KR20110085151A - Megastigmane derivatives isolated from ilex paraguariensis, preparation method thereof and anti-wrinkle composition containing the same as an active ingredient - Google Patents

Abstract

PURPOSE: A novel megastigmane derivative or pharmaceutically acceptable salt thereof is provided to prevent or treat wrinkling. CONSTITUTION: A megastigmane derivative or pharmaceutically acceptable salt thereof is denoted by chemical formula 1 or 2. A method for preparing the megastigmane derivative comprises: a step of adding water, organic solvent, or mixture solvent thereof to Ilex paraguariensis to prepare Ilex paraguariensis extract; a step of fractioning the Ilex paraguariensis extract with n-hexane and ethyl acetate to prepare an Ilex paraguariensis fraction; and a step of performing column chromatography.

Description

Method for preparing megastigman derivatives isolated from mate tea and composition for improving wrinkles containing the same as an active ingredient {Megastigmane derivatives isolated from Ilex paraguariensis, preparation method about and anti-wrinkle composition containing the same as an active ingredient}

The present invention relates to a megastigman derivative or a pharmaceutically acceptable salt thereof, a preparation method thereof and a composition for improving wrinkles containing the same as an active ingredient.

Skin, the largest organ in the human body, protects the body from physical and chemical stimuli from the outside and maintains the homeostasis of the body's humidity and body temperature. However, ageing or continuous exposure to external stimuli causes skin aging, such as damage to the skin and decreased physiological function. As the skin ages, the demand for quality of life increases due to natural phenomena or improvement of living standards that inevitably occur with age, and skin aging that has been increased due to various environmental factors. Countermeasures are urgently needed.

Skin aging is influenced by various factors such as genetic and environmental factors, and is classified into natural aging or endogenous aging and exogenous aging. Natural aging or intrinsic aging is an aging phenomenon that occurs over time according to genetic information. It is mainly observed in skin that is not exposed to sunlight. It is characterized by fine wrinkles and skin elasticity and is difficult to artificially control. Extrinsic aging is an aging phenomenon caused by external environmental factors, especially ultraviolet rays in sunlight. It is also called photoaging, and it appears in areas exposed to sunlight. Deep wrinkles, rough skin, pigmentation It is characterized by a decrease in skin tone and skin elasticity, and is easy to artificially adjust. Therefore, studies to prevent exogenous aging have been continued, and in particular, research on preventing wrinkle formation caused by exogenous photoaging caused by long-term ultraviolet exposure has been focused (Gilcher st BA, J. Am. Acad. Dermatol., 1989). : 21: 610-613).

The most prominent change in the appearance of the skin accompanying aging is wrinkles. Wrinkles occur on the face, neck, neck, elbow, armpits, hands, and feet, on the forehead, around the eyes, between the legs, and around the mouth. Most wrinkles start to appear around 30 years of age, and as they age, their number, depth, and range increase.

The causes of wrinkles according to the structure of the skin in more detail, as follows. Skin is divided into epidermis and dermis. The epidermis is a thin protective layer consisting mainly of keratinocytes, melanocytes and Langerhans cells, and the dermis is a thick and complex layer of fibrous and connective tissue composed of collagen and elastic fibers. Collagen is a major structural protein of the skin, which determines the tensile strength and toughness of the skin. Elastic fibers are important for skin resilience and skin elasticity.

In the epidermis, the turn-over of keratinocytes is reduced, resulting in the accumulation of stratum corneum, thereby thickening the stratum corneum, reducing the water-containing capacity of the stratum corneum, and curing the keratin to cause wrinkles. The epidermal and dermal boundaries are involved in epidermal cell support, adhesion, transport of nutrients, and regulation of epidermal differentiation.As a result of various aging factors, collagen IV and VII are reduced due to UV, resulting in reduced epidermal and dermal support. As a result, the selective permeation function is weakened and harmful ingredients easily affect the dermis, resulting in wrinkles (Keene, J., Cell Biol., 104, pp. 611-621, 1987).

The dermis is a tissue composed of connective tissue beneath the epidermis, which is not densely packed like epidermal keratinocytes, has a lot of extracellular space, and is filled by a network of macromolecules called extracellular matrix. This extracellular epilepsy is made from fibroblasts in the dermis and consists of fibrous proteins such as collagen and elastin and polysaccharides called acidic mucopolysaccharides such as hyaluronic acid. The connective tissue involved in skin wrinkle formation is mainly composed of fiber components such as collagen fiber, reticulated fiber, elastin fiber and the like. Collagen and elastin in the dermal tissue are important fibrous tissues that work closely together to maintain skin flexibility and elasticity.

Collagen is a fibrous tissue that gives skin flexibility and is a major component that accounts for about 70% of dermal tissue. It is involved in the mechanical firmness of the skin in the dermal layer, the adhesion of tissues, adhesion to cells and cell differentiation (Journal of the American Academy). of Dermatology, 2001, 17 (4): 610-613). Polypeptide chains of collagen fibers are synthesized in a state in which fibroblast granules have extended peptides (telopeptides) at both ends, and then secreted out of the cells in the form of procollagen combined in a triple helix structure. The secreted procollagen is digested with an extended peptide by a specific enzyme to produce tropocollagen, which is combined with each other in the extracellular matrix to synthesize collagen fibers. Synthesis of the collagen fibers is rapidly reduced as the skin ages, and as the collagen fibers are deformed by the external environment, a lot of wrinkles are generated and deepened in the skin. The collagen is involved in collagenase, which is a collagen degrading enzyme.

In addition, elastin, like collagen, is a major fibrous tissue that affects the elasticity of the skin, and its amount is about 2%, which is a small amount compared to collagen, but plays a very important role in skin elasticity because it forms a network tissue supporting the epidermis. Elastin is synthesized in a cell into a polypeptide called tropoelastin and then produced through cross-linking between tropoelastin extracellularly. Thus, elastin has secondary or three-dimensional elasticity by crosslinking of tropoelastin. Elastin is degraded by a degrading enzyme called elastase, and it continues to be produced by fibroblasts until the age of 18-19, but after that, elastin is weakened once it stops producing and must take care of skin elasticity with only elastin already produced. It is very difficult to recover its elasticity.

Such collagen or elastin is a major factor affecting skin wrinkle formation. That is, skin aging such as skin wrinkle formation is caused by destruction of collagen, decrease in collagen synthesis, loss of elastin, and the like, which occurs in the skin epidermis, but rather occurs in the dermis.

Human neutrophil elastase (HNE) is a serine protease that is primarily located in Hoazur granules of polymorphonuclear leukocytes. It has a substrate specificity that degrades connective tissue proteins such as cartilage issues. Biologically, elastase activity is noticeably increased with age and is known to reduce skin elasticity. Therefore, it is expected that skin wrinkles can be improved by inhibiting the elastase activity.

Active ingredients for skin wrinkle improvement or anti-aging currently being developed cannot be used for some cosmetic ingredients or are very unstable, and are not easy to transfer to the skin, requiring a special stabilization system and delivery system. There is a problem that is not visible, etc. Recently, attention has been gradually focused on skin protection agents containing retinoids. Retinoids are used as a means to solve photoaging phenomena such as wrinkles, skin thickening, sagging, and reduced elasticity, which are the result of daylight accumulation. However, retinoids are very unstable compounds and are sensitive to ultraviolet rays, moisture, heat, and oxygen, and easily cause chemical changes. Therefore, research is focused on developing active ingredients derived from natural products to solve them.

Mate (Ilex paraguariensis, mate) is an evergreen subfamily of persimmon tree, which is 6 m tall and is distributed in Paraguay and Brazil in South America. In the wild state, the upper part is originally rounded, but when it is grown, its shape is changed to become a shrub with many stems, and higher quality tea leaves can be grown than in the wild state, and harvesting is performed every two years. Flowers of mate are small, hanging on the axilla, 4 digit, green, fruits are red or reddish brown, round, 6 mm in diameter. The leaves of mate are drunk as tea, and the leaves are about 10 cm thick, egg or oval, and the edges are serrated. The leaves contain caffeine (about 2%) and have a good aroma. The leaves are separately harvested and drunk as water-drenched tea. Mate grows in mineral-rich soils such as calcium and iron, so it contains a lot of minerals such as iron, calcium and magnesium. In particular, mate is very high in iron and has five times the iron content of green tea. In particular, other teas contain tannins that interfere with iron absorption, while mate contains little tannin. Mate tea is made from dried leaves of mate and contains triterpenes, caffeine and caffeine-like compounds. Mate tea is known to be effective in the treatment of constipation, obesity, stiff shoulders, headaches.

Previous studies on mates have found the presence of many bioactive compounds such as xanthine, caffeoyl derivatives, flavonoids and triterpenoid saponins. Yerba mate (ilex paraguariensis) has antioxidant and cellular protective effects (Filip et al., 2000; Gugliucci and Stahl, 1995), anti-obesity and fever (Martinet et al., 1999), circulatory system and cholesterol lowering Various biological activities are known, such as effects (Gorgen et al., 2005) and bile stimulating action (Gorzalczany et at., 2001).

However, the effects of megastigmane derivatives isolated from mate on human neutrophil elastase are not known at all. Accordingly, the present inventors have been studying to develop a stable natural-derived wrinkle improving agent that does not easily cause chemical changes by ultraviolet rays, moisture, heat, oxygen, etc., while the Megastiggman derivative isolated from Mate effectively inhibits elastase activity. The present invention was completed by revealing an excellent skin wrinkle improvement effect and revealing that the composition containing it as an active ingredient can be used as a medicine, cosmetic or food for wrinkle improvement.

It is an object of the present invention to provide a novel megastigman derivative compound or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a method for producing the novel megastigman derivative.

Still another object of the present invention is to provide a pharmaceutical composition for preventing and treating wrinkles, which contains the novel megastigman derivative compound as an active ingredient.

It is another object of the present invention to provide a cosmetic composition for preventing and improving wrinkles containing the novel megastigman derivative compound as an active ingredient.

Another object of the present invention to provide a food composition for preventing and improving wrinkles containing the novel megastigman derivative compound as an active ingredient.

In order to achieve the above object, the present invention provides a Megastiggman derivative represented by the following formula (1) or formula (2) or a pharmaceutically acceptable salt thereof and a method for preparing the same.

[Formula 1]

[Formula 2]

In addition, the present invention provides a pharmaceutical composition for preventing and treating wrinkles, which contains the Megastiggman derivative compound represented by Formula 1 or Formula 2 as an active ingredient.

Furthermore, the present invention provides a cosmetic composition for preventing and improving wrinkles containing the Megastiggman derivative compound represented by Formula 1 or Formula 2 as an active ingredient.

In addition, the present invention provides a food composition for preventing and improving wrinkles, which contains the Megastiggman derivative compound represented by Formula 1 or Formula 2 as an active ingredient.

Since the megastiggman derivative compounds according to the present invention effectively inhibit human neutrophil elastase activity and exhibit excellent skin wrinkle improvement, the composition containing these as an active ingredient can be usefully used in medicines, cosmetics or foods for wrinkle improvement. Can be.

1 is data of ¹³ C-NMR spectrum, ¹ H-NMR spectrum and HMBC spectrum of Example 1 according to the present invention;
Figure 2 is the data of ¹³ C-NMR spectrum, ¹ H-NMR spectrum and HMBC spectrum of Example 2 according to the present invention.

The present invention provides a Megastiggman derivative represented by the following Chemical Formula 1 or Chemical Formula 2 or a pharmaceutically acceptable salt thereof.

Formula 1 and Formula 2 are named as Matenoside A and Matenoside B, respectively.

In addition, the present invention includes not only the compound of Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof, but also all possible solvates or hydrates that can be prepared therefrom.

Furthermore, the present invention provides a method for producing the megastiggman derivative represented by Formula 1 or Formula 2.

Megastigman derivatives of Formula 1 or Formula 2 according to the present invention

Adding a water, an organic solvent or a mixed solvent thereof to the mate to obtain a mate extract (step 1);

Sequentially fractionating the mate extract obtained in step 1 with n-hexane and ethyl acetate to obtain a mate fraction (step 2); And

It may be prepared by a manufacturing method comprising the step (step 3) of performing a column chromatography on the mate fraction obtained in step 2.

Hereinafter, the manufacturing method according to the present invention will be described in more detail step by step.

First, the step 1 according to the present invention is a step of obtaining a mat leaf extract as an extraction solvent.

The mate leaf extract can be obtained by extracting from mate or dried products thereof, mate can be used without limitation, such as harvested, cultured or commercially available.

The extract may be obtained by using a conventional extraction method such as a method using an extraction device such as supercritical extraction, subcritical extraction, high temperature extraction, high pressure extraction, or ultrasonic extraction, or using an adsorption resin including XAD and HP-20. It may be prepared, preferably warmed, and may be prepared by reflux extraction or extraction at room temperature, but is not limited thereto. The number of extraction is preferably 1 to 5 times, and more preferably 3 times of extraction, but is not limited thereto.

The extract may be extracted using water, an organic solvent or a mixed solvent thereof as an extraction solvent. The organic solvent is preferably any one or a mixed solvent selected from the group consisting of C ₁ to C ₄ alcohols, ethyl acetate, methylene chloride and chloroform, more preferably C ₁ to C ₄ alcohols, Most preferred is extraction with methanol or ethanol. The extraction solvent is preferably extracted by adding 2 to 10 times the weight of the mat, and more preferably by adding 3 to 5 times, but is not limited thereto.

As an example, mate extract according to the present invention may be crushed mate dried to an appropriate size, put into the extraction container, put the extraction solvent, left for a certain time and then filtered with a filter paper to obtain an alcohol extract. The extraction time is preferably 1 to 5 days, more preferably 2 to 4 days. Thereafter, a method such as vacuum concentration or lyophilization may be additionally performed.

Next, step 2 is a step of obtaining a fraction by dispersing the mate extract obtained in step 1 in water, and then sequentially fractionated with a solvent having a different polarity. As the solvent, n-hexane (n-hexane), ethyl acetate (ethylacetate) may be used sequentially.

Next, step 3 is a step of performing column chromatography on the fraction obtained in step 2.

The column chromatography may be performed using a column for silica gel chromatography or a column for size exclusion chromatography, and a column packed with Sephadex LH-20 may be used as the column for size exclusion chromatography.

Concentration gradient silica gel column chromatography was performed on the fractions obtained in step 2 using a mixed solvent of methylene chloride and methanol. At this time, the concentration gradient of the mixed solvent of methylene chloride and methanol is preferably 20: 1 to 1: 2, the flow rate is preferably 4.0 to 5.0 ml / min, the running time is preferably 20 to 25 hours.

Fractions obtained by the silica gel column chromatography may be chromatographed using a Sephadex LH-20 column. At this time. The mobile phase is preferably a mixed solvent (1: 1) of methylene chloride and methanol.

Fractional gradient silica gel column chromatography may be performed using a mixed solvent of n-hexane and acetone for the fraction obtained by performing chromatography using a Sephadex LH-20 column in step 3. At this time, the concentration gradient of the mixed solvent of n-hexane and acetone is preferably 10: 1 to 2: 8.

Fractions obtained by the silica gel column chromatography may be chromatographed using a Sephadex LH-20 column. At this time, the mobile phase is preferably methanol.

After performing the silica gel column chromatography and the chromatography using the Sephadex LH-20 column, high performance liquid chromatography may be performed to separate the material of the compound of Formula 1 or Formula 2. The high performance liquid chromatography may be performed using a mixed solvent (2: 3) of methanol and distilled water as a developing solvent. At this time, the flow rate of the mobile phase is preferably 3.0 to 5.0 ml / min, the running time is preferably 20 to 50 hours.

In addition, the present invention provides a pharmaceutical composition for preventing and treating wrinkles, which contains the megastiggman derivative represented by Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient.

The derivative compound of Formula 1 or Formula 2 according to the present invention can be used for preventing and treating wrinkles by inhibiting elastase activity. Their anti-wrinkle and therapeutic uses will be described based on specific experimental results.

Derivative compounds of the formula (1) or (2) according to the present invention inhibited the elastase activity in a concentration-dependent manner in the human neutrophil elastase inhibitory activity measurement experiment, and exhibited an excellent elastase by showing IC ₅₀ values of 50.4 μM and 11.1 μM, respectively. By showing the inhibitory effect, it can be seen that excellent skin wrinkle improvement effect (see Experimental Example 1 and Table 1).

Therefore, the derivative compound of formula 1 or 2 according to the present invention effectively inhibits human neutrophil elastase activity and exhibits an excellent skin wrinkle improvement effect, so that the composition containing them as an active ingredient is a medicine, cosmetic or food for wrinkle improvement. It can be usefully used.

Furthermore, the present invention provides a cosmetic composition for preventing and improving wrinkles, which contains the Megastiggman derivative compound represented by Formula 1 or Formula 2 or a cosmetically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a food composition for preventing and improving wrinkles, which contains the megastiggman derivative compound represented by Formula 1 or Formula 2 or a food acceptable salt thereof as an active ingredient.

According to the present invention, the pharmaceutical composition for preventing and treating wrinkles, which contains the megastiggman derivative compound represented by Chemical Formula 1 or Chemical Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient, may be various oral or parenteral dosage forms as follows. It may be formulated as, but is not limited thereto.

Formulations for oral administration include, for example, tablets, pills, hard / soft capsules. Liquid. Suspensions, emulsifiers, syrups. Granules, elixirs, and the like, these formulations may be used one or more diluents or excipients, such as fillers, extenders, wetting agents, disintegrants, lubricants, binders, surfactants, etc. that are commonly used in addition to the active ingredient. As a disintegrant, agar, starch, alginic acid or its sodium salt, calcium monohydrogen phosphate anhydride, etc. may be used, and as lubricant, silica, talc, stearic acid or its magnesium salt or calcium salt, polyethylene glycol, etc. may be used. As a binder, magnesium aluminum silicate. Starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine, low-substituted hydroxypropylcellulose, and the like can be used. In addition to lactose, dextrose, sucrose, mannitol, sorbitol, cellulose. Glycine or the like may be used as a diluent, and in some cases, generally known boiling mixtures, absorbents, colorants, flavors, sweeteners, and the like may be used together.

In addition, the anti-wrinkle and therapeutic pharmaceutical composition containing the megastiggman derivative compound of Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient can be parenterally administered, parenteral administration is a subcutaneous injection, By intravenous injection, intramuscular injection or intrathoracic injection. In this case, in order to formulate into a formulation for parenteral administration, the Megastiggman derivative compound of Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof is mixed with water with a stabilizer or a buffer to prepare a solution or suspension, and the ampoule Or in unit dosage forms of vials.

The composition may be sterile or contain preservatives, stabilizers, hydrating or emulsifiers, salts for controlling osmotic pressure, buffers and other auxiliaries and other therapeutically useful materials, which are conventional methods of mixing, granulating or coating methods. It can be formulated accordingly.

In the case of formulating a pharmaceutical composition for preventing and treating anti-wrinkle and containing the Megastiggman derivative compound of Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient, as an active ingredient, It is preferable to contain the Megastiggman derivative compound of Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof in a unit dose of about 0.1 to 1,500 mg. Dosage depends on the doctor's prescription depending on factors such as the patient's weight, age and the specific nature and severity of the disease. However, dosages required for adult treatment typically range from about 1 to 500 mg per day, depending on the frequency and intensity of administration. For intramuscular or intravenous administration to adults, a single dose separate from the usual dosage of about 5 to 300 mg per day would be sufficient, although for some patients a higher daily dose may be desirable.

The cosmetic composition for preventing and improving anti-wrinkle and improvement of the megastigman derivative compound of Formula 1 or Formula 2 according to the present invention or a cosmetically acceptable salt thereof as an active ingredient includes a basic cosmetic composition with a dermatologically acceptable excipient ( Face wash, packs, body oils, such as lotion, cream, essence, cleansing foam and cleansing water It may be prepared in the form of soap.

The excipients may include, but are not limited to, emollients, skin penetration enhancers, colorants, fragrances, emulsifiers, thickeners and solvents. In addition, fragrances, pigments, fungicides, antioxidants, preservatives and moisturizing agents may be further included, and may include thickeners, inorganic salts, synthetic polymer materials and the like for the purpose of improving the properties. For example, in the case of preparing the face wash and the soap with the cosmetic composition of the present invention, the megastigman derivative compound of Formula 1 or Formula 2 or a cosmetically acceptable salt thereof may be easily added to a conventional face wash and soap base. It can manufacture. When the cream is prepared, it may be prepared by adding the above-described megastiggman derivative compound of Formula 1 or Formula 2 or a cosmetically acceptable salt thereof to a cream base of general oil-in-water type (O / W). The fragrance, chelating agent, pigments, antioxidants, preservatives, etc. may be added to synthetic or natural materials such as proteins, minerals, vitamins and the like for the purpose of improving the physical properties.

The content of the Megastiggman derivative compound of Formula 1 or Formula 2 or a cosmetically acceptable salt thereof contained in the cosmetic composition of the present invention is preferably 0.001 to 10% by weight, and 0.01 to 5% by weight based on the total weight of the composition. It is more preferable to be, but is not limited thereto. If the content is less than 0.001% by weight can not expect the desired wrinkle improvement effect, if it is more than 10% by weight may be difficult to manufacture safety or formulation.

In addition, the cosmetic composition of the present invention is a fatty acid, an organic solvent, a dissolving agent, a thickening agent, a gelling agent, an emollient, an antioxidant, in addition to the megastiggman derivative compound of Formula 1 or Formula 2 or a cosmetically acceptable salt thereof Suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes , Adjuvant conventionally used in the cosmetic or dermatological field, such as pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients conventionally used in cosmetics. In addition, the above components may be introduced in an amount generally used in the dermatology field.

The anti-wrinkle and improvement food composition containing the megastiggman derivative compounds of the formula (1) or (2) according to the present invention or a food-acceptable salt thereof as an active ingredient is in a health supplement or health functional food such as food, beverage Can be added. In this case, the Megastiggman derivative compound of Formula 1 or Formula 2 or a food acceptable salt thereof is added in an amount of 0.01 to 20% by weight, preferably 0.1 to 5% by weight, based on the raw materials when used as a food additive. can do. The blending amount of the active ingredient may be suitably determined according to the purpose of use (prevention, health or therapeutic treatment). However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and since there is no problem in terms of stability, the active ingredient may be used in an amount above the above range. The Megastiggman derivative compound of Formula 1 or Formula 2 or a food acceptable salt thereof may be used together with other food or food ingredients, and may be suitably used according to a conventional method.

There is no particular limitation on the kind of food. Examples of the food to which the Megastiggman derivative compound of Formula 1 or Formula 2 or a food acceptable salt thereof may be added include meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, Dairy products including gums and ice creams, various soups, beverages, teas, drinks, alcoholic beverages and vitamin complexes, etc., and include all healthy foods in the usual sense.

The food supplement additive of the present invention may use various flavors or natural carbohydrates. The natural carbohydrates are sugars such as glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, xylitol, sorbitol, and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The proportion of said natural carbohydrates is generally about 0.01 to 0.04% by weight, preferably 0.02 to 0.03% by weight per 100% by weight of the composition according to the invention.

In addition to the above, the composition according to the present invention contains various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the compositions according to the invention may contain pulp for the production of natural fruit juices and vegetable beverages. These components can be used independently or in combination, and the proportion of the additive is not critical, but is usually selected in the range of 0.01 to 0.1% by weight per 100% by weight of the composition according to the invention.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following examples are merely to illustrate the present invention, but the content of the present invention is not limited by the following examples.

< Example  1> Matenoside  Manufacture of A

Step 1: Mate  Preparation of Methanol Extracts

8 kg of matatee purchased from Yerba Korea was put in an extraction container, 20 L of methanol was added thereto, extracted at room temperature for 3 days, and filtered through a filter paper to obtain an extract. The extraction process was repeated three times, after which the solvent was concentrated under reduced pressure and dried to obtain 570 g of methanol extract.

Step 2: Mate Fraction  Produce

In order to fractionate the methanol extract obtained in Preparation Example 1 according to the polarity, a systematic fraction using an organic solvent was performed. Specifically, after adding a certain amount of water to the ethanol extract and suspended, hexane (hexane), ethyl acetate (ethylacetate) was added to obtain a fraction. First, 2 L of water was added to 570 g of the concentrate obtained by concentrating the ethanol extract, and then 2 L of hexane was added and the hexane layer was separated to obtain a hexane fraction. 2 L of ethyl acetate was added to the water layer from which the hexane fraction was removed, and the ethyl acetate layer was separated to obtain an ethyl acetate fraction. The ethyl acetate fraction was removed and the remaining water layer was lyophilized to obtain a water fraction. The fractionation process was performed at room temperature, and each fraction was left for 5 hours. In the fractionation process, an organic solvent was added to the water layer, and the organic solvent layer was separated and repeated three times to obtain a fraction extract. The obtained fractions were filtered, concentrated under reduced pressure, and dried. Fraction yield according to the fractionation solvent was 22% for the hexane fraction, 42% for the ethyl acetate fraction, 36% for the water fraction.

Step 3: Matenoside  Manufacture of A

The ethyl acetate fraction obtained in step 2 of Preparation Example 1 was used as a mobile phase by using a mixed solvent of methylene chloride and methanol (20: 1 → 1: 2 concentration gradient), and then performing chromatography using silica gel to perform fraction 1 to fraction 1. 9 was obtained. Dissolve fraction 5 (12.8 g) in the fraction in methanol, and then perform Sephadex LH-20 column chromatography using methylene chloride (1: 1) and 70% methanol as a mobile phase to obtain fractions 5-1 to 5-5. It was. Fraction 5-1 to fraction 5-1 of the above fractions were used as a mobile phase by using a mixed solvent of n-hexane and acetone (10: 1 → 2: 8 concentration gradient) and performing chromatography using silica gel. 1-7 was obtained. After dissolving fraction 5-1-6 in methanol, the fractions 5-1-6-1 and 5 were subjected to Sephadex LH-20 column chromatography using methylene chloride (1: 1) and 70% methanol as a mobile phase. -1-6-2 was obtained. Fraction 5-1-6-1 in the fractions was subjected to preparative high performance liquid chromatography to give Matenoside A. At this time, using the mixed solvent of water and methanol (2: 3) as a mobile phase to obtain a peak appearing in 30 minutes. The flow rate was 4 ml / min and a YMC-Pack ODS (20 × 250 mm) column was used.

Appearance: white powder;

: +117.2 (c=0.04, MeOH);

: +117.2 (c = 0.04, MeOH);

CD (MeOH): +165.0 (244 nm);

UV (MeOH) λ _max nm (logε): 241 (3.94);

HR-ESI-TOF-MS (positive ion mode) m / z : 413.2159 [M + H] ⁺ (calcd. For C ₂₁ H ₃₃ O ₈ , 413.2175);

NMR measured ¹³ C-NMR (100 MHz), ¹ H-NMR (400 MHz) and 2D-NMR (HMBC) using a Bruker Avance II 900 spectrometer, and TMS was used as an internal standard. Data of ¹³ C-NMR spectrum, ¹ H-NMR spectrum and HMBC spectrum are shown in FIG. 1.

< Example  2> Matenoside  Manufacture of B

Fraction 5-1-6-1 obtained in step 3 of Preparation Example 1 was subjected to preparative high-performance liquid chromatography to obtain Matenoside B. At this time, using a mixed solvent (2: 3) of water and methanol as a mobile phase to obtain a peak appearing in 35 minutes. The flow rate was 4 ml / min and a YMC-Pack ODS (20 × 250 mm) column was used.

Appearance: white powder;

: +50.3 (c=0.01, MeOH);

: +50.3 (c = 0.01, MeOH);

CD (MeOH): +8.8 (244 nm);

UV (MeOH) λ _max nm (logε): 244 (3.67);

HR-ESI-TOF-MS (positive ion mode) m / z : 415.2319 [M + H] ⁺ (calcd. For C ₂₁ H ₃₅ O ₈ , 415.2332);

NMR measured ¹³ C-NMR (100 MHz), ¹ H-NMR (400 MHz) and 2D-NMR (HMBC) using a Bruker Avance II 900 spectrometer, and TMS was used as an internal standard. Data of ¹³ C-NMR spectrum, ¹ H-NMR spectrum and HMBC spectrum are shown in FIG. 2.

< Experimental Example  1> human Neutral sphere Elastase  Determination of Inhibitory Effect

When elastase, an enzyme that breaks down elastin, is active, elastin, an elastic fiber, is broken down to accelerate skin wrinkle formation. The following experiment was carried out to determine the effect of the neutrophil elastase inhibitory effect on humans of the compounds according to the invention.

Experimental solutions were prepared by dissolving the compounds of Examples 1 and 2 at 500 μl DMSO at concentrations of 1, 3, 10, 30 and 100 μM and then adding Tris-HCl buffer. 60 μl of the test solution and N-methoxysuccinyl-alanine-alanine-proline-valine- p -nitroanilide (N-methoxysuccinyl-ala-ala-pro-val- p- nitroanilide), which is a substrate of elastase. 125 [mu] l, 390 [mu] l of Tris-HCl buffer were mixed and 25 [mu] l of human neutrophil elastase (0.18 units) were added and incubated at 37 [deg.] C. for 1 hour. After 1 hour, the reaction was stopped by adding a soybean trypsin inhibitor. When the substrate of elastase N- methoxy-carbonyl-seat - the spectrometer the coloring degree of the nitroaniline-alanine-alanine-proline-valine-p-nitro when the fluoride is not decomposition of the yellow p-nitroaniline is a, p The elastase inhibitory activity rate can be measured by measuring the absorbance at 405 nm. Elastase inhibitory activity was calculated by Equation 1 below, and the IC ₅₀ value was calculated by measuring the elastase inhibitory effect of each compound concentration. The results are shown in Table 1 below.

A: absorbance in the absence of an elastase inhibitor

B: absorbance in the presence of an elastase inhibitor

sample
Elastase inhibitory effect (%) IC ₅₀ (μM)
1 μM 3 μM 10 μM 30 μM 100 μM Example 1 2.0 ± 3.5 8.6 ± 1.2 11.4 ± 1.6 34.5 ± 6.4 68.2 ± 1.8 50.4 Example 2 20.4 ± 6.5 36.1 ± 1.8 47.5 ± 3.7 62.2 ± 1.7 65.0 ± 0.7 11.1

Referring to Table 1, it can be seen that the compound according to the present invention inhibits elastase activity in a concentration-dependent manner. In particular, it can be seen that the compound of Example 1 exhibited an excellent inhibitory effect of elastase by showing an IC ₅₀ value of 50.4 μM and Example 2 of 11.1 μM.

It can be seen that the compound according to the present invention exhibits an excellent inhibition rate of elastase activity, thereby exhibiting an excellent skin wrinkle improvement effect.

Therefore, since the derivative compound according to the present invention has excellent human neutrophil elastase inhibitory effect, the composition containing these as an active ingredient can be usefully used for medicines, cosmetics or foods for wrinkle improvement.

On the other hand, the derivative represented by Formula 1 or Formula 2 according to the present invention can be formulated in various forms according to the purpose. Examples of preparations for the compositions of the present invention are illustrated below.

Formulation example  1: Preparation of Pharmaceutical Formulations

1-1. Powder  Produce

2 g of Megastiggman derivatives of Formula 1 or Formula 2

1g lactose

The above components were mixed and packed in airtight bags to prepare powders.

1-2. Manufacture of tablets

100 mg of Megastiggman derivative of Formula 1 or Formula 2

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

1-3. Preparation of Capsules

100 mg of Megastiggman derivative of Formula 1 or Formula 2

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

1-4. Injection preparation

100 mg of Megastiggman derivative of Formula 1 or Formula 2

Mannitol 180 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

Distilled water 2974 mg

According to a conventional method for preparing an injection, an injection was prepared by containing the above components in the contents shown.

Formulation example  2: manufacture of cosmetics

2-1. Manufacture of cream

4.6 wt.% Megastiggman Derivatives of Formula 1 or Formula 2

Cetostearyl alcohol 2.8 wt%

Beeswax 2.6 wt%

Stearic acid 1.4 wt%

2% by weight of glycerol phosphate monostearate

Fiji-100 Stearate 1% by weight

Sesquioleate sorbitan 1.4 wt%

4% by weight of jojoba oil

3.8% by weight of squalene

Polysorbate 60 1.1 wt%

2% by weight of macadamia oil

Tocopherol Acetate 0.2 wt%

0.4% by weight of methylpolysiloxane

0.1% by weight of ethyl paraben

Propylparaben 0.1 wt%

Euxyl K-400 0.1 wt%

1,3-butylene glycol 7% by weight

Methylparaben 0.05 wt%

Glycerin 6 wt%

d-panthenol 0.2 wt%

0.2% by weight of triethanolamine

pt 41891 0.2 wt%

pH ₂ O 46.05 wt%

2-2. Lotion

3.5% by weight of Megastiggman derivatives of Formula 1 or Formula 2

Cetostearyl alcohol 1.6 wt%

Stearic acid 1.4 wt%

Hydrophilic glycerol monostearate 1.8% by weight

Fiji-100 Stearate 2.6% by weight

Sesquioleate sorbitan 0.6 wt%

4.8% by weight of squalene

2% by weight of macadamia oil

2% by weight of jojoba oil

Tocopherol Acetate 0.4 wt%

0.2% by weight of methylpolysiloxane

0.1% by weight of ethyl paraben

Propylparaben 0.1 wt%

1,3-butylene glycol 4% by weight

Methylparaben 0.1 wt%

Xanthan gum 0.1 wt%

Glycerin 4 wt%

d-panthenol 0.15% by weight

Allantoin 0.1 wt%

4% by weight of carbone (2% aq.Sol)

0.15 wt% triethanolamine

Ethanol 3 wt%

pt 41891 0.1 wt%

pH ₂ 0 48.3 wt%

Formulation example  3: manufacture of food

3-1. Manufacture of beverages

522 mg of honey

Thioctamide 5 mg

Nicotinamide 10 mg

Riboflavin Sodium Hydrochloride 3 mg

Pyridoxine hydrochloride 2 mg

Inositol 30 mg

50 mg orotic acid

Megastiggman Derivatives of Formula 1 or Formula 2 0.48-1.28 mg

200 ml of water

A beverage was prepared using the above-mentioned composition and content by a conventional method.

3-2. Of chewing gum  Produce

Gum Base 20%

Sugar 76.36-76.76%

Megastiggman Derivatives of Formula 1 or Formula 2 0.24-0.64%

1% of fruit flavor

Water 2%

Chewing gum was prepared using the above-mentioned composition and content by a conventional method.

3-3. Candy

50-60% sugar

Starch syrup 39.26-49.66%

Megastiggman Derivatives of Formula 1 or Formula 2 0.24-0.64%

Orange flavor 0.1%

The composition and the content of the candy were prepared using a conventional method.

3-4. Manufacture of flour food products

0.5 to 5% by weight of the Megastiggman derivative of Formula 1 or Formula 2 according to the present invention is added to 100% by weight of wheat flour, and the bread, cake, cookies, crackers and noodles are prepared using the mixture to prepare food for health promotion. Prepared.

3-5. dairy product( dairy products Manufacturing

5 to 10% by weight of the Megastiggman derivative of Formula 1 or Formula 2 according to the present invention was added to 100% by weight of milk, and various dairy products such as butter and ice cream were prepared using the milk.

3-6. Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted. Black soybeans, black sesame seeds, and perilla were also steamed and dried in a known manner, and then roasted to prepare a powder having a particle size of 60 mesh. The grains and seeds prepared above and the Megastigman derivative of Formula 1 or Formula 2 according to the present invention were prepared by combining the following ratios.

30% brown rice

15% rate

Barley 20%

Perilla 7%

Black Bean 7%

Black Sesame 7%

Megastiggman derivatives of formula 1 or formula 3%

Ganoderma 0.5%

Foxglove 0.5%

Claims (8)

Megastiggman derivative represented by the following formula (1) or (2) or a pharmaceutically acceptable salt thereof.
[Formula 1]

(2)

Adding a water, an organic solvent or a mixed solvent thereof to the mate to obtain a mate extract (step 1);
Sequentially fractionating the mate extract obtained in step 1 with n-hexane and ethyl acetate to obtain a mate fraction (step 2); And
A method for preparing the megastiggman derivative of claim 1, comprising performing column chromatography on the mate fraction obtained in step 2 (step 3).
The method of claim 2, wherein the organic solvent of step 1 is a C ₁ to C ₄ alcohol, characterized in that the manufacturing method of megastiggman derivatives.
4. The method of claim 3, wherein the C ₁ to C ₄ alcohol is methanol or ethanol.
A pharmaceutical composition for preventing and treating wrinkles, which contains the megastiggman derivative represented by Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

(2)

6. The pharmaceutical composition for preventing and treating wrinkles according to claim 5, wherein the megastigman derivative or a pharmaceutically acceptable salt thereof inhibits elastase activity.
A cosmetic composition for preventing and improving wrinkles, which contains the megastiggman derivative represented by the following Chemical Formula 1 or Chemical Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

(2)

A food composition for preventing and improving wrinkles, comprising a megastigman derivative represented by the following Chemical Formula 1 or Chemical Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

(2)

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