KR101337280B1 - Composition for preventing and treating of pulmonary emphysema and pulmonary hypertension, comprising an extract of Sepiae Os as an effective component - Google Patents

Abstract

The present invention relates to a composition for the treatment and prevention of emphysema and pulmonary hypertension containing the seaweed extract as an active ingredient, specifically, using an alcohol solution of 2 to 5 carbon atoms of 10 to 100% by volume as the extraction solvent and effective from seaweed It relates to a composition for the prevention and treatment of emphysema and pulmonary hypertension, containing the seaweed extract extracted components as an active ingredient.
Since the composition according to the present invention is an extract extracted from natural substances, there are almost no side effects, and it effectively inhibits elastase activity and has excellent scavenging effect of DPPH and NO, and thus it can be used as a medicine for preventing or treating emphysema and pulmonary hypertension. In this case, excellent effects can be expected.

Description

Composition for preventing and treating of pulmonary emphysema and pulmonary hypertension, comprising an extract of Sepiae Os as an effective component

The present invention relates to a composition for the treatment and prevention of emphysema and pulmonary hypertension containing the seaweed extract as an active ingredient, specifically, using an alcohol solution of 2 to 5 carbon atoms of 10 to 100% by volume as the extraction solvent and effective from seaweed It relates to a composition for the prevention and treatment of emphysema and pulmonary hypertension, containing the seaweed extract extracted components as an active ingredient.

Emphysema is an abnormal and permanent condition of peripheral airway and alveolar expansion due to destruction of the terminal bronchiole distal airspace. Pulmonary hypertension refers to abnormally high blood pressure in the pulmonary artery.

Emphysema, also known as chronic obstructive pulmonary disease, is a long-term disease that causes significant damage to the alveoli and capillaries, small sacs that exchange oxygen and carbon dioxide in the lungs. do.

Several substances for preventing and treating such emphysema and pulmonary hypertension are known, but researches to develop more effective medicines are lacking in kind and effect.

Emphysema has recently been associated with elastase.

Neutrophil elastase (or leukocyte elastase) is a serine protease belonging to the same family as chymotrypsin and exhibits various substrate specificities. Along with other serine proteases, it has a charge transfer system consisting of catalytic triads consisting of histidine, aspartate and serine residues. These histidine, aspartate and serine residues In the primary sequence of a polypeptide, they are dispersed from each other, but in proteins folded into a tertiary structure, they are clustered together.

Neutrophil elastase, along with collagen, forms elastic fibers to break down elastin, which represents the physical properties of connective tissue, and is a powerful nonspecific serine protease that acts as a bactericidal agent in the immune system-depleted state by internal vasculature. Promotes emphysema, chronic obstructive disease.

Known developmental causes of emphysema include a combination of inflammation, elastase, matrix metalloprotease imbalance, self-killing and oxidative stress. When exposed, neutrophils and macrophages have been shown to trigger an acute lung inflammatory response.

In addition, the production of reactive oxygen species (ROS) is induced by neutrophil elastase. Studies have shown that the increase in MUC5AC messenger RNA levels in neutrophil elastase is dependent on the production of intracellular oxides or changes in cellular redox status, indicating that ROS play a role in elastase-mediated inflammation. it means. Nitric oxide also plays a pivotal role in elastase-mediated disease.

The present inventors conducted various studies on natural seaweeds in order to develop a material having fewer side effects and excellent prevention and treatment effect of emphysema and pulmonary hypertension. In particular, as a result of studies on the inhibitory activity and DPPH and NO scavenging activity of the elastase that causes emphysema and pulmonary hypertension, the seaweed extract has excellent preventive and therapeutic effects against emphysema and pulmonary hypertension. It confirmed and completed this invention.

Therefore, the main object of the present invention is to provide a composition for preventing and treating emphysema and pulmonary hypertension with little side effects and excellent effect.

According to one aspect of the invention, the present invention provides a composition for the prevention and treatment of emphysema and pulmonary hypertension containing seaweed extract as an active ingredient.

In the composition of the present invention, the preparation of the seaweed vinegar extract is preferably prepared by using 10 to 100% by volume of a C 2 to 5 alcohol solution of carbon atoms as the extraction solvent and heating, specifically, 10 to 100 parts by weight of seaweed vinegar 100 to 10000 parts by weight of ethanol solution of 100% by volume is preferably prepared by heating for 10 minutes to 30 hours, more preferably, 1000 to 3000 parts by weight of 50 to 90% by volume of ethanol solution is added to 100 parts by weight of seaweed. It is good to manufacture by heating for 4 hours. More preferably, it is preferable to concentrate the extracted extract, wherein the concentration method may be a reduced pressure concentration method and a freeze-drying method, it is preferable to freeze-dried after concentration under reduced pressure. The temperature at the time of heating is preferably 70 to 130 ° C, more preferably 90 to 110 ° C, and most preferably 100 ° C.

In the composition of the present invention, the composition preferably contains 0.1 to 50% by weight of seaweed extract, and more preferably 0.1 to 20% by weight. This is a content of the seaweed extract necessary for the composition of the present invention to effectively exhibit the effect of preventing and treating emphysema and pulmonary hypertension, and is determined based on the experimental results according to the concentration of the seaweed extract.

Sepiae Os (Sepiae Os), also called cuttlefish bone, is the bone of Sepiella maindroni De Rochebrune or Sepia esculenta Hoyle. It is discarded in the process of grooming cuttlefish, and in Asia, it produces about 40,000 to 50,000 tons a year. Haechocho has been used traditionally for medical purposes for a long time and is mainly used to stop bleeding of the lungs, stomach and uterus due to the qi deficiency caused by the inability of the spleen to maintain vascular blood circulation. It became. It was also used to strengthen worms and stop leukorrhea. It is known to neutralize gastric acid, relieve pain, and resolve chronic incurable inflammation, ulcers and rashes in the condition of stomach pain caused by acid reflux. However, there have been no studies on elastase, DPPH and NO of these seaweeds.

In the present invention, in order to confirm the prophylactic and therapeutic effects of emphysema and pulmonary hypertension on the seaweed extract, the effects on the inhibition of elastase activity and the elimination of DPPH and NO were investigated.

According to the present invention, seaweed herb extract inhibits elastase activity, effectively removes DPPH and NO, and exhibits an excellent preventive and therapeutic effect on emphysema and pulmonary hypertension.

The composition of the present invention can be used for medicines and foods.

At this time, the composition according to the present invention can be formulated based on the formulation criteria of the Food and Drug Administration (KFDA) to a conventional pharmaceutical formulation or a dietary supplement of dietary supplements.

The composition of the present invention may be diluted in a conventional manner, mixed with a pharmaceutically acceptable carrier, or encapsulated in a container form according to the administration method, dosage form, and purpose.

When the carrier is used as a diluent, oral administration with at least one carrier selected from the group consisting of saline, buffer, dextrose, water, glycerol, Ringer's solution, lactose, sucrose, calcium silicate, methyl cellulose and ethanol For parenteral administration it is prepared in formulations such as powders, granules, injections, syrups, solutions, tablets, suppositories, pesaries, ointments, creams or aerosols. However, the carrier of the present invention is not limited to the above carrier. In this case, parenteral administration means administration of the active ingredient through rectal, intravenous, peritoneal, muscular, arterial, transdermal, nasal, inhalation, etc. in addition to orally.

The formulations may further comprise a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, an antiseptic, etc. to formulate the composition so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. And the dosage of the present invention can be adjusted according to the condition of the patient, the route of administration and the dosage form is not limited and those skilled in the art according to the symptoms can be obviously used within various ranges, In general, in the present invention, it is determined that 0.1 to 2000 mg per 1 kg of body weight of the seaweed extract according to the present invention may be continuously or intermittently administered in an experimentally effective amount.

In addition, on the basis of the effective amount, the present invention provides a food comprising a composition containing the Haechocho extract itself or a food acceptable carrier as a basic dietary material (其 本 食餌 素材), the food is processed meat, Fish products, tofu, jelly, porridge, noodles such as ramen or noodles, seasoned foods such as soy sauce, miso, red pepper paste, mixed soy sauce, dairy products such as sauces, sweets, fermented milk and cheese, pickles such as kimchi and pickles, fruit, It can be used in foods of beverages such as vegetables, soy milk and fermented beverages. This is obvious to those skilled in the art of the present invention will not be described the specific cooking method or production method thereof. In addition, the pharmaceutically acceptable carrier may also be used as the pharmaceutically acceptable carrier.

As described above, since the composition according to the present invention is an extract extracted from natural substances, there are almost no side effects, and it effectively inhibits elastase activity and has an excellent scavenging effect of DPPH and NO, thereby preventing or treating emphysema and pulmonary hypertension. When used as a medicine to achieve excellent effects can be expected.

1 is a graph showing the inhibitory effect of elastase activity of the seaweed extract according to the present invention. C is a control group treated with distilled water instead of seaweed extract, 0.1, 1, 10 is an experimental group treated with a seaweed extract of 0.1, 1, 10mg / ㎖ concentration, respectively. Each result is expressed as the mean ± standard error (SEM) for the results of three replicates. * Indicates significant difference compared to the control (p <0.05).
Figure 2 is a graph showing the DPPH free radical scavenging activity of the seaweed extract according to the present invention. C is a control group treated with distilled water instead of seaweed extract, 0.4, 2, 10, 50 is an experimental group treated with a seaweed extract of 0.4, 2, 10, 50mg / ㎖ concentration, respectively. Each result is expressed as the mean ± standard error (SEM) for the results of three replicates. * Indicates significant difference compared to the control (p <0.05).
Figure 3 is a graph showing the nitrite radical scavenging activity of the seaweed extract according to the present invention at pH 1.2. C is a control group treated with distilled water instead of seaweed extract, 0.4, 2, 10, 50 is an experimental group treated with a seaweed extract of 0.4, 2, 10, 50mg / ㎖ concentration, respectively. Each result is expressed as the mean ± standard error (SEM) for the results of three replicates. * Indicates significant difference compared to the control (p <0.05).
Figure 4 is a graph showing the nitrite radical scavenging activity of the seaweed extract according to the present invention at pH 3.0. C is a control group treated with distilled water instead of seaweed extract, 0.4, 2, 10, 50 is an experimental group treated with a seaweed extract of 0.4, 2, 10, 50mg / ㎖ concentration, respectively. Each result is expressed as the mean ± standard error (SEM) for the results of three replicates. * Indicates significant difference compared to the control (p <0.05).
5 is a graph showing the nitrite radical scavenging activity of the seaweed extract according to the present invention at pH6.0. C is a control group treated with distilled water instead of seaweed extract, 0.4, 2, 10, 50 is an experimental group treated with a seaweed extract of 0.4, 2, 10, 50mg / ㎖ concentration, respectively. Each result is expressed as the mean ± standard error (SEM) for the results of three replicates.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1. Preparation of Haechocho extract

Haechocho was purchased from Omniherb, Korea.

2,000 ml of 70% ethanol was added to 100 g of seaweed vinegar and heated to 100 ° C. in a heat extractor for 3 hours to obtain an extract. The extract was filtered through a filter and concentrated using a rotary evaporator.

In addition, the concentrated extract was lyophilized using a lyophilizer to obtain 0.31 g of lyophilisate, and the lyophilisate was dissolved in water or a buffer solution at different concentrations, and the microfilter paper (Whatman no. 2, 0.45 ~ 0.2 μm) was filtered three times to prepare a sample for the experiment. Samples were stored in sterile glass bottles sealed.

Experimental Example 1

Elastase, p-nitroaniline, DPPH and Greries reagents used in this Example were purchased from Sigma-Aldrich, St. Louis, Mo., USA.

The experimental results are expressed as standard error of mean ± mean. The significance of the change was determined using one-way ANOVA as Dunnett's post hoc test. p <0.05 values indicate statistically significant.

1-1. Elastase Activity Assessment

A slightly modified method in the previously reported method of Kraunsoe et al. Evaluated the effect of inhibiting the elastase activity of the sample of Example 1 above. This is a method of measuring the amount of p-nitroaniline released at 410 nm with maximum absorbance. p-nitroaniline occurs by hydrolysis from the N-succinyl-Ala-Ala-Ala-p-nitroanilide substrate by elastase. 100 μl of a 2 mM N-succinyl-Ala-Ala-Ala-p-nitroanilide solution was prepared using 0.1M Tris-Cl buffer (pH8.0), and 50 μl of the sample of Example 1 was added to the solution. Each sample solution has a final concentration. Dilutions were made to 0.01, 0.1 and 1 mg / ml. The substrate solution to which the sample was added was lightly mixed by hand, and 50 µl of elastase solution (0.1360 unit / ml) was added. The reaction solution was reacted at 37 ° C. for 10 minutes and absorbance was measured at 410 nm (Synergy2, BioTek Inc., USA). Inhibition capacity was calculated as a percentage according to the following formula.

[formula]

% Elastase inhibitory activity = [(OD 410 of control)-(OD 410 of sample) / (OD 410 of control)] × 100

Each sample showed an elastase inhibitory effect concentration dependently (see FIG. 1). Elastase activity was significantly inhibited (46.4 ± 4.4%, p <0.05) in the experimental group treated with the concentration of 10 mg / ml, and was not significant at the concentrations of 0.1 and 1 mg / ml (107.9 ± 9.0% And 116.7 ± 7.2%).

1-2. Investigation of DPPH elimination effect

The scavenging effect of the samples on DPPH radicals was analyzed according to Shimada et al. DPPH radicals appear dark purple according to unpaired electrons, and the radical scavenging ability can be measured with reduced absorbance at 540 nm. 1 ml of the sample of Example 1 was added to 1 ml of an ethanol solution with a final DPPH radical concentration of 0.2 mM. After 30 minutes in the dark, the absorbance of the reaction solution against the ethanol control was measured at 540 nm using an ELISA reader (Synergy2, BioTek Inc., USA). Erasure capacity was calculated as a percentage according to the following equation.

[formula]

DPPH free radical scavenging activity (%) = [(OD 540 of control)-(OD 540 of sample) / (OD 540 of control)] × 100

The free radical scavenging ability of each of the different concentration samples (0, 0.4, 2, 10 and 50 mg / ml) was measured and showed the highest scavenging power at the concentration of 50 mg / ml (38.3 ± 7.7%), The remaining concentrations of 0.4, 2 and 10 mg / mL were 12.5 ± 2.7%, 10.8 ± 2.2% and 15.5 ± 3.2%, respectively.

1-3. Investigation of nitrite scavenging effect

Example 1 nitrite scavenging activity according to the pH conditions of the sample was determined using a grease reagent. 1 ml of sample was mixed with 1 ml of 1 mM sodium nitrite solution. 8 ml of 0.2 M citrate buffer (pH1.2, 3.0 and 6.0) was added to the mixed solution, and the mixture was reacted at 37 ° C for 1 hour. Then, a 1 ml fraction was obtained, and 2 ml of 2% acetic acid solution and 0.4 ml of grease reagent (1% sulfanilic acid, 1% naphthylamine, methanol solution of 30% acetic acid) were added thereto. After mixing with vortex, the mixed solution was placed at room temperature (21 ° C.) for 15 minutes, and the absorbance was measured at 520 nm. Nitrite scavenging activity was calculated as a percentage according to the following formula.

[formula]

Nitrite scavenging activity (%) = [1-(A-C) / B] × 100

A is the absorbance of the sample treatment group, C is the absorbance of the sample, B is the absorbance of 1 mM NaNO ₂

In case of pH1.2, as shown in FIG. 3, two concentrations of 10 and 50 mg / ml showed statistical significance (49.2 ± 0.0% and 64.2 ± 4.9%).

In case of pH 3.0, statistical significance was shown at a concentration of 50 mg / ml as shown in FIG. 4 (85.0 ± 2.2%, p <0.05).

In the case of pH6.0 it was shown that no significance at all concentrations as shown in FIG.

The nitrite scavenging activity of the sample changed with increasing pH, indicating that it is pH dependent. Therefore, low pH conditions (1.2 ~ 3.0) may be the optimal conditions to expect the nitrite scavenging activity of seaweed extract.

Claims (5)

A pharmaceutical composition for preventing and treating emphysema and pulmonary hypertension, characterized by inhibiting elastase activity and containing free radical and nitrite scavenging effects by containing the extract of Sepiae Os (Sepiae Os) as an active ingredient. According to claim 1, The seaweed extract is
10 to 100% by volume of an alcoholic solution of 2 to 5 carbon atoms as an extraction solvent, the composition characterized in that the extract extracted from the active ingredient from seaweed. According to claim 1, The seaweed extract is
It is an extract obtained by adding 100-10000 weight part of 10-100 volume% ethanol solutions to 100 weight part of seaweed vinegar and heating for 10 minutes-30 hours. According to claim 1, The seaweed extract is
A composition, characterized in that the extract obtained by adding 100 to 10000 parts by weight of 10 to 100% by volume of ethanol solution to 100 parts by weight of seaweed vinegar and heated for 10 minutes to 30 hours. The composition of claim 1, wherein the composition contains 0.1-50% by weight of seaweed extract.

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