KR20160013323A - A New Scolopendrasin-5 peptide isolated from the Scolopendra subspinipes and its synthetic composition - Google Patents

Abstract

The present invention relates to a new scolopendrasin-5 peptide isolated from Scolopendra subspinipes and a composition of the same. In the present invention, provided is a peptide which is synthesized from amino acid deducted from a gene of a peptide isolated from Scolopendra subspinipes, and is expressed by SEQ ID NO: 1. According to the present invention, provided are a new scolopendrasin-5 peptide isolated from Scolopendra subspinipes and a composition of the same which can provide antibiotics, food preservatives, cosmetic preservatives, medicines, etc., which are safe to the human body while expressing antifungal activity without cytotoxicity.

Description

[0001] The present invention relates to novel scolopendrazine-5 peptides isolated from the genus Scolopendra-

The present invention relates to novel scolopendrazine-5 peptides and compositions thereof isolated from Wangzine, and more particularly to novel scolopendrazine-5 peptides isolated from Wang Ji Nee and exhibiting antifungal and antifungal activity without cytotoxicity. And compositions thereof.

 Infection with pathogenic microorganisms is one of the most common and fatal causes of human disease, unfortunately the antibiotic resistance of pathogenic microorganisms has been caused by the abuse of antibiotics.

In fact, the rate at which pathogenic microorganisms are resistant to new antibiotics is much faster than the rate at which analogs of new antibiotics are developed.

For example, pathogenic microbial species such as Enterococcus faecalis , Mycobacterium tuberculosis , and Pseudomonas aeruginosa , which may pose a life threat, I have developed resistance to all antibiotics.

Tolerance to antibiotics is distinct from resistance to antibiotics, which was first discovered in the 1970s by Pneumococcus sp. And provided important clues to the mechanism of action of penicillin.

Resistant species stop growing in the presence of the usual concentration of antibiotics but do not eventually die.

This resistance occurs when the antibiotic inhibits the cell wall synthetase because the autolytic enzyme activity of the bacteria such as autolysin does not occur. This fact suggests that penicillin is an endogenous hydrolytic enzyme Activation kills bacteria and bacteria also inhibit their activity, resulting in the survival of antibiotics.

It is clinically very important that pathogenic microorganisms have resistance to antibiotics, because the inability to eradicate the resistant microorganisms is ineffective in antibiotic treatment in clinical infections.

In addition, tolerance is considered to be a prerequisite for resistance to antibiotics, which is due to the survival of strains despite antibiotic therapy.

These strains acquire new genetic elements that are resistant to antibiotics and continue to grow in the presence of antibiotics.

Since virtually all pathogenic microorganisms showing resistance are also known to be resistant, development of new antibiotics capable of killing pathogenic microorganisms resistant to these antibiotic resistance is urgent.

As described above, it is necessary to develop a new antibiotic to prevent damages caused by pathogenic microorganisms showing resistance to antibiotics, and to develop new antibiotics that act independently of autolysin activity.

In addition, there is a need to provide pharmaceutical compositions for effectively treating such new antibiotics with an infection of pathogenic microorganisms.

On the other hand, some of the substances that play an important role in maintaining the homeostasis of organisms are physiologically active substances derived from various organisms.

Many researches have been carried out on a number of biologically active substances, among which antimicrobial peptides isolated from various organisms are known to act in various ways, ranging from bacteria, fungi and viruses. Antimicrobial peptides are also known to play an important role in host defense and the innate immune system.

Korean Priority No. 10-0625875 (a novel peptide isolated from Aspergillus nidulans and a pharmaceutical composition containing it as an active ingredient) and Korean Patent No. 10-0964136 Antimicrobial and antifungal peptide genes, and synthetic peptides having antimicrobial and antifungal activity). However, no studies have been made on novel peptides isolated from Wang Ji Nee and novel uses thereof.

It is an object of the present invention to provide an amino acid sequence of a scolopendrazine-5 peptide isolated from Wang Ji-nee, which can be effectively used as a preventive and therapeutic agent for fungal diseases of the human body, as well as exhibiting antibacterial and antifungal activity.

In order to achieve the above object, the present invention provides a peptide synthesized from an amino acid deduced from a gene of a peptide isolated from Scolopendra subspinipes , which is represented by SEQ ID NO: 1.

The peptide is characterized by having antibacterial and antifungal activity.

The antimicrobial activity of the antibiotic-resistant Pseudomonas aeruginosa may be determined by measuring the concentration of the antimicrobial activity of the antibiotic-resistant Pseudomonas aeruginosa , which is expressed by at least one of Staphylococcus aureus , Escherichia coli 0-157 ( Escherichia coli 0-157) .

The antifungal activity is characterized in that it appears to Candida albicans .

The peptide is characterized by being non-toxic to human red blood cells.

The present invention provides a pharmaceutical composition for antibiotic and antifungal preparation containing the above peptide as an active ingredient, or an antibiotic, a food preservative, a cosmetic preservative or a pharmaceutical preservative.

The present invention provides novel scolopendrazine-5 peptides and compositions thereof isolated from Wang Ji Nee, and thus provides anticancer and antifungal activity without cytotoxicity, and at the same time provides antibiotics, food preservatives, cosmetic preservatives, And so on.

Fig. 1 is a graph showing the inhibitory activity of the novel scolopendrazine-5 peptide of the present invention isolated from Wang jingne against Gram-negative bacteria, positive bacteria and antibiotic-resistant bacteria.

Hereinafter, the present invention will be described in detail.

The present invention is represented by SEQ ID NO: 1 and provides a peptide synthesized from an amino acid deduced from a gene of a peptide isolated from Scolopendra subspinipes .

In the present invention, in order to identify a gene of a peptide showing antibacterial and antifungal activity from Scolopendra subspinipes , Wangzine is first rapidly frozen in liquid nitrogen to isolate total RNA, and then, using RNA seq analysis, Which is presumed to be a peptide exhibiting new antimicrobial and antifungal activity by screening amino acid sequences using the properties of antimicrobial peptides that have been discovered based on the sequence of amino acids translated from each transcript, I choose Jean.

Then, the amino acid sequence of the peptide thus produced was composed of 14 amino acids of YYGGGYKYKHWGCR-NH ₂ , which was named Scolopendrasin-5.

Such peptides exhibit antibacterial and antifungal activity, and in particular, are non-toxic to human erythrocyte cells. Accordingly, the present invention provides a pharmaceutical composition for antibacterial and antifungal preparation containing the antibacterial and antifungal activity as an active ingredient, or a pharmaceutical composition for antibiotic, food preservative, cosmetic preservative, .

Specifically, the present inventors used Scolopendrasin-5 (Scolopendrasin-5 and Staphylococcus aureus , Escherichia coli 0-157 as antibacterial activity bacteria to measure antifungal activity) RDA assay (radial diffusion assay) was performed on Candida albicans as the antibiotic-resistant Pseudomonas aeruginosa (Multi Drug Resistance Psudomonas aeruginosa ) and the antifungal active bacterium. As a result, a strong antibacterial or antifungal agent (See Fig. 1). The results are shown in Fig.

The inventors of the present invention also examined the cytotoxicity of the novel scolopendrathin-5 peptide of the present invention. As a result, it was found that the novel scolopendrazine-5 peptide Melitin, a bee venom used as a positive control, did not show cytotoxicity, but cytotoxicity was very high (see Table 2).

From the above results, it can be seen that the pharmaceutical composition containing the novel scolopendrazine-5 peptide of the present invention as an active ingredient exhibits excellent antifungal and antimicrobial effects as well as being free from cytotoxicity, so that it is useful as a safe antimicrobial agent and antifungal agent Can be used.

Such a pharmaceutical composition can be administered parenterally at the time of clinical administration and can be used in the form of a general pharmaceutical preparation.

That is, the novel peptide of the present invention can be administered in various forms of parenteral administration. In the case of formulation, the peptide is prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. As the base of the suppository, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

In addition, the novel peptide of the present invention can be used in combination with various carriers permitted as pharmaceutical agents, such as physiological saline or an organic solvent, and can be used in combination with carbohydrates such as glucose, sucrose or dextran, Antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers can be used as pharmaceuticals.

The effective dose of the novel peptide of the present invention is 0.01 to 100 mg / kg, preferably 0.1 to 10 mg / kg, and can be administered once to three times a day.

The total effective amount of the novel peptide of the present invention and the peptide having more than 95% homology thereto in the pharmaceutical composition of the present invention may be a single dose by a bolus form or by infusion for a relatively short period of time, , And may be administered by a fractionated treatment protocol in which multiple doses are administered over a prolonged period of time. Since the concentration of the effective dose of the patient is determined in consideration of various factors such as the route of administration and the number of treatments as well as the age and health condition of the patient, in view of this point, Lt; RTI ID = 0.0 > as < / RTI > the pharmaceutical composition of the novel peptide.

In addition, the present invention provides an antibiotic, a food preservative, a cosmetic preservative or a pharmaceutical preservative containing a novel peptide as an active ingredient.

In this case, food preservative, cosmetic preservative and pharmaceutical preservative are additives used to prevent deterioration, decay, discoloration and chemical change of foods or medicines, including bactericides and antioxidants, and proliferation of microorganisms such as bacteria, fungi and yeast As well as functional antibiotics such as inhibiting the growth of microorganisms or sterilizing the microorganisms in foods and medicines. Ideal conditions for these food preservatives, cosmetics, and medicine preservatives should be non-toxic and have a very small effect.

As shown in FIG. 1 and Table 2, the novel scolopendrazine-5 peptide of the present invention shows strong antimicrobial and antifungal activity and is not toxic, and thus it can be used as a preservative for foods, cosmetics and medicines.

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

Example  1: New from Wang Xin Peptides  Selection

In order to identify antimicrobial or antifungal activity peptides from Wang Zyun, firstly, Wang Ji Nee was rapidly frozen with liquid nitrogen to isolate total RNA. Using RNA seq analysis, it was analyzed for antimicrobial or antimicrobial activity based on the sequence of amino acids translated from Wangzine transcripts Peptides showing antifungal activity were selected (Table 1).

The selected amino acid sequence was composed of 14 amino acids with YYGGGYKYKHWGCR-NH ₂ and was named SP-5.

Scolopendrazine-5
Peptide sequence
(SEQ ID NO: 1) YYGGGYKYKHWGCR-NH ₂

Example  2: Scolopendrazine -5 Of peptide  Synthesis and separation purification

In Example 1, the scolopendrazine-5 peptide derived from Wang Zyene was synthesized and isolated and purified.

First, in order to synthesize the scolopendrathine-5 peptide, the present inventors produced peptides using the liquid phase solid phase method of Merrifield using a Fmoc amino group protecting container.

Method of the peptide synthesis the Fmoc (9-fluorenylmethoxycarbonyl) amino acids of the N _α were synthesized and used as a protecting group (protecting group) amino Groom (amino group) to the conventional method (solid phase method).

Concretely, the peptide having carboxyl terminal in the form of -NH ₂ used Rink Amide MBHA-Resin as the starting material, and the peptide having carboxyl terminal in the form of -OH used Fmoc-amino acid-Wang Resin as a starting material.

Elongation of the peptide chain by coupling of Fmoc-amino acid was performed by DCC ( N -hydroxybenzo-triazole (HOBt) -dicyclo-hexylcar-bodiimide) method.

After the Fmoc-amino acid of the amino terminal of each peptide was coupled, the Fmoc group was removed with 20% piperidine / N-methylpyrrolidone (NMP) solution, washed several times with NMP and dichloromethane (DCM) It was dried with gas.

A solution of TFA (trifluoroacetic acid) -phenol-thioanisole-H ₂ O-triisopropylsilane (85: 5: 5: 2.5: 2.5, vol./vol.) Was added and the reaction was carried out for 3 hours. The peptides were separated and the peptide was precipitated with diethyl ether. The crude peptide thus obtained was subjected to a reverse phase-HPLC column (Delta Pak, C ₁₈ 300 Å, 15 μ, 19.0 mm × 20 cm) using an acetonitrile gradient containing 0.1% TFA 30, Waters).

After the synthetic peptide was hydrolyzed with 6 N HCl at 110 ° C for 24 hours, the resulting residue was concentrated under reduced pressure, dissolved in 0.02 N HCl, and the amino acid composition was measured with an amino acid analyzer (Hitachi 8500 A).

The molecular weight was calculated based on the sequence of the synthesized peptides, and the exact molecular weight was measured using a MALDI mass spectrometer (MALDI).

As a result, it was confirmed that a peptide having an antimicrobial activity and an antifungal activity with the correct amino acid sequence was synthesized because the calculated molecular weight and the calculated molecular weight were identical.

Example  3: Scolopendrazine -5 Of peptide  Investigation of antimicrobial activity against pathogenic microorganisms

A radial diffusion assay (RDA) was performed to investigate the antimicrobial activity against the scolopendrazine-5 peptide. Melittin, a powerful antimicrobial peptide isolated from bees, was used as a positive control in the present invention. In the present invention, the antimicrobial peptide was stored at -20 ° C and dissolved in 0.01% acetic acid in consideration of the stability of the peptide.

[Antimicrobial activity measurement]

In order to measure the antimicrobial activity of the peptides of the present invention, the nasal pillow Stability kusu aureus (Staphylococcus aureus) First pathogenic bacteria, E. coli 0-157 (Escherichia coli 0-157) and an antibiotic resistant pseudomonas aeruginosa (Multi Drug Resistance Psudomonas aeruginosa ) were cultured in 3% (w / v) trypticase soy broth at 37 ° C and 200 rpm for 18 hours, And cultured for 2 hours and 30 minutes at a concentration of 4 × 10 ⁶ CFU (colony forming units) / ml.

Thereafter, a sterilized underlay gel consisting of citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4) and 1% (w / v) type I (low electroendosmosis) agarose and 0.03% TSB (4 × 10 ⁶ colony forming units / ml) were added to the underlay gel and poured into a square plate. When the underlay gel was hardened, a hole having a diameter of 3 mm was punched out to obtain a peptide having a concentration of 1 mg / ) Were added to the wells in 5 쨉 l portions.

peptide was allowed to diffuse for 3 hours at 37 ° C, 10 mL of an overlay gel (6% TSB, 1% agarose) was poured in and then re-cultured at 37 ° C to confirm formation of a clear zone. The results are shown in Fig.

[Measurement of antifungal activity]

The pathogenic fungus is the measurement of antifungal activity of the peptide of the present invention, Candida albicans (Candida albicans ) were grown in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) overnight at 30 ° C and 200 rpm, and then inoculated on fresh medium for 2 hours to become logarithmic phase.

Bacteria cultured on sterilized underlay gel consisting of citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4) and 1% (w / v) type agarose and 0.03% ⁶ colony forming units / mL) were mixed and poured into a square plate. When the underlay gel was hardened, a hole having a diameter of 3 mm was drilled and 5 μl of a peptide with a concentration of 1 mg / mL was punched.

peptide was allowed to diffuse for 3 hours at 37 ° C, 10 mL of overlay gel (6% TSB, 1% agarose) was poured in and the cells were cultured again at 37 ° C. to confirm formation of CLEAR ZONE. The obtained results are shown in FIG. .

[Results of antibacterial activity]

As shown in FIG. 1, it was confirmed that the scolopendrazine-5 peptide exhibited inhibitory activity against the pathogenic bacteria, positive bacteria and resistant bacteria, and this increased in a concentration-dependent manner.

In addition, when compared with melittin, a powerful antimicrobial peptide, it was confirmed that the inhibitory activity was higher than that of the negative bacteria and resistant strains, indicating that there is a possibility of being a therapeutic agent for existing antibiotic resistant strains.

[Results of antifungal activity]

As shown in FIG. 1, it was confirmed that CLEAR ZONE was formed as a result of measuring the antifungal activity of the scolopendrazine-5 peptide, and it was confirmed that the activity increased in a concentration-dependent manner as in the case of the negative bacteria and the positive bacteria.

In addition, this indicates that the peptide of the present invention exhibits potent antifungal activity, indicating that it exhibits an antifungal activity similar to melitin, a potent antifungal agent used as a control.

Example 4: Of the scopoledradin-5 peptide  Cytotoxicity measurement on human red blood cells

The cytotoxicity of human red blood cells was measured as follows.

Human erythrocytes were washed three times with phosphate-sodium chloride buffer, pH 7.4 (PBS), and then diluted with phosphate-sodium chloride buffer to prepare 8.0% red blood cell solution.

Then, 100 μl of the 8.0% red blood cell solution was dispensed into a 96-well plate, and then a stepwise diluted solution of the peptide was mixed.

Then, physiological saline (Saline 0.85% NaCl) was added to all wells so that the total amount became 200 μl, and the cells were cultured in a 37 ° C. incubator for 1 hour.

After the culture was completed, the supernatant was transferred to the side wells by centrifugation at a rotation speed of 1000 rpm for 10 minutes in a centrifuge.

The destructive activity against red blood cells was measured by measuring the absorbance at 414 nm using an ELISA reader.

100% destructive capacity was calculated for 0.1% Triton X-100 as a control, and 0% destructive capacity for only red cell.

At this time, the destructive ability of the peptide was calculated by the following equation (1), and the obtained results are shown in Table 2 below.

[Equation 1]

% Absorbance of solution treated with Triton X-100 414 nm absorbance of phosphoric acid-sodium chloride buffer 414 nm) * 100 (absorbance of peptide-treated solution 414 nm-absorbance of phosphoric acid-sodium chloride buffer 414 nm)

Measurement of cytotoxicity of scolopendrazine-5 peptide on human erythrocytes % Human erythropoietin ^a (占 퐂 / ml) 0 6.25 12.5 25 50 100 Scolopendrazine-5 0 3.3 0 1.1 2.1 1.1 Melitin 0 31.2 95.7 98.1 98.7 99.2 [Note] a: Cytotoxicity to humans is measured by the ability of peptides to destroy human red blood cells

As shown in Table 2, it was confirmed that the scallopendrazine-5 peptide did not show cytotoxicity at all concentrations. In contrast, melittin, a potent antimicrobial peptide, was highly cytotoxic at low concentrations.

These results indicate that the peptides found in the present invention are free from cytotoxicity, exhibit excellent antibacterial and antifungal activity, and can be used as safe antibiotics, food preservatives, cosmetic preservatives and pharmaceuticals.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Republic of korea <120> A new Scolopendra-5 peptide isolated from the Scolopendra          subspinipes and its synthetic composition <130> p2014-0038 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 1 Tyr Tyr Gly Gly Gly Tyr Lys Tyr Lys His Trp Gly Cys Arg   1 5 10

Claims (10)

Peptides synthesized from amino acids deduced from the genes of the peptides isolated from Scolopendra subspinipes , represented by SEQ ID No. 1 below.
SEQ ID NO: 1:
Tyr Tyr Gly Gly Gly Tyr Lys Tyr Lys His Trp Gly Cys Arg
The method according to claim 1,
Wherein said peptide has antibacterial and antifungal activity.
3. The method of claim 2,
The antimicrobial activity of the antibiotic-resistant Pseudomonas aeruginosa may be determined by measuring the concentration of the antimicrobial activity of the antibiotic-resistant Pseudomonas aeruginosa ( Sturmy ), which is expressed against at least one of Staphylococcus aureus , Escherichia coli 0-157 ( Escherichia coli 0-157) It is characterized by a peptide.
3. The method of claim 2,
Wherein said antifungal activity is exhibited against Candida albicans .
The method according to claim 1,
Wherein said peptide is non-toxic to human red blood cells.
A pharmaceutical composition for an antibacterial and antifungal preparation, which comprises the peptide of any one of claims 1 to 5 as an active ingredient.
An antibiotic comprising an effective ingredient of the peptide of any one of claims 1 to 5.
A food preservative containing the peptide of any one of claims 1 to 5 as an active ingredient.
A cosmetic preservative containing the peptide of any one of claims 1 to 5 as an active ingredient.
A pharmaceutical preservative containing the peptide of any one of claims 1 to 5 as an active ingredient.

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