30 minutes, surface sterilization with 70% ethanol for less than 30 seconds, and disinfection with a 1% sodium hypochlorite solution for less than 20 minutes, followed by washing with sterilized distilled water on an aseptic workbench.

[0088] In step 2 of the above method, approximately 1 cm long stem tissue fragments of disinfected lavender plants were plated on a callus induction medium. This induction medium was composed of Murashige & Skoog (MS) basal medium supplemented with B5 vitamins (Duchefa) 4.4 g / L, Sucrose 20 g / L, MES 0.5 g / L, 2,4-Dichlorophenoxyacetic acid 1.5 mg / L, α-Naphthaleneacetic Acid 0.1 mg / L, Kinetin 0.25 mg / L, Indole-3-Acetic Acid 0.1 mg / L, and Gelrite 4 g / L, and the pH was adjusted to 5.8.

[0089] In step 3 of the above method, calli were induced from lavender stem tissue 10 days after inoculation with the dedifferentiated cell induction medium, and these were propagated through subculture at 2-week intervals in the same medium (Fig. 1).

[0090] The callus derived from lavender stem tissue obtained through Example 1 was deposited as a patented resource at the Korea Center for Bioscience and Biotechnology (KCTC) of the Korea Research Institute of Bioscience and Biotechnology (Deposit No. KCTC 15497BP).

[0091]

[0092] Example 2: Liquid suspension culture and growth curve analysis of callus derived from lavender stem tissue.

[0093] In order to measure the degree of growth (proliferation, weight) according to the culture time of callus derived from lavender stem tissue, the callus derived from lavender stem tissue induced and proliferated on the solid medium of Example 1 was subjected to liquid suspension culture.

[0094] The medium used for liquid suspension culture was composed of Murashige & Skoog basal medium, B5 vitamins (Duchefa) 4.4 g / L, sucrose 20 g / L, MES 0.5 g / L, 2,4-Dichlorophenoxyacetic acid 1.5 mg / L, α-Naphthaleneacetic Acid 0.1 mg / L, Kinetin 0.25 mg / L, and Indole-3-Acetic Acid 0.1 mg / L, and the pH was adjusted to 5.8.

[0095] For the liquid suspension culture of callus derived from lavender stem tissue, 20 mL of the above liquid medium was dispensed into a 125 mL Erlenmeyer flask, and 2 g of callus (10%) was inoculated. The medium was cultured at 120 rpm in a rotating incubator under dark conditions at room temperature, and the dry weight was measured by harvesting the cells at daily intervals for 14 days.

[0096] The liquid suspension culture of callus derived from lavender stem tissue showed a log phase from the 2nd to the 9th day, and the maximum growth value (growth peak) was measured at about the 10th day, and thereafter, a stationary phase and a death phase were observed (Fig. 2).

[0097]

[0098] Example 3: Comparison of antioxidant activity of callus cell cultures derived from lavender stem tissues according to treatment with various activating agents.

[0099] To induce antioxidant and various activity enhancement in callus cell culture derived from lavender stem tissue, various activity elicitors were treated during the callus liquid suspension culture process.

[0100] The active inducers used were Methyl jasmonate (MJ), Ethephon (ET), Abscisic acid (ABA), Salicylic acid (SA), Flagellin 22 (Flg), and Chitosan (Chi). The concentrations of each treatment expected to exhibit an active induction effect were MJ 100 μM, ET 300 μM, ABA 300 μM, SA 100 μM, Flg 10 μM, and Chi 200 μg / ml.

[0101] In this experiment, for the liquid suspension culture of callus derived from lavender stem tissue, 20 mL of the liquid medium of Example 2 was dispensed into a 125 mL Erlenmeyer flask, and 2 g of callus was inoculated. This was cultured at 120 rpm using a rotating incubator under dark conditions at room temperature.

[0102] Liquid suspension cultures of callus derived from lavender stem tissue were pre-cultured for 7 days, treated with the above-mentioned inducer, and harvested 5 days after treatment. The harvested callus was freeze-dried, and the completely dried sample was used for material extraction. Material extraction was performed using 80% methanol, and a DPPH radical scavenging assay was performed to investigate antioxidant activity.

[0103] DPPH (2,2-diphenyl-1-picrylhydrazyl) is a substance that contains free radicals and is purple in color. When a spectrophotometer is used, it exhibits maximum absorbance at 520 nm. This can be visually confirmed by a yellow color change when the free radicals are eliminated by reducing power along with the addition of a sample, and the absorbance at 520 nm decreases. In other words, the more a sample with high antioxidant activity is reacted, the more yellow it changes and the lower the absorbance value.

[0104] In this analysis method, the standard sample, L-ascorbic acid (AsA), a vitamin C, and the measurement sample, the callus extract treated with an activation inducer, were dissolved in 80% methanol at concentrations of 7.813 μg / mL, 15.625 μg / mL, 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, and 1000 μg / mL, respectively, and the antioxidant activity of each sample was compared by DPPH radical scavenging activity through concentration-dependent measurement. As a result, the MJ treatment group sample showed a significantly higher DPPH radical scavenging activity than the untreated group and other activation inducer treatment groups. This means that the antioxidant activity by MJ treatment was the highest among various activation inducers (Fig. 3).

[0105]

[0106] Example 4: Scale-up (tank) culture of callus derived from lavender stem tissue and cytotoxicity test of extracts

[0107] Callus liquid suspension cultures derived from lavender stem tissue were scaled up from the Erlenmeyer flask stage to a 500-liter culture using a 1-ton tank bioreactor, with intermediate cultivation in a 20-liter bioreactor as the intermediate step. Through this, the feasibility of mass-cultivation of callus from untreated and MJ-treated groups was demonstrated, and cytotoxicity and various physiological activity tests of mass-cultivated callus extracts were conducted.

[0108] For the mass cultivation of this callus, 500 L of the mass cultivation callus harvest from the untreated and MJ treated groups were used as experimental materials by drying them with hot air (Fig. 4).

[0109] The extraction method used for cytotoxicity tests and physiological activities (antioxidant, wrinkle improvement, whitening) was to use 50% ethanol for higher extraction efficiency, refluxing and cooling three times for 2 hours each, and then concentrating under reduced pressure and freezing to prepare an extract powder. The prepared extract powder was suspended in distilled water and non-polar components such as lipids and chlorophyll were removed using hexane. Afterwards, the fraction obtained by repeatedly treating with ethyl acetate three times was concentrated under reduced pressure to completely remove the organic solvent, and the ethyl acetate fraction obtained was used (Fig. 5).

[0110] A cell viability assay was performed using the mass culture samples of lavender stem tissue-derived callus obtained from the above and the control lavender plant extracts at different concentrations for each extract. The lavender plant extract used as the control was prepared using a dried sample of the lavender plant in the same manner as the extraction and fractionation method described in Fig. 5.

[0111] The test substance group was prepared with the callus mass culture sample extracts of the untreated and MJ treated groups and the control lavender plant extract at concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively, and treated with WST reagent at a ratio of 10:1. If the concentration decreased to less than 80% of that of the unadded sample group after culture, it was judged to be potentially cytotoxic, and the cell viability was calculated using the following formula.

[0112]

[0113] Cell viability (%) = {(OD at 450 nm of sample-added group) / (OD at 450 nm of sample-free group)} × 100

[0114]

[0115] As a result, the untreated callus extract (CON) showed cell viability of 119.6±8.7, 86.8±3.8, and 73.4±5.3% at concentrations of 50, 100, and 200μg / mL, respectively, and the MJ treated callus extract (MJ) showed cell viability of 86.1±8.6, 82.3±5.6, and 67.4±3.9% at concentrations of 50, 100, and 200μg / mL, respectively. The control lavender plant extract (LA) showed cell viability of 94.7±6.2, 87.3±7.7, and 71.6±9.3% at concentrations of 50, 100, and 200μg / mL, respectively (Fig. 6).

[0116]

[0117] From these results, it was confirmed that the untreated group, MJ treated group, and control lavender plant extract did not exhibit cytotoxicity at concentrations below 100 μg / mL.

[0118]

[0119] Example 5: Antioxidant activity analysis of extracts from scale-up (tank) cultures of callus derived from lavender stem tissue.

[0120] The antioxidant activity of the mass-cultured cells was confirmed by performing DPPH radical scavenging assay and ABTS radical scavenging assay using the lavender callus mass-cultured sample extract obtained in Example 4 and the control lavender plant extract.

[0121] The DPPH radical scavenging assay and ABTS radical scavenging assay methods used in this antioxidant activity analysis were as follows: L-ascorbic acid, a vitamin C, as a standard sample, and the callus extract of the untreated group, the callus extract of the MJ treated group, and the control lavender plant extract were dissolved in 80% methanol at concentrations of 3.906 μg / mL, 7.813 μg / mL, 15.625 μg / mL, 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, and 1000 μg / mL, respectively, and the antioxidant activity of each sample was compared through concentration-dependent measurements using DPPH radical scavenging activity and ABTS radical scavenging activity.

[0122] The results show that both the DPPH (Fig. 7A) and ABTS (Fig. 7B) assays showed that the calli of both the untreated and MJ treated groups had higher antioxidant activity than the control lavender plant extract, and in particular, the antioxidant activity of the MJ treated group was significantly higher (Fig. 7).

[0123]

[0124] Example 6: Analysis of wrinkle improvement activity of callus scale-up (tank) culture extract derived from lavender stem tissue

[0125] To evaluate the wrinkle improvement activity of the mass-cultured sample extract derived from lavender stem tissue obtained in Example 4, a collagen synthesis assay was conducted using CCD-986sk cells.

[0126] The method of this collagen production test was performed in the following steps.

[0127] 1. Preparation steps for each concentration of test material (lavender callus mass culture sample extract)

[0128] 2. CCD-986sk cell dispersal and culture steps

[0129] 3. Control group (L-ascorbic acid) and concentration-based test substance treatment and culture steps

[0130] 4. Collagen and protein quantification step

[0131] 5. Step for calculating intracellular collagen production rate

[0132] The test materials of step 1 of the above method were prepared at concentrations of 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively, from the callus mass culture sample extracts of the MJ treatment group.

[0133] In step 2 of the above method, 1.5 Х 10 cells were seeded in a 96 well plate 5 Dispense 200 ㎕ at a concentration of cells / mL and culture in a 37℃, 5% CO2 incubator for 24 hours. 24 hours after cell dispensing, completely remove the medium inside the dish by washing with PBS.

[0134] In step 3 of the above method, 200 ㎕ of L-ascorbic acid and test substances at various concentrations were diluted in serum-free medium as a positive control and cultured for 24 hours in a 5% CO2 incubator at 37°C.

[0135] In step 4 of the above method, collagen quantification was performed by recovering the culture medium and measuring the content according to the Procollagen type Ⅰ Peptide EIA kit (TaKaRa, Japan) according to the collagen standard curve. Protein quantification was performed by removing all the culture medium, adding 200 ㎕ of cell lysis buffer to each well, reacting at 150 rpm for 10 minutes, placing the well in a microtube, centrifuging, and using the obtained supernatant, according to the BCA protein assay kit (Pierce, USA), and quantifying the protein according to the BSA standard curve.

[0136] In step 5 of the above method, the protein amount and collagen amount were finally calculated using their respective standard curves, and this was converted into the collagen amount (ng / ㎍) for the protein amount, and the intracellular collagen production rate was calculated according to the following formula.

[0137] Intracellular collagen production rate (%) = { (collagen / protein (ng / ㎍) of sample-added group) / collagen / protein (ng / ㎍) of sample-free group} × 100

[0138] As a result, the intracellular collagen production rates were 69.9±10.7, 98.2±9.0, and 127.5±3.0% at concentrations of 25, 50, and 100 μg / mL of callus extract (MJ) in the MJ treatment group, respectively (Fig. 8).

[0139]

[0140] Through this, it was confirmed that the MJ treated callus exhibited wrinkle improvement activity at an extract concentration of 50 μg / mL or higher, and in particular, it was confirmed that the MJ treated callus at a concentration of 100 μg / mL had stronger wrinkle improvement activity than the positive control group.

[0141]

[0142] Example 7: Analysis of whitening activity of callus scale-up (tank) culture extract derived from lavender stem tissue

[0143] To evaluate the whitening activity of the mass-cultured sample extract derived from lavender stem tissue obtained in Example 4 and the control lavender plant extract, a melanin inhibition assay was conducted using B16-F10 cells.

[0144] The method of this melanin production inhibition test was performed in the following steps.

[0145] 1. Preparation steps for each concentration of test material (lavender callus mass culture sample extract)

[0146] 2. B16-F10 cell division and culture steps

[0147] 3. Control group and concentration-specific test substance treatment and culture steps

[0148] 4. Melanin and protein quantification step

[0149] 5. Step for calculating the inhibition rate of intracellular melanin production

[0150] The test materials of step 1 of the above method were prepared at concentrations of 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively, from the callus mass culture sample extracts of the untreated group and the MJ treated group and the control lavender plant extract.

[0151] In step 2 of the above method, 5.0 Х 10 cells were seeded in a 96 well plate 4 2 mL was dispensed at a cell / well concentration and cultured for 24 hours in a 37°C, 5% CO2 incubator.

[0152] In step 3 of the above method, the medium inside the dish was completely removed, and 2 mL of α-MSH diluted with the medium and test substances at various concentrations were treated in total and cultured in a 5% CO2 incubator at 37°C for 72 hours. In order to confirm the inhibitory activity of intracellular melanin production, the medium was removed, washed with PBS, and cells were secured using tyrosine-EDTA, centrifuged at 1000 rpm for 3 minutes, the supernatant was removed, and 1 N NaOH dissolved in 10% DMSO was added to lyse the cells, and then boiled in a heat block at 80°C for 1 hour to dissolve melanin.

[0153] Melanin quantification in step 4 of the above method was performed by recovering the dissolved melanin, conducting an activity test, and then measuring the content according to a standard curve using a melanin standard substance. Protein quantification was performed according to the BCA protein assay kit (Pierce, USA), and then quantifying the protein according to the BSA standard curve. Absorbance was measured at a wavelength of 475 nm after dissolving for 1 hour.

[0154] In step 5 of the above method, the protein and melanin amounts were finally calculated using their respective standard curves. This was converted into melanin amount per protein amount, and the inhibition rate of intracellular melanin production was calculated according to the following equation.

[0155]

[0156] Inhibition rate of intracellular melanin production (%) = 100 - {(amount of melanin per protein in the sample addition group) × 100 / (amount of melanin per α-MSH protein)}

[0157]

[0158] As a result, the inhibition rate of intracellular melanin production was 38.5±1.2% at 100 μg / mL of the callus extract (CON) in the untreated group, 10.7±2.6, 37.6±7.4, and 78.8±0.7% at 25, 50, and 100 μg / mL of the callus extract (MJ) in the MJ treatment group, respectively. In addition, the inhibition rate was 20.6±3.4 and 56.0±1.0% at 50 and 100 μg / mL of the control lavender plant extract (LA) (Fig. 9).

[0159]

[0160] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

[0161]

Claims

1. A method for producing a cosmetic composition comprising an extract of callus derived from lavender stems or a fraction thereof as an active ingredient, The above production method (a) a step of inducing callus from lavender stem tissue; (b) a step of culturing the lavender stem-derived callus derived from step (a) in the presence of methyl jasmonate; and (c) A method comprising a step of obtaining an extract of callus derived from lavender stems cultured in step (b).

2. A method according to claim 1, wherein the concentration of methyl jasmonate is greater than 0 and less than 200 uM.

3. A method according to claim 1, wherein the cosmetic composition is a composition for improving skin.

4. A method according to claim 3, wherein the skin improvement has at least one characteristic selected from among anti-oxidation, anti-aging, skin whitening, and wrinkle improvement.

5. A method according to claim 1, wherein step (b) comprises culturing in a medium to which B5 vitamins (Duchefa), Sucrose, MES, 2,4-Dichlorophenoxyacetic acid, α-Naphthaleneacetic Acid, Kinetin, and Indole-3-Acetic Acid are added to Murashige & Skoog (MS) basic medium.

6. A method according to paragraph 1, wherein the lavender stem-derived callus is deposited as a patent resource (deposit number KCTC 15497BP) at the Korea Research Institute of Bioscience and Biotechnology (KCTC), a domestic patent depository for biological resources.

7. A method according to claim 3, wherein the skin improvement has at least one characteristic selected from skin whitening and wrinkle improvement.

8. A method for producing a pharmaceutical composition containing an extract of callus derived from lavender stems or a fraction thereof as an active ingredient, (a) a step of inducing callus from lavender stem tissue; (b) a step of culturing the lavender stem-derived callus derived from step (a) in the presence of methyl jasmonate; and (c) A method comprising a step of obtaining an extract of callus derived from lavender stems cultured in step (b).

9. A method according to claim 8, wherein the concentration of methyl jasmonate is greater than 0 and less than 200 uM.

10. A method according to claim 8, wherein the pharmaceutical composition is a composition for improving skin.

11. A method according to claim 10, wherein the skin improvement has at least one characteristic selected from among anti-oxidation, anti-aging, skin whitening, and wrinkle improvement.

12. A method according to claim 8, wherein step (b) comprises culturing in a medium to which B5 vitamins (Duchefa), Sucrose, MES, 2,4-Dichlorophenoxyacetic acid, α-Naphthaleneacetic Acid, Kinetin, and Indole-3-Acetic Acid are added to Murashige & Skoog (MS) basic medium.

13. A method according to paragraph 8, wherein the lavender stem-derived callus is deposited as a patent resource (deposit number KCTC 15497BP) at the Korea Research Institute of Bioscience and Biotechnology (KCTC), a domestic patent depository for biological resources.

14. A method according to claim 10, wherein the skin improvement has at least one characteristic selected from skin whitening and wrinkle improvement. 15.(a) Step of inducing callus from lavender stem tissue; (b) a step of culturing the lavender stem-derived callus derived from step (a) in the presence of methyl jasmonate; and (c) a step of obtaining an extract of callus derived from lavender stems cultured in step (b); A composition for improving skin, comprising a callus extract derived from lavender stems or a fraction thereof manufactured by as an active ingredient.

Images