KR20240077623A - External Composition Comprising the Plant Cell Culture of fragrant rose for Improving Skin - Google Patents

Abstract

The present invention relates to a composition for topical skin application comprising a plant cell culture of any one or more of 18R-120-11 and 15R 12-2 or an extract obtained therefrom; Fragrance composition; Plant cell culture method; And it relates to a method of producing the composition for external skin application.
The plant cell culture of any one or more of 18R-120-11 and 15R 12-2 according to the present invention, or the extract obtained therefrom, contains 2-ethyl-1-hexanol and has antioxidant, anti-aging, skin protection from ultraviolet rays, Since it has moisturizing and anti-aging effects, an external skin composition containing the plant cell culture or an extract obtained therefrom can be usefully used for skin improvement.

Description

External composition for skin application comprising plant cell culture of fragrant rose as an active ingredient {External Composition Comprising the Plant Cell Culture of fragrant rose for Improving Skin}

The present invention relates to a composition for external use for skin containing a plant cell culture of fragrant rose as an active ingredient, and specifically, a plant cell culture of any one or more of 18R-120-11 and 15R 12-2, which are fragrant rose strains. It relates to an external composition for improving skin containing antioxidant properties, anti-inflammatory properties, protecting skin from ultraviolet rays, moisturizing skin, and preventing skin aging, including as active ingredients, and a method for producing the same.

The skin is the largest organ in the body by weight and is made up of three layers: the keratinized outer epidermis, the dermis, which is an inner connective tissue rich in blood vessels, and the innermost subcutaneous tissue, and various appendages (hair) associated with the skin. , nails, sensory receptors, and glands) form the skin system (integumentary system). The skin forms the boundary between the inside and outside of the body to carry out the main functions of various organs within the body.

The skin is essential for maintaining homeostasis and is an important organ that performs a variety of functions, including regulating body temperature, suppressing moisture loss, containing sensory receptors, synthesizing various biochemical substances, and excreting small amounts of waste in addition to protective functions.

However, compared to other organs, the skin has many opportunities to come into direct contact with external stimulation or various pathogens, and in particular, when ultraviolet rays are absorbed into the skin, a photochemical reaction occurs, causing skin lesions. In addition, the number of patients with skin diseases caused by factors commonly occurring in modern society, such as mental stress, obesity, alcohol, and smoking, is rapidly increasing.

Accordingly, the development of various skin protectants to suppress skin damage and protect skin cells is actively being developed. However, the developed skin protectors mainly contain synthetic compounds as active ingredients, which may cause skin damage or cause itchiness and spots. It has the disadvantage of causing side effects. Therefore, there is a need to develop new natural materials that have no side effects on the skin and have excellent skin damage inhibition and skin strengthening functions.

Under this background, the present inventors introduced the aroma components 2-ethyl-1-hexanol and hexanal into plant cell cultures of new rose lines, 18R-120-11 and 15R 12-2. (hexanal), 1-heptyn-3-ol, and decane, and has other antioxidant, anti-inflammatory, skin protection from UV rays, skin moisturizing, and anti-aging effects. was confirmed, and the present invention was completed.

Republic of Korea Patent No. 10-2341932

The present invention aims to solve the above-described problems and other problems associated therewith.

An exemplary object of the present invention is to provide an external composition for skin improvement comprising as an active ingredient any one or more plant cell cultures of 18R-120-11 and 15R 12-2, or extracts obtained therefrom; To provide a fragrance composition.

Another exemplary object of the present invention is to provide a method for cultivating any one or more plant cells of 18R-120-11 or 15R 12-2.

Another exemplary object of the present invention is to provide a method for producing an external composition for skin improvement containing as an active ingredient any one or more plant cell cultures of 18R-120-11 and 15R 12-2, or extracts obtained therefrom. .

The technical problem to be achieved according to the technical idea of the invention disclosed in this specification is not limited to the problem to solve the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Additionally, the scope of the present application cannot be considered limited by the specific description described below.

In one aspect for achieving the above object, the present invention provides a plant cell culture of any one or more of 18R-120-11 and 15R 12-2, that is, 18R-120-11 or 15R 12-2, or a combination thereof, or Provided is an external composition for improving skin containing the extract obtained therefrom as an active ingredient.

In the present invention, the 18R-120-11 and 15R 12-2 are deposited at the Korea Research Institute of Bioscience and Biotechnology and are Rosa hybrida 18R-120-11 (KCTC 15143BP) and Rosa hybrida 15R-12-2 (KCTC 15142BP), respectively. A deposit number was assigned.

The above 180R-120-11 is a rose line obtained through a cross between 'Whisper' and 'Whisper' at the National Institute of Horticultural and Herbal Science, and 15R-12-2 is a rose line obtained through a cross between 'Marilyn' and 'Red Explorer' at the National Institute of Horticultural and Herbal Science. This is a rose strain obtained through

The term "plant cell culture" of the present invention refers to a culture result including a cell mass generated by culturing tissue cut from a plant in a medium, and in a broad sense, a cell mass that causes normal organ formation or tissue differentiation, such as callus or Agrobacterium, etc. It includes cultures of vegetative tumor tissues resulting from infection.

In the present invention, the plant cell culture of any one or more of 18R-120-11 and 15R 12-2 is a part of the plant of 18R-120-11 or 15R 12-2, such as leaves, stems, roots, etc. It refers to a part obtained through culturing in an induction medium.

In the present invention, 18R-120-11 or 15R 12-2 can induce plant cells from flowering rose petals. Here, bloomed roses can be divided into bud, middle, and full bloom stages according to the flowering stage, and bud stage refers to stage 1 flowering and intermediate stage based on the Agricultural Science and Technology Research and Analysis Standards published by the Rural Development Administration. means 4 to 5 stages of flowering, and full bloom means 8 to 9 stages of flowering, but is not limited thereto.

The term "extract" in the present invention refers to an extract obtained by extraction treatment of any one or more of the plant cell cultures of 18R-120-11 and 15R 12-2, a dilution or concentrate of the extract, a dried product obtained by drying the extract, the It includes extracts of the extract itself and all formulations that can be formed using the extract, such as crude extracts, purified products, or mixtures thereof.

In the present invention, “skin improvement” means any action that improves or benefits the skin using any one or more plant cell cultures of 18R-120-11 and 15R 12-2 of the present invention or extracts obtained therefrom. do.

The skin improvement of the present invention may include, but is not limited to, antioxidant, anti-inflammatory, skin protection from ultraviolet rays, skin moisturizing, and anti-aging as examples.

The term “skin aging” of the present invention is a concept that includes both intrinsic and extrinsic aging.

In the present invention, “intrinsic aging” is also referred to as physiological aging and is related to normal aging as one ages.

The intrinsic aging reduces the regeneration of skin cells, resulting in a clinically impaired appearance, such as a decrease in subcutaneous fat tissue, the appearance of fine lines and wrinkles, or an increase in the number and thickness of elastic fibers, from the elastic tissue membrane. It may be accompanied by histopathological changes, such as loss of plumb fibers and the development of large irregular fibroblasts from these elastic tissue cells.

In the present invention, “extrinsic aging” is usually aging caused by the environment, and is specifically a type of photo-aging phenomenon that occurs due to exposure to the sun, light, or other radiation, but is not limited thereto.

Anti-aging of the present invention includes, but is not limited to, increased TERT gene expression.

The TERT gene is known to perform functions such as telomere elongation, stabilization, and protection, and when treated with cells, it is known to promote cell growth as well as telomere elongation and have an anti-aging effect.

The term "containing as an active ingredient" in the present invention refers to an external composition for skin improvement containing an effective amount capable of exhibiting various skin improvement effects as described above.

In one embodiment, the plant cell culture of the present invention and the extract obtained therefrom contain the aroma components 2-ethyl-1-hexanol, hexanal, and 1-heptin-3ol. It includes, but is not limited to, one or more of (1-heptyn-3-ol) and decane. The fragrance ingredient is registered as a flavoring substance in the Food Additives Code, and has a fragrant odor, so it can be used in fragrance compositions, external skin preparation compositions, cosmetic compositions, etc.

In one embodiment, the plant cell culture of the present invention and the extract obtained therefrom may contain ellagic acid and gallix acid.

In the present invention, the external skin composition may be a cosmetic composition or a pharmaceutical composition.

The cosmetic composition may contain a carrier acceptable in cosmetic preparations. Here, “acceptable carrier in cosmetic preparations” refers to compounds or compositions already known and used that can be included in cosmetic preparations, or compounds or compositions to be developed in the future, which exhibit toxicity, instability or irritation beyond what the human body can adapt to upon contact with the skin. It says there is no such thing. The carrier may be included in the external skin preparation composition of the present invention in an amount of about 1% to about 99.99% by weight based on the total weight, preferably about 90% by weight to about 99.99% by weight of the composition. However, since the above ratio varies depending on the formulation in which the external skin composition of the present invention is manufactured, its specific application area (face, neck, etc.), and its preferred application amount, the above ratio does not in any way define the scope of the present invention. It should not be understood as limiting.

Examples of the carrier include alcohol, oil, surfactant, fatty acid, silicone oil, wetting agent, moisturizing agent, viscosity modifier, emulsion, stabilizer, ultraviolet scattering agent, ultraviolet absorber, coloring agent, fragrance, etc. Compounds or compositions that can be used as alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosity modifiers, emulsions, stabilizers, UV scattering agents, UV absorbers, coloring agents, and fragrances are already known in the art. Therefore, a person skilled in the art can select and use an appropriate material or composition.

The external skin composition according to the present invention can be prepared in various forms, such as lotion, essence, gel, emulsion, lotion, cream (oil-in-water type, water-in-oil type, multi-phase), solution, and suspension (anhydrous and aqueous). , anhydrous products (oil and glycol-based), gels, masks, packs, powders, or capsules (soft capsules, hard capsules) with a coating such as gelatin.

In addition, suitable cosmetic formulations include, for example, solutions, gels, solid or pasty anhydrous products, emulsions obtained by dispersing the oil phase in the water phase, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes), non-ionic. It may be provided in the form of a vesicular dispersant, cream, skin, lotion, powder, ointment, spray or stick. Additionally, it can be prepared in the form of foam or in the form of an aerosol composition further containing compressed propellant.

In addition, the external skin composition of the present invention further contains fatty substances, organic solvents, solubilizers, thickening and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, and ions. Formal or non-ionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any commonly used in cosmetics. It may contain auxiliaries commonly used in the fields of cosmetology or dermatology, such as other ingredients. Additionally, the above ingredients can be introduced in amounts commonly used in the field of dermatology.

In the present invention, skin is a concept that includes not only the face, but also the scalp and the whole body. External skin compositions that can be applied to the scalp include shampoos, rinses, treatments, hair growth agents, etc., and body cleansers that can be applied to the whole body. It can be manufactured in various forms for purposes such as:

Therefore, the external skin composition according to the present invention includes the form of functional cosmetics such as antioxidant, anti-inflammatory, skin protection from ultraviolet rays, skin moisturizing, and anti-aging.

In the present invention, the composition for topical skin application can function as a pharmaceutical composition for improving skin condition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent according to a conventional method. Pharmaceutically acceptable carriers are well known in the art depending on the administration route or dosage form, and for specifics, each country's pharmacopoeia, including the 'Korean Pharmacopoeia', can be referred to. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Examples include, but are not limited to, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Additionally, carriers, excipients, and diluents that may be included in the composition of the present invention may be unnatural carriers, but are not limited thereto.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, or sterile injectable solutions according to conventional methods. . In detail, it can be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are made by adding at least one excipient to the compound, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. It can be prepared by mixing. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, wethepsol, macrogol, Tween 61, cacao, laurin, glycerogelatin, etc. can be used. Regarding the specific formulation of pharmaceutical compositions, it is known in the art, and references can be made to, for example, Remington's Pharmaceutical Sciences (19th ed., 1995). The above documents are considered part of this specification.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The pharmaceutically effective amount refers to an amount that is sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects. The effective dose level refers to the patient's health status, type and severity of the disease, It can be determined based on factors including the activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, drugs combined or used simultaneously, and other factors well known in the medical field. The dosage and frequency of administration do not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, dogs, cats, cows, horses, pigs, and humans through various routes, and humans may be preferable. All modes of administration are contemplated and may include, but are not limited to, oral, intravenous, intramuscular or subcutaneous injection.

The composition for topical skin application of the present invention may be a quasi-drug composition.

In addition to the above ingredients, the quasi-drug composition of the present invention may further include pharmaceutically acceptable carriers, excipients, or diluents as needed. The pharmaceutically acceptable carrier, excipient, or diluent is not particularly limited as long as it does not impair the effect of the present invention, and includes, for example, fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, fragrances, preservatives, etc. may include.

Quasi-drug compositions may include, but are not limited to, disinfectant cleaners, shower foams, ointments, wet tissues, coating agents, etc., and the formulation method, dosage, usage method, and components of quasi-drugs can be determined from conventional techniques known in the technical field. It can be selected appropriately.

In another aspect of the present invention, the present invention provides a method for cultivating plant cells of 18R-120-11 or 15R 12-2, comprising the following steps: (a) culturing the plant cells of 18R-120-11 or 15R 12-2 Inducing plant cells by wounding the petals and culturing them in the dark; and (b) obtaining a plant cell culture by subculturing the induced plant cells.

For the above-mentioned culture of the present invention, the flowering stage of the plant, culture direction, culture site, etc. can be selected, and any method generally used for plant tissue culture in the technical field of the present invention can be used without limitation.

The flowering stage refers to the bud, middle, and full bloom stages, and in the case of the present invention, petals of the full bloom stage of 18R-120-11 or 15R 12-2 are collected and used for plant cell induction culture, but is not limited thereto.

In the step of inducing plant cells of the present invention, the flower petals at the full bloom stage of 18R-120-11 or 15R 12-2 are disinfected with 60 to 80% ethanol and NaOCl disinfectant, washed with sterilized water, and then added with 0.1 mg of the plant growth regulator auxin. This refers to the step of dark culturing under conditions of 25 ± 2°C in a medium containing /mL to 11 mg/mL.

The subculture step of the present invention refers to the step of culturing the induced plant cells in a medium of the same composition.

An appropriate medium can be used for the subculture, and any medium commonly used for plant tissue culture in the technical field of the present invention can be used without limitation.

As an example, SH medium can be used for subculture of plant cells of the present invention. The SH medium may contain 2,4-Dichlorophenoxyacetic acid (2,4-D), sucrose, and phytagel, preferably 5 to 15 It may include mg/L 2,4-dichlorophenoxyacetic acid, 1 to 5% sucrose, and 1 to 5 g/L phytagel.

As an example, SH medium can be used for subculture of plant cells of the present invention. The SH medium may contain 2,4-Dichlorophenoxyacetic acid (2,4-D), sucrose, and proline, preferably 1 to 5 mg. /L 2,4-dichlorophenoxyacetic acid, 1 to 5% sucrose and 200 to 400 mg/L proline.

In another aspect of the present invention, the present invention provides a method for producing the composition for external use comprising the following steps: (a) wounding the petals of the plants of 18R-120-11 or 15R 12-2 and culturing them in the dark; Inducing plant cells; (b) obtaining a plant cell culture by subculturing the induced plant cells, and (c) preparing an external composition containing the plant cell culture obtained in step (b) or an extract obtained therefrom as an active ingredient. .

For the above-mentioned culture of the present invention, the flowering stage of the plant, culture direction, culture site, etc. can be selected, and any method generally used for plant tissue culture in the technical field of the present invention can be used without limitation.

The flowering stage refers to the bud, middle, and full bloom stages, and in the case of the present invention, petals of the full bloom stage of 18R-120-11 or 15R 12-2 are collected and used for plant cell induction culture, but is not limited thereto.

In the step of inducing plant cells of the present invention, the petals of 18R-120-11 or 15R 12-2 at the full bloom stage are disinfected with 70% ethanol and NaOCl disinfectant solution, washed with sterilized water, and then added with 0.1 mg/mL of plant growth regulator auxin. Dark culture was performed under conditions of 25 ± 2°C in a medium containing 11 mg/mL.

Additionally, an appropriate medium can be used for the above-mentioned culture of the present invention, and any medium commonly used for plant tissue culture in the technical field of the present invention may be used without limitation.

As an example, SH medium can be used for subculture of plant cells of the present invention. The SH medium may contain 2,4-Dichlorophenoxyacetic acid (2,4-D), sucrose, and phytagel, preferably 5 to 15 It may include mg/L 2,4-dichlorophenoxyacetic acid, 1 to 5% sucrose, and 1 to 5 g/L phytagel.

As an example, SH medium can be used for subculture of plant cells of the present invention. The SH medium may contain 2,4-Dichlorophenoxyacetic acid (2,4-D), sucrose, and proline, preferably 1 to 5 mg. /L 2,4-dichlorophenoxyacetic acid, 1 to 5% sucrose and 200 to 400 mg/L proline.

In the present invention, the extraction is not particularly limited and can be extracted according to methods commonly used in the art. Non-limiting examples of the extraction method include hot water extraction, cold immersion extraction, solvent extraction, steam distillation, elution, compression, ultrasonic extraction, filtration, and reflux extraction, which are performed alone or using a combination of two or more methods. It can be performed by doing this.

In one embodiment, the extraction step of the present invention may be by hot water extraction. Preferably, hot water extraction may be performed at 90°C to 140°C for 10 to 1 hour, and more preferably, hot water extraction may be performed at 100°C to 130°C for 15 to 20 minutes.

The extract of the present invention is extracted from a plant cell culture of one or more of 18R-120-11 and 15R 12-2 using an appropriate solvent, for example, all crude extracts, polar solvent-soluble extracts, or non-polar solvent-soluble extracts. It can be included. The type of extraction solvent used to prepare the extract is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of the extraction solvent include water; Lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, propanol, isopropanol, butanol, propyl alcohol, and butyl alcohol; Polyhydric alcohols such as glycerin, butylene glycol, and propylene glycol; Hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane;　Or a mixture thereof can be used. Additionally, a solvent extract can be prepared by extracting the extract one or more times using the solvent.

The extract of the present invention can be used by filtering, for example, mesh filtration to remove floating solid particles, or by drying it using freeze-drying, hot air drying, spray drying, etc. Additionally, the extract may further undergo a conventional fractionation process or may be purified using a conventional purification method.

In another aspect of the present invention, the present invention provides a fragrance composition comprising as an active ingredient any one or more plant cell cultures of 18R-120-11 and 15R 12-2, or extracts obtained therefrom.

The fragrance composition of the present invention can be applied to cosmetic compositions, fragrance compositions, external skin preparation compositions, etc. for fragrance.

The fragrance composition of the present invention can be used for skin, lotion, essence, lotion, gel, nutritional cream, massage cream, hand cream, foot cream, foundation, makeup base, hair cream, hair tonic, hair conditioner, hair treatment, hair styling gel, It may be provided as a cosmetic composition such as a bath additive, liquid cleanser, soap, or shampoo, and is not limited to the above as long as it is an article to which a fragrance composition can be applied.

The fragrance composition of the present invention may be provided as a fragrance composition such as perfume, air freshener, deodorant, diffuser, or aroma candle.

The fragrance composition of the present invention can be provided as an external skin preparation composition such as ointment, lotion, soluble burn, suspension, emulsion, cream, gel, spray, patch, warning agent, patch, or water paste.

The fragrance composition of the present invention can be provided as an oral composition such as mouthwash, toothpaste, dental floss, oral spray, mouthwash, and wet tissue for oral cleaning. At this time, the compositions may additionally contain generally acceptable colorants, disinfectants, preservatives, antioxidants, moisturizers, thickeners, etc. to improve physical properties, and some fragrance ingredients and additional ingredients may be replaced with ingredients permitted for oral use. possible.

The plant cell culture of any one or more of 18R-120-11 and 15R 12-2 according to the present invention, or the extract obtained therefrom, has skin improvement effects such as antioxidant, anti-inflammatory, skin protection from ultraviolet rays, skin moisturizing, and anti-aging. , 18R-120-11, and 15R 12-2, a plant cell culture, or an extract obtained therefrom as an active ingredient can be usefully used for skin improvement.

Figure 1 is a diagram confirming the results of culturing 18R-120-11 and plant cells of the present invention.
Figure 2 is a diagram confirming the results of culturing 15R 12-2 and plant cells of the present invention.
Figure 3 is a diagram confirming the results of HPLC analysis of the 18R-120-11 plant cell culture extract of the present invention.
Figure 4 is a diagram confirming the results of HPLC analysis of the 15R 12-2 plant cell culture extract of the present invention.
Figure 5 is a diagram confirming the cytotoxicity evaluation results of plant cell culture extracts of 18R-120-11 and 15R 12-2 of the present invention.
Figure 6 is a diagram confirming the antioxidant effect (SOD1) of the plant cell culture extract of 18R-120-11 and 15R 12-2 of the present invention.
Figure 7 is a diagram confirming the antioxidant effect (CAT) of the plant cell culture extract of 18R-120-11 and 15R 12-2 of the present invention.
Figure 8 is a diagram confirming the antioxidant effect (NRF1) of the plant cell culture extract of 18R-120-11 and 15R 12-2 of the present invention.
Figure 9 is a diagram confirming the ultraviolet ray-induced inflammation suppressing effect (COX-2) of the plant cell culture extracts of 18R-120-11 and 15R 12-2 of the present invention.
Figure 10 is a diagram confirming the skin protection effect of the plant cell culture extracts of 18R-120-11 and 15R 12-2 of the present invention against cell damage induced by ultraviolet rays.
Figure 11 is a diagram confirming the moisturizing effect (AQP3) of the plant cell culture extract of 18R-120-11 and 15R 12-2 of the present invention.
Figure 12 is a diagram confirming the anti-aging effect (TERT) of the plant cell culture extract of 18R-120-11 and 15R 12-2 of the present invention.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1: Confirmation of optimal culture conditions for plant cells of 18R-120-11 or 15R 12-2

To confirm the optimal culture conditions for plant cells of 18R-120-11 and 15R 12-2, the degree of callus formation by flowering stage, culture direction, and medium was investigated.

The petals of 18R-120-11 and 15R 12-2 in the mid-flowering stage and at full bloom were disinfected and then cultured on each medium so that the surface and back of the petals were in contact with the medium. As a result of examining the degree of callus formation after 1 month of dark culture, as shown in Table 1, it was effective to use full bloom petals for the flowering stage, and callus was formed without significant difference in the culture direction on both the surface and the back side, and the medium was SH11DA [SH basic medium. + 11mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D) + 3% sucrose + 7.2g/L Agar], SH11DP [SH basic medium + 11mg/L 2, 4-Dichlorophenoxyacetic acid (2,4-D) + 3% sucrose + 2.4g/L Phytagel] and WPM11D [WPM basic medium + 11mg/L 2,4-dichlorophenoxy Among acetic acid (2,4-Dichlorophenoxyacetic acid: 2,4-D) + 3% sucrose + Agar 7.2 g/L], SH11DP medium was confirmed to be the most effective [(+) Callus formation in less than 20% of explants, ( ++) Callus formation in 50-60% of explants, (+++) Callus formation in more than 90% of explants, (×) No callus formation].

system flowering
step culture
direction Callus formation rate (%) SH11DA SH11DP WPM11D + ++ +++ X + ++ +++ X + ++ +++ X 15R 12-2 middle award 20 0 0 80 30 23 40 7 63 0 0 37 under 43 3 0 53 33 23 40 3 60 0 0 40 full bloom award 40 30 30 0 25 5 65 5 63 3 0 33 under 77 10 0 13 77 10 0 13 55 5 0 40 18R 120-11 middle award 87 13 0 0 83 17 0 0 7 0 0 93 under 90 10 0 0 83 13 0 3 13 0 0 87 full bloom award 77 10 0 13 80 20 0 0 13 0 0 87 under 93 3 0 3 87 13 0 0 27 3 0 70

Example 2: Preparation of plant cell culture of 18R-120-11 or 15R 12-2 and extract obtained therefrom

Petals were collected from rose plants of 18R-120-11 and 15R 12-2, then immersed in 70% ethanol for 30 seconds and washed with sterile water. After completely removing the water from the disinfected petal tissue, the petals of the plant are cut at once using a sharp knife and cultured in the dark under growth conditions at 25 ± 2°C in a medium containing 0.1 to 11 mg/mL of the plant growth regulator auxin. Initial plant cells were induced and subculture was performed with new medium every 3 to 6 weeks.

As shown in Figures 1 and 2, plant callus of plant cells was confirmed from 6 weeks, and 18R-120-11 plant cells were grown in SH medium with 3 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-Dichlorophenoxyacetic acid: 2,4-D), in a medium containing 300 mg/L proline and 3% sucrose, and 15R 12-2 in a medium containing 11 mg/L 2,4-dichlorophenoxyacetic acid (2) in SH medium. , 4-Dichlorophenoxyacetic acid: 2,4-D), 3% sucrose, and 2.4 g/L phytagel. Subculture was carried out under conditions of 25 ± 2°C.

The plant cell cultures of 18R-120-11 and 15R 12-2 obtained from the above culture were harvested, washed several times with purified water, sufficiently removed, and dried in a dryer at 60°C for 2 days. 1 L of purified water was added to 2 g of dried plant cell cultures of 18R-120-11 and 15R 12-2, and then subjected to hot water extraction at 100°C to 130°C for 15 minutes. After extraction, the plant cell culture extracts of 18R-120-11 and 15R 12-2 were separated and used by filtering through mesh.

Experimental Example 1: Confirmation of fragrance components through GC-MS analysis

The aroma components contained in the 15R 12-2 plant cell culture were extracted using a solvent (hexane, dichloromethane (DCM), hexane:dichloromethane=1:5, methanol, hexane:methanol=1:5). 3 ml of each solvent was added to 100 mg of the dried sample, vortexed for 5 minutes, and extracted for 3 hours. The extract was centrifuged, the supernatant was recovered, concentrated using a solvent freeze dryer, and then used for GC-MS analysis. GC-MS analysis was performed under the conditions shown in Table 2.

extraction Model Auto sampler (PAL System) Extraction HS-SPME (Headspace-Solid phase Micro Extraction) Fiber DVB/C-WR/PDMS (fiber Tickness 80um, fiber length 10mm) Agitators On time: 5s, off time: 2s, speed: 350rpm, Temperature: 40℃ Incubation time 10min Extraction time 30min GC-MS analysis Model GC-MS system (Agilent, 7890B GC system, 5977B MSD) Column HP-5MS, 30m x 250um x 0.25um Flow 1ml/min Inlet 230℃, splitless Oven temp. 40℃ (5min) → 3℃/min →240℃ (5min) Ingredient analysis Mass library NIST 14 library Match factor ≥80 Soft ware MassHunter Qualitative Analysis Workflows (Agilent)

As a result of GC-MS analysis, the components of the 15R 12-2 plant cell culture were shown in Table 3.

No. R.I. CAS designation chemical formula Relative content (%) Odor Description Identification
method Hexane DCM Hex:DCM
(1:5) MeOH Hex:MeOH
(1:5) One 758 2919-23-5 Cyclobutanol C ₄ H ₈ O - - - 0.08 - M.S. 2 801 66-25-1 Hexanal C ₆ H ₁₂ O 13.07 2.37 13.45 7.75 0.83 Fresh, green, fatty, aldehydic, grass, leafy, fruity, sweaty M.S., R.I. 3 833 553-90-2 Ethanedioic acid, dimethyl ester C ₄ H ₆ O ₄ - - - 22.83 - M.S. 4 873 13952-84-6 sec-Butylamine C ₄ H ₁₁ N - - 0.02 - - Fishy, ammonia M.S. 5 953 7383-19-9 1-Heptyn-3-ol C ₇ H ₁₂ O - 24.01 19.01 1.94 - M.S. 6 986 110-93-0 5-Hepten-2-one, 6-methyl- C ₈ H ₁₄ O - 3.76 3.09 - - Citrus, green, musty, lemongrass, apple M.S., R.I. 7 991 3173-53-3 Cyclohexane, isocyanato- C ₇ H ₁₁ NO - - - 1.98 0.19 M.S. 8 998 124-18-5 Decane C ₁₀ H ₂₂ - - - 17.89 0.47 M.S., R.I. 9 1011 17302-01-1 3-Ethyl-3-methylheptane C ₁₀ H ₂₂ - - - - 1.83 M.S. 10 1027 104-76-7 2-Ethyl-1-hexanol C ₈ H ₁₈ O 59.01 9.74 5.28 1.80 0.62 Citrus, fresh, floral, oily, sweet M.S., R.I. 11 1042 821-97-6 3-Undecene, (Z)- C ₁₁ H ₂₂ - - - - 0.05 M.S. 12 1058 1000309-20-1 Sulfurous acid, 2-ethylhexyl octadecyl ester C ₂₆ H ₅₄ O ₃ S - - - - 0.31 M.S. 13 1065 26001-58-1 2-Octen-1-ol, (Z)- C ₈ H ₁₆ O - - 1.19 - - Sweet, floral M.S. 14 1080 55170-80-4 1-Decene, 2,4-dimethyl- C ₁₂ H ₂₄ - - - - 6.82 M.S. 15 1096 1000309-24-3 Oxalic acid, allyl pentadecyl ester C ₂₀ H ₃₆ O ₄ - - - 0.27 - M.S. 16 1099 3913-02-08 1-Octanol, 2-butyl- C ₁₂ H ₂₆ O - - - 0.33 - M.S. 17 1101 124-19-6 Nonanal C ₉ H ₁₈ O - - - 0.30 - waxy, aldehydic, rose, fresh, orris, orange, peel, fatty, peely M.S. 18 1240 61141-72-8 Dodecane, 4,6-dimethyl- C ₁₄ H ₃₀ - - - 0.62 - M.S. 19 1276 31295-56-4 Dodecane, 2,6,11-trimethyl- C ₁₅ H ₃₂ - - - - 3.55 M.S., R.I. 20 1301 91337-07-4 2-Isopropyl-5-methyl-1-heptanol C ₁₁ H ₂₄ O - - - - 5.37 M.S. 21 1304 1000309-34-3 Oxalic acid, 6-ethyloct-3-yl isohexyl ester C ₁₈ H ₃₄ O ₄ - - - 0.27 - M.S. 22 1310 6750-34-1 1-Dodecanol, 3,7,11-trimethyl- C ₁₅ H ₃₂ O - - - 2.53 - M.S. 23 1315 13187-99-0 2-Bromo dodecane C ₁₂ H ₂₅ Br - - - - 0.57 M.S. 24 1318 18675-24-6 1-Decanol, 2-methyl- C ₁₁ H ₂₄ O - - - 1.36 - M.S. 25 1322 3891-98-3 Dodecane, 2,6,10-trimethyl- C ₁₅ H ₃₂ - - - 0.73 - M.S. 26 1366 1000406-39-2 Dodecyl heptyl ether C ₁₉ H ₄₀ O - - - - 0.18 M.S. 27 1425 1009-61-6 Ethanone, 1,1'-(1,4-phenylene)bis- C ₁₀ H ₁₀ O ₂ - - - - 0.30 M.S. 28 1475 195194-80-0 2-Piperidinone, N-[4-bromo-n-butyl]- C ₉ H ₁₆ BrNO - - - - 0.08 M.S. 29 1492 504-44-9 Crocetane C ₂₀ H ₄₂ - - - - 0.64 M.S. 30 1535 1000406-09-9 2-Heptenoic acid, tetradecyl ester C ₂₁ H ₄₀ O ₂ - - - - 0.08 　 M.S.

In particular, in the case of 15R 12-2 plant cell culture, not only the flower or leaf of the plant, but the aroma component 2-ethyl-1-hexanol, known as the rose scent component, as well as hexanal ( It was confirmed that it contained hexanal), 1-heptyn-3-ol, and decane.

Experimental Example 2: Confirmation of extract components through HPLC analysis

To confirm the indicator substances and active ingredients contained in the plant cell culture extracts of 18R-120-11 and 15R 12-2, HPLC analysis was performed under the analysis conditions shown in Table 4.

Instrument HPLC System (Agilent 1260 InfinityⅡ system (Agilent, U.S.A.) Column Shim-pack GIS C ₁₈ (4.6 × 250 mm, 5 μm, Shimadzu, Japan) Detector Diode Array Detector Mobile Phase Mobile Phase A: 0.1% Trifluoroacetic acid in water Mobile Phase B: 0.1% Trifluoroacetic acid in acetonitrile Flow 1mL/min Injection volume 20 μL Gradient Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 100 0 5 100 0 75 30 70

Specifically, during HPLC analysis, various peaks were detected as a result of checking the chromatogram in the UV range of 200 to 600 nm using a diode array detector. When confirmed at 255 nm, a peak of ellagic acid appeared at 33.6 minutes (Figures 3 and 4), many small peaks appeared at 210 nm, and a gallic acid peak appeared at 15.4 minutes. In particular, in the case of 18R-120-11 plant cell culture extract, it was confirmed that the extract contained 12.0 mg/L and 5.1 mg/L, respectively, as a result of quantification using ellagic acid and gallic acid standard substances. These two substances have been reported to have antioxidant and anti-inflammatory effects, and it was confirmed that they contribute to improving the antioxidant and anti-inflammatory effects of this 18R-120-11 plant cell culture extract.

Experimental Example 3: Confirmation of skin cell toxicity

To confirm whether the composition of the present invention is toxic, MTT assay was performed. For this purpose, HaCaT cells (keratinocytes, keratinocytes) were dispensed into a 96-well plate along with DMEM (Dubelcco's Modified Eagle's Medium) containing 10% FBS (Fetal Bovine Serum) and 1% Antibiotic-Antimycotic, and incubated at 37°C for 5 days. Cultured for 24 hours in a thermostat under % CO ₂ conditions. Afterwards, the cells were treated with a control group to which purified water was added and a test substance composition, and further cultured for 24 hours. Then, 4 μL of 5 mg/mL MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) solution was added to each well, and incubated for 4 hours to react. After removing the culture medium, 100 μL of DMSO (dimethyl sulfoxide) solution was added to lyse the cells, and the absorbance was measured at 540 nm.

As a result of measuring the cell survival rate in HaCaT cells treated with the composition of the present invention, as shown in Figure 5, 18R-120-11 did not show intracellular toxicity compared to 100% of the control group, and 15R 12-2 also showed a possibility of causing skin irritation as a result of the experiment. It was confirmed to be a safe material.

Experimental Example 4: Confirmation of antioxidant effect

In order to determine whether the composition of the present invention has an antioxidant effect, SOD1 (superoxide dismutase 1), CAT (catalase), which are antioxidant enzymes that play a key role in the process of converting harmful oxygen to the human body into water and oxygen, and external stress We examined changes in the expression level of Nrf1 (Nuclear Respiratory Factor 1), which is known to protect cells from reactions such as inflammation caused by . The positive control group (PC) was treated with 2mM NAC (N-acetyl-L-cysteine).

Specifically, HaCaT cells were dispensed into a 96-well plate and cultured for 24 hours under cell culture conditions. The cells were treated with the composition according to the present invention and further cultured for 24 hours. Real-time PCR was performed to confirm the expression of SOD1, CAT, and Nrf1 at the gene level, and the test sequence was as follows. SuperPrep ^TM cell lysis & RT Kit for qPCR (TOYOBO, Cat. SCQ-101) was used for RNA isolation and cDNA synthesis. To comparatively analyze gene expression, the cDNA synthesized above was used as a template, and real-time PCR analysis was performed using Thunderbird ^TM SYBR qPCR Mix (TOYOBO, Cat. QPS-201). The primers used in the experiment were QuantiTect® primer assays: GAPDH (Qiagen, Cat no. QT01192646, Germany), SOD1 (Qiagen, Cat no. QT01008651, Germany), CAT (Qiagen, Cat no. QT00079674, Germany), and NRF1 (Qiagen, Cat no. QT01192688, Germany), and the expression levels of SOD1, CAT, and NRF1 mRNA in the samples were quantified using GAPDH.

As a result of confirming the expression of SOD1, CAT, and NRF1 genes related to antioxidant effects by treating HaCaT cells with the extract of the present invention, the expression level of SOD1 increased in all concentration groups of 18R-120-11 and 15R 12-2 cell culture extracts ( 6), the expression level of CAT increased in the group treated with 18R-120-11 cell culture extract at 0.5% and 1% concentration and the group treated with 15R 12-2 cell culture extract at 0.5% concentration (Figure 7), and NRF1 was expressed in 18R-120 -11 and 15R 12-2 cell culture extracts The expression level increased in the group treated with 0.5% concentration (Figure 8), confirming that 18R-120-11 and 15R 12-2 cell culture extracts are substances that help with antioxidant effects (Figure 8) 7 to 9).

Experimental Example 5: Confirmation of anti-inflammatory effect

In order to confirm whether the composition of the present invention has an anti-inflammatory effect, it is expressed on skin exposed to UV and plays a role in changing arachidonic acid into prostaglandin, destroying the skin by prostaglandin and causing skin cancer, etc. We investigated changes in COX-2 expression. The positive control group (PC) was treated with 2mM NAC (N-acetyl-L-cysteine).

Specifically, HaCaT cells were dispensed into a 96-well plate and cultured for 24 hours under cell culture conditions. Afterwards, the medium was removed, an inflammatory response was induced with 5 mJ/cm ² UV irradiation using a UVP CL-1000 UV Crosslinking Chamber (Hyland Scientific, Stanwood, WA, USA), and then the cells were treated with the composition according to the present invention. Additional culture was performed for 24 hours. Real-time PCR was performed to confirm the expression of COX-2 at the gene level, and the test sequence was as follows. SuperPrep ^TM cell lysis & RT Kit for qPCR (TOYOBO, Cat. SCQ-101) was used for RNA isolation and cDNA synthesis. To comparatively analyze gene expression, the cDNA synthesized above was used as a template, and real-time PCR analysis was performed using Thunderbird ^TM SYBR qPCR Mix (TOYOBO, Cat. QPS-201). The primers used in the experiment were QuantiTect® primer assays GAPDH (Qiagen, Cat no. QT01192646, Germany), COX-2 (Qiagen, Cat no. QT00040586, Germany), and the COX-2 mRNA expression level of the sample was quantified using GAPDH.

As a result of measuring the COX-2 gene expression rate in HaCaT cells by treating them with the extract of the present invention, the expression level of COX-2 decreased in the 5% concentration treatment group according to treatment with the 18R-120-11 cell culture extract (FIG. 9), indicating anti-inflammatory activity. It was confirmed that it was a substance that helped the effect.

Experimental Example 6: Confirmation of skin protection effect against ultraviolet ray-induced cell damage

In order to determine whether the composition of the present invention has a skin protection effect against ultraviolet ray-induced cell damage, MTT assay was performed. 1x10 ⁵ HaCaT cells (keratinocytes, keratinocytes) per well were dispensed into each 96-well plate and cultured for 24 hours. Afterwards, starvation was performed for 4 hours using a medium not containing FBS, and 5 mJ/cm ² UV irradiation was performed using a UVP CL-1000 UV Crosslinking Chamber (Hyland Scientific, Stanwood, WA, USA). .

Afterwards, the cells were treated with a control group to which purified water was added and a test substance composition, and further cultured for 24 hours. Then, 4 μL of 5 mg/mL MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) solution was added to each well, and incubated for 4 hours to react. After removing the culture medium, 100 μL of DMSO (dimethyl sulfoxide) solution was added to lyse the cells, and the absorbance at 540 nm was measured using a Thermo Scientific Multiskan GO Microplate Spectrophotometer (Fisher Scientific Ltd., Vantaa, Finland).

As a result of measuring the cell viability of HaCaT cells by treating them with the extract of the present invention, the cell viability of the negative control group, which was reduced by UV irradiation, increased to 37% by treatment with 5% concentration of 18R-120-11 plant cell culture extract. A significant increase was confirmed (Figure 10), confirming that it protects skin cells from ultraviolet rays along with an anti-inflammatory effect.

Experimental Example 7: Confirmation of skin moisturizing effect

In order to confirm whether the composition of the present invention has a moisturizing effect, changes in the expression level of AQP3, an intracellular moisturizing-related gene and a transport membrane protein that allows water and glycerol to pass through, were examined. The positive control group (PC) used 1% Glyceryl glucoside.

Specifically, HaCaT cells were dispensed into a 96-well plate and cultured for 24 hours under cell culture conditions. The cells were treated with the composition according to the present invention and further cultured for 24 hours. Real-time PCR was performed to confirm the expression of AQP3 at the gene level, and the test sequence was as follows. SuperPrep ^TM cell lysis & RT Kit for qPCR (TOYOBO, Cat. SCQ-101) was used for RNA isolation and cDNA synthesis. To comparatively analyze gene expression, the cDNA synthesized above was used as a template, and real-time PCR analysis was performed using Thunderbird ^TM SYBR qPCR Mix (TOYOBO, Cat. QPS-201). The primers used in the experiment were QuantiTect® primer assays, GAPDH (Qiagen, Cat no. QT01192646, Germany) and AQP3 (Qiagen, Cat no. QT00212996, Germany), and the AQP3 mRNA expression level of the sample was quantified using GAPDH.

As a result of measuring the expression level of the AQP3 gene in HaCaT cells treated with the extract of the present invention, AQP3 was observed in the group treated with 1% concentration of 18R-120-11 plant cell culture extract and the group treated with 0.5% concentration of 15R 12-2 plant cell culture extract. It was confirmed that the expression level of the gene was significantly increased (Figure 11), confirming that it was a substance that helped with the moisturizing effect.

Experimental Example 8: Confirmation of anti-aging effect

In order to confirm whether the composition of the present invention has an anti-aging effect, changes in the expression level of TERT (Telomerase Reverse Transcriptase), an anti-aging related gene, were examined. The positive control group (PC) used 1mM Shikimic acid (SA).

Specifically, HaCaT cells were dispensed into a 96-well plate and cultured for 24 hours under cell culture conditions. The cells were treated with the composition according to the present invention and further cultured for 24 hours. Real-time PCR was performed to confirm the expression of TERT at the gene level, and the test sequence was as follows. SuperPrep ^TM cell lysis & RT Kit for qPCR (TOYOBO, Cat. SCQ-101) was used for RNA isolation and cDNA synthesis. To comparatively analyze gene expression, the cDNA synthesized above was used as a template, and real-time PCR analysis was performed using Thunderbird ^TM SYBR qPCR Mix (TOYOBO, Cat. QPS-201). The primers used in the experiment were QuantiTect® primer assays, GAPDH (Qiagen, Cat no. QT01192646, Germany), Telomerasereversetranscriptase (TERT) Cat.#QT00073409, and the TERT mRNA expression level in the sample was quantified using GAPDH.

As a result of measuring the expression level of the TERT gene in HaCaT cells treated with the extract of the present invention, TERT was found in the group treated with 1% concentration of 18R-120-11 plant cell culture extract and the group treated with 0.5% concentration of 15R 12-2 plant cell culture extract. It was confirmed that the expression level of the gene was significantly increased (Figure 12), confirming that it is a substance that helps with anti-aging effects.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC15142BP 20221018 Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC15143BP 20221018

Claims (15)

An external composition for skin improvement comprising any one or more plant cell cultures of 18R-120-11 and 15R 12-2, or extracts obtained therefrom, as an active ingredient. According to paragraph 1,
The plant cell culture of any one or more of the 18R-120-11 and 15R 12-2, or the extract obtained therefrom, contains 2-ethyl-1-hexanol, hexanal, 1 -A composition for external use containing at least one of 1-heptyn-3-ol and decane. According to paragraph 1,
The skin improvement is an antioxidant or anti-inflammatory composition for external use. According to paragraph 1,
The skin improvement is a composition for external use for protecting the skin from ultraviolet rays. According to paragraph 1,
A composition for external use wherein the skin improvement is moisturizing improvement. According to paragraph 1,
The skin improvement is an anti-aging, external composition. The plant cell culture method according to claim 1 comprising the following steps:
(a) wounding the petals of plants of 18R-120-11 or 15R 12-2 and culturing them in the dark to induce plant cells; and
(b) Subculturing the induced plant cells to obtain a plant cell culture. In clause 7,
The method of step (a) is to wound the rose petals of 18R-120-11 or 15R 12-2 at the full bloom stage and perform dark culture. In clause 7,
Step (b) is a method of culturing in SH medium containing 5 to 15 mg/L 2,4-dichlorophenoxyacetic acid, 1 to 5% sucrose, and 1 to 5 mg/L phytagel. In clause 7,
Step (b) is a method of culturing in SH medium containing 1 to 5 mg/L 2,4-dichlorophenoxyacetic acid, 1 to 5% sucrose, and 200 to 400 mg/L proline. A method for producing the composition for external use according to claim 1 comprising the following steps:
(a) wounding the petals of plants of 18R-120-11 or 15R 12-2 and culturing them in the dark to induce plant cells;
(b) obtaining a plant cell culture by subculturing the induced plant cells; and
(c) Preparing a composition for external use containing the plant cell culture obtained in step (b) or the extract obtained therefrom as an active ingredient. According to clause 11,
The method of step (a) is to wound the rose petals of 18R-120-11 or 15R 12-2 at the full bloom stage and perform dark culture. According to clause 11,
Step (b) is a method of culturing in SH medium containing 5 to 15 mg/L 2,4-dichlorophenoxyacetic acid, 1 to 5% sucrose, and 1 to 5 mg/L phytagel. According to clause 11,
Step (b) is a method of culturing in SH medium containing 1 to 5 mg/L 2,4-dichlorophenoxyacetic acid, 1 to 5% sucrose, and 200 to 400 mg/L proline. A fragrance composition comprising any one or more plant cell cultures of 18R-120-11 and 15R 12-2, or extracts obtained therefrom, as an active ingredient.