KR101807215B1 - Antidote composition for inhibiting toxic protease of natural venom - Google Patents

Abstract

The present invention relates to an antidote composition for inhibiting a toxic protease of natural venom comprising mangiferin as an active ingredient, which inhibits and reduces lethality of skin cells due to natural venom of jellyfish, snake and the like and inhibits activity and protein expression of substrate metalloproteinase, which is one of skin necrosis causing substances, and thus can be used as an antidote cytotoxicity by natural venom, wherein the mangiferin is a naturally occurring substance to have high stability and has an excellent cytotoxic effect, thereby being useful in the field of wound healing agents.

Description

TECHNICAL FIELD The present invention relates to an antidote composition for inhibiting toxic protease of natural venom,

The present invention relates to an antidote to cytotoxicity by natural poison containing maculopyrine as an active ingredient.

Jellyfish stinging accidents on the waterfront due to the rise of jellyfish due to rising sea water temperature are increasing each year. In 2016, damage to jellyfish stones in the beaches in Busan has tripled from 2105, and it is expected to increase steadily in the future. In addition, the number of snake-bite patients in Korea for the past five years is about 20,000. The number of jellyfish stinging accidents increases year by year, and many patients are attacked by toxic organisms such as snakes, scorpions and spiders. However, there are no drugs or first aid products available in the country.

The protease of natural poison is recognized as an important cause of skin necrosis when poisoned or shot by toxic organisms. Previously used steroid ointments and antihistamines have side effects such as atrophy of the skin tissue and enlargement of the capillaries of the skin. However, there is no side effect yet, and there is no therapeutic agent with excellent therapeutic effect.

Accordingly, the present inventor searched for a substance having a neutralizing effect against a natural poison that expresses a native protease in the case of a snake, a scorpion, a jellyfish, or a saw or biter.

Meanwhile, Korean Patent No. 1487224 discloses a 'composition for combating jellyfish' using an extract of anthocyanin and / or a camphor tree extract that inhibits the action of toxin from jellyfish jelly, However, the cytotoxic antidote of natural poisons containing the macroreticular perine of the present invention as an active ingredient directly suppresses the toxicity of the components of the toxin solution, and its mechanism of action is different.

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above needs, and it is an object of the present invention to provide a therapeutic agent for skin diseases, which is characterized by neutralizing the protease activity and skin cell toxicity contained in seven different natural poisons such as jellyfish and snakes, The present inventors have completed the present invention by confirming that skin necrosis and activity of matrix metalloproteinase are inhibited when macroreticular perine is treated on scars of injected rats.

In order to solve the above problems, the present invention provides an antidote to cytotoxicity by natural poison containing maculiferin or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides an external preparation for skin for treating wounds injured or shot by jellyfish, snakes, scorpions or spiders containing maculopyrine or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a pharmaceutical composition for treating wounds injured or injured by a jellyfish, a snake, a scorpion or a spider containing maculopyrine or a pharmaceutically acceptable salt thereof as an active ingredient.

The composition of the present invention containing macaferrin as an active ingredient inhibits and alleviates lethality of skin cells caused by natural poisons such as jellyfish and snakes and inhibits the activity and protein expression of matrix metalloproteinase It can be used as an antidote to cytotoxicity by natural toxin. Since manganese perrine is a naturally occurring substance, its stability is high and cell necrosis inhibiting effect is excellent, it can be usefully used in the field of wound treatment.

FIG. 1 shows the results of confirming that macel ferrin shows a protease inhibitory activity in various natural poisons. -: natural poison treatment group; +: Additional treatment of natural poison with manganese perrine.
FIG. 2 shows the results of confirming the neutralization effect of manganese perrine on human skin cell toxicity of various natural poisons. M: Mackerel Perrin.
FIG. 3 shows the results of confirming the mitigation effect of Nomura embryonic jellyfish venom of mangiferin on experimental animal skin toxicity.
4 is a result of confirming the change of skin necrosis factor (MMP) in experimental animal tissues by Nomura embryo jellyfish venom of mangiferin.

In order to achieve the object of the present invention, the present invention provides an antidote to cytotoxicity by natural poison containing maculiferin or a pharmaceutically acceptable salt thereof as an active ingredient.

The natural venom can be a jellyfish, a snake, a scorpion or a spider in Germany, preferably a jellyfish or a snake in Germany, but is not limited thereto.

According to one embodiment of the present invention, the jellyfish may be, but not limited to, Nomura embryo jellyfish, curtain eel jellyfish, ghost jellyfish, forest root jellyfish, mora cubic jellyfish and the like.

According to one embodiment of the present invention, the snake may be, but is not limited to, a Western diamond rattlesnake ( Crotalus atrox ) or Agkistrodon contortrix laticinctus .

According to one embodiment of the present invention, the macelferrin inhibits the activity of a toxic protease contained in a natural poison and can neutralize human skin cell toxicity caused by a natural poison.

The cytotoxic antidote of the present invention can protect cells by inhibiting the activity of matrix metalloproteinase in cells exposed to natural toxin, which is an active ingredient, mangiferin.

The macrorpin compounds of the present invention may exist in the form of pharmaceutically acceptable salts. Salts are useful as acid addition salts formed by pharmaceutically acceptable free acids. The term "pharmaceutically acceptable salt" of the present invention means a concentration that has a relatively nontoxic and innocuous beneficial effect on a patient, and that the side effect due to this salt does not impair the beneficial effects of the netting perine Or an inorganic addition salt.

The addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The same molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration. As the free acid, an organic acid and an inorganic acid can be used. As the inorganic acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acid include methanesulfonic acid, p- toluenesulfonic acid, acetic acid, trifluoroacetic acid, Maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycollic acid, Gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid and the like can be used But are not limited to these.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal salt or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble salt salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically acceptable to produce sodium, potassium, or calcium salt, but not limited thereto. The corresponding silver salt can also be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

The pharmaceutically acceptable salts of the macroreticular perineal compounds, unless otherwise indicated, include salts of acidic or basic groups that may be present in the macroreticular perineal compounds. For example, pharmaceutically acceptable salts may include sodium, calcium and potassium salts of hydroxy groups, and the other pharmaceutically acceptable salts of amino groups include hydrobromides, sulphates, sulphates, phosphates, hydrogen phosphates (Hydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts and the like, ≪ / RTI >

The present invention also provides an external preparation for skin for treating wounds injured or shot by jellyfish, snakes, scorpions or spiders containing maculopyrine or a pharmaceutically acceptable salt thereof as an active ingredient.

The external preparation for skin for treating wounds injured or injured by a jellyfish, a snake, a scorpion or a spider of the present invention is prepared by the formulation of a cream, a gel, a patch, an ointment, a warning agent, a lotion agent, a liquid agent, an emulsion, a powder agent or a spray But is not limited thereto.

When the formulation of the present invention is a cream or a gel, animal fibers, plant fibers, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

When the formulation of the present invention is a liquid or an emulsion, a solvent, a solvent or an emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

The present invention also provides a pharmaceutical composition for treating wounds injured or injured by a jellyfish, a snake, a scorpion or a spider containing maculopyrine or a pharmaceutically acceptable salt thereof as an active ingredient.

The pharmaceutical compositions for treating jellies, snake, scorpion or spider-borne lesions of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent in addition to the maculopyrine. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, textol sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, and a mixture of methylhydroxybenzo.

Dosage forms of the compositions according to the present invention may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set.

The pharmaceutical compositions for treating wounds injured or injured by jellyfish, snake, scorpion or spider according to the present invention can be used for oral administration such as capsules, powders, granules, tablets, suspensions, emulsions, syrups and aerosols And may be formulated in the form of a formulation, external preparation, suppository or sterile injection solution. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used diluents, various excipients such as wetting agents, suspending agents, emulsions, freeze- And the like. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include tapestry, macrogol, tween 61, cacao paper, laundromat, glycerogelatin and the like.

The invention also provides a method of treating a jellyfish, snake, scorpion, or spider-bitten or thrown object comprising administering a jellyfish, a snake, a scorpion, or a spider to a mating subject.

As used herein, the term "individual" refers to all animals, including humans, monkeys, dogs, goats, pigs, or rats, that can be treated with jugular, periwinkle, For example, mammals other than humans.

In the method according to the present invention, the route of administration of buckwheat perennial may be administered via any conventional route so long as it can reach the target tissue. The method of administration is not particularly limited, and it can be administered by contents, external application, and injection. The preferred dosage of the macula perrine of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. The administration may be carried out once a day or divided into several times.

Hereinafter, the present invention will be described in detail with reference to Examples. The following examples are illustrative of the present invention, but the present invention is not limited to the following examples.

Materials and methods

1. Assessment of natural toxicity of mangiferin to inhibit toxic protease activity

Among the various organisms with toxic proteases, five kinds of jellyfish (Nomura, jellyfish, curtain, jellyfish, forest jellyfish, or mora cubic jellyfish) Experiments were carried out using a Western diamond rattlesnake ( Crotalus atrox ) or Agkistrodon contortrix laticinctus venom ( Agkistrodon contortrix laticinctus ). The toxic protease is known to have skin necrosis, haemolysis and cytotoxicity. In the present study, we investigated the effect of manganese perrine on inhibition of toxic protease activity by extracting each venom with PBS and using gelatin zymography method. 1% gelatin was mixed with 10% SDS-PAGE gel to obtain gelatin zymograpy gel And a total of seven kinds of extracted natural poisons (five species of jellyfish venom (Nomura's Jellyfish, curtains pelagic jellyfish, ghostly jellyfish, forest roots jellyfish or Mora cubic jellyfish-derived poisons) and snake venom of two (Western diamondback rattlesnake (Crotalus atrox) Or Agkistrodon contortrix laticinctus ) were loaded, respectively, and electrophoresis was performed at 4 ° C and 60 V. The gel was then diluted 2.5% in Triton X-100 solution for 30 minutes The reaction buffer (pH 7.4) was prepared by mixing the geranium perlein and the enzyme was inhibited by incubating the gel in the reaction buffer at 37 ° C for 16 hours.

2. Neutralization effect of maculopyrine on human skin cytotoxicity of natural poison

First, to determine whether toxicity of natural toxins in human skin cells, HaCaT human cell keratinocyte was cultured in a 24-well plate at 1 × 10 ⁵ cells / ml for 24 hours. After washing the cells with clean PBS, the cells were replaced with fresh culture medium. Natural toxin was treated at different concentrations and cultured for 24 hours. Cell viability was determined by MTT assay, and the cytotoxicity of each natural toxin was investigated. To investigate the cytotoxic neutralizing effect of manganese perrine on the natural venom, each natural venom was treated with cells, and at the same time, manganese perrine was treated by concentration. Twenty - four hours later, cell viability was determined by MTT assay.

3. Mitigation effects of manganese perrine on animal skin toxicity of Nomura marine jellyfish

Animal experiments were carried out to investigate the effect of Nomura agate jellyfish on skin necrosis. SD rats were supplied and used for the experiment after 1 week of laboratory purification. The experimental animals were conditioned at 23 ± 2 ℃, 55 ± 10% relative humidity and 12 hours of illumination. This experiment was conducted with approval of the Animal Ethics Committee of Gyeongsang National University. The day before the test substance treatment, the animal was previously depilated using a clipper on the back surface of the animal. As a negative control, 200 μl of PBS was injected intradermally, and 200 μl of Nomura cane jellyfish was injected intradermally.

In order to confirm the toxic neutralization effect of the manganese perrine, mango perine cream was prepared by mixing mango perrine with lanolin cream. Immediately after the injection of the Nomura Ecket jellyfish poison solution, the injection site was coated with a macroreticular perine cream, and the cream was applied at regular intervals every day. On the 6th day after injection, the animals were euthanized and the change of skin necrosis factor was confirmed. The skin tissue at each injection site was collected and homogenized in RIPA buffer. The supernatant was then centrifuged and mixed with sample buffer and loaded onto 10% gelatin gel. The gel was loaded at 4 ° C to maintain enzyme activity.

Example 1. Effect of maculopyrine inhibiting toxic protease activity

The inhibitory activity of manganese perrine in various natural poisons exhibiting toxic protease activity was confirmed using gelatin zymography (Fig. 1). The toxic protease activity was observed in each natural poison, but the toxic protease activity shown by the natural poison was abruptly decreased in the treated group (+) in which the candidate substance, manganese perrine, was added to the reaction buffer (Fig. 1).

Example 2. Neutralizing effect of maculoprine on human skin cytotoxicity of natural poisons

Because cytotoxicity of natural poisons is known to be positively correlated with toxic protease activity, cytotoxicity can also be inhibited if protease activity is inhibited. Toxic protease has been identified as a component showing toxicity to skin cells, and it has been confirmed that maculopyrine inhibits cytotoxicity through this experiment (Fig. 2). To examine the toxicity of skin cells, HaCaT, a human skin keratinocyte, was treated with each natural poison, and as a result, it was confirmed that natural poisons cause cytotoxicity. To investigate the cytotoxic neutralizing effect of macroreticular perine by natural venom, each natural venom was treated with cells and at the same time treated with maculliferin. After 24 hours, the cytotoxic neutralization effect was confirmed using the MTT method as shown in FIG. In comparison with the cytotoxicity observed when natural poison was treated, it was confirmed that the cell survival rate was increased from 10% to as much as 40% as a result of treatment with the macroreticular perinein, so that the macroreticular perinein neutralized the toxicity of the natural poison.

Example 3 Mitigation Effect of Manganese Perrin on Animal Skin Toxicity of Nomura Asset Jellyfish Poisonous Liquid

As a first aid model, the experiment was conducted using Nomura cetacean jellyfish, which is the easiest toxic creature around us. After the day, the Nomura embryo was injected intradermally and artificially created a jellyfish model. 200 [mu] l of the solution was injected intradermally and PBS was used as a negative control. In case of jellyfish, 200 μg of protein was injected.

Immediately after the injection, one group was coated with maculoprine and the other group was coated with creme, the vehicle control of maculopyrine. The results of fresh cream application and observation at the same time every day are shown in Fig. In the negative control PBS, no scars or any skin lesions were observed. In the Nomura embryo jellyfish group, the skin necrosis developed gradually after 24 hours. After 48 hours, skin necrosis completely occurred and wound formation was observed there was. This also occurred in the cream control group as a vehicle control. On the other hand, there was no scar on the wound area and the injection site was confirmed but the scar was insignificant.

On the 6th day of experiment, the skin tissue of the experimental animals injected with the Nomura embryo jellyfish poisonous euthanized the experimental animals and the skin tissue treated with the mitigating substances were treated with matrix metalloproteinase (MMP), which is a factor related to skin necrosis The result of the gelatin self-expression for confirming the change was as shown in FIG. The MMP9 dimer, pro MMP9, active MMP9, and MMP2 activities were significantly increased in the Nomura agar jellyfish poison injection site and cream treated group compared to the skin tissue of the PBS injection site, It was observed that the degree of activity was weak.

Claims (5)

The present invention relates to a method for inhibiting the activity of matrix metalloproteinase in cells exposed to Nomura marine jellyfish poison, Toxic antidote. delete delete Which is characterized by inhibiting the activity of a matrix metalloproteinase in cells exposed to Nomura embryo jellyfish poison, or a pharmaceutically acceptable salt thereof, as an active ingredient. An external skin preparation for treating wounds. Which is characterized by inhibiting the activity of a matrix metalloproteinase in cells exposed to Nomura embryo jellyfish poison, or a pharmaceutically acceptable salt thereof, as an active ingredient. A pharmaceutical composition for treating a wound.

Images