JP3777010B2 - Novel peptide and drug containing the peptide as an active ingredient - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an extract extracted from a marine organism belonging to the mollusc genus, Dolabella auricularia , and a novel peptide separated and purified therefrom.
More specifically, the present invention relates to a novel peptide that is chemically stable to acids, heat, or organic solvents such as chloroform, has strong antifungal activity and antibacterial activity, and a drug containing the peptide as an active ingredient.
[0002]
[Prior art]
Mycosis caused by fungal infection is roughly divided into superficial mycosis caused by skin fungi and deep-seated mycosis caused by yeast or biphasic fungi, Causes systemic mycosis due to decreased immunity or immune deficiency. In such cases, it is known to cause opportunistic infections such as sepsis, brain / liver / lung tumors, cystitis, vaginitis, etc., but no effective therapeutic agent for fungal-derived infection has been reported yet.
In recent years, deep mycosis due to the use of antibiotics, steroids, immunosuppressants, anticancer agents and the like has become a problem. Conventionally, as pharmaceuticals that inhibit fungi, for example, amphotericin B, griseofulvin, tricomycin, siccanin and the like are considered as antibiotics, and miconazole, econazole, flucytosine and the like are studied as synthetic drugs. However, the actual condition is that a satisfactory effect is not always obtained by the treatment with these pharmaceuticals.
[0003]
In recent years, contamination by various microorganisms has become a problem. For example, harmful foods such as E. coli O-157 have caused mass food poisoning with death. Such contamination by microorganisms is thought to be related to changes in the living environment, but there are concerns that such problems will increase in the future. Especially when the cause of the occurrence is unknown, the impact on the general public (anxiety) is great, and people's interest is increasing, but there are few useful means for decontamination, treatment, prevention, etc. for these microbial contaminations. Is the current situation.
[0004]
On the other hand, the search target of active ingredients such as antifungal agents related to natural ingredients has been mainly plants and soil microorganisms, but since the 1980s, research on components of marine organisms such as sponges, sea squirts, snails and soft corals has been conducted. As the process progressed, unique compounds that were completely different from those derived from terrestrial plants and microorganisms were found one after another. Furthermore, various physiological activities are recognized in these compounds, and many seed chemical compounds and chemical tools for elucidating physiological activities are known from among them. Furthermore, compounds expected as anticancer agents, antibacterial substances, and anti-AIDS agents are being found from them.
[0005]
[Problems to be solved by the invention]
An object of the present invention is to extract a new component effective for treating or preventing a disease caused by microbial contamination, which is expected to increase in the future, from natural products, particularly marine organisms, and to elucidate the chemical structure of the main body of the active component There is.
[0006]
[Means for Solving the Problems]
In view of the above-mentioned objectives, the present inventors have intensively studied marine organisms to search for substances having high antifungal activity against fungi such as Saccharomyces sp. And Candida sp. As a result, antifungal activity was found in the extract of Dolabella auricularia .
Furthermore, it has been found that a fraction obtained by fractionating a sea urchin extract by centrifugation, precipitation, chromatography, ultrafiltration, etc. has extremely excellent antifungal activity, and this fraction has a chemical composition consisting of 15 amino acids. It was confirmed that the peptide was highly stable.
It was also confirmed that the peptide had an extremely excellent antibacterial action.
[0007]
Based on the above findings, the present invention provides a novel peptide useful as a therapeutic agent for fungal diseases, particularly preferably mycosis caused by Candida sp. , And as an antibacterial agent, and the peptide as an active ingredient. A pharmaceutical product is provided.
According to the present invention, it is possible to treat or prevent diseases caused by fungi, and sterilize and disinfect food contamination caused by Escherichia coli, Gram-negative bacilli, Gram-positive anaerobic bacilli, Staphylococcus, Pseudomonas aeruginosa, Salmonella and the like. Therefore, it is possible to prevent the contamination. Furthermore, it can also be used as a therapeutic agent for diseases caused by these fungi, such as dysentery, typhoid fever, cholera, anthrax, brucellosis, tuberculosis and the like.
[0008]
[Structure of the invention]
The present invention
1) A peptide represented by the amino acid sequence of Sequence 1 described below:
SerHisGlnAspCysTyrGluAlaLeuΗisLysCysMetAlaSer,
2) The peptide according to 1 above, which is obtained by extraction from a marine organism Dolabella auricularia and related organisms,
3) A drug containing the novel peptide according to 1 as an active ingredient,
4) The drug according to 3 above, which comprises an extract from marine organisms, Dolabella auricularia and related organisms, as an active ingredient,
5) The drug according to 3 or 4 above, which is an antifungal agent,
6) The agent according to 3 or 4 above, which is an antibacterial agent.
[0009]
The present invention will be described in detail below.
The novel peptide according to the present invention is extracted and isolated from sea cucumber ( Dolabella auricularia ) by the method described below, and the structure thereof is confirmed, but is not limited to that extracted and isolated from sea urchin ( Dolabella auricularia ), It includes those produced by chemical synthesis, and those extracted and isolated from systems containing the peptide by other natural products or by means of microbial production.
[0010]
One example of the method for preparing the peptide of the present invention will be described.
That is, first, buffer salt solution (PBS) is added to the skin and body wall of mussel ( Dolabella auricularia ), homogenized, and then centrifuged to obtain a precipitate. The solvent used for extraction may be PBS, water, or an isotonic solution other than PBS.
The resulting precipitate was extracted with acetonitrile-trifluoroacetic acid-water system, centrifuged, and after removing fat from the supernatant, fractionated by ultrafiltration followed by reverse-phase Η PLC and ion-exchange Η PLC, The peptide compound of the present invention can be obtained by re-purifying with the compatible PLC. As the reversed-phase HPLC carrier to be used, one having a C18 chain bonded is usually used, but one having C8, C4, C1, phenyl or the like bonded thereto may be used.
As a result of amino acid sequence analysis, the active fraction thus obtained was obtained by the following formula:
SerHisGlnAspCysTyrGluAlaLeuΗisLysCysMetAlaSer
The peptide consisting of 15 amino acids shown in the above, and this peptide was confirmed to be novel as a result of searching the prior art by “SWISS-PROT, EMBL” which is a peptide / protein sequence database. The present inventor named it dolaverin B2 (Dolabellanin B2, hereinafter abbreviated as DA 2).
[0011]
The physicochemical properties of DA 2 are as follows.
(1) Elemental analysis (2) Molecular weight: 1.72 KDa
(3) Ultraviolet absorption spectrum (4) Infrared absorption spectrum (5) Solubility in solvent: It dissolves well in acetonitrile and water.
(6) Stability Thermal stability: stable even when heated at 100 ° C. for 10 minutes.
Solvent stability: It is stable even after adding chloroform for 5 minutes at room temperature.
Stability to acid: Can be stored for a long time in a state dissolved in a trifluoroacetic acid solution (pH 3 or lower).
(7) Inhibition, activation and stabilization
(8) Antibacterial activity As shown in the examples below, DAB2 is effective against fungi Saccharomyces sp. And Candida sp. , And also against gram-negative bacteria Echerichia sp. .) showed very good results in the antibacterial test against.
[0013]
Therefore, according to the present invention, an extract of the marine organism Dolabella auricularia and a peptide obtained by fractionating the antifungal activity and antibacterial activity are pharmaceutical products against diseases caused by fungi and bacteria. Available as Furthermore, it can be used as food additives, cosmetics, quasi-drugs and the like for the purpose of sterilizing and removing pathogenic microorganisms.
[0014]
Application to pharmaceuticals:
For the purpose of treating or preventing diseases caused by fungi, or diseases caused by Escherichia coli, Gram-negative bacilli, Gram-positive anaerobes, Staphylococcus, Pseudomonas aeruginosa, Salmonella, etc., such as dysentery, typhoid fever, cholera, anthrax, For the purpose of treating or preventing brucellosis and the like, the peptide of the present invention is usually administered systemically or locally, generally in an oral or parenteral form.
The dose varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but it is usually administered orally once to several times a day in the range of 100 μg to 100 mg per adult. In addition, it is administered parenterally once or several times a day in the range of 10 μg to 100 mg per adult per person.
In order to administer the compound of the present invention, it is used as solid compositions, liquid compositions and other compositions for oral administration, injections for parenteral administration, external preparations, suppositories and the like.
[0015]
Solid compositions include tablets, pills, capsules, powders, granules and the like.
Liquid compositions include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, etc., and generally used inert diluents (eg, purified water, ethanol, etc.). May be included. Further, in addition to the inert diluent, auxiliary agents such as wetting agents and suspending agents, sweetening agents, flavoring agents, fragrances and preservatives may be contained.
Injections include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. As an aqueous or non-aqueous solution or suspension, the peptide of the present invention is mixed with an inert diluent. The injection may further contain auxiliary agents such as commonly used preservatives, wetting agents, emulsifying agents, dispersing agents, stabilizing agents and solubilizing agents. These are used after being sterilized by filtration through a bacteria-retaining filter, blending of a bactericidal agent, or irradiation.
Compositions for parenteral administration include solutions for external use, ointments, coating agents, suppositories for intrarectal administration, pessaries and the like which contain the peptide of the present invention and are formulated by conventional methods.
[0016]
Acute toxicity:
The toxicity of the peptide compound of the present invention is very low, and can be judged to be sufficiently safe for use as a medicine.
[0017]
【Example】
Hereinafter, the present invention will be described with reference to examples.
[0018]
Examples Skin of mussels ( Dolabella auricularia ) collected in the Pacific Ocean off the Miura Peninsula and 200 g (wet weight) of the body surface were added with equal amounts of PBS and homogenized. After centrifugation at 2000 × g for 20 minutes, the supernatant was removed. An equal amount of acetonitrile-trifluoroacetic acid-water (60: 1: 39) was added to 180 g of the resulting precipitate, homogenized, and centrifuged again at 2000 × g for 20 minutes. This supernatant was degreased with chloroform and then lyophilized, redissolved in water containing 0.1% trifluoroacetic acid, fractionated with reverse phase PLC (gel: C18), and 50 mg fraction eluting at 43 minutes was obtained. . Further, this fraction was fractionated with an ion-exchanged PLC (resource S), and 10 mg of fraction eluted at around 6 minutes was collected. This fraction was fractionated by an ultrafiltration membrane having a fractional molecular weight of about 10,000, and the membrane passage portion was further fractionated by reversed-phase PLC (gel: C18) to obtain 2 mg of a fraction eluting around 43 minutes. As a result of amino acid sequence analysis, the obtained compound was a peptide consisting of 15 amino acids (DAB2).
[0019]
The entire sequence is
SerHisGlnAspCysTyrGluAlaLeuΗisLysCysMetAlaSer
It is.
[0020]
When supplementing the physicochemical properties of DA-2, please describe the results together with the measurement method (means) below.
(Elemental analysis, molecular weight, ultraviolet absorption spectrum, infrared absorption spectrum, solvent solubility, inhibition, activation, stabilization, etc.)
[0021]
Antibacterial activity The antifungal activity of DAB2 was measured using Saccharomyces cerevisiae A581A and Candida albicans TCC 36232. That is, Saccharomyces cerevisiae A581A and Candida albicans ATCC 36232 were pre-cultured in ΡYD broth medium to 1 × 10 ⁸ cells / ml and diluted in ＹYD broth medium to 2 × 10 Prepared to ⁵ cells / ml. DAB2 (10 μl) adjusted to 0-100 μg / ml was added to 10 μl, and Saccharomyces cerevisiae A581A was shaken at 30 ° C. for 0 and 120 minutes, and Candida albicans Α TCC 36232 After shaking culture at 37 ° C. for 0 and 120 minutes, the culture was stopped with 980 μl of ice-cold water, 50 μl was cultured in PYD-ampicillin agar medium for 2 days, and the number of colonies obtained was measured. The results are shown in FIGS. The vertical axis in the figure is the colony forming unit (CFU). In the figure, in addition to the colony number (black square in the figure) of 0 minute shaking culture, the number of colonies when only the cells were cultured without DAB2 (vertical axis in ● in the figure) The above) is also shown, so that the relative decrease in the number of cells by DAB2 treatment can be seen. FIG. 1 shows the antifungal activity of DAB2 against Saccharomyces cerevisiae A581A, and FIG. 2 shows the antifungal activity of DAB2 against Candida albicans ATCC 36232.
[0022]
As a result of measurement of these antifungal activities, it was confirmed that the peptide according to the present invention showed no bactericidal action against Saccharomyces cerevisiae A581A at a concentration of 100 μg / ml or more and showed a bactericidal action. . On the other hand, Candida albicans ΑTCC 36232 shows a bacteriostatic effect because the number of colonies is constant at a concentration of 25 μg / ml to 100 μg / ml and is almost equal to the number of cells before culture. I understand that.
[0023]
Also, the antifungal activity against Saccharomyces cerevisiae A581A was changed with time in the culture time, with DAB2 concentration of 30 μg / ml (the amount of DA 2 that reduces the number of colonies to half of the control after incubation for 2 hours) as 1 U. The result of measurement is shown in FIG. FIG. 3 shows the changes over time in the number of cells (black squares in the figure) after treatment with Saccharomyces cerevisiae A581A without DAB2 treatment (● in the figure) and DAB2 (10 U). As is clear from FIG. 3, a decrease in colonies was confirmed after 15 minutes of culture by DAB2 (10 U) treatment, and almost no colonies were observed after 30 minutes. From this, it was clarified that Saccharomyces cerevisiae A581A was killed in about 30 minutes by the above-mentioned concentration of DAB2.
[0024]
Measurement of antibacterial activity <br/> antibacterial activity was carried out using Escherichia coli (Escerichia coli) JΜ109.
Escherichia coli (Escerichia coli) JΜ109, after incubation for 1 day at 37 ° C. using a LB broth medium and cultured for 2 hours in antibiotic medium 3 medium. This was diluted with an antibiotic medium 3 so that the absorption at a wavelength of 650 nm was 0.005, 0 to 100 μg / ml of DAB2 was added, and the mixture was cultured at 37 ° C. for 8 hours, and then the absorption at 650 nm was measured. The result is shown in FIG. 4, against Escherichia coli (Escerichia coli) JΜ109, showing the DAB2 (0~100μg / ml) anti-bacterial activity by treatment. The vertical axis in the figure represents the absorption at 650 nm (data on the horizontal axis “0 hour” in the figure). The figure also shows the absorption of the medium before culturing at 37 ° C. for 8 hours as a control.
As apparent from FIG. 4, as compared to the case of culturing with no intervening DAB2, it was recognized that inhibit the growth of Escherichia coli (Escerichia coli) JΜ109 at a concentration of DAB2 (10μg / ml) or .
[0025]
Thermal Stability and Stability to Chloroform The thermal stability of DAB2 was investigated. That is, after heat-treating DAB2 at 100 ° C. for 10 minutes, the thermal stability was examined using antifungal activity as an index. The result is shown in FIG. FIG. 5 shows antifungal activity against Saccharomyces cerevisiae A581A at various concentrations of DAB2 when heat-treated (black square in the figure) and without heat-treatment (● in the figure).
[0026]
FIG. 5 shows that the antifungal activity using heat-treated DAB2 shows almost the same dose dependency as compared with the case without heat treatment, so that DAB2 is stable to heat treatment.
[0027]
The stability of chloroform treatment was also examined. That is, chloroform was added to DAB2, stirred, allowed to stand at room temperature for 5 minutes, and then dried. The results of examining the antifungal activity of this dried product are shown in FIG.
[0028]
From FIG. 6, the antifungal activity using black-treated DAB2 (black squares in the figure) shows almost the same dose dependency as the antifungal activity using DAB2 not treated with chloroform (● in the figure). As can be seen, DAB2 is a stable peptide against chloroform treatment.
[Brief description of the drawings]
FIG. 1 is a graph showing the results of measuring the antifungal activity of DAB2 against Saccharomyces cerevisiae .
FIG. 2 is a graph showing the results of measuring the antifungal activity of DAB2 against Candida albicans .
FIG. 3 is a graph showing the relationship between the bactericidal action of DAB2 on Saccharomyces cerevisiae and the culture time.
FIG. 4 is a graph showing the antibacterial activity of DA 2 against Escherichia coli .
FIG. 5 is a graph showing the results of measuring the antifungal activity of heat-treated DAB2 and non-heat-treated DAB2 against Saccharomyces cerevisiae .
FIG. 6 is a graph showing the results of measuring the antifungal activity of chloroform-treated DAB2 and non-chloroform-treated DAB2 against Saccharomyces cerevisiae .

Claims (6)

Novel peptide represented by the following amino acid sequence:
SerHisGlnAspCysTyrGluAluLeuΗisLysCysMetAlaSer. The peptide according to claim 1, which is obtained by extraction from a marine organism, Dolabella auricularia or its related organisms. A drug comprising the novel peptide according to claim 1 as an active ingredient. The agent according to claim 3, comprising an extract from a marine organism, Dolabella auricularia, or a related organism. The drug according to claim 3 or 4, which is an antifungal agent. The drug according to claim 3 or 4, which is an antibacterial agent.

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