KR20090112528A - Composition comprising deer velvet extract as an effective component for improving gastrointestinal disease or wound healing - Google Patents

Abstract

PURPOSE: A composition containing Cervi parvum cornu extract is provided to suppress gastric acid and colony formation of Helicobacter pylory. CONSTITUTION: A method for extracting Cervi parvum cornu comprises: a step of extracting powder of Cervi parvum cornu using ultra sonic wave with chloroform:methanol (2:1) at 30 minutes; a step of centrifuging the extract at 15 °C, 500 x g for 30 minutes to separate supernatant and precipitate; a step of collecting supernatant (organic layer); a step of concentrating at 0.5 bar to obtain 20 mg of oil soluble powder of Cervi parvum cornu; and a step of maintaining at -20 °C.

Description

Composition comprising deer velvet extract as an effective component for improving gastrointestinal disease or wound healing}

The present invention relates to a composition having gastrointestinal disease improvement or wound healing efficacy containing the antler extract as an active ingredient.

Gastritis and gastric ulcer refers to a condition in which the defects caused by damage to the upper digestive tract due to the action of gastric acid and pepsin invade below the gastric mucosal layer. Gastritis and gastric ulcers are one of the most common diseases in the world, and more than half of the US adult population and 90% of adults in many developing countries, including Korea, are infected with H. pylori (Scrip, 1993, 1820, 18).

This disease is characterized by acute and ulceration, which can be treated with mental and physical stability, diet, medication, etc., but it is not likely to be cured because of the high probability of recurrence within a certain period of time. , Complications such as pyloric stenosis and perforation (Jeen, YT and Hyun, JH 1991). Gastritis and gastric ulcers are not caused by one cause, but a combination of factors. Shay and Sun (Shay et al., 1945) in 1963 argued that gastric mucosal damage is caused by a breakdown of attack and defense factors, and this "Balance theory" has been accepted to date. The identification of Helicobacter pylori (H. pylori) by Marshall has led to new findings on the pathophysiology of gastritis, gastric ulcer and duodenal ulcer.

In the past, anti-ulcer drugs have been mainly focused on pharmacological action that inhibits attack factors, but when gastric ulcers are treated only by controlling the production and secretion of hydrochloric acid, the recurrence rate is high. Required. Recently, a Gram-negative bacillus called Helicobacter pylori has been identified as a pathogenic factor of peptic ulcer. This pathogenic microorganism generates neutrophils to generate reactive oxygen radicals. It is newly discovered that tissue damage is caused by the production of lipids.

The velvet antlers are made from freshly grown, unosteolyzed young horns of a stag, which are rusted old horns that fall from mid-March each year, or fall off spontaneously in the following year. Used differently.

Various antlers are classified into manok, plum, siberian, and new zealand, according to the type of mountain and deer. Recently, the effect of promoting growth and development, the effect of inhibiting aging, the effect of hematopoietic function, heart function It is known that it contains a large amount of ingredients that exhibit improvement effects, anti-muscular fatigue effects, and anti-pain effects. In particular, oriental medicine has been considered as the best blood tonic for a long time, for this purpose, thinly simmered in water and then ingested for a long time or sometimes added to foods such as samgyetang.

The present inventors, along with the analysis of the active ingredient of the antler extract, in order to determine whether it can be applied to gastrointestinal diseases or wound healing, through the in vivo and in vitro tests to verify the efficacy on the improvement of gastric function and the proliferation of skin fibroblasts The new use of the extract was identified and the present invention was completed.

An object of the present invention is to provide a new use of the antler extract by presenting a composition having gastrointestinal disease improvement or wound healing efficacy containing the antler extract as an active ingredient.

It is also an object of the present invention to provide a health food and pharmaceutical composition comprising the composition.

In order to achieve the above object,

According to one aspect of the invention,

It is possible to provide a composition having gastrointestinal disease improvement or wound healing efficacy containing the antler extract as an active ingredient.

In addition, according to another aspect of the present invention, it is possible to provide a pharmaceutical composition for improving gastrointestinal diseases or wound healing comprising the composition.

In addition, according to another aspect of the present invention, it is possible to provide a health food for improving gastrointestinal diseases or wound healing comprising the composition.

Hereinafter, the present invention will be described in detail.

Deer antler extract of the present invention can be prepared by the following production method.

The antler powder was dissolved in a mixed solvent of chloroform and methanol (chloroform: methanol = 2: 1, v / v) and ultrasonically extracted for 30 minutes at room temperature. After 30 minutes of ultrasonic extraction, the extract was placed in a centrifuge container and centrifuged at 15 ° C. and 500 × g for 30 minutes to separate supernatant and residue. The supernatant (organic layer) was collected and concentrated to nitrogen gas at 0.5 bar at room temperature to finally obtain 20 mg of dried powder. Samples were stored at −20 ° C. (rust soluble powder).

The antler extract of the present invention may be prepared by dissolving the antler powder in sterilized distilled water as well as the preparation method, ultrasonically extracting for 30 minutes, filtering, and lyophilizing the filtrate (an antler water-soluble powder),

In addition, the antler extract of the present invention may be prepared by adding papain and purified water to the antler powder and subjecting the hydrolysis reaction at 32 ± 3 ° C. for 15 to 24 hours, followed by filtration and lyophilization after heating at 85 ° C. to 95 ° C. for 2 to 3 hours. (Deer Antler Powder).

The present inventors evaluated whether the antler extract has gastrointestinal disease improvement or wound healing effect by using the antler extract powder prepared by various methods.

Gastritis and gastric ulcer refers to a condition in which the defects caused by damage to the upper digestive tract due to the action of gastric acid and pepsin invade below the gastric mucosal layer. In the past, anti-ulcer drugs have been mainly focused on pharmacological action that inhibits attack factors, but when gastric ulcers are treated only by controlling the production and secretion of hydrochloric acid, the recurrence rate is high. Required. Recently, a Gram-negative bacillus called Helicobacter pylori has been pointed out as a pathogenic factor of peptic ulcer disease.

The present inventors performed an antacid test, colony formation inhibitory test against Helicobacter pylori bacteria in vitro to determine whether the antler extract of the present invention can be applied to gastrointestinal diseases such as gastritis and gastric ulcer.

The pH change of the samples in the antacid test was determined by the NaOH consumption measured after preparing and treating the gastric juice in the same state as gastritis, wherein the antler extract of the present invention had an inhibitory effect of approximately 14 to 50%. It was confirmed (Table 1). As a result of the test of the samples (Examples 1 to 3) according to the change in the treatment method, it was confirmed that there were antacids of 46.74, 14.49, and 48.19%, respectively. This means that the antler extract of the present invention sufficiently possesses the ability to neutralize gastric acid.

In addition, antimicrobial experiments with H. pyroli at concentrations of 10, 50 and 100 μg / mL were performed on all samples, resulting in a reduction of colonies up to 50 μg / mL and complete production of colonies at 100 ug / mL. It was confirmed that it was suppressed (Table 2).

Therefore, it was confirmed that the placental extract of the present invention can be effectively used to improve gastrointestinal diseases such as gastritis and gastric ulcer by effectively inhibiting gastric acid and effectively inhibiting colony formation of Helicobacter pylori, which is a factor of gastric ulcer.

On the other hand, it was confirmed that the antler extract of the present invention also has a function of promoting the proliferation of skin fibroblasts. In other words, the antler extract promoted the growth of dermal fibroblasts even at low concentrations of both water-soluble and fat-soluble components. In particular, it can be seen that the fat-soluble component promotes 1.5 times the growth of the control at a concentration of 25 ug / ml. This fact can be thought that the phospholipid component of deer antler (particularly, phosphatidyl choline) binds to the cell membrane receptor along with the growth factor of the serum medium used for cell culture to promote cell growth through signal transduction.

The antler extract of the present invention may be prepared as a pharmaceutical composition for improving gastrointestinal disease or wound healing by having the same effect as described above. The pharmaceutical composition may further contain one or more active ingredients exhibiting the same or similar function in addition to the antler extract. The composition of the present invention may contain at least one active ingredient exhibiting another function in addition to the antler extract.

The pharmaceutical composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and the dosage may be based on the weight, age, sex, The range varies depending on the state of health, diet, time of administration, method of administration, rate of excretion and the severity of the disease. The daily dosage is 0.1 to 10 mg / kg, preferably 0.1 to 3 mg / kg, and more preferably administered once to several times a day.

The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of gastrointestinal diseases or wounds.

In addition, the antler extract of the present invention may be added to health food for the purpose of improving gastrointestinal diseases or wound healing. When the antler extract of the present invention is used as a food additive, the antler extract may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). Generally, in the preparation of foods or beverages, the extract of the present invention is added in an amount of up to 100% by weight, preferably up to 50% by weight relative to the raw material. However, in the case of long-term intake for health and hygiene purposes or for health control purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range. have.

There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The proportion of the natural carbohydrate is generally about 0.1-20 g, preferably about 1-10 g per 100 ml of the composition of the present invention.

In addition to the above components, the composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, And a carbonation agent used for the carbonated beverage. In addition, the composition of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is usually selected in the range of 0.05 to 50 parts by weight per 100 parts by weight of the composition of the present invention.

As described above, the antler extract of the present invention effectively inhibits the formation of gastric acid and Helicobacter pylori bacteria and promotes the growth of dermal fibroblasts, which is useful for the manufacture of pharmaceutical compositions or health foods having the effect of improving gastrointestinal diseases or wound healing. Can be used.

Hereinafter, the configuration and operation of the present invention through the preferred embodiment of the present invention will be described in more detail. However, this is presented as a preferred example of the present invention and in no sense can be construed as limiting the present invention. Details that are not described herein will be omitted since those skilled in the art can sufficiently infer technically.

Example 1

1 g of antler powder was added to 30 ml of chloroform: methanol (2: 1, v / v) and ultrasonically extracted for 30 minutes at room temperature. The extracts after 30 minutes of extraction were placed in a centrifuge container and centrifuged at 15 ° C. and 500 × g for 30 minutes to separate supernatant and residue. The supernatant (organic layer) was collected and concentrated to nitrogen gas at 0.5 bar at room temperature to finally obtain 20 mg of dried rust-soluble soluble powder. Samples were stored at -20 ° C.

Example 2

1 g of antler powder was dissolved in 100 ml of sterilized distilled water, sonicated for 30 minutes, filtered, and the filtrate was lyophilized (detergent soluble powder).

Example 3

To 1 g of antler powder, papain 300mU (Sigma, 30U / mg) and 10 ml of purified water were added thereto, followed by hydrolysis reaction at 33 ° C. for 18 hours, heating at 90 ° C. for 3 hours, and filtration and lyophilization (detoxification enzyme decomposition powder).

Test Example 1: Antacid (acid neutralizing ability) test

The antacid test method is a test method for determining the antacid power of an agent that reacts with gastric acid and changes the antacid test method specified in the Korean Pharmacopoeia to 0.1 N hydrochloric acid (Examples 2 to 4 of the present invention). After taking mg, the mixture was reacted for 1 hour in a shaking incubator at 37 ° C, and titrated with 0.1 N aqueous sodium hydroxide solution. Methyl orange was used as an indicator and hydrotalcite was used as a positive control (KEPCO Amendment, 2003). The results are shown in Table 3. The negative control group is titrated with 0.1 N NaOH in gastric juice conditions, that is, 0.1 N hydrochloric acid. As a result of the test of the samples (Examples 1 to 3) according to the change in the treatment method, it was confirmed that there were antacids of 46.74, 14.49, and 48.19%, respectively. This means that the placental extract of the present invention has the ability to neutralize stomach acid.

TABLE 1

sample NaOH consumption (volume) control(%) Negative control 92.0 ± 1.73 - Example 1 49.00 ± 1.00 46.74 Example 2 78.67 ± 5.03 14.49 Example 3 47.67 ± 2.52 48.19 hydrotalcite 78.67 ± 1.15 14.5

Test Example 2: Test for inhibiting colony formation against Helicobacter pylori

To the test sample 10 for both, 50, 100 ㎍ / mL · concentration of H ₂ 0 of H. pyroli As a result of testing the inhibition of colony formation (Table 3), the number of colonies was reduced up to 50 µg / mL, and colony production was completely inhibited at the concentration of 100 µg / mL (see Table 2). Negative controls are on H. pyroli It is a medium which is not treated with a sample sample.

Accordingly, the present invention is an extract of deer velvet H. By strongly inhibited the growth of degradation-resistant pyroli on the gastric ulcer and gastric cancer pathogenesis of H. pyroli carcinogen, hyperproliferation induced mucosal, carcinogenic N-nitroso compound, and relieve such as an increase in secondary It is thought to be able to effectively prevent the symptoms of chronic infection.

TABLE 2

sample dose (ug / ml) Degree of colony formation Negative Control - ++++ Ampicillin 100 -  Example 1 10 ++++ 50 +++ 100 -  Example 2 10 ++++ 50 ++ 100 -  Example 3 10 ++++ 50 ++ 100 -

Test Example 3 Analysis of Lipid Components of Deer Antler Powder

3-1: Separation of Neutral and Phospholipids

After dissolving the total lipid (rust paper soluble powder) in chloroform (10mg / 1ml), solid phase extraction was performed. After equilibrating the column with 4 ml of hexane using an aminopropyl column (Alltech), 1 ml (10 mg) of total lipid was injected into the column to obtain solvent A; Chloroform: washed with 6 ml of 2-propanol (15: 1) to give neutral lipid, solvent B; Ether: washed with 5 ml of acetic acid (100: 2) to give free fatty acid. Phospholipids in turn are solvent C; 4 ml of methanol and solvent D; Hexane containing 2-%: 2-propanol: ethanol: 0.1 M ammonium acetate salt: formic acid (420: 350: 100: 50: 0.5) was washed by successively with 4 ml. The obtained neutral lipids and phospholipids were concentrated with nitrogen and stored at -20 ° C.

3-2: Analysis of composition of neutral lipid and phospholipid

Neutral lipid and phospholipids obtained above were dissolved in chloroform and separated and quantified by high performance liquid chromatography. Lipid composition was analyzed by Hitachi pump (model L-6200) and ELS detector (SEDEX 75). The column was Recrosspair DIOL-100 and the column temperature was fixed at 50 ° C. using a column heater (FIAtron). Neutral lipid is 0 min-12% B, 25 min.- solvent A (hexane: acetic acid, 99: 1) and solvent B (hexane: 2-propanol: acetic acid, 84: 15: 1) at a flow rate of 0.8 ml / min. Isolation was carried out using a gradient program of 100% B, 26 min-100% B, 29 min-12% B. Phospholipids were flow rate 1 ml / min with solvent A (hexane: 2-propanol: acetic acid, 82: 17: 1) and solvent B (2-propanol: water: acetic acid, 85: 14: 1) 0 min-5% B, 25 min-40% B, 35 min-100% B, 46 min-5% B was isolated using a gradient program. Each lipid component was identified by comparison with the retention time of the standard material of lipids, and a calibration curve was prepared using the surface area value of each standard material to quantify the lipid components of the sample. The content of triglyceride and phospholipid is expressed as the content of each lipid for 10 mg of total lipid.

3-3: Analysis of Lipid Components

For deer antler, 37.4% of the total lipids included cholesterol esters (cholesterol ester, 63.3%), triglycerides (6.8%), free fatty acids (11.7%), cholesterol (cholesterol, 18.2%) and It was confirmed that there is a trace amount of monoglyceride (monoglyceride) (Fig. 2). 14.9% of phospholipids for total lipids include phosphatidic acid (PA, 6.3%), phosphatidyl ethanolamine (PE, 21.9%), phosphatidyl choline (PC, 33.4%), sphingomyelin (SM, 15.9%), phosphatidyl serine (PS, 7.2%) and unidentified phospholipids (UK, 15.3%) were confirmed (FIG. 3). Neutral lipid was found to contain the most cholesterol esters, phospholipids phosphatidyl choline. Phospholipids are known as the major constituents of cell membranes. These phospholipids are known to participate in cell signal transduction, cellular metabolic processes, and play important roles in the fatty acid and lysoform phospholipids of each phospholipid, as well as several pharmacological, biological activities, or precursors thereof.

Test Example 4: Fibroblast cell proliferation experiment

Laying the skin fibroblasts in 1% FBS DMEM starvation media to kiwoseo in 10% FBS DMEM medium 96 cell plate 1x10 ³ gae After 24 hours, the sample (Examples 1 to 3) for each of 1, 10, dissolved in 1% FBS DMEM, Treated at 25, 50 μg / ml concentration (concentration in final medium). 72 hours after the sample treatment, the cell counting (CCK-8) reagent was added, and after 2 hours and 30 minutes, cell proliferation was measured using a UV spectrometer at 450 nm. (The control is a comparison of cells grown in 1% FBS DMEM with 1% FBS DMEM without sample processing and cells treated with the sample. Compares growth with)

As shown in Figure 4, the antler extract promoted the growth of fibroblast at low concentrations of both water-soluble and fat-soluble components. In particular, it can be seen that the fat-soluble component promotes 1.5 times the growth of the control at a concentration of 25 ug / ml. This fact suggests that the phospholipid component of deer antler (particularly, phosphatidyl choline) binds to the cell membrane receptor along with the saint factor of serum medium used for cell culture, thereby promoting cell growth through signal transduction.

Therefore, the antler extract of the present invention can be used for healing wounds such as burns and wounds by promoting the growth of dermal fibroblasts.

While the embodiments of the present invention have been described with reference to the accompanying tables, the present invention is not limited to the above embodiments, but may be embodied in various forms, and a person of ordinary skill in the art to which the present invention pertains. It will be appreciated that the present invention may be embodied in other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Figure 1 is a schematic diagram showing a process of separating the lipid component from antler fat-soluble powder according to an embodiment of the present invention.

Figure 2 is a graph showing the result of separating the neutral lipid from the lipid component of antler using high-speed liquid chromatography.

CE: cholesterol ester, TG: triglycerides, CHOL: cholesterol

Figure 3 is a graph showing the result of separating each phospholipid from the lipid component of antler using high-speed liquid chromatography.

PA: phosphatidic acid, U.K: unidentified substance, PE: phosphatidyl ethanolamine,

PC: phosphatidyl choline, SM: sphingomyelin, PS: phosphatidyl serine

Figure 4 is a graph showing the results of skin fibroblast proliferation test by the antler extract according to an embodiment of the present invention.

(y-axis: cell viability (%), x-axis: left, control, water-soluble powder sample, enzyme digestion powder sample, fat-soluble powder sample 1, 10, 25, 50 ㎍ / ml, respectively)

0.01> *, 0.001> **, 0.0001> ***

Claims (3)

A composition having a gastrointestinal disease improvement or wound healing effect containing the antler extract as an active ingredient. A pharmaceutical composition for improving gastrointestinal disease or wound healing, comprising the composition of claim 1. Gastrointestinal disease improvement or wound healing health food comprising the composition of claim 1.

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