KR102339984B1 - An Antibacterial and Antioxidant Composition using Scenedesmus intermedius Extract - Google Patents

Abstract

Senendesmus intermedius ( Scenedesmus intermedius ) processed extract of the present invention is antibacterial against Gram-positive bacteria (Staphylococcus aureus, Streptococcus mutans and Bacillus cereus) Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) bacteria and fungi (Aspergillus parasiticus and Candida albicans) As a result, the minimum inhibitory concentration (MIC) for Gram-negative bacteria was 12-24 μg/mL, and the MIC for Gram-positive bacteria was 24-48 μg/mL and fungi Since it was confirmed that the MIC shows 60-192 μg/mL and at the same time has excellent antioxidant activity, it can be seen that it can exhibit an excellent effect in diseases induced by microbial infection. Therefore, the composition of the present invention is effective for preventing and treating infectious diseases induced by microorganisms such as gram-negative bacteria, gram-positive bacteria and fungi, or as an active ingredient of an antibacterial and antioxidant composition.

Description

An Antibacterial and Antioxidant Composition using Scenedesmus intermedius Extract as an active ingredient

The present invention relates to an antibacterial and antioxidant composition containing a Senendesmus intermedius extract, and more particularly, a cosmetic, food and feed composition and a natural preservative containing the Senendesmus intermedius extract having antibacterial and antioxidant effects. is about

Antibiotics are metabolites produced by microorganisms and are substances that inhibit or kill the growth of other microorganisms in small amounts. It is a drug that treats infections caused by pathogens and is used worldwide because it shows excellent efficacy in the symptoms of infection.

However, many of the antibacterial compounds developed so far have developed the ability to resist antibiotics by themselves due to misuse and abuse, and the resistance is getting stronger. As it becomes more difficult to do, it is emerging as a global problem. Therefore, there is an urgent need for new materials and development for new antibiotic products that can replace conventional antibiotics.

Most of the main components of drugs are those derived from natural products. In particular, antibacterial compounds recovered from microorganisms or plants are occupied. Recently, many efforts are being made to screen biological agents, biological nanoparticles, bio-nanocomposites, etc. for the development of antibiotics. In this context, microalgae are also used in a wide variety of fields such as food, pigment, and feed, but surprisingly, investigations or studies on antibacterial or antioxidant activity have not been conducted.

The present inventors have tried to develop microalgae as a renewable natural resource of fatty acid methyl ester (FAME) with excellent antibacterial and antioxidant activity. It has been confirmed that it has an excellent effect as an antibacterial agent against By doing so, the present invention was completed.

Accordingly, the present inventors completed the present invention by confirming that, among microalgae, Scenedesmus intermedius has antibacterial and antioxidant activity, as a result of searching for natural substances that have excellent antibacterial and antioxidant activity and can be safely applied. did

Accordingly, it is an object of the present invention to provide an antibacterial and antioxidant composition containing the processed extract of Scenedesmus intermedius as an active ingredient.

Another object of the present invention is to provide an antibacterial and antioxidant food composition containing a processed extract of Senendesmus intermedius.

Another object of the present invention, Senendesmus intermedius ( Scenedesmus intermedius ) Prevention of infectious diseases caused by any one or more bacteria selected from the group consisting of Gram-negative bacteria, Gram-positive bacteria and fungi using the processed extract as an active ingredient or a therapeutic pharmaceutical composition.

In order to achieve the above object, the present invention provides an antibacterial and antioxidant composition comprising a processed extract of Scenedesmus intermedius as an active ingredient.

In addition, the present invention provides an antibacterial and antioxidant food composition comprising the processed extract of Scenedesmus intermedius as an active ingredient.

Another object of the present invention, Senendesmus intermedius ( Scenedesmus intermedius ) Prevention of infectious diseases caused by any one or more bacteria selected from the group consisting of Gram-negative bacteria, Gram-positive bacteria and fungi using the processed extract as an active ingredient Or it provides a pharmaceutical composition for treatment.

The Senendesmus intermedius ( Scenedesmus intermedius ) processed extract may be extracted with any one or more solvents selected from the group consisting of water, C1 to C4 lower alcohols, hexane and chloroform.

The Senendesmus intermedius ( Scenedesmus intermedius ) The processed extract is Senendesmus intermedius ( Scenedesmus intermedius ) Cells or Senendezmus intermedius ( Scenedesmus intermedius ) Water, C1 to C4 lower alcohol, hexane and After obtaining the extract with any one or more solvents selected from the group consisting of chloroform, it may be prepared by adding alcohol and a base catalyst to the extract to perform a transesterification reaction.

0.5 to 5 parts by weight of the solvent may be added based on 1 part by weight of the cells of the Senendesmus intermedius or Senendesmus intermedius (Scenedesmus intermedius) culture solution.

The Senendesmus intermedius ( Scenedesmus intermedius ) As a result of analyzing the processed extract by GC, based on the total dry weight of the extract, 20-25 wt% palmitic acid methyl ester (C16: 0), 0.1-0.5 wt% cis-10 -Heptadecanoic acid methyl ester (C17: 1), 2-10 wt% stearidonic acid methyl ester (C19:2), and 1-5 wt% linolenic acid methyl ester (C18:3) may be included.

The composition has an antibacterial activity for inhibiting the growth of any one or more bacteria selected from the group consisting of gram-negative bacteria, gram-positive bacteria and fungi, and the composition includes gram-positive bacteria of Staphylococcus aureus, Streptococcus mutans and Bacillus cereus and Escherichia. coli and Pseudomonas aeruginosa gram-negative bacteria Aspergillus parasiticus and Candida albicans fungi may have antibacterial activity.

Therefore, the present invention is Senendesmus intermedius ( Scenedesmus intermedius ) Antibacterial and antioxidant composition, food composition, and Gram-negative bacteria, Gram-positive bacteria and fungi comprising the processed extract as an active ingredient. By any one or more bacteria selected from the group consisting of Provided is a pharmaceutical composition for preventing or treating an induced infectious disease.

Senendesmus intermedius ( Scenedesmus intermedius ) processed extract of the present invention is antibacterial against Gram-positive bacteria (Staphylococcus aureus, Streptococcus mutans and Bacillus cereus) Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) bacteria and fungi (Aspergillus parasiticus and Candida albicans) As a result, the minimum inhibitory concentration (MIC) for Gram-negative bacteria was 12-24 μg/mL, and the MIC for Gram-positive bacteria was 24-48 μg/mL and fungi Since it was confirmed that the MIC shows 60-192 μg/mL and at the same time has excellent antioxidant activity, it can be seen that it can exhibit an excellent effect in diseases induced by microbial infection. Therefore, the composition of the present invention is effective for preventing and treating infectious diseases induced by microorganisms such as gram-negative bacteria, gram-positive bacteria and fungi, or as an active ingredient of an antibacterial and antioxidant composition.

1 is a view showing the comparison of each measurement of the total lipid content in the extracts of a total of 20 unidentified microalgae. 1a is a graph showing the analysis of the lipid content relative to the total cell weight for each extract of NTEB01 to NTEB20. This is the average value of a total of three experiments, and is expressed as the mean ± SE.
Figure 2 shows the analysis of the composition of the Senendesmus intermedius ( Scenedesmus intermedius ) processed extract confirmed through gas chromatography.
Figure 3 shows the comparison of the antibacterial activity and composition of Senendesmus intermedius ( Scenedesmus intermedius ) processed extract of the present invention and other known conventional extracts of microalgae.
Figure 4 is Senendesmus intermedius for various pathogens ( Scenedesmus intermedius ) Agar diffusion analysis results of processed extracts.
Figure 4 (a) is for E. coli, Figure 4 (b) is for P. aeruginosa, Figure 4 (c) is for S. aureus. 4(d) is for B. cereus, FIG. 4(e) is for C. albicans, and FIG. 4(f) is for A. parasiticus.
5 is a graph showing the inhibitory activity of Senendesmus intermedius ( Scenedesmus intermedius ) processing extract against various human pathogens.
Figure 5a is a measure of the inhibition zone for each pathogen and expressed as a percentage, Figure 5b shows the measurement of the MIC for each pathogen.
6 is a graph showing DPPH radical scavenging activity (bold text) and hydroxyl radical scavenging activity (dotted line) as a result of measuring the antioxidant activity of a processed extract of Scenedesmus intermedius. It is the average value of a total of three experiments.

Hereinafter, the present invention will be described in detail.

As described above, the search for a substance that has excellent antibacterial and antioxidant activity and can be safely applied is still required, but the processed extract of Scenedesmus intermedius inhibits the growth of Gram-negative bacteria, Gram-positive bacteria and fungi. In addition to the antibacterial effect, studies on having an excellent antioxidant effect have not yet been reported.

Accordingly, one aspect of the present invention relates to an antibacterial and antioxidant composition comprising the processed extract of Scenedesmus intermedius as an active ingredient.

In addition, another aspect of the present invention relates to an antibacterial and antioxidant food composition comprising the processed extract of Scenedesmus intermedius as an active ingredient.

In addition, another aspect of the present invention is Senendesmus intermedius ( Scenedesmus intermedius ) Prevention of infectious diseases caused by any one or more bacteria selected from the group consisting of Gram-negative bacteria, Gram-positive bacteria, and fungi using the processed extract as an active ingredient or to a pharmaceutical composition for treatment.

Senendezmus intermedius (Scenedesmus intermedius) processed extract according to the present invention against Gram-positive bacteria (Staphylococcus aureus, Streptococcus mutans and Bacillus cereus) Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) bacteria and fungi (Aspergillus parasiticus and Candida albicans) , can achieve significantly excellent antibacterial activity at a low concentration, as well as exhibit a very strong antioxidant effect at the same time, and is effective as an active ingredient in antibacterial and antioxidant compositions, food compositions, and pharmaceutical compositions for the prevention or treatment of infectious diseases.

The processed extract used in the present invention refers to a product made by obtaining an extract from Scenedesmus intermedius and transesterifying it and processing it.

FAME as used in the present invention means all fatty acids methyl esterified with fatty acid methyl esters.

The Senendesmus intermedius (Scenedesmus intermedius) is a novel strain having excellent antibacterial and antioxidant activity, FAME production capacity, and selected as a strain showing the highest lipid content among a total of 20 types of microalgae isolated from Indian freshwater became

Furthermore , as a result of obtaining an extract from Scenedesmus intermedius and transesterifying it to recover the processed extract, it is significantly superior to the processed extract obtained from conventional microalgae and has a wide range of bacteria (gram-negative, gram-positive) and human pathogens such as fungi) was confirmed to exhibit antibacterial activity, and it was also confirmed to have an antioxidant activity equivalent to that of ascorbic acid (positive control), which is known to have the highest antioxidant effect.

In addition, Scenedesmus intermedius of the present invention is isolated from a freshwater habitat in India, and its morphological characteristics are single-celled thalli, spherical to ellipsoidal, and elongate. ) or elongated fusiform, fusiform, cell poles capitates, obtuse, or long tapering, with a single pyrenoid uninucleate cell have.

As such , the processed extract of Scenedesmus intermedius has a minimum inhibitory concentration (MIC) of 12-24 μg/mL for Gram-negative bacteria, and a MIC of 24-48 μg/mL for Gram-positive bacteria, The MIC for fungi was 60-192 μg/mL, and at the same time, it was confirmed that it also has excellent antioxidant activity, so it can exhibit an excellent effect in diseases induced by microbial infection. Therefore , the processed extract of Scenedesmus intermedius is an antibacterial and antioxidant composition, and it can be used as a natural preservative in various foods. At the same time, since it has an excellent antioxidant function, a composition containing it can be developed as an active ingredient in a cosmetic composition, food, health functional food, food additive, and animal feed that can prevent or alleviate oxidative and bacterial infections.

The type and content of the fatty acid or fatty acid methyl ester (FAME) component included in the processed extract varies significantly depending on the variety, the local environment in which the microalgae grows, and the extraction process, which makes the effect of the extract different. A detailed comparison thereof is shown in FIG. 3 ,

The main compounds identified from the Scenedesmus intermedius processed extract are palmitic acid methyl ester (C16: 0) (23.08% w/w), cis-10-heptadecanoic acid methyl ester (C17: 1) ( 0.44 % w/w), stearidonic acid methyl ester (C19: 2) (5.04 % w/w) and linolenic acid methyl ester (C18: 3) (3.76 % w/w).

The Senendesmus intermedius (Scenedesmus intermedius) processed extract may be one extracted with any one or more solvents selected from the group consisting of water, C1 to C4 lower alcohols, hexane and chloroform, preferably methanol and chloroform It may be a mixed solvent. The mixed solvents of chloroform and the mixture is most preferable 1 to 3 parts by weight based on 1 part by weight of methanol, the active ingredient (fatty acid) of the said Sennen des mousse inter Medi-house (Scenedesmus intermedius), the extract in the case out of the above range This can be extracted in small amounts.

The Senendesmus intermedius ( Scenedesmus intermedius ) The processed extract is Senendesmus intermedius ( Scenedesmus intermedius ) Cells or Senendezmus intermedius ( Scenedesmus intermedius ) Water, C1 to C4 lower alcohol, hexane and After obtaining the extract with any one or more solvents selected from the group consisting of chloroform, it may be prepared by adding alcohol and a base catalyst to the extract to perform a transesterification reaction.

The base catalyst may be at least one selected from anhydrous sodium sulfate, sulfate hydroxide, and potassium hydroxide, and the alcohol may be at least one selected from the group consisting of methanol, ethanol, propanol, butanol, and mixtures thereof. have.

The transesterification reaction is preferably performed at 50 to 70° C. for 1 to 5 hours.

Also preferred that the Sennen des mousse inter Medi-house mixed bacterial cells or Sennen des mousse inter Medi-house (Scenedesmus intermedius) culture medium 0.5 to 5 parts by weight of solvent based on 1 part by weight of (Scenedesmus intermedius), the Sennen des mousse inter Medi-house ( Scenedesmus intermedius ) When the weight ratio of the cell or Senendesmus intermedius culture medium and the solvent is out of the above range, the active ingredient of the Senendesmus intermedius in the extract may be extracted in a small amount. can

As used herein, the term 'processed extract' includes a fraction obtained by additionally fractionating the processed extract. That is, the Senendesmus intermedius extract includes not only the one obtained through the transesterification reaction after extraction using the above-described solvent, but also the one obtained by additionally applying a purification process thereto. In addition, the fraction obtained by passing the processed extract through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (prepared for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through various purification methods are also included in the processed extract of Senendesmus intermedius of the present invention.

In the present specification, the term 'process extract' used while referring to the processing of Senendesmus intermedius (Scenedesmus intermedius) refers to a fraction thereof as well as a reduced pressure distillation and freeze-drying or spray-drying of the processed extract of Senendesmus intermedius (Scenedesmus intermedius) It may be prepared in a powder state by an additional process, such as.

In addition, the Senendesmus intermedius (Scenedesmus intermedius) processed extract of the present invention in a broad sense Senendesmus intermedius (Scenedesmus intermedius) Senendesmus intermedius formulated so that the processed extract can be administered to animals ( Scenedesmus intermedius ) Processed extracts, for example, Senendesmus intermedius ( Scenedesmus intermedius ) It has a meaning including powders of processed extracts. Although in the present invention, Senendesmus intermedius ( Scenedesmus intermedius ) Although the experiment was conducted with the processed extract, it is possible to achieve the desired effect in the same form as the formulated Senendesmus intermedius ( Scenedesmus intermedius ) processed extract. It will be expected by those skilled in the art.

Component analysis of the processed extract of Senendesmus intermedius of the present invention is preferably analyzed using thin-layer chromatography, high-performance liquid chromatography and gas chromatography, more preferably thin-layer chromatography-flame ionization It is preferable to analyze the composition using a detector (TLC-FID) and a gas chromatography-flame ionization detector (GCFID).

As a result of analyzing the Senendesmus intermedius processed extract by GC or GCFID, 20-25 wt% palmitic acid methyl ester (C16:0), 0.1-0.5 wt% cis based on the total dry weight of the extract -10-heptadecanoic acid methyl ester (C17: 1), 2-10 wt% stearidonic acid methyl ester (C19:2) and 1-5 wt% linolenic acid methyl ester (C18:3), specific The components are as shown in FIG. 3 . It can be confirmed through experimental examples described below that the processed extract of Scenedesmus intermedius having such a component has significantly superior antibacterial activity and antioxidant effect compared to the conventional processed extract of microalgae.

The “infectious disease” of the present invention is characterized in that it is infected with any one selected from the group consisting of gram-negative bacteria, gram-positive bacteria, and fungi, but is not limited thereto. For the purpose of the present invention, it refers to all infectious diseases caused by gram-negative bacteria, gram-positive bacteria and fungi. For example, the infectious disease of the present invention includes respiratory diseases, digestive diseases, and inflammatory diseases caused by gram-negative bacteria, gram-positive bacteria and fungi, and specifically, pneumonia, peritonitis, meningitis, wound infection, osteoarthritis, cholecystitis, urinary tract Infectious disease, meningitis, endocarditis, myocarditis, epicarditis, arthritis, population infection, gonorrhea, bacterial dysentery, enteritis, conjunctivitis, gastritis, otitis media, cystitis, lymphangitis, sepsis, etc., but are not limited thereto

The gram-negative bacteria is Escherichia O (Escherichia) genus Pseudomonas (Pseudomonas) genus Acinetobacter (Acinetobacter) genus, Salmonella (Salmonella), A keurep when Ella (Klebsiella) genus Neisseria (Neisseria) genus Enterobacter ( Enterobacter) genus Shigella (Shigella), a morak Cellar (Moraxella) genus Helicobacter (Helicobacter), a to stacking notes Pomona's (Stenotrophomonas), a the group consisting of genus Vibrio (Bdellovibrio) in and Legionella (Legionella) as beudel It may be at least one selected from the group consisting of Escherichia coli and Pseudomonas aeruginosa , preferably any one or more selected from the group consisting of.

The Gram-positive bacteria are Staphylococcus (Stahpylococcus), A Streptococcus (Streptococcus), A pneumophila Rhodococcus (Pneumococci) genus Bacillus (Bacilli) genus Corynebacterium (Corynebacteria) genus Listeria (Listeria) genus Clostridia ( Clostridia ) It may be at least one selected from the group consisting of the genus and Treponema , and preferably, it may be at least one selected from the group consisting of Staphylococcus aureus, Streptococcus mutans and Bacillus cereus .

The fungus may be any one selected from the group consisting of the genus Candida, the genus Aspergillus and the genus Cryptococcus, preferably selected from the group consisting of Aspergillus parasiticus and Candida albicans It may be any one or more.

In the present invention, "prevention" means any action that suppresses or delays the onset of an infectious disease by administration of the composition, and "treatment" means that the symptoms caused by an infectious disease are improved or beneficially changed by administration of the composition means any action.

The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food, food additives and feed, and includes animals including humans or livestock. target for eating. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

The food composition according to the present invention can be prepared in various forms according to conventional methods known in the art. Common foods include, but are not limited to, beverages (including alcoholic beverages), fruits and their processed foods (eg, canned fruit, bottled, jam, marmalade, etc.), fish, meat and their processed foods (eg, ham, sausages) Corned beef, etc.), breads and noodles (eg udon noodles, soba noodles, ramen, spagate, macaroni, etc.), fruit juice, various drinks, cookies, syrup, dairy products (eg butter, cheese, etc.), edible vegetable oils and fats, margarine , vegetable protein, retort food, frozen food, various seasonings (eg, soybean paste, soy sauce, sauce, etc.) can be prepared by adding the processed extract of the present invention to Senendesmus intermedius ( Scenedesmus intermedius ). In addition, the nutritional supplement is not limited thereto, but it can be prepared by adding the processed extract of the present invention to the capsule, tablet, pill, etc. Senendesmus intermedius ( Scenedesmus intermedius ). In addition, the processed extract of Scenedesmus intermedius of the present invention can be prepared in the form of tea, juice, and drink and liquefied, granulated, encapsulated and powdered so that it can be consumed (health drink) and ingested. have. In addition, in order to use the processed extract of Scenedesmus intermedius of the present invention in the form of a food additive, it may be prepared and used in the form of a powder or a concentrate. Further, it is possible to mix with the invention Sennen des mousse inter Medi-house (Scenedesmus intermedius) processing chuchulmulwa antimicrobial active ingredients and the known known to have antioxidant properties of the prepared in the form of a composition.

When the processed extract of Scenedesmus intermedius of the present invention is used as a health drink, the health drink composition may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional drink. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as taumatine, stevia extract; A synthetic sweetener such as saccharin or aspartame may be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the composition of the present invention.

In addition, the processed extract of Scenedesmus intermedius of the present invention may be contained as an active ingredient in an antibacterial and antioxidant composition or a food composition, the amount of which is selected from the group consisting of gram-negative bacteria, gram-positive bacteria and fungi. Although it is not particularly limited to an effective amount to achieve antibacterial activity and antioxidant action that inhibits or kills the growth of one or more bacteria, it is preferably 0.01 to 100% by weight based on the total weight of the total composition. The food composition of the present invention can be prepared by mixing the processed extract with other active ingredients known to be effective in antibacterial and antioxidant compositions with Scenedesmus intermedius.

In addition to the above, the health food of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid, pectic acid salts, alginic acid, alginic acid salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, It may contain glycerin, alcohol or a carbonation agent and the like. In addition, the health food of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The antibacterial and antioxidant composition, food composition, and pharmaceutical composition for the prevention or treatment of infectious diseases of the present invention contains only Senendesmus intermedius ( Scenedesmus intermedius ) Processed extract, or Senendesmus intermedius ( Scenedesmus intermedius ) Processed extract and It may contain one or more types of active ingredients exhibiting similar functions. When an additional component is included, the muscle function improvement effect of the composition of the present invention may be further enhanced. When adding the above ingredients, skin safety according to combined use, ease of formulation, and stability of active ingredients can be considered.

Therefore, the processed extract of Scenedesmus intermedius of the present invention has excellent and broad antibacterial activity against any one or more selected from the group consisting of gram-negative bacteria, gram-positive bacteria and fungi, along with a remarkable antioxidant effect Therefore, it can exhibit a preventive or therapeutic effect in an infectious disease induced by infection with any one or more selected from the group consisting of gram-negative bacteria, gram-positive bacteria and fungi, and can be used as an active ingredient in a pharmaceutical composition thereof.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable salt of the processed extract of Senendesmus intermedius. As used herein, the term "pharmaceutically acceptable" means that it is physiologically acceptable and does not normally cause an allergic reaction or a reaction similar thereto when administered to humans, and the salt includes a pharmaceutically acceptable free acid (free acid). acid) is preferred.

In addition, the pharmaceutical composition for preventing or treating infectious diseases caused by any one or more bacteria selected from the group consisting of gram-negative bacteria, gram-positive bacteria and fungi of the present invention may further include a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Carriers for parenteral administration may also include water, suitable oils, saline, aqueous glucose and glycols, and the like. In addition, it may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. As other pharmaceutically acceptable carriers, reference may be made to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The pharmaceutical composition of the present invention may be administered to mammals including humans by any method. For example, it may be administered orally or parenterally, and parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal. , intranasal, enteral, topical, sublingual or rectal administration.

The pharmaceutical composition of the present invention may be formulated as a formulation for oral administration or parenteral administration according to the administration route as described above. When formulated, one or more buffers (eg, saline or PBS), antioxidants, bacteriostatic agents, chelating agents (eg, EDTA or glutathione), fillers, bulking agents, binders, adjuvants (eg, aluminum hydroxide) side), suspending agents, thickening agents, wetting agents, disintegrating agents or surfactants, diluents or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, liquids, gels, syrups, slurries, suspensions or capsules, and such solid preparations include at least one excipient in the pharmaceutical composition of the present invention, for example , starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose , methyl cellulose, sodium carboxymethyl cellulose, and hydroxypropylmethyl-cellulose or gelatin may be mixed and prepared. For example, tablets or sugar tablets can be obtained by blending the active ingredient with a solid excipient, grinding it, adding a suitable adjuvant, and processing it into a granule mixture.

In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, or syrups. In addition to water or liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, flavoring agents, or preservatives may be included. .

In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant, and an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, and a preservative may be additionally included. .

When administered parenterally, the pharmaceutical composition of the present invention may be formulated according to methods known in the art in the form of injections, transdermal administrations and nasal inhalants together with suitable parenteral carriers. In the case of the injection, it must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. For injection, examples of suitable carriers include, but are not limited to, water, ethanol, polyols (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof, and/or a solvent or dispersion medium containing vegetable oil. can More preferably, suitable carriers include Hanks' solution, Ringer's solution, PBS (phosphate buffered saline) with triethanolamine or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose. etc. can be used. In order to protect the injection from microbial contamination, it may further include various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In addition, in most cases, the injection may further contain an isotonic agent such as sugar or sodium chloride.

In the case of transdermal administration, forms such as ointment, cream, lotion, gel, external solution, pasta, liniment, and air are included. In the above, 'transdermal administration' means that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin by topically administering the pharmaceutical composition to the skin.

In the case of administration by inhalation, the compounds for use according to the invention may be administered in pressurized packs or using a suitable propellant, for example, dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be conveniently delivered in the form of an aerosol spray from the nebulizer. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. For example, gelatin capsules and cartridges for use in inhalers or insufflators may be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a recipe commonly known to all pharmaceutical chemistry.

The pharmaceutical composition for the prevention or treatment of infectious diseases caused by any one or more bacteria selected from the group consisting of gram-negative bacteria, gram-positive bacteria and fungi of the present invention contains Senendesmus intermedius processed extract in an effective amount It can provide a desirable effect of preventing and treating infectious diseases when In the present specification, the term 'effective amount' refers to an amount that exhibits a higher reaction than that of a negative control group, and preferably to inhibit the growth or function of any one or more bacteria selected from the group consisting of gram-negative bacteria, gram-positive bacteria and fungi. say enough The pharmaceutical composition of the present invention may contain 0.01 to 99.99% of the processed extract of Senendesmus intermedius , and the remaining amount may be occupied by a pharmaceutically acceptable carrier. The effective amount of the processed extract of Senendezmus intermedius contained in the pharmaceutical composition of the present invention will vary depending on the form in which the composition is commercialized.

The total effective amount of the pharmaceutical composition of the present invention may be administered to a patient as a single dose, or may be administered by a fractionated treatment protocol in which multiple doses are administered for a long period of time. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease. When administered parenterally, it is preferably administered in an amount of 0.01 to 50 mg, more preferably 0.1 to 30 mg per 1 kg of body weight per day, based on the processed extract of Scenedesmus intermedius, and oral When administered, it is preferably administered in an amount of 0.01 to 100 mg, more preferably 0.01 to 10 mg per 1 kg of body weight per day based on the processed extract of Senendesmus intermedius, divided into 1 to several times administered. can do. However, the dose of the Senendesmus intermedius (Scenedesmus intermedius) processed extract depends not only on the administration route and number of treatments of the pharmaceutical composition, but also on various factors such as the age, weight, health status, sex, severity of the disease, diet and excretion rate of the patient. Since the effective dosage for the patient is determined in consideration of this, those of ordinary skill in the art in consideration of this point will use the Senendesmus intermedius (Scenedesmus intermedius) processed extract for the prevention and treatment of muscle diseases. It will be possible to determine an appropriate effective dosage according to the The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route and administration method as long as the effect of the present invention is exhibited.

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

The pharmaceutical composition for preventing and treating infectious diseases of the present invention may also be provided in the form of an external preparation comprising a Senendesmus intermedius processed extract as an active ingredient.

When the pharmaceutical composition for the prevention and treatment of infectious diseases caused by any one or more bacteria selected from the group consisting of gram-negative bacteria, gram-positive bacteria, and fungi of the present invention is used as an external preparation for skin, a fatty substance, an organic solvent, a solubilizing agent, Thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic emulsifiers, nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives , vitamins, blockers, humectants, essential oils, dyes, pigments, hydrophilic actives, lipophilic actives or any other ingredients commonly used in external preparations for the skin, such as lipid vesicles. can In addition, the above ingredients may be introduced in an amount generally used in the field of dermatology.

When the pharmaceutical composition for preventing and treating infectious diseases caused by any one or more bacteria selected from the group consisting of Gram-negative bacteria, Gram-positive bacteria and fungi of the present invention is provided as an external preparation for the skin, but is not limited thereto, ointments, patches, It may be in the form of a gel, cream or spray.

In addition, the processed extract of the present invention Senendesmus intermedius ( Scenedesmus intermedius ) Since it can exhibit a high level of antibacterial activity as well as an antioxidant effect to inhibit or kill the growth of Gram-positive bacteria, Gram-negative bacteria and fungi, Gram-negative bacteria, Gram Infectious diseases caused by any one or more bacteria selected from the group consisting of gram-negative bacteria, gram-positive bacteria and fungi, which can exhibit an effect in infectious diseases caused by any one or more bacteria selected from the group consisting of positive bacteria and fungi It can be used as an active ingredient in a food composition for improvement.

Another aspect of the present invention relates to a method for preparing a processed extract of Senendesmus intermedius comprising the following steps.

1) culturing Senendesmus intermedius ( Scenedesmus intermedius ) to obtain a culture solution or cells thereof;

2) preparing an extract by adding a solvent to the Senendesmus intermedius (Scenedesmus intermedius) culture medium or cells; and

3) transesterifying the extract to prepare a processed extract of Senendesmus intermedius (Scenedesmus intermedius).

After step 1), 1a) may further include a step of pulverizing through drying or freeze-drying. The freeze-drying is preferably made at minus 100 to minus 60 ℃.

The solvent may be any one or more solvents selected from the group consisting of water, C1 to C4 lower alcohols, hexane and chloroform, preferably a mixed solvent of methanol and chloroform. The mixed solvents of chloroform and the mixture is most preferable 1 to 3 parts by weight based on 1 part by weight of methanol, the active ingredient (fatty acid) of the said Sennen des mousse inter Medi-house (Scenedesmus intermedius), the extract in the case out of the above range This can be extracted in small amounts.

It is preferable to mix 0.5 to 5 parts by weight of a solvent based on 1 part by weight of the cells of the Senendesmus intermedius or the culture medium of Senendesmus intermedius, the Senendesmus intermedius ( Scenedesmus intermedius ) When the weight ratio of the cells or Senendesmus intermedius culture medium and the solvent is out of the above range, the active ingredient of the Senendesmus intermedius ( Scenedesmus intermedius ) can be extracted in a small amount in the extract have.

After preparing an extract containing a fat component from Scenedesmus intermedius through steps 1) and 2) above, transesterification of the fat component of the extract is performed to prepare a processed extract.

The step of washing 1 to 5 times with distilled water to remove the water-soluble component from the extract may be further included.

In the transesterification reaction in step 3), it is preferable to perform the transesterification reaction by adding alcohol and a base catalyst to the extract.

The base catalyst may be at least one selected from anhydrous sodium sulfate, sulfate hydroxide, and potassium hydroxide, and the alcohol may be at least one selected from the group consisting of methanol, ethanol, propanol, butanol, and mixtures thereof. have.

The transesterification reaction is preferably performed at 50 to 70° C. for 1 to 5 hours.

Hereinafter, the present invention will be described in more detail through Examples and Preparation Examples. These Examples and Preparation Examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these Examples and Preparation Examples. .

Example 1. Lipid content analysis of various microalgal extracts

Twenty unidentified microalgae (NTEB01 ~ NTEB20) to be used in the present invention are provided from Bharathidasan University (Fresh water (NRMC-F)) in India, which is a National Repositor related to microalgae and cyanobacteria. did Each of the microalgae was cultured, and the recovered culture solution was centrifuged at 10,000 rpm for 10 minutes (TGL-20MC, PINGFAN, China) to harvest the cells (biomass). The cells were freeze-dried, and methanol:chloroform solvent mixed in a weight ratio of 1:2 was added thereto to prepare a mixture. At this time, 100 mg of a solvent (a weight ratio of 1:1) was treated per 100 mg of the freeze-dried cells. The mixture was extracted using a mortar and pestle, and then treated with distilled water to remove water-soluble impurities from the extract. , The two layers were separated using a separatory funnel, and the lower lipid layer was collected. The collected lipid layer was extracted from the 20 microalgal cells by evaporating the solvent through a rotary vacuum evaporator, respectively. The total lipid content in each extract was measured by gravimetric method (AUY 220, Shimadzu, Japan).

1 is a view showing the comparison of each measurement of the total lipid content in the extracts of a total of 20 unidentified microalgae. 1a is a graph showing the analysis of the lipid content relative to the total cell weight for each extract of NTEB01 to NTEB20. This is the average value of a total of three experiments, and is expressed as mean ㅁ SE.

Figure 1b is a micrograph of the extract of NTEB15, the table on the right shows the total biomass weight, dry biomass weight and lipid productivity of Scenedesmus sp. In this case, the numerical value represents the average value of a total of three experiments.

As shown in FIG. 1 , arbitrarily 20 types of microalgae were selected, and extracts were obtained and analyzed therefrom. As a result, it was confirmed that about 10 types of extracts of microalgae exhibiting a lipid content of 20% or more relative to the total weight of the cells.

In particular, the NTEB15 extract had an average lipid content of 27.9 ㅁ 0.69% relative to the total weight of the dried cells, and was confirmed to be the best lipid producer. Furthermore, the morphological features of NTEB15 microalgae are single-celled thalli, spherical to ellipsoidal, elongate or elongated spindle to fusiform, cell poles capitates, It was confirmed to have an obtuse or long tapering, uninucleate cell with a single pyrenoid, which is morphology with Scenedesmus intermedius, a high-lipid accumulator isolated from a freshwater habitat. It was confirmed that they are very similar academically. Therefore, NTEB15, which was confirmed morphologically to be Scenedesmus intermedius while producing the most lipids among a total of 20 unidentified microalgae, was selected.

Example 2. Senendesmus intermedius ( Scenedesmus intermedius ) composition analysis of processed extracts

The composition of the extract derived from the NTEB15 strain, Scenedesmus intermedius , selected in Example 1 was analyzed through GC analysis.

Specifically, Senendesmus intermedius ( Scenedesmus intermedius ) Microalgae were cultured, and the recovered culture solution was centrifuged at 10,000 rpm for 10 minutes (TGL-20MC, PINGFAN, China) to harvest the cells (biomass, biomass). . The cells were freeze-dried, and methanol:chloroform solvent mixed in a weight ratio of 1:2 was added thereto to prepare a mixture. At this time, 100 mg of a solvent (a weight ratio of 1:1) was treated per 100 mg of the freeze-dried cells. The mixture was extracted using a mortar and pestle, and then treated with distilled water to remove water-soluble impurities from the extract. , The two layers were separated using a separatory funnel, and the lower lipid layer was collected. The collected lipid layer was evaporated by evaporating the solvent through a rotary vacuum evaporator, thereby preparing a processed extract of Scenedesmus intermedius.

In order to methyl esterify the extract through transesterification, 10 to 20 times of methanol containing anhydrous sodium sulphate dl to act as a catalyst was added to the extract. The reaction was vortexed for 10 minutes (KMC). -1300 V), put in an incubator at 100 ° C for 3 hours to carry out transesterification reaction, cooled at room temperature, mixed with 1 mL of distilled water, and centrifuged (4000 g) to separate the upper and lower layers from the lower layer The bottom of the was removed using a syringe, and the upper layer was recovered. In order to analyze the FAME composition in the processed extract of Senendesmus intermedius of the upper layer, a gas chromatography (GC 2014, Shimadzu, Japan) equipped with a flame lonization detector (FID) was used. Prepared Senendesmus intermedius ( Scenedesmus intermedius ) 1 μL of the processed extract sample was placed on a FAME WAX column (Restek, USA) (30 m × 32 mm ID × 25 μm film thickness). The temperature conditions of the program were as follows: initially maintained at 140 °C for 5 minutes, then at a ramp rate of 2 °C/min, held at ~ 230 °C for 5 minutes. The column flow was set at 22.2 mL/min. The instrument conditions were as follows: Carrier gas nitrogen: FID was set at 260 °C, split ratio was 10: 1. The run time (run time) of a single sample was 55 minutes.

FAME-MIX (C4-C24, Supelco Analytical, USA) was used as a standard material for composition analysis of the processed extract of Scenedesmus intermedius, and it was measured through GC-FID.

Figure 2 shows the analysis of the composition of the Senendesmus intermedius ( Scenedesmus intermedius ) processed extract confirmed through gas chromatography.

As shown in Figure 2, the main compounds identified from the processed extract of Scenedesmus intermedius were palmitic acid methyl ester (C16: 0) (23.08% w / w), cis-10-heptadecanoic acid methyl ester ( Confirmed to contain C17:1) (0.44% w/w), stearidonic acid methyl ester (C19:2) (5.04% w/w) and linolenic acid methyl ester (C18:3) (3.76% w/w) became In the extract, it was confirmed that, in addition to the main FAME, FAME having various pharmacological activities was present at a relatively low concentration.

In the present invention, Senendesmus intermedius (Scenedesmus intermedius) processed extract is GC analysis, palmitic acid accounts for 23.08% of the total weight percent of FAME, it was confirmed that a large amount of palmitic acid (C16: 0) is present.

Based on the above, the antibacterial activity and weight of palmitic acid were compared between the processed extract of Scenedesmus intermedius of the present invention and other known conventional microalgal extracts, and the results are shown in FIG. . The known conventional microalgae extract was also compared based on the same processed extract (FAME) as the present invention.

[S.No 1] L.A. Meireles, A.C. Guedes, F.X. Malcata, Lipid class composition of the microalga Pavlova lutheri: Eicosapentaenoic and Docosahexaenoic Acids, Agri. Food Chem. 51 (2003) 2237-2241.

[S.No 2] Y. Ghasemi, A. Moradian, A. Mohagheghzadeh, S. Shokravi, M.H. Morowvat, Antifungal and antibacterial activity of the microalgae collected from paddy fields of Iran: characterization of antimicrobial activity of Chroococcus disperses, J. Biol. Sci. 7 (2007) 904-910.

[S.No 3] D. MubarakAli, R. PraveenKumar, T. Shenbagavalli, T. MariNivetha, A. ParveezAhamed, A. NaifAbdullah, N. Thajuddin, New reports on anti-bacterial and anti-candidal activities of fatty acid methyl esters (FAME) obtained from Scenedesmus bijugatusvar. bicellularis biomass, RSC Adv. 2 (2012) 11552-11556.

[S.No 4] H.M. Najdenski, G. GigovaLiliana, I. Iliev Ivan, S. Pilarski Plamen, T. Lukavsky Jaromir, V. Iva, S. Ninova Mariana, K. Kussovski Vesselin, Antibacterial and antifungal activities of selected microalgae and Cyanobacteria, Int. J. Food Sci. Technol.48 (2013) 1533-1540.

[S.No 5] R. Thangam, V. Suresh, W. Asenath Princy, M. Rajkumar, N. Senthilkumar, P. Gunasekaran, R. Rengasamy, C. Anbazhagan, K. Kaveri, S. Kannan, CPhycocyanin from Oscillatoria tenuis exhibited an antioxidant and in vitro antiproliferative activity through induction of apoptosis and G0/G1 cell cycle arrest, Food Chem. 140 (2013) 262-272.

[S.No 6] D. Surendhiran, M. Vijay, Exploration on bioflocculation of Nannochloropsis oculata using response surface methodology for biodiesel production, Sci. World J. 2014 (2014) Article ID 202659, 9 pages https://doi.org/0.1155/2014/202659.

[S.No 7] Y.S. Cakmak, M. Kaya, M. Asan-Ozusaglam, Biochemical composition and bioactivity screening of various extracts from Dunaliella salina, a green microalga, EXCLI J. 13 (2014) 679-690.

[S.No 8] O.M.A. Salem, E.M. Hoballah, M. SafiaGhazi, N. Suzy, N. Hanna, Antimicrobial activity of microalgal extracts with special emphasize on Nostoc sp, Life Sci. J. 11 (2014) 752-758.

[S.No 9] E. Baldev, D. MubarakAli, M. Dhivya, M. Kanimozhi, T. ShakenaFathima, N.S. Alharbi, C. Arunachalam, S.A. Alharbi, N. Thajuddin, Facile and novel strategy for methods of extraction of biofuel grade lipids from microalgae- an experimental report, Int. J. Biotechnol. Wellness Ind. 3 (2014) 121-127.

[S.No 10] G. Sibi, Inhibition of lipase and inflammatory mediators by Chlorella lipid extracts for antiacne treatment, J. Adv. Pharm. Technol. Res 6 (2015) 7-12.

[S.No 11] V.R. Sushanth, M. Rajasekar, Antioxidant and Antimicrobial activities in the four species of marine microalgae isolated from Arabian Sea of Karnataka coast, Indian J. Geo Mar. Sci. 44 (2015) 69-75.

[S.No 12] Senendesmus intermedius of the present invention ( Scenedesmus intermedius ) Processed extract

Microalgae have been considered as a major renewable resource of natural FAME (Fatty acid methyl esters), but the FAME profiles that can be obtained from each organism are different, and obtained from the organism according to the characteristics of the available fatty acids. It can be confirmed that the functional properties of one FAME compound are determined. In addition to FIG. 3, it is known that short-chain and long-chain fatty acid methyl esters exist in the green algae H. pluvialis and S. obliques, but they have antibacterial activity only against E. coli and S. aureus. As described above, it can be seen that the content of palmitic acid is completely different even for extracts of the same genus Senendesmus, and in particular, the effect of MIC on bacteria is also very different.

Therefore, as well as the content of palmitic acid, the effect and effect varies depending on the overall composition of the Senendesmus intermedius ( Scenedesmus intermedius ) processed extract, and in the present invention, the Senendesmus intermedius ( Scenedesmus intermedius ) processed extract is It can be seen that, compared to other microalgae in the prior art, it exhibits a significantly superior MIC against gram-negative and gram-positive bacteria as well as fungi, and has statistically significantly superior antibacterial activity.

Example 3. Senendesmus intermedius ( Scenedesmus intermedius ) Analysis of Antibacterial Efficacy of Processed Extracts

1) Human pathogen culture for antibacterial activity evaluation

The antibacterial activity of Scenedesmus intermedius processed extracts includes Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, Streptococcus mutans (bacteria), Candida albicans (unicellular yeast) and Aspergillus parasiticus (unicellular yeast) and Aspergillus parasiticus A variety of human pathogens were analyzed. To this end, each of the human pathogens was prepared by culturing as follows. First, the bacteria were cultured in Luria-Bertani medium (LB; yeast extract 0.5%, NaCl 0.5%, tryptone 1%) at 37 ° C, and C. albicans was cultured in yeast peptone medium (YPD; yeast extract 1%, peptone 2%; Dextrose 2%) incubated at 30 ℃. A. parasiticus was cultured in potato dextrose medium (PD; potato dextrose 2.4%) at 24 °C. When performing the solid culture, agar (1.5%) was added to each medium to prepare a solid medium, and then cultured using it.

2) Agar diffusion analysis

The antibacterial activity of the processed extracts of Scenedesmus intermedius was evaluated using the Kirby-Bauer agar diffusion method. ^{Briefly, about 10 4} colonies (CFUs) of the newly cultured human pathogens were uniformly spread on a muller-hinton agar plate (beef extract 0.2%, casein 1.75%, strarch 0.15%, agar 1.7%). Two wells were made using a sterile cork bore, and approximately 35 μl of FAME was placed in one well and methanol was placed in the other well to serve as a control. Agar plates in which bacteria were widely cultured were cultured at 37 °C for 24 hours, in the case of C. albicans at 30 °C for 48 hours, and in the case of A. parasiticus at 25 °C for 3-5 days. Measure and compare growth inhibition zones before and after incubation.

3) Minimum Inhibitory Concentration (MIC) Assay

The antibacterial activity of the processed extract of Senendesmus intermedius was quantitatively analyzed by measuring the MIC against the pathogen. Mix cultures (50 mL) from all the above pathogens ( E. coli, P. aeruginosa, S. aureus, S. mutans, B. cereus, C. albicans and A. parasiticus ) in 5 mL of LB, YPD or PD broth. A solution of the processed extract (13.5 mg mL-1) of various concentrations of Senendesmus intermedius was added and placed in a sample analysis vessel. After overnight incubation, the lowest FAME concentration without growth (no turbidity) was determined as the MIC for each pathogen cultured under appropriate conditions.

4) Analysis result

Figure 4 is Senendesmus intermedius for various pathogens ( Scenedesmus intermedius ) Agar diffusion analysis results of processed extracts. Figure 4 (a) is for E. coli, Figure 4 (b) is for P. aeruginosa, Figure 4 (c) is for S. aureus. 4(d) is for B. cereus, FIG. 4(e) is for C. albicans, and FIG. 4(f) is for A. parasiticus.

As shown in FIG. 4 , the processed extract of Scenedesmus intermedius was tested against the various human pathogens, and as a result, it was confirmed that it had both antibacterial and antibacterial activity through the formation of the preceding inhibition zone. Senendesmus intermedius ( Scenedesmus intermedius ) Processed extract against Gram-positive (S. aureus and B. cereus), Gram-negative (E. coli and P. aeruginosa) bacteria and fungi (A. parasiticus and C. albicans) It was analyzed that it had very good activity as an antibacterial agent. In particular , the processed extract of Scenedesmus intermedius had significantly higher inhibitory activity on Gram-negative bacteria than on Gram-positive bacteria. In agar diffusion assay, E. coli and P. aeruginosa (gram-negative bacteria) showed inhibition regions of about 21 and 17 mm, respectively, and Gram-positive bacteria S. aureus, B. cereus and S. mutans were identified with zones of inhibition of 16.5, 15 and 22 mm, respectively. In particular, it was confirmed that S. mutans has a large inhibitory region as large as that of E. coli. Previously, it has been reported that extracts from Scenedesmus sp. have very weak or no antibacterial activity. And it can be seen that it has a very good antibacterial activity against human pathogens such as fungi.

5 is a graph showing the inhibitory activity of Senendesmus intermedius ( Scenedesmus intermedius ) processing extract against various human pathogens. Figure 5a is a measure of the inhibition zone for each pathogen and expressed as a percentage, Figure 5b shows the measurement of the MIC for each pathogen.

As shown in FIG. 5 , the MIC for Gram-negative bacteria of the processed extract of Scenedesmus intermedius was 12-24 μg/mL, and the MIC for Gram-positive bacteria was 24-48 μg/mL. The MIC against the fungus was measured as high as 60-192 μg/mL.

Fatty acid methyl esters (FAMEs) have been found to exhibit potent antimicrobial activity when compared to lipids or free fatty acids. This results from a unique function influenced through modification of the carbonyl species in fatty acids. In addition to this, it is known that polyhydric alcohols whose biological activity is increased through transesterification are also formed in the above modification. Nevertheless, in most processed extracts (FAME) extracted from microalga, it was confirmed that the MIC for bacteria was 50,000 μg/mL (see S. NO 8). In addition, D. salina had a MIC of 1250-2500 μg/mL against bacteria and MIC of 2500 μg/mL or more against fungi (see S. NO 7).

After the development of antibiotics, mankind expected to be able to easily conquer bacterial infections, but faced a new problem of 'antibiotic resistance'. made it difficult In particular, the main resistance mechanism showing resistance to third-generation cephalosporin antibiotics in enterobacteriaceae such as Escherichia coli and Pneumococcus pneumoniae is generation of extended-spectrum beta-lactamase (ESBL). ESBL is an enzyme produced by bacteria, and it should be noted that the MIC may vary depending on the type and production level of ESBL.

Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa are important pathogens that can cause urinary tract infections, abdominal infections, and pneumonia. In particular, Pseudomonas aeruginosa and Acinetobacter are frequently resistant to carbapenem, which is a major clinical problem. As a result, it was confirmed that the processed extract of Scenedesmus intermedius has very good antibacterial activity against the above pathogens. You can see that it can be used. In addition, it can be seen that the fatty acid methyl ester (FAME), which is known to have excellent antibacterial activity, exhibits remarkably excellent antibacterial activity against ESBL or multidrug-resistant bacteria, which have little effect on conventional various microalgae extracts including the same.

Example 4. Senendesmus intermedius ( Scenedesmus intermedius ) Analysis of antioxidant activity of processed extracts

1) DPPH scavenging ability analysis

The effect of the processed extract of Senendesmus intermedius on DPPH radicals was evaluated through DPPH radical scavenging activity analysis. Was to prepare a DPPH solution was added to 0.135mM DPPH in methanol solution, Sennen des mousse inter Medi-house (Scenedesmus intermedius) distributing the processed extract at different concentrations in methanol solution sample 1.0 mL (1.2, 2.5, 5 , 10, 20, 40 μg / mL) was prepared, and 1 mL of the DPPH solution and 1 mL of the sample were mixed 1:1 to prepare a reaction solution. The reaction solution was sufficiently vortexed and left for 30 minutes in a dark place at room temperature. The absorbance of the reaction solution was measured with a spectrophotometer at 517 nm. The ability to remove DPPH radicals was calculated through Equation 1 below.

[Equation 1]

DPPH scavenging activity (%) = (absorbance of control group (Abs Control) - absorbance of experimental group (Abs Test))/(absorbance of control group (Abs Control))×100

2) Hydroxyl radical scavenging ability test

The hydroxyl radical scavenging ability was measured by the deoxyribose method. Specifically, 2.8 mM 2-deoxy-2-ribose, 0.1 mM phosphate buffer (pH 7.4), 20 μM ferric chloride, EDTA 100 μM, hydrogen peroxide 500 μM, as Corbic acid (ascorbic acid) 100 μM and Senendesmus intermedius ( Scenedesmus intermedius ) Processed extracts were mixed at various concentrations (1.25 to 40 μg/mL), but the final volume thereof was prepared to be 1 mL. Then, the mixture was reacted at room temperature for 45 minutes. 800 μL of the reaction was recovered, 1.5 mL of TCA solution (2.8%), 1 mL of TBA solution (1% in 50 mM NaOH) and 200 μL of sodium dodecyl sulphate were added. Finally, the reaction solution was heated at 90° C. for 30 minutes to develop color, cooled and the absorbance of the blank solution was measured at 532 nm. The hydroxyl free radical scavenging activity (%) was calculated according to Equation 2 below.

Hydroxyl radical scavenging activity (%) = (absorbance of control group (Abs Control) - absorbance of experimental group (Abs Test))/(absorbance of control group (Abs Control))×100

3).Analysis Results

6 is a graph showing DPPH radical scavenging activity (bold text) and hydroxyl radical scavenging activity (dotted line) as a result of measuring the antioxidant activity of a processed extract of Scenedesmus intermedius. It is the average value of a total of three experiments.

As shown in FIG. 6 , it was confirmed that the processed extract of Scenedesmus intermedius had excellent scavenging activity against DPPH radicals. DPPH scavenging activity continued to increase as the concentration of the processed extract of Senendesmus intermedius increased, and when the processed extract of Scenedesmus intermedius was treated at a maximum concentration of 40 μg/mL It was confirmed that the DPPH scavenging activity was 80%. This can be said to be very similar to the maximum activity level (80 to 82%) of ascorbic acid, which is the best natural antioxidant in the prior art.

As shown in FIG. 7 , as the concentration of the processed extract of Scenedesmus intermedius increased from 1.25 to 40 μg/mL, the hydroxyl radical scavenging ability also continuously increased. That is, as a result of evaluating the hydroxyl radical scavenging activity through reduction of 2-deoxyribose , when the concentration of the processed extract of Scenedesmus intermedius was 20 μg/mL, the hydroxyl radical scavenging activity was 60%. , it was confirmed that the highest hydroxyl radical scavenging activity (70%) was exhibited at 40 μg/mL. Hydroxyl radicals cause oxidative damage to DNA, lipids and proteins, and the processed extract of Scenedesmus intermedius of the present invention evaluated the hydroxyl radical scavenging ability through reduction of deoxyribose. It was found that the concentration had a very remarkably excellent antioxidant effect.

Combining the above experimental results, it was confirmed that the processed extract of Scenedesmus intermedius in the present invention exhibited the best antibacterial and antifungal activity among microalgae. As a result of analyzing the composition of the processed extract of Scenedesmus intermedius by gas chromatography, it was confirmed that palmitic acid (C16: 0) was present in a high ratio, and other fatty acid methyl esters (FAMEs) were appropriate. It was confirmed that the ratio exists. Fatty acids exist in different amounts depending on the microalgae, and accordingly, a composition of fatty acid methyl ester (FAME) is formed, and various effects are exerted through the combination of these constituents. Palmitic acid is known to have strong antibacterial activity, but when compared to other microalgal extracts in which only a large amount of palmitic acid is present , the processed extracts of Scenedesmus intermedius have higher and more extensive bacterial (gram-negative) , Gram-positive and fungi), it can be seen that the content and combination of the lipid components of the extract are very important when looking at the achievement of the antioxidant effect as well as the remarkable antibacterial activity.

Through the present invention, it can be seen that the processed extract of Scenedesmus intermedius is a new source of antibacterial agents and exhibits excellent effects in treating human pathogens and further multidrug-resistant pathogens.

Hereinafter, an example of the preparation of a drug or food containing the processed extract of Scenedesmus intermedius according to the present invention as an active ingredient will be described, but the present invention is not intended to limit it, but to describe it in detail. . With the processed extract of Scenedesmus intermedius having excellent antibacterial activity and antioxidant effect, the pharmaceutical or food compositions of Preparation Examples 1 and 2 were prepared according to a conventional method according to the following composition components and composition ratios.

<Preparation Example 1> Preparation of pharmaceutical formulations

<1-1> Preparation of powders

Senendesmus intermedius of the present invention ( Scenedesmus intermedius ) 0.1 g of processed extract

1.5 g lactose

0.5 g of talc

The above ingredients were mixed and filled in an airtight bag to prepare a powder.

<1-2> manufacture of tablets

Senendesmus intermedius of the present invention ( Scenedesmus intermedius ) 0.1 g of processed extract

7.9 g of lactose

1.5 g of crystalline cellulose

0.5 g magnesium stearate

After mixing the above ingredients, tablets were prepared by direct tableting method.

<1-3> manufacture of capsules

Senendesmus intermedius of the present invention ( Scenedesmus intermedius ) 0.1 g of processed extract

5 g cornstarch

4.9 g of carboxycellulose

After mixing the above ingredients to prepare a powder, the powder was filled in a hard capsule according to a conventional capsule preparation method to prepare a capsule.

<1-4> Preparation of injection

Senendesmus intermedius of the present invention ( Scenedesmus intermedius ) 0.1 g of processed extract

Appropriate amount of sterile distilled water for injection

Appropriate amount of pH adjuster

According to a conventional injection preparation method, the content of the above ingredients per 1 ampoule (2 mL) was prepared.

<1-5> Preparation of liquids

Senendesmus intermedius of the present invention ( Scenedesmus intermedius ) 0.1 g of processed extract

10 g isomerized sugar

5 g of mannitol

Purified water appropriate amount

Each component was added and dissolved in purified water according to a conventional liquid preparation method, and an appropriate amount of lemon flavor was added, followed by mixing the above components. Then, purified water was added to adjust the total to 100, filled in a brown bottle and sterilized to prepare a solution.

<Production Example 2> Preparation of food

<2-1> Manufacture of wheat flour food

0.5 to 5.0 parts by weight of the processed extract of Scenedesmus intermedius of the present invention was added to wheat flour, and the mixture was used to prepare bread, cakes, cookies, crackers and noodles.

<2-2> Preparation of soups and gravies

By adding 0.1 to 5.0 parts by weight of the processed extract of Scenedesmus intermedius of the present invention to soup and broth, processed meat products for health promotion, soup and broth of noodles were prepared.

<2-3> Preparation of ground beef

Ground beef for health promotion was prepared by adding 10 parts by weight of the processed extract of Scenedesmus intermedius of the present invention to ground beef.

<2-4> Production of dairy products

5 to 10 parts by weight of the processed extract of Scenedesmus intermedius of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

<2-5> Manufacturing of health supplements

Senendesmus intermedius of the present invention ( Scenedesmus intermedius ) Processed extract 100 mg

appropriate amount of vitamin mixture

Vitamin A Acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

0.5 mg of vitamin B6

0.2 μg of vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

50 μg of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture appropriate amount

ferrous sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

potassium phosphate monobasic 15 mg

Dibasic calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

The composition ratio of the above vitamin and mineral mixture was prepared by mixing components suitable for relatively healthy food in a preferred embodiment, but the mixing ratio may be arbitrarily modified, and the above components are mixed according to a conventional health food manufacturing method. , to prepare granules, and can be used for preparing health food compositions according to a conventional method.

<2-6> Manufacture of health drinks

Senendesmus intermedius of the present invention ( Scenedesmus intermedius ) Processed extract 100 mg

100 mg citric acid

100 mg of oligosaccharides

Plum Concentrate 2 mg

Taurine 100 mg

Add purified water to total 500 mL

After mixing the above ingredients according to a conventional health drink manufacturing method, after stirring and heating at 85° C. for about 1 hour, the resulting solution is filtered and acquired in a sterilized 1 L container, sealed and sterilized, and then stored in a refrigerator according to the present invention used in the manufacture of health beverage compositions.

Although the composition ratio is prepared by mixing ingredients suitable for relatively favorite beverages in a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as demand class, demanding country, and use.

Claims (15)

In the antibacterial composition comprising the processed extract of Scenedesmus intermedius as an active ingredient,
Based on the total dry weight of the processed extract of Scenedesmus intermedius, 20-25 wt% palmitic acid methyl ester (C16:0), 0.1-0.5 wt% cis-10-heptadecanoic acid methyl ester (C17) :1), 2-10 wt% stearidonic acid methyl ester (C19:2) and 1-5 wt% linolenic acid methyl ester (C18:3),
one or more gram-positive bacteria selected from the group consisting of Staphylococcus aureus, Bacillus cereus and Streptococcus mutans;
one or more gram-negative bacteria selected from the group consisting of Escherichia coli and Pseudomonas aeruginosa; or
An antibacterial composition, characterized in that it has antibacterial activity against one or more fungi selected from the group consisting of Aspergillus parasiticus and Candida albicans.
According to claim 1,
The Senendesmus intermedius ( Scenedesmus intermedius ) The processed extract is a composition characterized in that extracted with any one or more solvents selected from the group consisting of water, C1 to C4 lower alcohols, hexane and chloroform.
According to claim 1,
The Senendesmus intermedius ( Scenedesmus intermedius ) The processed extract is Senendesmus intermedius ( Scenedesmus intermedius ) Cells or Senendezmus intermedius ( Scenedesmus intermedius ) Water, C1 to C4 lower alcohol, hexane and After obtaining the extract with any one or more solvents selected from the group consisting of chloroform, a composition characterized in that it is prepared by transesterification by adding alcohol and a base catalyst to the extract.
According to claim 1,
The Sennen des mousse inter Medi mouse cells or Sennen des mousse (Scenedesmus intermedius) inter Medi-house (Scenedesmus intermedius) composition, characterized in that 1 part by weight of the culture medium by adding 0.5 to 5 parts by weight based on the solvent is added.
delete delete In the antibacterial and/or antioxidant food composition comprising the processed extract of Scenedesmus intermedius as an active ingredient,
It characterized in that it contains 20 to 30% by weight of palmitate (C16:0) relative to the total dry weight of the processed extract of Scenedesmus intermedius,
one or more Gram-positive bacteria selected from the group consisting of the Staphylococcus aureus, Bacillus cereus and Streptococcus mutans;
one or more gram-negative bacteria selected from the group consisting of Escherichia coli and Pseudomonas aeruginosa; or
A food composition, characterized in that it has antibacterial activity against one or more fungi selected from the group consisting of Aspergillus parasiticus and Candida albicans.
8. The method of claim 7,
The Senendesmus intermedius ( Scenedesmus intermedius ) The processed extract is a composition characterized in that extracted with any one or more solvents selected from the group consisting of water, C1 to C4 lower alcohols, hexane and chloroform.
8. The method of claim 7,
The Senendesmus intermedius ( Scenedesmus intermedius ) The processed extract is Senendesmus intermedius ( Scenedesmus intermedius ) Cells or Senendezmus intermedius ( Scenedesmus intermedius ) Water, C1 to C4 lower alcohol, hexane and After obtaining the extract with any one or more solvents selected from the group consisting of chloroform, a composition characterized in that it is prepared by transesterification by adding alcohol and a base catalyst to the extract.
8. The method of claim 7,
The Sennen des mousse inter Medi mouse cells or Sennen des mousse (Scenedesmus intermedius) inter Medi-house (Scenedesmus intermedius) composition, characterized in that 1 part by weight of the culture medium by adding 0.5 to 5 parts by weight based on the solvent is added.
delete delete One or more gram-negative bacteria selected from the group consisting of Escherichia coli and Pseudomonas aeruginosa using a Scenedesmus intermedius processed extract as an active ingredient, Staphylococcus aureus, one selected from the group consisting of Bacillus cereus and Streptococcus mutans A pharmaceutical composition for preventing or treating infectious diseases caused by any one or more bacteria selected from the group consisting of one or more fungi selected from the group consisting of Gram-positive bacteria and Aspergillus parasiticus and Candida albicans.
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