KR102496590B1 - A composition for tissue repair that exhibits anti-inflammatory effects in tissues and excellent skin repair effects by inhibiting toxic substances of crosslinking agents - Google Patents

Abstract

The present invention relates to a composition for tissue repair that exhibits an anti-inflammatory effect in tissues and an excellent skin repair effect due to the inhibitory activity of toxic substances of a crosslinking agent. , Inflammation due to toxicity generated by the cross-linking agent added during cross-linking of hyaluronic acid can be prevented, thereby providing a safer filler.

Description

A composition for tissue repair showing an anti-inflammatory effect and an excellent skin repair effect due to the inhibitory activity of toxic substances in the tissue of a crosslinking agent

The present invention relates to a composition for tissue repair that exhibits an anti-inflammatory effect in tissue and an excellent skin repair effect due to the inhibitory activity of toxic substances of a crosslinking agent. More specifically, it relates to a composition for tissue repair that exhibits better anti-inflammatory and tissue repair effects by further including a natural extract in addition to hyaluronic acid.

Hyaluronic acid is a linear polymer polysaccharide in which β-D-N-acetylglucosamine and β-D-glucuronic acid are alternately bonded. It is known that hyaluronic acid is present in connective tissues such as subcutaneous tissue and cartilage tissue of mammals, as well as in the vitreous body of the eye, the umbilical cord, and the like, and also in the capsular membranes of streptococci and bacilli. Common methods for obtaining hyaluronic acid include a method of extracting from chicken combs, umbilical cords, etc., and a method of culturing streptococci and bacilli, followed by extraction and purification from them.

Natural hyaluronic acid, which has excellent biocompatibility, has no specificity between species and does not have tissue or organ specificity. It enhances skin moisturizing power, maintains skin elasticity, and reduces damage to the lower layer of the skin when skin is damaged. It also acts as a lubricant to facilitate the movement between the spaces. However, when natural hyaluronic acid is used as it is, not only does it have poor mechanical properties, but it is easily decomposed and removed by an enzyme called hyaluronidase present in the body, so there are limitations in various applications. In order to compensate for the disadvantages of natural hyaluronic acid, many studies have been conducted to form hydrogels through chemical modification or crosslinking using various crosslinking agents.

Hydrogel formation through chemical modification and crosslinking of hyaluronic acid is generally achieved through an alcohol group and a carboxylic acid group present in the main chain of hyaluronic acid. The carboxylic acid group of the main chain of hyaluronic acid is mainly chemically modified by esterification (D. Campoccia et al., Biomaterials, 19, 1998, 2101-2127), and the crosslinking reaction for hydrogel formation is dihydrazide (K. P. Vercruysse et al., Bioconjug. Chem., 8, 1997, 686-694), dialdehydes (Y. Luo et al., J. Control. Release, 69, 2000, 169-184), or disulfides ( X. Z. Shu et al., Biomacromolecules, 3, 2002, 1304-1311) was used for research. In addition, research on preparing a hydrogel through photocrosslinking by introducing methacrylamide into a carboxylic acid group has also been conducted (Y. D. Park et al., Biomaterials, 24, 2003, 893-900). On the other hand, the alcohol group of the hyaluronic acid backbone is divinylsulfone (A. Ramamurthi et al., J. Biomed. Master Res., 60, 2002, 195-205) or diglycidyl ether (T. Segura et al., Biomaterials, 26, 2005, 359-371) was used for research.

On the other hand, fillers were the first to be treated with autologous fat grafting in the 1890s, and later, paraffin and liquid silicone were used, but due to serious side effects, today, many countries including the United States and Korea are regulating illegal medical practices. After that, fillers using collagen, represented by Zyderm and Zyplast, were invented, but collagen fillers use animal-derived collagen, so an allergy test must be performed one month before the procedure, and the lifespan is relatively short, about 3 to 6 months (Baumann, L., J. Kaufman, and S. Saghari, Collagen fillers. Dermatologic therapy, 2006. 19(3): p. 134-140.). To compensate for this, hyaluronic acid-based fillers have been developed. Representatively, there are Hylaform and Restylane/Perlane, etc., and the lifespan is about twice as long as 6 to 12 months compared to collagen, and it is widely used now because it has the advantage of not having an allergy test.

However, hyaluronic acid itself has a short half-life of only several hours in the human body, which limits its application, and studies have been conducted to increase the half-life (persistence in the body) through cross-linking. For example, U.S. Patent No. 4,582,865 discloses a cross-linked hyaluronic acid derivative using divinylsulfone (DVS) as a cross-linking agent, and its hydrogel form is commercially available under the trade name Hylaform®, and U.S. Patent No. 5,827,937 is a multifunctional A method for preparing a cross-linked hyaluronic derivative using an epoxy compound as a cross-linking agent is disclosed, and hyaluronic acid prepared using 1,4-butanediol diglycidyl ether (BDDE) as a cross-linking agent as a double-functional epoxy compound. Restylane®, a hydrogel form of a cross-linked product, has been approved by the US FDA and is commercially available worldwide as a filler for tissue augmentation.

Such a cross-linked hyaluronic acid filler includes a filler made of a single phase (monophasic HA filler) and a filler made of two phases (biphasic HA filler). Since the monophasic hyaluronic acid filler is prepared using a homogenate containing crosslinked hyaluronic acid, it generally has low elasticity and high cohesive force. Accordingly, when the monophasic hyaluronic acid filler is injected into the skin, it is unlikely to escape from the injected site, but it does not maintain the injected shape for a long time, and the shape (shape) maintenance period is only about 2 months after the procedure. there is.

Bi-phasic hyaluronic acid fillers are prepared by using cross-linked hyaluronic acid particles alone or by mixing liquid-like non-cross-linked hyaluronic acid (untreated non-cross-linked hyaluronic acid, linear HA), so they generally have high elasticity. low cohesion. Accordingly, when the biphasic HA filler is injected into the skin, it can maintain its shape for a long time, but there is a problem in that it is highly likely to depart from the injected site. A representative example of such a biphasic HA filler is the aforementioned Restylane® (available from Galderma).

As such, the monophasic HA filler and the biphasic HA filler have advantages and disadvantages, respectively. An example of mixing the fillers to have both the characteristics of the conventional monophasic hyaluronic acid filler and biphasic hyaluronic acid filler is However, in this case, the advantages of both the monophasic hyaluronic acid filler and the biphasic hyaluronic acid filler are reduced together, making it unsuitable as a filler. Therefore, there is a need for a filler capable of maintaining its shape for a long time while having a low possibility of departing from the injected site.

Therefore, in order to prepare a filler that has an excellent skin repair effect while preventing inflammation caused by toxicity that may be caused by a crosslinking agent added for crosslinking hyaluronic acid by additionally including a natural extract in hyaluronic acid or showing an effect of improving inflammation did

KR 10-1856435 B1 KR 10-2020-0006509 A

An object of the present invention is to provide a tissue repair composition comprising hyaluronic acid and a natural extract and having an excellent skin repair effect.

An object of the present invention is to provide a composition for tissue repair comprising hyaluronic acid and a natural extract and exhibiting an anti-inflammatory effect against the toxicity of a small amount of a cross-linking agent added for cross-linking hyaluronic acid.

Another object of the present invention relates to a functional product including the tissue repair composition, skin repair effect and anti-inflammatory activity, and it is possible to provide a filler without side effects.

In order to achieve the above object, the composition for tissue repair according to an embodiment of the present invention is byeongjohipul extract; Mincuji tree extract; black ginseng extract; chamomile extract; And an extract selected from the group consisting of mixtures thereof and hyaluronic acid may be included as active ingredients.

The hyaluronic acid may be crosslinked by a crosslinking agent.

The crosslinking agent is butanediol diglycidyl ether (1,4-butandiol diglycidyl ether: BDDE), ethylene glycol diglycidyl ether (EGDGE), and hexanediol diglycidyl ether (1,6- hexanediol diglycidyl ether), propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, Neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether ), trimethylpropane polyglycidyl ether, bis-epoxypropoxyethylene (1,2- (bis(2,3-epoxypropoxy)ethylene), pentaerythritol polyglycidyl ether (pentaerythritol polyglycidyl ether) and sorbitol polyglycidyl ether (sorbitol polyglycidyl ether).

The hyaluronic acid has a molecular weight of 2,000,000 Da to 3,000,000 Da.

The extract is extracted using an extraction solvent selected from the group consisting of water, alcohol having 1 to 6 carbon atoms, and mixtures thereof.

A filler for tissue repair according to another embodiment of the present invention may include the composition for tissue repair.

Method for preparing a composition for tissue repair according to another embodiment of the present invention includes a pulverization step of pulverizing the dried natural product; An extract preparation step of extracting the pulverized product to prepare a natural extract; A cross-linking step of cross-linking hyaluronic acid using a cross-linking agent; Mixing step of mixing the natural extract and hyaluronic acid; And a stirring step of stirring the natural extract and hyaluronic acid; A first stirring step of stirring at 100 to 300 rpm for 20 to 30 minutes; And it may include a second stirring step of stirring at 100 to 150 rpm for 10 to 20 minutes, wherein the natural product is selected from the group consisting of buckwheat plant, mint chinensis, black chickweed, chamomile, and mixtures thereof. can

The composition for tissue repair may further include a buffer solution.

Hereinafter, the present invention will be described in more detail.

As used herein, the term 'extract' has a meaning commonly used in the art as a crude extract in the art as described above, but also includes fractions obtained by additional fractionation of the extract in a broad sense. That is, the extract includes not only those obtained using the above-described extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the extract through an ultrafiltration membrane having a certain molecular weight cut-off value, separation by various chromatography (made for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through the purification method are also included in the extract of the present invention.

The extract used in the present invention may be prepared in a powder state by additional processes such as distillation under reduced pressure and freeze drying or spray drying.

In the present invention, tissue repair may mean regenerating tissue such as temporarily or semi-permanently improving or restoring wrinkles on the face or body, improving contour, forming a sense of volume of tissue, or healing scars by injecting the composition.

The tissue may mean a face or a part of the body.

The composition for tissue repair may be filled in a syringe and injected into the skin layer.

In the present invention, hyaluronic acid is used as a meaning including both salts and derivatives thereof as well as hyaluronic acid itself. Therefore, hyaluronic acid used herein is a concept including all aqueous solutions of hyaluronic acid, aqueous solutions of salts of hyaluronic acid, aqueous solutions of hyaluronic acid derivatives, and aqueous mixtures thereof.

In order to achieve the above object, the composition for tissue repair according to an embodiment of the present invention is byeongjohipul extract; Mincuji tree extract; black ginseng extract; chamomile extract; And an extract selected from the group consisting of mixtures thereof and hyaluronic acid may be included as active ingredients.

The byeongjohuipul (Clematis heracleifolia DC.) is a deciduous shrub growing in the forest, reaching a height of 1m, and the lower part is woody, but the upper part dies. Leaves are opposite and consist of 3 leaflets, and leaflets are broad ovate, pointed, acute or axillary, and have teeth-like sawtooth. Flowers are heterogeneous and bloom in August-September. The fruit is an achene and has a flat oval shape. The root is used as a medicinal material, and has the effect of relieving pain and inflammation, so it is medicinal for gouty arthritis. However, byeongjohuipul is slightly toxic, so you should not eat more than the prescribed amount.

The Minkuji tree (Broussonetia papyrifera) is a deciduous broad-leaved tree of the dicotyledonous plant Nettle neck Moraceae and grows on the sunny side of the mountain at an altitude of 100 to 700 m. It is around 12m in height. The bark is dark gray, and the hairs grow densely on the small branches. Leaves are broad egg-shaped or round-shaped and alternate. The length is 7~20cm, the surface is dark green, hairless and glossy. There are sawtooth on the edge, the end is long and deeply dented like a tail, and the petiole is 3-10cm long and has hairs. Stipules are purple, egg-shaped, and fall early.

The black chickweed (Comarum palustre L.) is a perennial plant of the dicotyledonous plant Rosaceae and grows in the mountains or field 　 wetlands. It has short hairs all over, the stem stands straight, and the height is 30~60cm. Leaves are alternate, pinnate compound leaf once, and the petiole is long. Small leaves are long oval, 2.5-6cm long, 1-2cm wide, with sawtooth. Dark purple flowers bloom in June-July, forming cyme inflorescences, and sepals spread horizontally. Petals are egg-shaped, purplish, and shorter than sepals. The flower jaw is hairless and becomes flesh after the flower withers. The stems of the root part of the black chickweed are effective in pulmonary tuberculosis, diarrhea, thrombophlebitis, jaundice, neuralgia, toothache, stomach cancer, and mammary cancer.

The line deung rake (Vicia heptajuga Nakai) is a leguminous perennial plant that grows in mountains and fields. Leaves are alternate phyllotaxis, even pinnate compound leaves composed of 4-6 pairs of leaflets, and there are traces of tendrils at the ends. Leaflets are lanceolate, 2-6cm long, 8-10mm wide, dull at the base, pointed at the end, flat at the edges, triangular in shape, with sharp sawtooth. It has a slightly ridged angle, sometimes branched, and has a few short hairs.

The hyaluronic acid (HA) is a biosynthetic natural substance that is abundant in the skin of animals, etc., and is a hydrophilic substance because it contains many hydroxyl groups (-OH), and acts as a moisturizing agent in the skin of animals. Because it has a moisturizing effect, it is often added to cosmetics, etc., and reacts with CD44 protein expressed in various epithelial cells to regulate various physiological actions.

Hyaluronic acid is degraded in two ways in the body. First, it is degraded by hyaluronidase. can

The hyaluronidase is an enzyme that hydrolyzes hyaluronic acid and is a carbohydrate enzyme that hydrolyzes glycosidic bonds by acting on carbohydrates or glycosides. Hyaluronidase can be divided into three types, the first is an enzyme made in the testes of mammals (sheep and cows), the second is an enzyme made in the salivary glands of leeches or crustaceans, and the third is an enzyme made in bacteria.

It is known that the biodegradation of hyaluronic acid is very fast and all of them are degraded within 0.5 to several days. Therefore, to prevent rapid degradation of hyaluronic acid, it is recommended to reduce the speed of action of hyaluronidase by using cross-linked hyaluronic acid. desirable.

The composition for tissue repair of the present invention may be used by further including an extract selected from the group consisting of the above hyaluronic acid extract, mint extract, black perilla extract, sagebrush extract, and mixtures thereof,

More preferably, it contains 5 to 10 parts by weight of japonica extract, 5 to 10 parts by weight of chinensis extract, 1 to 5 parts by weight of black foxglove extract, and 1 to 5 parts by weight of rake extract, based on 100 parts by weight of hyaluronic acid. can

When used as a complex extract within the above weight part range, the hyaluronidase inhibitory effect is increased due to the complex action between each component, and as a result, the tissue repair effect is further improved, and the crosslinking agent is present in a small amount of hyaluronic acid. Inflammation caused by toxicity can be ameliorated.

The hyaluronic acid may be crosslinked by a crosslinking agent.

As described above, in order to overcome the short biodegradation of hyaluronic acid, cross-linking of hyaluronic acid was introduced, and when the molecular weight is increased by cross-linking, phagocytosis by leukocytes, especially monocytes, can be suppressed, and hyaluronic acid can be hydrolyzed. The rate of action of hyaluronidase, an enzyme that degrades it, is also reduced.

The cross-linking agent is also referred to as a bridge-crossing agent, and is a material that serves as a bridge between chains of chain-like polymers, and is an additive that can give mechanical strength and chemical stability such as hardness and elasticity to a resin.

The crosslinking agent is butanediol diglycidyl ether (1,4-butandiol diglycidyl ether: BDDE), ethylene glycol diglycidyl ether (EGDGE), and hexanediol diglycidyl ether (1,6- hexanediol diglycidyl ether), propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, Neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether ), trimethylpropane polyglycidyl ether, bis-epoxypropoxyethylene (1,2- (bis(2,3-epoxypropoxy)ethylene), pentaerythritol polyglycidyl ether It may be at least one selected from the group consisting of (pentaerythritol polyglycidyl ether), sorbitol polyglycidyl ether, and (tetraethylene glycol dimethacrylate).

More preferably, it is preferable to use butanediol diglycidyl ether (1,4-butandiol diglycidyl ether: BDDE) because of its low toxicity compared to other crosslinking agents.

However, even if a small amount of the cross-linking agent added for cross-linking of hyaluronic acid is included, a toxin reaction may occur, which may cause an inflammatory reaction in tissues caused by the toxin.

Therefore, the present invention, in order to prevent the inflammatory reaction due to the toxicity, further extracts selected from the group consisting of hyaluronic acid, rhododendron japonica extract, chinensis extract, black foxglove extract, chamomile extract, and mixtures thereof. to include

In addition to the above extract, it may further preferably further include a cottonseed hemp extract and a dandelion extract.

The dog cotton horse (Onoclea orientalis Hook.) is a perennial plant of the cotton horse family, which is also known as Mogwanjung and lives in the forests of mountains. The rhizome is thick and grows obliquely, the lower part of the dry petiole remains, and the leaves are clumped. The trophic leaf is larger than the spore leaf, and the petiole of the trophic leaf is 20 to 60 cm long and has dense scales. Scale pieces are string-shaped lanceolate or narrow egg-shaped, 7-20mm long, 1.5-4mm wide, pointed at the end, light brown, membranous (translucent like thin paper), glossy, with flat edges. The leaf body of the trophic leaf is long egg-shaped or long elliptical, 30-50 cm long and 20-30 cm wide, and the tip suddenly narrows to become sharp and split into a pinnate once. The leaf pieces are 8-20 pairs, narrow lancet-shaped, without a stalk, and narrow scales hanging on the axis in the middle of the back side, split into feathers. The cracked pieces are egg-shaped, 4-7mm wide, and have fine teeth on the edge. The dried rhizome of Gamyeonma has the effect of lowering fever, detoxifying, and stopping coughing.

The dandelion (Crepis tectorum L.) is an annual plant of the Asteraceae family, which means that it is similar to a dandelion. there is. Stem leaves become narrower as they go up, becoming about 1 cm long, and the lower part covers the stem, and the edge is flat. When the stem or leaves are cut, a white sap comes out. Flowers bloom in yellow at the ends of stems and branches from May to July, and several capitulums gather to form corymbose inflorescences. The diameter of the capitulum is about 2 cm, and there are only several tongue-shaped flowers without tubular flowers. There are hairs and glandular hairs on the peduncle and involucre. The gun pieces are arranged in two rows, the outer piece being shorter than the inner piece.

In the case of further including the Gamyeon hemp extract and the Dandelion extract, it is possible to increase the content of the active ingredient in the extract of the activated active ingredient in the buckwheat plant, mint chinensis, black chinensis, and chamomile, and further reduce the hyaluronidase activity. Accordingly, a more excellent skin repair effect can be exhibited through the effect of inhibiting hyaluronic acid degradation, and inflammation can be further improved due to toxicity that may exist in hyaluronic acid cross-linked by the cross-linking agent.

Even more preferably, the tissue repair composition may include 1 to 5 parts by weight of Gamyeon hemp extract and 0.1 to 2 parts by weight of nadodandelion extract, based on 100 parts by weight of hyaluronic acid.

The hyaluronic acid has a molecular weight of 2,000,000 Da to 3,000,000 Da.

If the average molecular weight of the hyaluronic acid is less than 2,000,000 Da, the effect of inhibiting phagocytosis by monocytes is insufficient, so when administered for tissue repair, the tissue repair effect (filler function) may be insufficient due to rapid biodegradation. That is, when the molecular weight is high compared to when the molecular weight is small, the area exposed to the attack of the degrading enzyme is reduced. Therefore, hyaluronic acid has excellent tissue repair effect durability when the molecular weight is 2,000,000 Da or more, more preferably in the range of 2,000,000 Da to 3,000,000 Da. If applicable, the tissue repair durability is better, but is not limited thereto.

The extract is extracted using an extraction solvent selected from the group consisting of water, alcohol having 1 to 6 carbon atoms, and mixtures thereof.

More specifically, water is used as an extraction solvent, but it is not limited to the above examples and can be used without limitation.

A method for preparing the extract may be a conventional extraction method in the art, such as a reflux extraction method, an leaching method, and an ultrasonic extraction method. Specifically, it may be an extract obtained by extracting a natural product from which foreign substances are removed by washing and drying with water, alcohol having 1 to 6 carbon atoms, or a mixed solvent thereof, or may be an extract extracted by sequentially applying the solvents to a sample.

The reflux extraction method is based on 100 mL of alcohol having 1 to 6 carbon atoms, 10 to 30 g of pulverized natural product, 1 to 3 hours of reflux, and 50 to 100% of alcohol having 1 to 6 carbon atoms. More specifically, based on 100 mL of alcohol having 1 to 6 carbon atoms, 10 to 20 g of pulverized natural product, 1 to 2 hours of reflux, and 70 to 90% of alcohol having 1 to 4 carbon atoms.

The leaching method is by 15 to 30 ℃, for 24 to 72 hours and 50 to 100% of alcohol having 1 to 6 carbon atoms. More specifically, at 20 to 25° C. for 30 to 54 hours and 70 to 80% of an alcohol having 1 to 6 carbon atoms.

The ultrasonic extraction method is by 30 to 50 ℃, 0.5 to 2.5 hours and 50 to 100% of alcohol having 1 to 6 carbon atoms. Specifically, at 40 to 50° C. for 1 to 2.5 hours and 70 to 80% of an alcohol having 1 to 6 carbon atoms.

The extraction solvent may be used in an amount of 2 to 50 times, more specifically, 2 to 20 times, based on the weight of the sample. For extraction, the sample may be left for leaching in the extraction solvent for 1 to 72 hours, more specifically 24 to 48 hours.

After extraction, the extract can be fractionated by sequentially applying a fresh fractionation solvent. The fractionation solvent used in the fractionation is at least one selected from the group consisting of water, hexane, butanol, ethylacetic acid, ethyl acetate, methylene chloride, and mixtures thereof, and is preferably ethyl acetate or methylene chloride. After obtaining the extract or fraction, a method such as concentration or lyophilization may be additionally used.

A filler for tissue repair according to another embodiment of the present invention may include a composition for tissue repair.

Method for producing a composition for tissue repair according to another embodiment of the present invention comprises a pulverization step of pulverizing the dried natural product; An extract preparation step of extracting the pulverized product to prepare a natural extract; A cross-linking step of cross-linking hyaluronic acid using a cross-linking agent; Mixing step of mixing the natural extract and hyaluronic acid; And a stirring step of stirring the natural extract and hyaluronic acid; A first stirring step of stirring at 100 to 300 rpm for 20 to 30 minutes; And it may include a second stirring step of stirring at 50 to 150 rpm for 10 to 20 minutes, wherein the natural product is selected from the group consisting of buckwheat plant, mint chinensis, black chickweed, chamomile, and mixtures thereof. can

The crushing step and the manufacturing step are a method for preparing a natural extract, which may include a natural product selected from the group consisting of buckwheat plant, minkuna tree, black chickweed, chamomile, and mixtures thereof.

The cross-linking step is a step of adding a cross-linking agent for cross-linking of hyaluronic acid, and is a step for increasing the molecular weight to 2,000,000 Da to 3,000,000 Da.

The extract prepared in the mixing step, Minkuji tree extract, Black Cyperus extract, chamomile extract and hyaluronic acid may be mixed.

Thereafter, the extract and hyaluronic acid may be uniformly mixed through a stirring step. The stirring step includes a first stirring step and a second stirring step, specifically, a first stirring step of stirring at 100 to 300 rpm for 20 to 30 minutes, and a second stirring step of stirring at 50 to 150 rpm for 10 to 20 minutes. A stirring step may be included.

In the case of stirring in two steps as described above, the mixed extract and hyaluronic acid in the mixing step can be mixed at a more uniform concentration, and more specifically, when the rotation speed is less than 100 rpm in the first stirring step. Bubbles may occur in the composition, and when mixing at a rotational speed of more than 300 rpm, the temperature of the composition increases due to the rotational speed, which may cause denaturation in the case of temperature-sensitive hyaluronic acid, so preferably 100 to 300 rpm It is preferable to stir for 20 to 30 minutes.

In the second stirring step, the extract and hyaluronic acid mixed in the first stirring step are stirred at a lower rotational speed to lower the temperature rise of hyaluronic acid, and it is preferable to stir at 50 to 150 rpm for additional mixing. After the step, when mixing at a speed of less than 50 rpm, there is a possibility of additional foaming of the composition, and precipitation may occur due to the combination of hyaluronic acid and natural extract, and when mixing at a speed of more than 150 rpm, the temperature is affected by the rotation speed. Hyaluronic acid denaturation problems may occur due to elevation, so it is preferable to stir for 10 to 20 minutes at a speed of 50 to 150 rpm in the second stirring step.

In addition, when stirring at the preferred speed described above, the viscosity of the tissue repair composition corresponds to 20,000 cP to 50,000 cP, thereby providing a tissue repair composition containing hyaluronic acid that exhibits both monophasic and biphasic hyaluronic acid properties. there is.

The monophasic hyaluronic acid and biphasic hyaluronic acid correspond to cross-linked hyaluronic acid, and monophasic hyaluronic acid has low elasticity and less lumps, enabling medium volume increase and duration, and is used for relatively thick skin. The ideal hyaluronic acid has high elasticity and forms lumps well, so it is used for a large amount of volume increase, long-term persistence, and relatively thin skin.

The composition for tissue repair may further include a buffer solution.

In preparing the composition for tissue repair, a buffer solution may be further included, and other conventional additives, dispersants, lubricants, binders, diluents, etc. may be additionally added and used, as required by those skilled in the art. The configuration to be may be additionally included, but is not limited thereto.

The present invention provides a composition for tissue repair comprising hyaluronic acid and a natural extract and having an excellent skin repair effect.

The present invention provides a composition for tissue repair comprising hyaluronic acid and a natural extract and exhibiting an anti-inflammatory effect against the toxicity of a small amount of a cross-linking agent added for cross-linking hyaluronic acid.

A filler according to another embodiment of the present invention relates to a functional product including the tissue repair composition, skin repair effect and anti-inflammatory activity, and provides a filler without side effects.

1 is a flow chart of a method for manufacturing a composition for tissue repair according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein.

[Preparation Example 1: Preparation of extract]

1. Manufacture of B. chinensis extract (CH)

After washing and drying the bottled greens, it was pulverized. The pulverized product was mixed with purified water, maintained at 98 to 100° C. for 2 hours, cooled, and then filtered with Whatman filter paper to obtain the filtrate to prepare an extract (CH) of the pulverized plant.

2. Preparation of other natural extracts

Minkuji extract (BD), black foxglove extract (PS), and chamomile extract (VH) were prepared in the same manner as the B. chinensis extract (CH).

[Preparation Example 2: Preparation of composition for tissue repair]

1. Preparation of cross-linked hyaluronic acid (HA)

Dissolve sodium hyaluronate at a concentration of 100 mg/ml in 0.25 N sodium hydroxide (NaOH) aqueous solution, and mix with 50 mM 1,4-butanediol diglycidyl ether (BDDE) as a cross-linking agent. (Mixer) After mixing homogeneously, the mixer was placed in a constant temperature water bath and crosslinked at a temperature of 35 ° C. for 18 hours to proceed with the crosslinking step.

2. Preparation of composition for tissue repair

Cross-linked hyaluronic acid (HA), chinensis extract (CH), chinensis extract (BD), black lentil extract (PS), and chamomile extract (VH) were mixed in the composition shown in Table 1 below.

TR1 TR2 TR3 TR4 TR5 TR6 HA 100 100 100 100 100 100 CH - One 5 7 10 15 BD - One 5 7 10 15 PS - 0.1 One 3 5 10 VH - 0.1 One 3 5 10

(unit weight parts)

Thereafter, the stirring step was performed, the first stirring step was performed at 200 rpm for 20 minutes, and the second stirring step was performed at 100 rpm for 10 minutes.

[Preparation Example 3: Preparation of Filler Containing Composition for Tissue Repair]

The tissue repair composition TR4 prepared in Preparation Example 1 was transferred to a tank containing a buffer solution, washed and swollen, and the washing solution was removed.

Thereafter, after confirming that the pH of the cleaning solution is in the neutral range, the tissue repair composition that has undergone the washing and swelling steps is transferred to a sterilization tank, heat-treated at a temperature of 100 ° C or higher for about 90 seconds, vacuum filled in a syringe by a predetermined amount, and washed And a syringe containing the tissue repair composition subjected to the swelling step was steam sterilized at a temperature of 100 ° C. or higher for about 10 minutes to prepare a filler.

[Experimental Example 1: Evaluation of inhibition of hyaluronic acid degradation]

50 μL of hyaluronidase solution (1 mg/mL) (sigma-aldrich) was placed in a microtube, 50 μL of each of TRs 1 to 6 prepared in Preparation Example 2 was added, and incubated at 37° C. for 10 minutes. The reaction was stopped by adding 50 μl of aqueous sodium hydroxide solution (0.2 M). After boiling this solution for 3 minutes, it was cooled with running water, 300 μL of 10% p-DMAB (dimethylaminobenzaldehyde) solution was added, and the mixture was incubated at 37° C. for 20 minutes. After returning to room temperature, 100 μL each was transferred to a 96 well plate, and OD585 nm was measured using POWERSCAN HT. After setting the absorbance value to 100% with TR1 not containing the natural extract as a control, the relative results of the comparison are shown in Table 2 below.

TR1
(control group) TR2 TR3 TR4 TR5 TR6 Activity of hyaluronidase on TR1 (%) 100 97.46 93.31 92.16 93.72 96.55

(unit %)

According to Table 2, compared to TR1, which does not contain natural extracts and contains only hyaluronic acid, B. chinensis extract (CH), chinensis extract (BD), black foxglove extract (PS), and chamomile extract (VH ), it can be confirmed that the activity of hyaluronidase is lowered in the case of TR2 to TR6 containing the natural extract, and in the case of TR3 to TR5 containing a preferred weight part of the natural extract, the activity of hyaluronidase is further lowered. In the case of TR1, the hyaluronidase activity for TR1 was 93.31%, which was the lowest level, so that preferred parts of hyaluronic acid, A. It was confirmed that the hyaluronic acid decomposition inhibitory effect was excellent when the rake extract (VH) was included.

[Experimental Example 2: Evaluation of toxicity and anti-inflammatory effect of cross-linked hyaluronic acid]

1. MTT evaluation for TR1

Toxicity evaluation of TR1 was performed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (3-(4,5-dimethyl-2-thiazolyl)-2,5 -diphenyl-2H-tetrazolium, MTT) analysis method was used. For the MTT evaluation, toxicity analysis was performed on 100 μl of 1 ml of the eluate released from TR1 for 72 hours, and the toxicity evaluation results of the eluate released from Teflon, a positive control group, were compared and analyzed. A toxicity evaluation of the dosage composition was performed.

More specifically, in order to evaluate cytotoxicity, 100 μl of 1 ml of the eluate obtained by elution in a buffer solution for 72 hours in vitro for TR1 was injected into cultured fibroblasts, and 24 hours after MTT, Cell viability was quantified and evaluated through the assay.

As a result of evaluating the viability of fibroblasts through the MTT assay, a result showing cell viability was obtained compared to the viability of fibroblasts in the positive control group (P<0.05)

2. MTT evaluation for TR2 to 6

The MTT evaluation for TRs 2 to 6 was performed in the same manner as the MTT evaluation for TR1.

The MTT evaluation of TR1 was fixed at an index of 5 and used as a standard, and the result of including the natural extract was compared with this and expressed as an index of 1 to 10. The higher the index, the better the anti-inflammatory activity, and the results are shown in Table 3 below.

TR1 TR2 TR3 TR4 TR5 TR6 MTT evaluation 5 6 7 8 8 6

(unit exponent)

According to Table 3, compared to TR1 containing only hyaluronic acid, TR2 additionally containing chinensis extract (CH), chinensis extract (BD), black foxglove extract (PS) and sagebrush extract (VH) It can be confirmed that MTT evaluation values are more excellent in the case of TR6 to TR6, and in particular, as TR3 to TR5 show higher MTT evaluation, a small amount is included in the hyaluronic acid crosslinked by the crosslinking agent when the preferred weight part of the extract is included. It was confirmed that it can exhibit inhibitory activity against toxic substances, and through this, the occurrence of inflammation caused by toxicity can be prevented.

[Experimental Example 3: Evaluation of Bubble Formation, Viscosity and Precipitation of Tissue Repair Composition]

The tissue repair composition TR4, which proceeded from Preparation Example 2 to the mixing step, was stirred under the stirring conditions of AS1 to AS10 in Table 4 below, and the viscosity, bubble formation, and precipitation according to the stirring speed of the tissue repair composition were observed.

The tissue repair composition according to the stirring conditions was placed in a transparent container and bubble formation and viscosity were observed with the naked eye, and after 18 hours, whether or not precipitation occurred was observed, and the observation results are comprehensively shown in Table 5 below.

<Whether bubbles are formed>

bubbles are formed. : ○

No bubble formation. :X

<Viscosity>

Suitable for 20,000 to 50,000 cP.

<Whether sedimentation occurs>

Precipitation occurs. : ○

No precipitation occurs. :X

stirring process 1st agitation rate 2nd agitation rate AS1 50 rpm - AS2 100 rpm - AS3 200 rpm - AS4 300 rpm - AS5 400 rpm - AS6 200 rpm 30 rpm AS7 200 rpm 50 rpm AS8 200 rpm 100 rpm AS9 200 rpm 150rpm AS10 200 rpm 200 rpm

(unit rpm)

viscosity Whether bubbles are formed Precipitation occurs or not AS1 12543.8 cP ○ ○ AS2 16543.8 cP X ○ AS3 17909.4 cP X ○ AS4 18154.4 cP X ○ AS5 12248.6 cP X ○ AS6 18624.6 cP ○ ○ AS7 35161.5 cP X X AS8 35798.1 cP X X AS9 34983.2 cP X X AS10 19316.4 cP X X

As shown in Table 5, as a result of confirming the formation of bubbles in the tissue repair composition of TR4, the first stirring was performed at 50 rpm, and the first stirring was performed at 200 rpm, but the second stirring was performed at 30 rpm. It can be confirmed that bubbles are formed.

In addition, as a result of checking the viscosity according to the stirring process, it was confirmed that in the case of AS1 to 5 in which only the first stirring was performed, the viscosity was less than 20,000 Cp, indicating a viscosity in an unsuitable range. Among them, the first stirring speed was 100 rpm to 300 rpm, it was confirmed that the viscosity was close to 20,000 cP.

In the case of AS6 to AS10 in which the second stirring was further performed after the first stirring, the viscosity was higher than that of AS6 to AS10. It is preferable to proceed with both the primary stirring and the secondary stirring, and it was confirmed that the most preferred viscosity was obtained when stirring at a speed of 100 rpm to 300 rpm and 50 to 150 rpm for the secondary stirring.

In addition, when only the first stirring, which is the stirring condition of AS1 to AS5, was not mixed uniformly, it was confirmed that precipitation occurred due to the combination of hyaluronic acid and natural extract, and AS6 to AS10 with the second stirring In the case of AS6 stirring with a secondary stirring speed of 50 rpm or less, precipitation also occurred, and in the case of AS7 to AS10 with a secondary stirring speed of 50 rpm or more, it was confirmed that no precipitation occurred.

[Preparation Example 4: Preparation of Composition for Tissue Repair Containing Cabbage Hemp Extract and Dandelion Extract]

1. Preparation of cotton hemp extract and dandelion extract

In the same manner as in Preparation Example 1 for preparing the extract (CH) of B. chinensis extract (OH) and dandelion extract (CT), respectively, were prepared.

2. Preparation of Composition for Tissue Repair Containing Komyeon Hemp Extract and Dandelion Extract

A composition for tissue repair containing a composite extract was prepared by mixing the prepared Gaeyeon hemp extract (OH) and dandelion extract (CT) containing TR4 and mixing them as shown in Table 6 below.

MTR1 MTR2 MTR3 MTR4 MTR5 TR4 TR4 TR4 TR4 TR4 TR4 OH 0.1 One 3 5 10 CT 0.01 0.1 One 2 5

(unit weight parts)

[Experimental Example 4: Evaluation of tissue repair composition containing composite extract]

The effects of MTR1 to MTR5 in Table 6 were compared and shown in the same method as the experimental method of the tissue repair composition shown in Experimental Examples 1 and 2.

The results are comprehensively shown in Table 7 below, and for objective comparison, the effect of TR4 is represented by an index of 5, and the higher the index, the better.

The index was expressed as a number from 1 to 10.

TR4 MTR1 MTR2 MTR3 MTR4 MTR5 hyaluronic acid
Degradation inhibition degree 5 6 7 8 8 6 anti-inflammatory effect 5 6 7 8 7 6

(unit exponent)

According to Table 7, it can be confirmed that when TR4 further includes a cottonseed hemp extract (OH) and a dandelion extract (CT), the improvement is further compared to the composition for tissue repair containing only TR4, especially in the case of MTR2 to MTR4. It can be seen that the activity is more excellent.

Therefore, in the case of the composition for tissue repair of the present invention, 5 to 10 parts by weight of hyaluronic acid, 5 to 10 parts by weight of japonica japonica extract, 5 to 10 parts by weight of cinnamon plant extract, 1 to 5 parts by weight of black cinnamon plant extract, In the case of including 1 to 5 parts by weight of sagebrush extract, 1 to 5 parts by weight of Gamyeon hemp extract, and 0.1 to 2 parts by weight of Dandelion extract,

As the activity of hyaluronidase that decomposes hyaluronic acid is lowered, the sustained tissue repair effect is more excellent, and the anti-inflammatory effect can be exhibited against inflammation caused by toxicity that may appear from the crosslinking agent included by crosslinking hyaluronic acid.

Compared to commercially available fillers, it is possible to provide a filler with excellent tissue repair effect and high stability.

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concept of the present invention defined in the following claims are also made according to the present invention. falls within the scope of the rights of

Claims (8)

chinensis extract, mint extract, black chickweed extract, sagebrush extract and containing hyaluronic acid
A composition for tissue repair. According to claim 1,
The hyaluronic acid is cross-linked by a cross-linking agent
A composition for tissue repair. According to claim 2,
The crosslinking agent is butanediol diglycidyl ether (1,4-butandiol diglycidyl ether: BDDE), ethylene glycol diglycidyl ether (EGDGE), and hexanediol diglycidyl ether (1,6- hexanediol diglycidyl ether), propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, Neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether ), trimethylpropane polyglycidyl ether, bis-epoxypropoxyethylene (1,2- (bis(2,3-epoxypropoxy)ethylene), pentaerythritol polyglycidyl ether At least one selected from the group consisting of (pentaerythritol polyglycidyl ether) and sorbitol polyglycidyl ether
A composition for tissue repair. According to claim 1,
The hyaluronic acid has a molecular weight of 2,000,000 Da to 3,000,000 Da
A composition for tissue repair. According to claim 1,
The extract is extracted using an extraction solvent selected from the group consisting of water, alcohol having 1 to 6 carbon atoms, and mixtures thereof.
A composition for tissue repair. Claims 1 to 5, comprising a tissue repair composition according to any one of the preceding
Filler for tissue repair. A pulverization step of pulverizing the dried natural product;
An extract preparation step of extracting the pulverized product to prepare a natural extract;
A cross-linking step of cross-linking hyaluronic acid using a cross-linking agent;
Mixing step of mixing the natural extract and hyaluronic acid; and
Stirring step of stirring the natural extract and hyaluronic acid; Including,
The stirring step may include a first stirring step of stirring at 100 to 300 rpm for 20 to 30 minutes; And a second stirring step of stirring at 50 to 150 rpm for 10 to 20 minutes,
The natural product is to include buckwheat grass, minkuna tree, black chickweed and seondeung rake
A method for preparing a composition for tissue repair. According to claim 7,
The composition for tissue repair further comprises a buffer solution
A method for preparing a composition for tissue repair.

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