FR3117034A1 - Callus lysate comprising high content of callus metabolite and process for its preparation - Google Patents

Abstract

Callus lysate comprising high content of callus metabolite and process for its preparation Disclosed are a callus lysate containing a callus metabolite without loss of callus metabolite and its preparation method, a capsule containing a callus lysate prepared by coating encapsulation of the callus lysate with a natural polymer material and its preparation method, and a composition for external skin application and a cosmetic composition which includes the callus lysate or the capsule containing the callus lysate as an active ingredient. The callus lysate of the present invention contains a large amount of a callus metabolite without loss of callus metabolite and thus enables penetration or absorption of callus metabolite directly into the skin, thereby providing effects of callus metabolite for prevention and improvement of various skin symptoms. No figures

Description

Callus lysate comprising high content of callus metabolite and process for its preparation

FIELD OF THE INVENTION

The present invention relates to a callus lysate having a high content of callus metabolites, a method for preparing the callus lysate, and a composition including the callus lysate.

BACKGROUND OF THE INVENTION

In botany, a callus generally refers to an unorganized mass of undifferentiated plant parenchyma cells. Callus is also called plant stem cell because of its ability to differentiate into a whole plant through treatment with plant growth regulators or equivalent. In recent years, plant tissue culture techniques have been developed to produce various metabolites of plant origin by mass culture of callus cells without damaging plants. Callus culture is therefore of great interest to industry.

A callus is a cell of plant origin and is therefore surrounded by a cell wall composed of cellulose, pectin, and lignin which cannot degrade in the human body. For this reason, even if the callus itself is applied to human skin, the metabolites in the callus cannot penetrate into the human skin, because the human skin has no cellulase to break down the cell wall of the callus, and the physical force of pressing the callus on the skin with the hands for application to the skin is not strong enough to rupture the cell wall. In other words, the metabolites in the callus are not available for use as a beneficial component directly on human skin simply by applying the callus itself to the skin.

Therefore, various methods have been tried to utilize the metabolites contained in callus as beneficial components in the human body. One such method is to use extraction, separation, and purification of beneficial components from callus with an organic solvent that has high solubility in a specific component. However, the method is problematic due to the difficulty of extracting beneficial components with high yield and the use of a large amount of organic solvents unsuitable for human skin in a repetitive process of fractionation and concentration to obtain a given content of beneficial components. Another process involves supercritical extraction or the like that does not use organic solvents. However, the supercritical extraction process requires expensive equipment and consumes a lot of energy to carry out the process, so it is complicated to achieve an economic feasibility that is advantageous with this process.

Furthermore, conventional extraction processes cannot utilize all of a plant's nutrients, but involve very high integration of only a portion of the beneficial components to lose or eliminate beneficial components other than the highly integrated ones. Eventually, they cannot make full use of the metabolites contained in the callus, but do not make sufficient benefits in relation to the cost.

All of the classic callus patents such as Registered Korean Patent Nos. 10-1791641 and 10-0931768 and Published Korean Patent No. 10-2020-0011239 involve solvent extraction or supercritical callus extraction and application of beneficial components contained in callus to human skin and also have the above-mentioned problems, which still need to be solved.

Brief summary of the invention

Therefore, the inventors of the present invention endeavored to develop a method for using metabolites contained in callus as useful components in the human body and found that carrying out mechanical homogenization of callus at a pressure specific not only makes it possible to apply metabolites in callus to the human body as beneficial components without loss of callus metabolites, but markedly increases the penetration and absorption of metabolites in callus into human skin, significantly increasing the various beneficial effects of metabolites in callus on human skin, thereby completing the present invention.

An object of the present invention is to provide a method for preparing a callus lysate containing callus metabolites without loss of callus metabolites, the method including a step of performing mechanical homogenization of a cal at a predetermined pressure.

Another object of the present invention is to provide a callus lysate prepared by the method.

Yet another object of the present invention is to provide a method of encapsulating callus lysate to enhance preservation and stability of callus lysate.

Another object of the present invention is to provide a capsule containing the encapsulated callus lysate.

Yet another object of the present invention is to provide a composition with improved dermal penetration and dermal absorption of callus metabolites, the composition including the callus lysate or the capsule containing a callus lysate.

Another object of the present invention is to provide a composition for external skin application for improving skin hydration, preventing or improving skin wrinkles, skin lightening, improving skin elasticity, skin, or complexion enhancement, the composition for external skin application including the aforementioned composition.

Another object of the present invention is to provide a cosmetic composition for improving skin hydration, preventing or improving skin wrinkles, skin lightening, improving skin elasticity, or an improvement in skin tone, the cosmetic composition including the aforementioned composition.

Yet another object of the present invention is to provide a method for improving skin hydration, preventing or improving skin wrinkles, skin lightening, improving skin elasticity, or improvement in skin tone, the method including administration of the composition for external skin application or the cosmetic composition to an individual biological subject.

Another object of the present invention is to provide the use of the callus lysate as defined above or the capsule containing a callus lysate as defined above as an active ingredient, for an improvement of hydration of the skin, prevention or improvement of skin wrinkles, lightening of the skin, improvement of the elasticity of the skin, or improvement of the complexion.

In one aspect, the present invention provides a method for preparing a callus lysate containing a callus metabolite without loss of callus metabolites which includes a step of performing mechanical homogenization of a callus at a predetermined pressure .

As used herein, the term "callus" means an unorganized mass of undifferentiated plant parenchyma cells, including callus isolated from plant tissue, callus obtained by induction of dedifferentiation of a plant section, or a callus formed by the culture of the callus.

The term "plant", as used herein, refers to both woody plants and herbaceous plants. Examples of the plant may include fruit trees, e.g., apple, pear, persimmon, vine, and ginkgo, conifers, e.g., pine, nut pine, and metasequoia, hardwoods, e.g., forsythia, magnolia, and cherry, annual herbs, e.g., cosmos, purslane, and peas, biennial herbs, e.g. barley, wheat, shepherd's purse, and thalius cress, and herbaceous perennials, eg, dandelion, bellflower, and mugwort.

In a specific example, a large amount of callus can be produced in a process of sterilizing the branches of a plant, placing in a favorable environment for the induction of callus, induction of callus, then transplanting the induced callus.

The medium favorable to the induction of a callus can be a basic medium known in the related field, such as an MS, SH, N6, or B5 medium. The medium conducive to callus induction, a solid medium, may include agar, gelling gum, and the like in addition to the basic medium. The medium may further include sugar, inorganic salts, vitamins, amino acids, growth regulators, etc., if necessary. Examples of the growth regulator may include, but are not limited to, indole acetic acid (AA), naphthalene acetic acid (NAA), zeatin, 6-benzylaminopurine (BA), and kinetin. The growth regulator can be used alone or in combination with one or more other growth regulators.

Cal induction can be performed under temperature conditions known in the related field, e.g., 20-30°C, for an induction period, e.g., 5-10 weeks.

Callus transplanting can be performed in a callus culture medium well known in the related field, preferably a liquid medium contained in an air-stirred bioreactor. Examples of the liquid medium include MS medium or SH medium. The medium may further include sugar, inorganic salts, vitamins, amino acids, growth regulators, etc., in addition to MS or SH medium, if necessary.

Callus transplanting can be performed under temperature conditions known in the related field, eg, 20 to 30°C, for a transplanting period, eg, 2 to 6 weeks.

Therefore, the method for preparing a callus lysate containing a callus metabolite according to the present invention may further include collecting a callus prior to the mechanical homogenization of the callus under high pressure.

The step of removing a callus includes, as described above, removing a callus isolated from plant tissue and/or removing a callus produced by inducing dedifferentiation of a plant section.

Further, the step of removing a callus may include removing an adult callus obtained by callus culture using the callus isolated from plant tissue or the callus produced by induced dedifferentiation of a section vegetable.

Callus culture can be carried out, as described above, in a callus culture medium well known in the related field, in particular, a liquid medium contained in an air-stirred bioreactor. Examples of the liquid medium include MS medium or SH medium. The medium may additionally include sugar, inorganic salts, vitamins, amino acids, growth regulators, etc., in addition to MS or SH medium, if necessary.

In addition, the callus of the present invention includes somatic embryos resulting from callus differentiation. In this disclosure, callus includes somatic embryos obtained by differentiation of callus, unless otherwise specified.

The method of preparing a callus lysate containing a callus metabolite without loss of callus metabolite according to the present invention may include performing mechanical homogenization of callus under high pressure.

The term “mechanical homogenization,” as used herein, means disturbing and homogenizing a target material with a ball/bead mill, rotor-stator, or blade-type homogenizer using a mechanical approach.

The mechanical homogenization is carried out under high pressure conditions, i.e., 200 to 2000 bar, more preferably 300 to 1800 bar, and even more preferably 500 to 1500 bar. When the pressure range is below 200 bar, the efficiency of exposing the metabolites in the callus outside the cell wall is too low to hope to obtain the effects of the callus metabolites. When the pressure range is over 2000 bar, the excessive pressure causes change in properties and appearance or even destruction of metabolites, especially, beneficial components, in the callus, making it difficult to achieve the effects of metabolites. callus, and rendering the callus metabolites susceptible to a change in color or odor qualities and therefore unsuitable for use in the compositions mentioned below.

In addition, the mechanical homogenization process is carried out under high pressure conditions for a cycle time of 15 to 30 minutes, preferably 18 to 22 minutes, more preferably 19 to 21 minutes, and most preferably 20 minutes. Further, the mechanical homogenization process is carried out 2 to 5 times, preferably 2 to 4 times, and more preferably 3 times. If the frequency and cycle time of the mechanical homogenization process are below the set range, the cell wall which prevents the elution and dispersal of callus metabolites is not sufficiently disturbed, and the callus metabolites do not can therefore be eluted or dispersed only to a small extent. If the frequency and cycle time of mechanical homogenization are above the set range, the cell wall is sufficiently fragmented as long as the frequency and cycle time are within the set range, and it will not be further disturbed , which is inefficient.

As such, mechanical homogenization of callus at high pressure weakens and breaks the binding strength of constituent components (e.g., cellulose, pectin, and lignin or cutin) of the cell wall preventing elution callus metabolites by mechanical shearing, cavitation, or the like. This not only facilitates the elution and dispersal of callus metabolites but also allows callus metabolites to exist naturally as a callus lysate along with the remnants of the constituent components of the destroyed cell wall. Additionally, mechanical homogenization results in destruction of not only part of the cell wall, but at least 50%, preferably at least 60%, more preferably at least 70%, even more more preferably at least 80%, and even more preferably at least 90% of the cell wall, which accelerates the elution and dispersal of callus metabolites and causes the callus metabolites to exist naturally as callus lysate with the remains of the constituent components of the destroyed cell wall.

The term "metabolite of callus", as used herein, means a substance containing components beneficial to human skin that are produced by the organelles of a callus, including, but not limited to, a component for a skin hydration improvement, a component for prevention or improvement of skin wrinkles, a component for skin lightening, a component for skin elasticity improvement, and a component for the complexion.

The term "loss of callus metabolite", as used herein, refers to the fact that the content of a specific reference substance contained in a callus lysate prepared according to the method of the present invention is 1.3 times, preferably 1.25 times, and more preferably 1.2 times lower than the content of the specific reference substance in a callus lysate obtained by a method different from the method of the present invention, such as a method carried out in the same manner as the method of the present invention with the exception of carrying out the mechanical homogenization of the callus under the pressure of 100 bar, or in a hot water extract of the callus prepared by another method different from the method of the present invention. Accordingly, the term "without loss of callus metabolite", as used herein, refers to the fact that the content of a specific reference substance contained in a callus lysate prepared according to the method of the present invention is 1.2 times, preferably 1.25 times, and more preferably 1.3 times higher than the content of the specific reference substance contained in a callus lysate obtained by a method different from the method of the present invention, such as a process which is identical to the process of the present invention with the exception of carrying out the mechanical homogenization of the callus under the pressure of 100 bar, or in a hot water extract of callus prepared by a process different from the process of the present invention. Examples of the specific reference substance may include, but are not limited to, flavonoids and terpenoids.

The term "callus lysate", as used herein, refers to the fact that callus metabolites and callus (preferably callus organelles) whose cell walls are destroyed by the process described above are present in a dispersed or dissolved state. In the present invention, the callus lysate is characterized by being prepared according to the method described above and is thus also called "high pressure callus homogenate" for convenience.

Furthermore, the method of preparing a callus lysate containing a callus metabolite according to the present invention may further include, if necessary, removing from the callus lysate cellular organelles not beneficial to human skin, such as nuclei, mitochondria, and a vacuole, after performing mechanical homogenization of the callus under high pressure.

Removal of components not beneficial to human skin from the callus lysate can be accomplished by any suitable method selected based on the target component to be removed, such as filtration, centrifugation, enzymatic treatment, or column chromatography.

In another aspect, the present invention provides a callus lysate prepared as described above.

The callus lysate of the present invention is characterized by being prepared by mechanical homogenization of callus under high pressure. In particular, the callus lysate of the present invention is a substance in which the bonds of the components constituting the callus cell wall are broken by mechanical shearing force and cavitation to shear different parts of the cell wall. This facilitates the elution of callus metabolites without loss of callus metabolites.

The term "loss of callus metabolite(s)," as used herein, refers to the fact that the content of specific reference substance contained in a callus lysate prepared according to the method of the present invention is 1.3 times, preferably 1.25 times, and more preferably 1.2 times lower than the content of specific reference substance contained in a callus lysate obtained by a method different from the method of the present invention, such as a method which is identical to the method of the present invention except for carrying out the mechanical homogenization of the callus under the pressure of 100 bar, or in a hot water extract of callus prepared by a method different from the method of the present invention . Accordingly, the term "without loss of callus metabolite", as used herein, refers to the fact that the content of specific reference substance contained in a callus lysate prepared according to the method of the present invention is 1.2 times , preferably 1.25 times, and more preferably 1.3 times higher than the content of the specific reference substance contained in a callus lysate obtained by a method different from the method of the present invention, such as a method which is identical to the method of the present invention except for carrying out the mechanical homogenization of the callus under the pressure of 100 bar, or in a hot water extract of callus prepared by a method different from the method of the present invention. Examples of the specific reference substance may include, but are not limited to, flavonoids and terpenoids.

More specifically, the classic callus extract has a problem that only the beneficial components soluble in a specific solvent are extracted into the callus extract, so it is not possible to use the beneficial components insoluble in the solvent. specific. In addition, conventional supercritical callus extraction may involve insufficient cell wall destruction, making it difficult to elute callus metabolites, and require high energy consumption and expensive equipment to disrupt the cell wall to facilitate elution of callus metabolites. Furthermore, conventional ultrasonic homogenization of callus is not effective in reducing plant cells to a size small enough to allow plant cells to be absorbed into human skin, and even if callus lysate can be prepared , its preparation requires a very long processing time and has a limitation in the processing of calluses in large quantities available for commercial use.

On the other hand, the callus lysate prepared according to the present invention has the following advantages over the conventional techniques described above using calli.

(1) The callus lysate prepared according to the present invention is obtained by disrupting different parts of the callus cell wall with low energy consumption for a short time, facilitating the elution of callus metabolites.

In a specific example, the callus lysate prepared according to the present invention has a significantly higher content of flavonoids and terpenoids compared to a callus lysate prepared by the conventional method (e.g., a hot water extract of callus ) under other pressure conditions.

(2) In the callus lysate prepared according to the present invention, callus metabolites are present in a general state naturally dispersed or dissolved with the constituent components of the disrupted cell wall due to the disruption of the callus cell wall. This advantageously makes it possible to use all of the components in callus metabolites beneficial to human skin.

In a specific example, the callus lysate of the present invention is much more effective in improving skin hydration, preventing or improving skin wrinkles, skin lightening, improving skin elasticity, skin, and improved skin tone than callus lysate obtained by a conventional method (eg, hot water extract of callus) under other pressure conditions.

(3) In the callus lysate prepared according to the present invention, callus metabolites are present in a dispersed or dissolved state due to the disruption of the callus cell wall, ensuring high penetration and absorption in human skin.

In a specific example, the callus lysate of the present invention is 2.04 to 8 times more permeable in the skin than a callus lysate prepared by a conventional method (e.g., hot water extract of callus). ) under other pressure conditions.

In yet another aspect, the present invention provides a capsule containing a callus lysate which is prepared by encapsulation by coating the callus lysate described above with a natural polymeric material.

The callus lysate contained in the capsule of the present invention is as described above.

In the capsule of the present invention, the natural polymeric material is a material derived from plants, minerals, or microorganisms, preferably a thermoplastic material that is solid at room temperature and melts at body temperature, frictional heat, or pressure when applied to the skin. Examples of the natural polymeric material may include, but are not limited to, at least one selected from agar, pectin, alginic acid, alginate, carrageenan, curdran, Starch, Gellan Gum, Glucomannan, Locust Bean Gum, Guar Gum, Tara Gum, Gum Arabic, Xanthan Gum, Hyaluronic Acid, Karaya Gum, tragacanth gum, larch gum, dextran, cellulose, and a mixture thereof.

The encapsulation of the present invention is characterized by the emulsification and dispersion of the callus lysate in a natural polymer in a liquid state.

Emulsification can consist of preparing the callus lysate in a solubilized formulation or an emulsion formulation such as a water-oil (W/O) emulsion, an oil-water (O/W) emulsion, a water-in-water emulsion oil-in-water (W/O/W), or an oil-in-water-in-oil (O/W/O) emulsion.

Emulsification may involve the use of a surfactant or an emulsifier, which may be ionic or non-ionic. Examples of the surfactant or emulsifier may include a poloxamer or a pluronic, polyethylene glycol, polyethylene glycol monostearate, sodium lauryl sulfate, polyoxyl castor oil, and hydrogenated castor oil. Here, the surfactant or emulsifier may be used in an amount of 5 to 30 parts by weight, preferably 5 to 20 parts by weight, based on 100 parts by weight of the callus lysate.

The natural polymeric material, such as a thermoplastic material, can be liquefied at elevated temperatures of 50°C or more, preferably 50°C to 90°C, more preferably 60 to 90°C, so even more preferably 70 to 90°C, and most preferably 80 to 90°C.

In the capsule of the present invention, the weight ratio between the natural polymeric material and the callus lysate is 1:0.001 to 1:10, specifically 1:0.005 to 1:10, and more specifically 1:0.05 at 1:10. If the weight ratio is within the range defined above, the capsule can increase the preservation and stability of the active ingredient, a callus lysate, and provide favorable qualities for better skin application when used on the skin, while increasing the retention of the active ingredient, callus lysate, in the storage environment.

The capsule of the present invention may have an average particle size of 0.1 to 30 mm, specifically 0.5 to 10 mm, and more specifically 1 to 10 mm. If the average particle size is within the range defined above, it could improve the preservation and stability of the callus lysate contained in the capsule.

In order to improve melting and spreadability for better dermal application, the capsule of the present invention may have a melting point of 30 to 50°C, preferably 35 to 45°C, and more preferably 36°C or higher, which aids capsule stability, and 45°C or lower, which aids compatibility and ease of capsule spread.

The capsule containing a callus lysate according to the present invention increases the preservation and stability of the active ingredient, a callus lysate, and provides favorable qualities for better dermal application when used on the skin, while increasing preservation of the active ingredient, callus lysate, in the storage environment.

In yet another aspect, the present invention provides a method of preparing a capsule containing callus lysate which includes preparing the above-specified callus lysate and then coating the prepared callus lysate with a natural polymeric material to encapsulate the callus lysate.

The callus lysate is as described above, and a description thereof will be omitted hereinafter.

The callus lysate preparation step, as described above, is defined as performing mechanical homogenization of a callus at a pressure of 200 to 2000 bar; or performing mechanical homogenization of a callus, and removing a component not beneficial to human skin from the resulting mechanical homogenate of the callus; or removing a callus, performing mechanical homogenization of the removed callus, and removing components not beneficial to human skin from the resulting mechanical homogenate of the callus. The callus lysate preparation step is described above, and the above description will be taken as reference.

The step of coating the prepared callus lysate with a natural polymeric material to encapsulate the callus lysate includes emulsifying the callus lysate and dispersing an emulsion of the callus lysate in a natural polymeric material in a liquid state to encapsulate the lysate cal.

Emulsification can consist of preparing the callus lysate in a solubilized formulation or an emulsion formulation such as a water-oil (W/O) emulsion, an oil-water (O/W) emulsion, a water-in-water emulsion oil-in-water (W/O/W), or an oil-in-water-in-oil (O/W/O) emulsion.

Emulsification may involve the use of a surfactant or an emulsifier, which may be ionic or non-ionic. Examples of the surfactant or emulsifier may include a poloxamer or a pluronic, polyethylene glycol, polyethylene glycol monostearate, sodium lauryl sulfate, polyoxyl castor oil, and hydrogenated castor oil. Here, the surfactant or emulsifier may be used in an amount of 5 to 30 parts by weight, preferably 5 to 20 parts by weight, based on 100 parts by weight of the callus lysate.

The natural polymeric material is a material derived from plants, minerals, or microorganisms, preferably a thermoplastic material that is solid at room temperature and melts at body temperature, frictional heat, or pressure when applied to the skin. Examples of the natural polymeric material may include, but are not specifically limited to, at least one selected from agar, pectin, alginic acid, alginate, carrageenan, curdran, starch, gellan gum, glucomannan, locust bean gum, guar gum, tara gum, gum arabic, xanthan gum, hyaluronic acid, Karaya gum , gum tragacanth, larch gum, dextran, cellulose, and a mixture thereof.

The natural polymeric material in a liquid state can be prepared by liquefying the natural polymeric material of the properties described above at elevated temperatures of 50°C or more, preferably 50°C to 90°C, more preferably 60°C to 90°C, even more preferably 70°C to 90°C, and most preferably 80°C to 90°C.

The weight ratio between the natural polymeric material and the callus lysate can be 1:0.001 to 1:10, specifically 1:0.005 to 1:10, and more specifically 1:0.05 to 1:10. If the weight ratio is within the range defined above, the capsule can increase the preservation and stability of the active ingredient, a callus lysate, and provide favorable qualities for better skin application when used on the skin, while increasing the retention of the active ingredient, callus lysate, in the storage environment.

In order to increase the stability of the capsule, the encapsulation step can additionally include a dispersion of an emulsion of the callus lysate in a natural polymer material in the liquid state and then a regulation of the external temperature at 20° C or less, specifically -10°C to 15°C, and more specifically 0°C to 10°C. In this regard, the method of temperature control may include, but is not limited to, temperature control with a device including frozen refrigerants, such as silicone oil, mineral oil, paraffin oil, liquid nitrogen, liquid oxygen, and liquid air, where the device is disposed outside a reactor. In this case, it is possible to prepare a capsule containing a callus lysate which is not only easy to regulate in terms of the weight ratio of the natural polymeric material forming the capsule to the callus lysate contained in the capsule, but also has an excellent formability.

The capsule thus prepared may have an average particle size of 0.1 to 30 mm, specifically 0.5 to 10 mm, and more specifically 1 to 10 mm. If the average particle size is within the range defined above, it could further improve the preservation and stability of the callus lysate contained in the capsule.

In yet another aspect, the present invention provides a composition with increased dermal penetration and dermal absorption of callus metabolites, the composition including the callus lysate or the capsule containing a callus lysate.

The callus lysate or the capsule containing a callus lysate used in the composition of the present invention are as described above.

The composition with increased skin penetration and skin absorption of callus metabolites according to the present invention may contain, as an active ingredient, the callus lysate or the capsule containing a callus lysate in an amount of 0.01 to 100 % by weight, preferably from 1 to 90% by weight, and more preferably from 5 to 80% by weight, relative to the total weight of the composition.

In a specific example, the active ingredient, i.e., the callus lysate itself or the callus lysate contained in the capsule, is a substance containing callus metabolites and rendering the elution and dispersion of easier callus metabolites without loss of callus metabolites, as described above. Therefore, as demonstrated in a permeability test in a dermal equivalent, the callus lysate of the present invention exhibits a permeability in a dermal equivalent 2.04 to 8 times higher than that of a conventional callus lysate obtained in the form of a hot water extract of cal or equivalent.

Accordingly, the composition of the present invention enables rapid dermal penetration of a large amount of the callus lysate containing callus metabolites in an eluted or dispersed state and increases the dermal absorption of the callus lysate. Thus, the composition may provide beneficial effects due to the action of the eluted and dispersed callus metabolites on the skin, including but not limited to improvement of skin hydration, lightening of the skin, an improvement in skin wrinkles, skin elasticity, and skin tone.

In a specific aspect, the present invention provides a composition for external dermal application which includes the callus lysate or capsule containing a callus lysate.

The callus lysate or the capsule containing a callus lysate according to the present invention are as described above.

The composition intended for external cutaneous application according to the present invention may include, as an active ingredient, in particular as a cosmetic active ingredient, a composition exhibiting improved cutaneous penetration and cutaneous absorption of callus metabolites, which composition may be included alone or in combination with an excipient if required.

The excipient is not specifically limited as long as it is an excipient suitable for external use. For example, the excipient can include hydrophilic solvents, including purified water, ethanol, etc. ; vegetable oils including jojoba oil, etc. ; animal oils, including squalane, etc. ; lubricants, including liquid paraffin, etc. ; aliphatic alcohols, including cetylaryl alcohol, etc. ; skin treatment products including propylene glycol, butylene glycol, glycerin, tocopheryl acetate, polyglyceryl-3 methylglucose distearate, etc. ; surfactants, including triethanolamine, glyceryl stearate, etc. ; thickeners, including carboxy vinyl polymers, etc. ; emulsifiers; curators; and perfumes.

The composition for external skin application according to the present invention may include the callus lysate or the capsule containing a callus lysate in an amount of 0.1 to 80% by weight, preferably 1 to 50% by weight, and more preferably 10 to 50% by weight. If the content of the callus lysate or the capsule containing a callus lysate is less than 0.1% by weight, it results in rendering the effects of the callus lysate to a lesser extent. If the content of the callus lysate or the capsule containing a callus lysate is more than 80% by weight, the effects of the callus lysate are very small for the content.

The composition for external application shows excellent effects due to the action of callus metabolites contained in callus lysate, including but not limited to improving skin hydration, prevention or improvement of skin wrinkles, lightening of the skin, improvement of the elasticity of the skin, or improvement of the complexion.

In another specific aspect, the present invention provides a composition comprising the callus lysate or the capsule containing the callus lysate, as an active ingredient.

The callus lysate or the capsule containing a callus lysate according to the present invention are as described above.

Specifically, the compositions can include formulations for external dermal application and personal care products. More specifically, compositions can include, but are not limited to, disinfectant cleansers, shower foams, mouthwashes, disinfectant wet wipes, detergents, hand soaps, or ointments.

When the callus lysate or the capsule containing a callus lysate of the present invention is used as the active ingredient, the callus lysate or the capsule containing a callus lysate can be used alone or in combination with other ingredients. active ingredients and applied appropriately according to conventional methods. The added amount of the active ingredient can be suitably determined depending on the purpose of use.

The composition of the present invention may include the callus lysate or the capsule containing a callus lysate in an amount of 0.1 to 80% by weight, specifically 1 to 50% by weight, and more specifically 10 to 50% in weight. If the content of the callus lysate or the capsule containing the callus lysate is less than 0.1% by weight, it results in rendering the effects of the callus lysate to a lesser extent. If the content of the callus lysate or the capsule containing the callus lysate is more than 80% by weight, the effects of the callus lysate are very small for the content.

The composition shows excellent effects due to the action of callus metabolites contained in the callus lysate, including but not limited to improving skin hydration, preventing or improving skin wrinkles, lightening of the skin, improvement of the elasticity of the skin, or improvement of the complexion.

In another specific aspect, the present invention provides a cosmetic composition including the callus lysate or the capsule containing a callus lysate.

The cosmetic composition of the present invention may further include ingredients commonly used in cosmetic compositions in addition to the callus lysate or the capsule containing a callus lysate. For example, the cosmetic composition can include typical adjuvants, such as antioxidants, stabilizers, solubilizing agents, vitamins, pigments, and fragrances, and carriers.

The cosmetic composition of the present invention can be prepared in any formulation typical of the related field, including but not limited to solution, suspension, emulsion, paste, gel, cream, lotion, a powder, a soap, a cleansing oil containing a surfactant, a powder foundation, an emulsion foundation, a wax foundation, and a spray. In more detail, the cosmetic composition can be prepared in a formulation such as a nourishing cream, an astringent, a tonic, a lotion, an essence, a nourishing gel, or a massage cream.

When the formulation of the cosmetic composition is a paste, a cream, or a gel, the carrier component in the composition can be an animal oil, a vegetable oil, a wax, paraffin, starch, tragacanth , cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, and the like.

When the formulation of the cosmetic composition is a powder or a spray, the carrier component in the composition can be lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder. . In particular, in the case of a spray formulation, the cosmetic composition can additionally include a propellant such as chlorofluorohydrocarbon, propane/butane, or dimethyl ether.

When the formulation of the cosmetic composition is a solution or an emulsion, the carrier component in the composition can be a solvent, a solubilizing agent, or an emulsifier, including but not limited to water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl acetate, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, a aliphatic ester of glycerol, polyethylene glycol, or an aliphatic ester of sorbitan.

When the formulation of the cosmetic composition is a suspension, the carrier component in the composition may be a liquid diluent such as water, ethanol, or propylene glycol, a suspending agent such as as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, or the like.

When the formulation of the cosmetic composition is a detergent containing a surfactant, the carrier component in the composition may be an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate , imidazolinium derivative, methyl taurate, sarcosinate, aliphatic amide ether sulfate, alkylamidobetaine, aliphatic alcohol, aliphatic glyceride, aliphatic diethanolamide, vegetable oil, derivative lanolin, an aliphatic ester of ethoxylated glycerol, and so on.

The cosmetic composition of the present invention may include the callus lysate or the capsule containing a callus lysate in an amount of 0.1 to 80% by weight, specifically 1 to 50% by weight, and more preferably 10 to 50%. % in weight. If the content of the callus lysate or the capsule containing a callus lysate is less than 0.1% by weight, the effects of the callus lysate can only be rendered to a lesser extent. If the content of the callus lysate or the capsule containing a callus lysate is more than 80% by weight, it makes the effects of the callus lysate very weak for the content.

The cosmetic composition exhibits excellent effects due to the action of callus metabolites contained in the callus lysate, including but not limited to skin hydration improvement, prevention or improvement skin wrinkles, skin lightening, skin elasticity improvement, or skin tone improvement.

In another specific aspect, the present invention provides a method for improving skin hydration, preventing or improving skin wrinkles, skin lightening, improving skin elasticity, or improving skin tone, the method comprising the step of administering the composition including a callus lysate or a capsule containing a callus lysate to an individual biological subject.

The term "prevent", as used herein, is defined as the suppression of the occurrence of a symptom in an individual biological subject who has never been diagnosed with the symptom but is prone to the symptom.

The term "ameliorate" or "treat", as used herein, means (a) the suppression or (b) the alleviation of a symptom in an individual biological subject.

The term "individual biological subject", as used herein, refers to mammals, including humans, capable of achieving the aforementioned objects by administration of the composition containing a callus lysate or a capsule containing a callus lysate callus as an active ingredient according to the present invention, examples of such mammals including humans may include monkey, cow, horse, pig, sheep, dog, cat, rat, mouse, or a chimpanzee.

The term "administration", as used herein, means the introduction of a defined substance into an individual biological subject by any suitable method. The mode of administration of the composition according to the present invention is oral or parenteral, preferably parenteral, by any general route as long as the composition can reach the target tissue. Additionally, the composition of the present invention can be administered by any device designed to move the active substance to a target subject, such as a cell.

Appropriate dosage of the composition of the present invention varies depending on the individual biological subject's condition and weight, the dosage form of the active ingredient, and the frequency. But it can be appropriately selected by the specialist in the field. The callus lysate or the capsule containing a callus lysate can be administered, if necessary, from one to five times a day, at a daily dose of 1.0 to 3.0 ml for an adult.

The composition comprising the callus lysate or the capsule containing a callus lysate according to the present invention may contain the callus lysate or the capsule containing a callus lysate in an amount of 0.1 to 80% by weight, specifically 1 to 50% by weight, and more preferably 10 to 50% by weight, relative to the total weight of the composition. If the content of the callus lysate or the capsule containing a callus lysate is less than 0.1% by weight, it results in rendering the effects of the callus lysate to a lesser extent. If the content of the callus lysate or the capsule containing the callus lysate is more than 80% by weight, it makes the effects of the callus lysate very weak for the content.

The method for preparing a callus lysate according to the present invention is a method for preparing a callus lysate having a high content of callus metabolites even without using a solvent harmful to the human body, but avoiding a loss of metabolites. of callus, compared to conventional callus extract or equivalent. The callus lysate thus prepared by the method contains callus metabolites, which are eluted from a callus and dispersed in the callus lysate, and thus enable rapid penetration and absorption of callus metabolites directly into the skin. In addition, the capsule containing the callus lysate also enables rapid penetration and absorption of callus metabolites directly into the skin, increases the retention and stability of the active ingredient, a callus lysate, and provides favorable qualities for improved dermal application when used on the skin, while increasing retention of the active ingredient, a callus lysate, in the storage environment. In addition, the composition comprising the callus lysate or the capsule containing a callus lysate contains callus metabolites, which are eluted from a callus and dispersed in the callus lysate, and thus exhibit excellent performance of improving skin hydration, preventing or improving skin wrinkles, brightening skin, improving skin elasticity, or improving skin tone, due to the action of callus metabolites. Therefore, the composition can be beneficially used as a preparation for skin application and a cosmetic preparation.

Detailed description of the invention

Hereinafter, a detailed description will be given to better understand the present invention by referring to examples, which may be subject to various modifications and variations and not intended to limit the scope of the present invention. The examples of the present invention disclosed herein are provided to enable those skilled in the art to better understand the present invention in its entirety.

Example of preparation 1

Apple tree branches grown in that year (scientific name: Malus pumila , variety name: Fuji apple, harvested part: part including cambium in branches, growth time: 1 to 6 months) were immersed in 35% v/v hydrogen peroxide for 10 minutes then 70% v/v ethanol for 1 minute, and then stirred for 30 minutes in a 2% v/v solution of sodium hypochlorite for sterilization.

Sterilized apple tree branches were washed with sterile water five or more times and cut into small sections of about 1 cm. A section thus obtained was placed in a basal medium containing 30 g/l of sucrose, 4.0 g/l of gelling gum, 1 ppm of NAA used as a growth regulator, 4.4 g/l of MS powder, and the rest of purified water and cultured for 8 weeks in a 25°C incubator to induce callus formation.

The induced callus was subcultured at 5 week intervals for proliferation and purification. Callus induced as described above was inoculated into an air-flow shaker bioreactor, grown by cell suspension culture for 6 weeks in a 25°C incubator in the dark, and then harvested. In this regard, the culture medium was an MS medium prepared in the same volume of the basal medium by excluding the gelling gum of the basal medium, and the suspension cell culture was carried out for 6 weeks in a 25° incubator. C in the dark.

Example of preparation 2

The procedures were carried out in the same manner as described in Preparation Example 1, except that branches of Lycium chinense grown in that year (scientific name: Lycium chinense , part removed: part including the cambium in branches, growth time: 1 to 6 months) were used instead of apple branches grown that year.

Example of preparation 3

The procedures were carried out in the same manner as described in Preparation Example 1, except that vine branches grown in that year (scientific name: Vitis vinifera L. , variety name: Campbell, harvested part: part comprising the cambium in branches, growth period: 1 to 6 months) were used in place of apple tree branches grown that year.

Example 1

500 ml of the apple callus obtained in Preparation Example 1 was taken out and subjected to 3 cycles of homogenization at 200 bar with each cycle time of 20 minutes in a high-pressure homogenizer (product name: Scientz-150, manufacturer: Ningbo Scientz Biotechnology Co., Ltd.). The apple callus high-pressure homogenate thus obtained by high-pressure homogenization was treated at 75°C for 30 minutes for pasteurization and enzyme inactivation in a further treatment process and then used.

Example 2

The procedures were carried out to prepare a high pressure homogenate of apple callus in the same manner as described in Example 1, except that the pressure of the high pressure homogenizer was 500 bar.

Example 3

The procedures were carried out to prepare a high pressure homogenate of apple callus in the same manner as described in Example 1, except that the pressure of the high pressure homogenizer was 1000 bar.

Example 4

The procedures were carried out to prepare a high pressure homogenate of apple callus in the same manner as described in Example 1, except that the pressure of the high pressure homogenizer was 2000 bar.

Example 5

500 ml of Lycium chinense callus culture obtained in Preparation Example 2 was taken out and subjected to 3 cycles of homogenization at 200 bar with each cycle time of 20 minutes in a high pressure homogenizer (product name: Scientz -150, Manufacturer: Ningbo Scientz Biotechnology Co., Ltd.). The plant callus high-pressure homogenate thus obtained by high-pressure homogenization was treated at 75°C for 30 minutes for pasteurization and enzyme deactivation in a further treatment process and then used.

Example 6

The procedures were carried out to prepare a high pressure homogenate of Lycium chinense callus in the same manner as described in Example 5, except that the pressure of the high pressure homogenizer was 500 bar.

Example 7

The procedures were carried out to prepare a high-pressure homogenate of Lycium chinense callus in the same manner as described in Example 5, except that the pressure of the high-pressure homogenizer was 1000 bar.

Example 8

The procedures were carried out to prepare a high pressure homogenate of Lycium chinense callus in the same manner as described in Example 5, except that the pressure of the high pressure homogenizer was 2000 bar.

Example 9

500 ml of grape callus culture obtained in Preparation Example 3 was taken and subjected to 3 cycles of homogenization at 200 bar with each cycle time of 20 minutes in a high-pressure homogenizer (product name: Scientz- 150, manufacturer: Ningbo Scientz Biotechnology Co., Ltd.). The grape callus high-pressure homogenate thus obtained by high-pressure homogenization was treated at 75°C for 30 minutes for pasteurization and enzyme deactivation in a further treatment process and then used.

Example 10

The procedures were carried out to prepare a high pressure homogenate of grape callus in the same manner as described in Example 9, except that the pressure of the high pressure homogenizer was 500 bar.

Example 11

The procedures were carried out to prepare a high pressure homogenate of grape callus in the same manner as described in Example 9, except that the pressure of the high pressure homogenizer was 1000 bar.

Example 12

The procedures were carried out to prepare a high pressure homogenate of grape callus in the same manner as described in Example 9, except that the pressure of the high pressure homogenizer was 2000 bar.

Comparative example 1

500 ml of the apple callus culture obtained in Preparation Example 1 was taken out, removed from the culture medium, immersed in 1 l of sterile water, and then subjected to hot water extraction at 80°C for 60 minutes. The extracted solution thus obtained was filtered through a filter paper (Advantes, No. 2) to prepare a hot water extract of cal.

Comparative Example 2

500 ml of the Lycium chinense callus culture obtained in Preparation Example 2 was taken out, removed from the culture medium, immersed in 1 l of sterile water, and then subjected to extraction with hot water at 80 ° C for 60 minutes. The extracted solution thus obtained was passed through a filter paper (Advantes, No. 2) to prepare a hot water callus extract of Lycium chinense.

Comparative example 3

500 ml of the grape callus culture obtained in Preparation Example 3 were taken out, removed from the culture medium, immersed in 1 l of sterile water, and then subjected to hot water extraction at 80 ° C for 60 minutes. The extracted solution thus obtained was passed through a filter paper (Advantes, No. 2) to prepare a hot water grape callus extract.

Comparative example 4

The procedures were carried out to prepare a high pressure homogenate of grape callus in the same way as described in Example 9, except that the pressure of the high pressure homogenizer was 100 bar.

Comparative Example 5

The procedures were carried out to prepare a high pressure homogenate of grape callus in the same manner as described in Example 9, except that the pressure of the high pressure homogenizer was 2100 bar.

Example 13

30% by weight of the high pressure grape callus homogenate prepared in Example 12, 65% by weight of purified water, and 5% by weight of an emulsifier were mixed, and the resulting mixture was emulsified at 60-70°C to prepare an oil-water emulsion (O/W).

20 ml of the emulsion was added to 80 ml of a solution prepared by dissolving 2% by weight of agar used as natural polymeric material in 98% by weight of purified water at high temperatures of 80-90°C and dispersed for encapsulation for 2 hours under the same temperature conditions. The capsule thus obtained had an average particle size of about 5 mm.

Example 14

The ingredients given by the composition of the following Table 1 were mixed in a reactor and emulsified at an elevated temperature of 70°C. At the end of the emulsification, the reactor temperature was reduced to 45°C. The capsule containing the grape callus as prepared in Example 13 was mixed and dispersed in a composition, which was then cooled to 30°C to prepare a cosmetic formulation of nutrient cream.

Comparative example 6

The ingredients given by the composition of the following Table 1 were mixed in a reactor and emulsified at an elevated temperature of 70°C. At the end of the emulsification, the reactor temperature was reduced to 45°C. The capsule callus extract as prepared in Example 3 was mixed and dispersed into a composition, which was then cooled to 30°C to prepare a cosmetic nutrient cream formulation.

Comparative example 7

The ingredients given by the composition of the following Table 1 were mixed in a reactor and emulsified at an elevated temperature of 70°C. At the end of the emulsification, the reactor temperature was reduced to 45°C. The emulsion was dispersed and then cooled to 30°C to prepare a cosmetic formulation of nourishing cream.

[Table 1] Ingredients (% by weight) Example 14 Comparative Example 6 Comparative Example 7 Vine callus homogenate of Example 12 15 - - Extract of vine callus of comparative example 3 - 15 - Jojoba oil 5 5 5 Liquid paraffin 7 7 7 Cetylaryl alcohol 2 2 2 Polyglyceryl-3 methylglucose distearate 2 2 2 Glyceryl Stearate 0.5 0.5 0.5 Squalane 3 3 3 Propylene glycol 4 4 4 Glycerin 5 5 5 Triethanolamine 0.3 0.3 0.3 Carboxy Vinyl Polymer 0.3 0.3 0.3 Tocopheryl acetate 0.2 0.2 0.2 Preservative, perfume - 0.1 - Purified water 65.7 55.6 70.7 Total 100 100 100

Experimental example 1: Evaluation of the effectiveness of skin penetration

A dermal equivalent (DE) was used to measure the skin penetration effect of the samples of examples 1 to 12 and comparative examples 1 to 5.

The dermal equivalent was prepared by culturing normal human fibroblasts in a medium containing collagen and DMEM (Dulbecco's modified Eagle's medium) in a 5% CO ₂ incubator at 37°C for 24 hours and then culturing epidermal keratinocyte on the surface of normal human fibroblasts in K-SFM (serum-free keratinocyte culture medium) containing EGF (epidermal growth factor) and BPE (bovine pituitary gland extract) for 7 days to form a multi-layered epidermis-dermis structure.

Each sample of Examples 1-12 and Comparative Examples 1-5 was applied to the top layer of the dermal equivalent prepared under the same incubation conditions. After 4 hours of incubation, the amount of sample entering the dermal equivalent was analyzed by HPLC. The experimental results are shown in Table 2 below. Substances used as reference for HPLC analysis of permeability were chlorogenic acid, rutin, resveratrol, etc.

[Table 2] Div. Permeability for dermal equivalent (ug/ml) Example 1 Apple callus homogenate (200 bar) 13.2 Example 2 Apple callus homogenate (500 bar) 19.4 Example 3 Apple callus homogenate (1000 bar) 33.7 Example 4 Apple callus homogenate (2000 bar) 38.4 Example 5 Lycium chinense callus homogenate (200 bar) 12.7 Example 6 Lycium chinense callus homogenate (500 bar) 19.4 Example 7 Lycium chinense callus homogenate (1000 bar) 32.5 Example 8 Lycium chinense callus homogenate (2000 bar) 37.2 Example 9 Vine callus homogenate (200 bar) 10.4 Example 10 Vine callus homogenate (500 bar) 19.5 Example 11 Vine callus homogenate (1000 bar) 32.8 Example 12 Vine callus homogenate (2000 bar) 37.8 Comparative example 1 Apple callus extract
4.8 Comparative example 2 Lycium chinense callus extract 5.3 Comparative example 3 Vine callus extract 5.1 Comparative example 4 Vine callus homogenate (100 bar) 8.4 Comparative Example 5 Vine callus homogenate (2100 bar) 40.0

As can be seen from Table 2, the samples of Examples 1 to 12 including plant callus homogenates prepared from apple callus, Lycium chinense callus, or grapevine callus by a high pressure homogenizer at 200-2000 bar were 2.04 (10.4/5.1) to 8 (38.4/4.8) times significantly higher in permeability for the dermal equivalent than those of Comparative Examples 1, 2 and 3 using hot water extracts of the same plant calluses.

In addition, the sample of Comparative Example 4 where the pressure for homogenization was less than 200 bar showed lower skin permeability than that of the examples of the present invention and showed a large amount of residues, including cell walls remaining not broken. The sample of Comparative Example 5 where the pressure for homogenization was higher than 2000 bar showed sufficiently high permeability, but showed a change in properties and appearance such as color and odor qualities, showing that the sample was unsuitable for use in compositions for external application.

Experimental example 2: Evaluation of the effectiveness of cutaneous hydration

A total of 30 male and female participants between the ages of 20 and 50 were recruited to participate in an evaluation of skin hydration efficacy.

Each participant was asked to apply 10 g of each sample prepared in Examples 1 to 13 and Comparative Examples 1 to 5 to the surface of an abdominal skin region with an area of 2.5 cm x 2.5 cm. After 24 hours, the transepidermal water loss (TEWL) was measured using a TEWA evapometer (Tewameter TM 210, Germany). The TEWA evapometer has been designed to automatically calculate and provide an average value of the measurements acquired for a predetermined measurement time. Transepidermal water loss (TEWL) was measured in a room with constant temperature and humidity maintained at 22°C and 40% relative humidity (RH) with no air movement or direct sunlight. In addition, the measurements for a total of 30 participants were averaged for each sample of examples and comparative examples. The experimental results are shown in Table 3 below.

[Table 3] Div. TEWL (g/m ² /h) Example 1 Apple callus homogenate (200 bar) 15.2 Example 2 Apple callus homogenate (500 bar) 13.7 Example 3 Apple callus homogenate (1000 bar) 11.1 Example 4 Apple callus homogenate (2000 bar) 10.4 Example 5 Lycium chinense callus homogenate (200 bar) 16.3 Example 6 Lycium chinense callus homogenate (500 bar) 14.1 Example 7 Lycium chinense callus homogenate (1000 bar) 11.5 Example 8 Lycium chinense callus homogenate (2000 bar) 10.1 Example 9 Vine callus homogenate (200 bar) 14.3 Example 10 Vine callus homogenate (500 bar) 12.1 Example 11 Vine callus homogenate (1000 bar) 11.2 Example 12 Vine callus homogenate (2000 bar) 10.5 Example 13 Grape callus homogenate capsule formulation (2000 bar) 9.2 Comparative example 1 Apple callus extract
28.4 Comparative example 2 Lycium chinense callus extract 25.4 Comparative example 3 Vine callus extract 26.7 Comparative example 4 Vine callus homogenate (100 bar) 20.3 Comparative Example 5 Vine callus homogenate (2100 bar) 19.7

As can be seen from Table 3, the samples of Examples 1 to 12 including plant callus homogenates prepared from apple callus, Lycium chinense callus, or grapevine callus by a high pressure homogenizer at 200-2000 bar showed significantly higher skin hydration efficiency than Comparative Examples 1, 2 and 3 using hot water extracts of the same plant calli and Comparative Examples 4 and 5 where the pressure for the homogenization was outside the defined range of the present invention.

In particular, the sample of Example 13, a capsule formulation containing an agar-coated grape callus homogenate, had a further improved TEWL due to the combined action since the outer shell of the capsule was also applied to the skin during sample application.

Experimental Example 3: Evaluation of Wrinkle Prevention Efficacy

Antibodies were used to measure the activity of collagenase, an enzyme responsible for the breakdown of collagen.

Normal human fibroblasts (Pacific Co., Ltd.) were inoculated into a 6-well microplate containing DMEM medium at a rate of 2 x 10 ⁵ cells/well and cultured in a 5% CO ₂ incubator at 37°C for 24 hours. After 24 hours of incubation, the culture medium was removed from each well. Samples were treated by concentration and cultured for an additional 24 hours. After 24 hours of incubation, the cell culture medium was removed to prepare a test sample.

10 ng/ml of a tumor necrosis factor alpha (TNF-alpha, Sigma, USA), known as a substance inducing collagenase activity, was added to the 6-well microplate in which the normal human fibroblasts were placed. in culture. Using a collagenase type 1 assay kit (Amersham Biosciences, RPN2629), absorbance at 450 nm was determined with an ELISA reader (Bio-Tek ELx808™ Series Ultra Microplate Reader, UK). The control was distilled water. Absorbance measurements were used to calculate collagenase inhibition rate according to equation 1 below. The results are shown in Table 4 below.

[Equation 1]

Collagenase inhibition rate (%) = 100 – {[(A1 - B1) - (C1 - D1)]/(A1 - B1)} x 100

A1: Control absorbance

B1: Absorbance of the control blank

C1: Absorbance of experimental group

D1: Absorbance of the blank of the experimental group

[Table 4] Div. Collagenase inhibition rate (%) Example 1 Apple callus homogenate (200 bar) 30.2 Example 2 Apple callus homogenate (500 bar) 32.7 Example 3 Apple callus homogenate (1000 bar) 45.2 Example 4 Apple callus homogenate (2000 bar) 47.7 Example 5 Lycium chinense callus homogenate (200 bar) 33.5 Example 6 Lycium chinense callus homogenate (500 bar) 37.4 Example 7 Lycium chinense callus homogenate (1000 bar) 44.2 Example 8 Lycium chinense callus homogenate (2000 bar) 47.8 Example 9 Vine callus homogenate (200 bar) 33.4 Example 10 Vine callus homogenate (500 bar) 39.6 Example 11 Vine callus homogenate (1000 bar) 40.6 Example 12 Vine callus homogenate (2000 bar) 43.8 Example 13 Grape callus homogenate capsule formulation (2000 bar) 51.4 Comparative example 1 Apple callus extract
14.8 Comparative Example 2 Lycium chinense callus extract 15.4 Comparative example 3 Vine callus extract 14.5 Comparative example 4 Vine callus homogenate (100 bar) 21.8 Comparative Example 5 Vine callus homogenate (2100 bar) 29.4

As can be seen from Table 4, the samples of Examples 1 to 12 including plant callus homogenates prepared from apple callus, Lycium chinense callus, or grapevine callus by a high pressure homogenizer at 200-2000 bar exhibited significantly higher wrinkle prevention efficacy than Comparative Examples 1, 2 and 3 using hot water extracts of the same plant calli and Comparative Examples 4 and 5 where the pressure for the homogenization was outside the defined range of the present invention.

In particular, the sample of Example 13, a capsule formulation containing an agar-coated grape callus homogenate, had a further improved collagenase inhibition efficacy due to the combined action since the outer shell of the capsule was also applied to the skin during application of the sample.

Experimental example 4: Evaluation of tyrosinase inhibition efficacy

In this experiment, murine melanoma cells (B-16 F1, Korea Cell Line Bank) were inoculated in a 6-well plate with DMEM medium containing 10% fetal bovine serum (FBS) at a rate of 1 x 10 ⁵ cells/well and placed in culture under conditions of 5% CO ₂ and 37° C. until approximately 80% or more of the cells are immobilized at the bottom of the wells. Then, the sample was removed from the medium, transferred to another culture medium diluted to an appropriate concentration, and then cultured under conditions of 5% CO ₂ and 37°C for 3 days. Cells removed from the medium were washed with phosphate buffered saline (PBS), treated with trypsin and harvested. The cells thus collected were counted using a hemocytometer (Tiefe Depth Depth 0.100 mm, Paul Marienfeld GmbH & Co. KG, Germany), centrifuged at 5,000 to 10,000 rpm for 10 minutes, and then removed from the supernatant liquid to obtain a nerve. The cell pellet was pulverized using lysis buffer and centrifuged at 12,000 rpm for 10 minutes to obtain the supernatant.

The supernatant thus obtained was measured for absorbance at 492 nm with a microplate reader (Bio-Tek ELx8081U, USA) to determine tyrosinase activity per constant cell number. The absorbance was used to calculate the tyrosinase inhibition rate according to equation 2 below. The experimental results are shown in Table 5 below.

The blank for the experimental group was reagents other than tyrosinase solution, i.e., reagents consisting of tyrosine, callus homogenate, callus extract, and sodium phosphate buffer solution. The blank of the control group was prepared by replacing the callus homogenate and the callus extract with methanol in the blank of the experimental group.

[Equation 2]

Tyrosinase inhibition rate (%) = 100 – {[(A2 - B2) - (C2 - D2)]/(A2 - B2)} x 100

A2: Control absorbance

B2: Absorbance of the control blank

C2: Absorbance of experimental group

D2: Absorbance of the experimental group blank

[Table 5] Div. Tyrosinase inhibition rate (%) Example 1 Apple callus homogenate (200 bar) 24.4 Example 2 Apple callus homogenate (500 bar) 27.1 Example 3 Apple callus homogenate (1000 bar) 30.4 Example 4 Apple callus homogenate (2000 bar) 33.5 Example 5 Lycium chinense callus homogenate (200 bar) 26.7 Example 6 Lycium chinense callus homogenate (500 bar) 27.4 Example 7 Lycium chinense callus homogenate (1000 bar) 28.6 Example 8 Lycium chinense callus homogenate (2000 bar) 32.4 Example 9 Vine callus homogenate (200 bar) 25.4 Example 10 Vine callus homogenate (500 bar) 29.7 Example 11 Vine callus homogenate (1000 bar) 30.1 Example 12 Vine callus homogenate (2000 bar) 33.4 Example 13 Grape callus homogenate capsule formulation (2000 bar) 31.7 Comparative example 1 Apple callus extract
12.6 Comparative example 2 Lycium chinense callus extract 9.7 Comparative example 3 Vine callus extract 12.1 Comparative example 4 Vine callus homogenate (100 bar) 22.3 Comparative Example 5 Vine callus homogenate (2100 bar) 25.9

As can be seen from Table 5, the samples of Examples 1 to 12 including plant callus homogenates prepared from apple callus, Lycium chinense callus, or grapevine callus by a high pressure homogenizer at 200-2000 bar exhibited significantly higher tyrosinase inhibition efficacy than Comparative Examples 1, 2 and 3 using hot water extracts of the same plant calli and Comparative Example 4 where the pressure for the homogenization was less than 200 bar. In addition, the sample of Comparative Example 5 where the pressure for homogenization was more than 2000 bar showed sufficiently high tyrosinase inhibition efficiency, but change in properties and appearance such as color qualities and odor, which demonstrated that the sample was unsuitable for use in formulations for external application or cosmetic compositions.

In addition, the sample of Example 13, a capsule formulation containing an agar-coated grape callus homogenate, also exhibited excellent tyrosinase inhibition efficacy.

Experimental Example 5: Evaluation of Skin Lightening Efficacy

100 healthy adult male and female participants with melasma, freckles, or hyperpigmentation were asked to use the nutrient cream formulations prepared in Example 14 and Comparative Examples 6 and 7 and then measure the variation skin color tint (∆L) with a chromameter (Minolta CR300).

In this experiment, the 100 healthy adult male and female participants with melasma, freckles or hyperpigmentation were randomly divided into three groups (A, B, C) of 20 people. Groups A, B, and C of participants were asked to apply Experimental Group 1, Control Group, and Comparative Group 1, respectively, to the skin twice daily for 12 weeks and measure the change in the color of the skin. The experimental results are shown in Table 6 below.

[Table 6] Participant example 14 (Group A) Comparative Example 6 (Group B) Comparative Example 7 (Group C) 1 6.2 5.7 4.4 2 5.4 4.9 3.4 3 5.6 4.8 3.0 4 6.6 4.5 2.4 5 5.7 4.1 3.6 6 6.6 5.7 2.4 7 6.3 5.9 4.1 8 5.1 5.2 3.3 9 6.8 4.7 3.4 10 6.1 4.2 3.8 11 4.9 3.8 2.6 12 6.2 5.4 4.5 13 6.3 6.2 4.2 14 5.6 4.1 3.4 15 5.8 5.0 3.7 16 6.3 5.3 2.2 17 6.7 5.4 2.5 18 6.9 5.2 3.5 19 6.5 4.9 2.9 20 6.1 5.1 2.7 Mean 6.1 5.0 3.3

As can be seen from Table 6, the nutrient cream formulation of Example 14 including plant callus homogenate prepared from grape callus by a high pressure homogenizer at 200-2000 bar exhibited a significantly higher skin lightening efficacy than the control using vehicle (Comparative Example 7) alone. In particular, it even exhibited significantly higher skin lightening efficacy than that of the nourishing cream of Comparative Example 6 using a hot water extract of grape callus.

Experimental Example 6: Evaluation of Antioxidant Efficacy

Antioxidant activity was assessed by measuring the free radical scavenging capacity, DPPH (1,1-diphenyl-2-picrylhydrazyl). DPPH, a color radical, was used to directly determine the radical scavenging ability of the sample.

50 µl of each sample from Examples 1 to 13 and Comparative Examples 1 to 5 were mixed with 1 ml of a 100 µM DPPH solution. The resulting mixture was reacted and incubated at 20°C (room temperature) for 30 minutes. Then, the amount of remaining DPPH was determined by measuring absorbance at 517 nm.

At the end of the reaction, the free radical scavenging activity (%) of the sample was calculated according to equation 3 below. The experimental results are shown in Table 7 below. In this regard, the positive control was 400 µg/ml ascorbic acid, and the blank was purified water.

[Equation 3]

Radical scavenging capacity (%) = {1 – (group absorbance to sample / group absorbance without sample) x 100}

[Table 7] Div. Free radical scavenging capacity (%) Example 1 Apple callus homogenate (200 bar) 80.2 Example 2 Apple callus homogenate (500 bar) 83.6 Example 3 Apple callus homogenate (1000 bar) 88.5 Example 4 Apple callus homogenate (2000 bar) 90.3 Example 5 Lycium chinense callus homogenate (200 bar) 77.2 Example 6 Lycium chinense callus homogenate (500 bar) 80.9 Example 7 Lycium chinense callus homogenate (1000 bar) 84.3 Example 8 Lycium chinense callus homogenate (2000 bar) 89.6 Example 9 Vine callus homogenate (200 bar) 82.6 Example 10 Vine callus homogenate (500 bar) 85.3 Example 11 Vine callus homogenate (1000 bar) 87.3 Example 12 Vine callus homogenate (2000 bar) 90.1 Example 13 Grape callus homogenate capsule formulation (2000 bar) 88.8 Comparative example 1 Apple callus extract
50.4 Comparative example 2 Lycium chinense callus extract 38.1 Comparative Example 3 Vine callus extract 40.4 Comparative example 4 Vine callus homogenate (100 bar) 37.5 Comparative Example 5 Vine callus homogenate (2100 bar) 61.2 Witness Ascorbic acid (400 µg/ml) 80.7 White Distilled water 0

As can be seen from Table 7, the samples of Examples 1 to 12 including plant callus homogenates prepared from apple callus, Lycium chinense callus, or grapevine callus by a high pressure homogenizer at 200-2000 bar and Example 13 prepared as a capsule formulation showed significantly higher antioxidant efficacy than Comparative Examples 1, 2 and 3 using hot water extracts of the same plant calli and Comparative Examples 4 and 5 prepared at a pressure outside the defined range of the present invention.

In particular, all samples from Examples 1 to 13 showed significantly higher radical scavenging efficiency even than the positive control, ascorbic acid.

Experimental example 7: Evaluation of the flavonoid content

10 ml of each sample of Examples 1 to 13 and Comparative Examples 1 to 5 were filtered through a 0.2 µm syringe filter to prepare a test sample. The total polyphenol content was determined using a Folin-Denis method (Gutfinger R. 1981). 1 mg of each sample was dissolved in 1 ml of distilled water. A tenth dilution was performed to prepare a dilute solution. 0.2 ml of a phenol reagent from Folin-Ciocalteu, was added to 2 ml of the diluted solution, which was then kept at room temperature for 3 minutes. 2 ml of 2 M Na ₂ CO ₃ were added to the dilute solution to activate a reaction, which lasted one hour. The resulting supernatant was subjected to a microplate reader for measurement at 725 nm. The content was determined from a standard curve obtained using the gallic acid standard. The experimental results are shown in Table 8 below.

Experimental example 8: Evaluation of the terpenoid content

10 ml of each sample of Examples 1 to 13 and Comparative Examples 1 to 5 were filtered through a 0.2 µm syringe filter to prepare a test sample. Each sample was freeze-dried at -20°C for 48 hours and then extracted with petroleum ether (60-80°C). The extract thus obtained was filtered and concentrated. The concentrate fraction was then analyzed by thin layer chromatography (TLC) on silica gel C using benzene-ethyl acetate solvent (99:1). The sample was analyzed using a glass column by gas-liquid chromatography (Shimadzu GC07A, Kyoto, Japan). Quantitation was performed by standard curve using artemisinin standard, and total terpenoid content was determined by HPLC using methanol-water eluent (80:20)(0.8ml/min) . The measurement results are shown in Table 8 below.

[Table 8] Flavonoid content (mg/l) Terpenoid content (mg/l) Example 1 178.55 42.15 Example 2 223.44 45.48 Example 3 267.89 52.22 Example 4 303.21 56.31 Example 5 163.63 38.66 Example 6 186.59 42.11 Example 7 200.06 57.67 Example 8 254.64 73.32 Example 9 201.66 33.54 Example 10 264.38 54.22 Example 11 297.69 66.45 Example 12 321.28 78.83 Example 13 110.06 31.22 Comparative example 1 83.32 10.23 Comparative Example 2 61.55 11.25 Comparative Example 3 88.54 12.82 Comparative Example 4 91.32 22.33 Comparative Example 5 93.27 27.13

As shown in Table 8, all samples of the present invention according to Examples 1 to 13 had a significantly higher content of skin-beneficial components, such as flavonoids and terpenoids, than those of Comparative Examples 1 to 5.

Experimental Example 9: Evaluation of Formulation Stability

The samples of Examples 12 and 13 and Comparative Example 3 were evaluated for light stability as follows. First, 4 aliquots of each sample, with a volume of 30 ml per aliquot, were placed in a light-tight container and a light-permeable container, stored in a thermostat maintained at an illumination of 1000 ± 200 lux. and a temperature of 25 ± 2°C (room temperature) followed by rapid liquid chromatography at 2 week intervals. For analysis of resveratrol by rapid liquid chromatography, 0.1 g of each sample was dissolved in 10 ml of 100% pure methanol and subjected to three cycles of spraying with each cycle duration of 10 seconds followed by extraction for 12 hours. . The extract thus obtained was centrifuged and filtered through a 0.2 μm nylon filter for an organic solvent to prepare a test sample solution.

Each 10 µl of test sample solution and standard solution was used to determine the peak areas of resveratrol (C ₁₄ H ₁₂ NO ₃ ), AT (Test area: the peak area for the sample) and AS (Standard area: the area of the peaks for the standard sample), according to the liquid chromatography method under the following operating conditions.

[Equation 4]

Amount of resveratrol (C ₁₄ H ₁₂ NO ₃ )(mg) = amount of resveratrol standard product (mg) x AT/AS

[Working conditions]

Detector: Ultraviolet spectrophotometer

Column: A column with approximately 5 µm of octadecylsilyl silica gel for liquid chromatography packed in a stainless steel tube with an inside diameter of 4.6 mm and a length of 15 cm, or equivalent

Column temperature: room temperature

Mobile phase: Water with 0.1% TFA: acetonitrile = 70:30

Flow rate: 1.0ml/min

[Table 9] Light Duration (week) Amount of resveratrol remaining (%) Comparative Example 3 Example 12 Example 13 Blocked Light/Light Exposure 0 100 100 100 occulted light 2 84 91 100 occulted light 4 69 85 98 Exposure to light 2 55 77 93 Exposure to light 4 22 55 76

As can be seen from Table 9, it was observed that the grape callus homogenate of Example 12 retained a higher residual amount of resveratrol than that of the hot water extract of a grape callus of Comparative Example 3. In addition, the stability of resveratrol was significantly improved in a capsule formulation featuring the agar-coated plant callus homogenate as in Example 13.

Simple variations or modifications of the present invention can readily be made by those skilled in the art, and such variations or modifications are all intended to be included within the scope of the present invention.

Claims (18)

Method for preparing a callus lysate containing a callus metabolite without loss of callus metabolite, the method comprising a step of mechanically homogenizing a callus at a pressure of 200 to 2000 bar. Process according to claim 1, further comprising, before the mechanical homogenization step, at least one step selected from the group consisting of:
(a) isolation of callus from plant tissue and removal of the isolated callus;
(b) inducing dedifferentiation of a plant section to produce callus and harvesting the produced callus; And
(c) culturing the callus taken according to step (a) or (b) to grow the callus and removing the callus. A method according to claim 1 or 2, further comprising a step of removing a component not beneficial to human skin from a mechanical homogenate of callus after the step of mechanical homogenization. A method according to any one of claims 1 to 3, wherein the loss of callus metabolite consists of having the content of a specific reference substance contained in the callus lysate 1.3 times lower than the content of the reference substance specific in a callus lysate prepared by a method different from the method according to claim 1. A method according to any one of claims 1 to 4, wherein the callus metabolite is a substance containing a component beneficial to human skin produced by a callus organelle. A method according to claim 5, wherein the callus lysate exhibits at least one effect selected from the group consisting of improving skin hydration, preventing or improving skin wrinkles, improving skin elasticity, skin, lightening of the skin, and improvement of the complexion, due to the action of the component beneficial to human skin. Callus lysate containing a callus metabolite without loss of callus metabolite, the callus lysate being prepared by mechanical homogenization of a callus at a pressure of 200 to 2000 bar. Callus lysate according to claim 7, wherein the loss of callus metabolite consists of having the content of a specific reference substance contained in the callus lysate 1.3 times lower than the content of the specific reference substance in a lysate of callus prepared with a process different from the process according to claim 1. A callus lysate according to claim 7 or 8, wherein the callus metabolite is a substance containing a component beneficial to human skin produced by a callus organelle. A capsule containing a callus lysate, the capsule being prepared by coating encapsulation of the callus lysate according to any one of claims 7 to 9 with a natural polymeric material. A capsule containing a callus lysate according to claim 10, wherein the weight ratio between the natural polymeric material and the callus lysate is from 1:0.001 to 1:10. Capsule containing callus lysate according to claim 10 or 11, wherein the natural polymeric material is at least one selected from the group consisting of agar, pectin, alginic acid, alginate, carrageenan, curdran, starch, gellan gum, glucomannan, gum bean gum, guar gum, tara gum, gum arabic, xanthan gum, hyaluronic acid, Karaya gum, tragacanth gum, larch gum, dextran, cellulose, and a mixture thereof. A method of preparing a capsule containing a callus lysate, the method comprising coating the callus lysate according to any of claims 7 to 9 with a natural polymeric material to encapsulate the callus lysate. A method according to claim 13, wherein the natural polymeric material is at least one selected from the group consisting of agar, pectin, alginic acid, alginate, carrageenan, curdran, starch, gellan gum, glucomannan, locust bean gum, guar gum, tara gum, gum arabic, xanthan gum, hyaluronic acid, karaya gum, tragacanth gum, larch gum, dextran, cellulose, and a mixture thereof. A method according to claim 13 or 14, wherein in the encapsulation step, the weight ratio between the natural polymeric material and the callus lysate is from 1:0.001 to 1:10. Use of the callus lysate according to any one of claims 7 to 9 or a capsule containing a callus lysate according to any one of claims 10 to 12 as an active ingredient for improving skin hydration, preventing or improving skin wrinkles, brighten skin, improve skin elasticity, or improve skin tone. Cosmetic composition for improving skin hydration, preventing or improving skin wrinkles, lightening skin, improving skin elasticity, or improving skin tone, the cosmetic composition comprising the callus lysate according to any one of the claims 7 to 9 or the capsule containing a callus lysate according to any one of claims 10 to 12. A cosmetic process for improving skin hydration, preventing or improving skin wrinkles, lightening skin, improving skin elasticity, or improving skin tone, the process including administering the cosmetic composition according to claim 17 to the skin of an individual biological subject.