CN107899000A - The spiral antibacterial peptides of α and surfactant complex and its application - Google Patents

Abstract

The invention provides a complex of α-helical antibacterial peptide and surfactant and application thereof. The complex of α-helical antibacterial peptide and surfactant includes: anionic α-helical peptide and cationic surfactant. The antibacterial composition of the present invention, because the anionic α-helical antibacterial peptide that has almost no antibacterial activity is combined with the cationic surfactant DTAB of low concentration, makes the compound of the two produce antimicrobial effect on various bacteria and fungi at extremely low concentrations. good inhibitory effect.

Description

Compound of α-helical antibacterial peptide and surfactant and its application

technical field

The invention relates to a complex of α-helical antibacterial peptide and surfactant and application thereof, belonging to the technical field of medicine preparation.

Background technique

The discovery and widespread use of antibiotics has greatly improved the health status of human beings. However, the problems of drug resistance, side effects and residues caused by the long-term abuse of antibiotics have aroused great concern from the whole society. Drug-resistant bacteria have seriously threatened human health and safety, so the development of new antibiotics has become a major problem that needs to be solved urgently worldwide. In recent years, small molecular peptides with antibacterial activity have gradually attracted widespread attention, and its mechanism of action is different from traditional antibiotics, making it a hot spot in the field of new antibiotics. Traditional antibiotics mainly use drugs to target bacteria, thereby inhibiting the replication and pairing of bacterial DNA, hindering protein synthesis and destroying the normal functions of the cell wall. However, bacterial chromosomal variation or flowing genetic fragment variation can lead to the loss of the target that originally binds to the antibiotic, resulting in drug resistance and causing the antibiotic to fail. Studies have found that antimicrobial peptides mainly play a bactericidal role by destroying the integrity of biofilms and causing the outflow of cell contents. Compared with traditional antibiotics, bacteria are less likely to produce drug resistance to the physical destruction of this missing targeting site, so antimicrobial peptides have broad application prospects in solving the problem of drug resistance.

The current research direction focuses on structural modification of natural antimicrobial peptides through site-specific substitution of amino acid residues, interception of partial sequences of natural antimicrobial peptides, or de novo design and synthesis of new antimicrobial peptides, which are currently the main means of obtaining superior antimicrobial peptides. However, regardless of chemical synthesis, microbial synthesis or gene expression, the synthesis and purification of polypeptides are complicated and difficult after all.

Contents of the invention

The object of the present invention is to provide a new antibacterial composition.

The present invention adopts following technical scheme:

A complex of α-helical antibacterial peptide and surfactant, characterized in that it comprises: anionic α-helical peptide and cationic surfactant.

Furthermore, the complex of the α-helical antimicrobial peptide and the surfactant of the present invention may also have the following characteristics: wherein, the sequence of the anionic α-helical antimicrobial peptide is: Ac-CWVRLGRYLLRRLKTPFT-NH ₂ .

Further, the complex of α-helical antimicrobial peptide and surfactant of the present invention can also have such characteristics: wherein, the cationic surfactant is dodecyltrimethylammonium bromide, tetradecyltrimethylammonium Any one of ammonium bromide or cetyltrimethylammonium bromide.

Furthermore, the complex of α-helical antimicrobial peptide and surfactant of the present invention may also have such a feature: wherein, the cationic surfactant is dodecyltrimethylammonium bromide.

Further, the α-helical antimicrobial peptide and surfactant complex of the present invention may also have such a feature: wherein, the concentration ratio of the cationic surfactant to the anionic α-helical peptide is 1 μM/L:5 μM/L ~100μM/L: 5μM/L.

Further, the α-helical antimicrobial peptide and surfactant complex of the present invention may also have the following characteristics: wherein, the concentration ratio of the anionic α-helical peptide and the cationic surfactant is: 50 μM/L: 25 μM/ L.

The invention also provides the application of the complex of α-helical antibacterial peptide and surfactant in the preparation of antibacterial drugs.

Furthermore, the application provided by the present invention may also have the following feature: wherein, the sequence of the anionic α-helical peptide is: Ac-CWVRLGRYLLRRLKTPFT-NH ₂ .

Further, the application provided by the present invention can also have such characteristics: wherein, the cationic surfactant is dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide or cetyl Any of the base trimethylammonium bromide.

Further, the application provided by the present invention may also have the following characteristics: wherein, the concentration ratio of the anionic α-helical peptide and the cationic surfactant is: 50 μM/L:25 μM/L.

The present invention also provides an application of the above-mentioned α-helical antimicrobial peptide and surfactant complex in preparing cosmetics.

When the α-helical antibacterial peptide and surfactant complex is applied to cosmetics, after adding the basic raw materials of cosmetics, the final concentration range of the ratio of anionic α-helical peptide and cationic surfactant should be maintained at 1 μM/L: 5μM/L～100μM/L: 5μM/L.

Preferably, the sequence of the α-helical antimicrobial peptide is: Ac-CWVRLGRYLLRRLKTPFT-NH ₂ .

Preferably, the surfactant is lauryltrimethylammonium bromide.

Preferably, the final concentration ratio of α-helical antimicrobial peptide and surfactant in cosmetics is 50 μM/L: 25 μM/L.

Beneficial Effects of the Invention

The α-helical antimicrobial peptide of the present invention and surfactant compound and its application, because the anionic α-helical antibacterial peptide with almost no antibacterial activity is combined with the cationic surfactant DTAB of low concentration, make the compound of the two in It has a good inhibitory effect on a variety of bacteria and fungi at extremely low concentrations.

When the α-helical antibacterial peptide and surfactant complex of the present invention is used in cosmetics, since the antibacterial components used are peptide substances, it has no chemical toxicity and is safe and reliable.

Detailed ways

The technical solutions of the present invention are further explained below through specific examples.

<Example 1>

Antibacterial effect of 1 μM DTAB and 5 μM AMP-18 compound system on Escherichia coli (ATCC8739) and Candida albicans (ATCC10231).

1. Use PBS buffer solution to prepare 4 μM/L DTAB solution and 20 μM/L AMP-18. The PBS solution for preparing the polypeptide must be sterilized in advance, and after cooling, filter it into the Erlenmeyer flask with a 0.45uM filter membrane.

The sequence of antimicrobial peptide AMP-18 of the present invention is as follows:

Ac-CWVRLGRYLLRRLKTPFT-NH ₂

2. The culture medium is lowered to a suitable temperature (before solidification), and poured onto the plate.

3. Experimental grouping:

(1) Take 100 μL of DTAB solution with a concentration of 4 μM/L in a 1.5ml centrifuge tube, then add 100 μL of AMP-18 with a concentration of 20 μL, and finally add 200 μL of Escherichia coli with different colony numbers, and react 30 minutes.

(2) Add 200 μL of DTAB with a concentration of 2 μM/L and 200 μL of different concentrations of Escherichia coli bacteria liquid respectively.

(3) Add 200 μL of AMP-18 with a concentration of 10 μM/L and 200 μL of different concentrations of Escherichia coli bacterial liquid, respectively. The negative control sample is 200 μL PBS solution + 200 μL Escherichia coli bacteria solution of different concentrations; prepare at least 3 parallel samples for each sample.

4. Use a pipette gun to take out 100 μL of the mixture from the above system, put it on the surface of the solidified medium, and spread it evenly with a spreader.

5. Count the colonies after culturing at 37°C for 24 hours, and compare with the negative control to calculate the inhibition rate

Wherein A is the average number of colonies of the negative control sample, and B is the average number of colonies of the experimental samples.

6. In the experimental procedure of Candida albicans, the cultivation time needs to be at least 48 hours, and the remaining operation steps, grouping and reagent concentrations are the same as those of Escherichia coli.

7. The experimental results are shown in Table 1:

Table 1 Antibacterial effect of DTAB and α-helical antimicrobial peptide AMP-18

<Example 2>

Antibacterial effect of 25μM/L DTAB and 50μM/LAMP-18 compound system on Escherichia coli (ATCC8739) and Candida albicans (ATCC10231).

1. Use PBS buffer solution to prepare 100 μM/L DTAB solution and 200 μM/L AMP-18. The PBS solution for preparing the polypeptide must be sterilized in advance, and after cooling, filter it into the Erlenmeyer flask with a 0.45uM filter membrane.

The sequence of antimicrobial peptide AMP-18 of the present invention is as follows:

Ac-CWVRLGRYLLRRLKTPFT-NH ₂

2. The culture medium is lowered to a suitable temperature (before solidification), and poured onto the plate.

3. Experimental grouping:

(1) Take 100 μL of DTAB solution with a concentration of 100 μM/L in a 1.5ml centrifuge tube, then add 100 μL of AMP-18 with a concentration of 200 μL, and finally add 200 μL of Escherichia coli with different colony numbers, and react 30 minutes.

(2) Add 200 μL of DTAB with a concentration of 50 μM/L and 200 μL of different concentrations of Escherichia coli bacterial liquid respectively.

(3) Add 200 μL of AMP-18 with a concentration of 100 μM/L and 200 μL of Escherichia coli bacteria liquid with different concentrations, respectively. The negative control sample is 200 μL PBS solution + 200 μL Escherichia coli bacteria solution of different concentrations; prepare at least 3 parallel samples for each sample.

4. Use a pipette gun to take out 100 μL of the mixture from the above system, put it on the surface of the solidified medium, and spread it evenly with a spreader.

5. Count the colonies after culturing at 37°C for 24 hours, and compare with the negative control to calculate the inhibition rate

Wherein A is the average number of colonies of the negative control sample, and B is the average number of colonies of the experimental samples.

6. In the experimental procedure of Candida albicans, the cultivation time needs to be at least 48 hours, and the remaining operation steps, grouping and reagent concentrations are the same as those of Escherichia coli.

7. The experimental results are shown in Table 2:

Table 2 Antibacterial effect of DTAB and α-helical antimicrobial peptide AMP-18

<Example Three>

Antibacterial effect of 100 μM DTAB and 5 μM AMP-18 compound system on Escherichia coli (ATCC8739) and Candida albicans (ATCC10231).

1. Use PBS buffer solution to prepare 400 μM/L DTAB solution and 20 μM/L AMP-18. The PBS solution for preparing the polypeptide must be sterilized in advance, and after cooling, filter it into an Erlenmeyer flask with a 0.45 μm filter membrane.

2. The culture medium is lowered to a suitable temperature (before solidification), and poured onto the plate.

3. Experimental grouping:

(1) Take 100 μL of DTAB solution with a concentration of 400 μM/L in a 1.5ml centrifuge tube, then add 100 μL of AMP-18 with a concentration of 20 μL, and finally add 200 μL of Escherichia coli with different colony numbers, and react 30 minutes.

(2) Add 200 μL of DTAB with a concentration of 200 μM/L and 200 μL of different concentrations of Escherichia coli bacteria liquid respectively.

(3) Add 200 μL of AMP-18 with a concentration of 10 μM/L and 200 μL of different concentrations of Escherichia coli bacterial liquid, respectively. The negative control sample is 200 μL PBS solution + 200 μL Escherichia coli bacteria solution of different concentrations; prepare at least 3 parallel samples for each sample.

4. Use a pipette gun to take out 100 μL of the mixture from the above system, put it on the surface of the solidified medium, and spread it evenly with a spreader.

5. Count the colonies after culturing at 37°C for 24 hours, and compare with the negative control to calculate the inhibition rate

a)

Wherein A is the average number of colonies of the negative control sample, and B is the average number of colonies of the experimental samples.

6. The cultivation time needs to be at least 48 hours, and the rest of the operation steps are the same as those of bacteria.

7. The experimental results are shown in Table 3:

Table 3 Antibacterial effect of DTAB and α-helical antimicrobial peptide AMP-18

In other embodiments, tetradecyltrimethylammonium bromide or cetyltrimethylammonium bromide can also be used as the cationic surfactant, which can also achieve better bactericidal effect.

Function and effect of embodiment

The minimum inhibitory concentration MIC of surfactant DTAB is 250μM. The α-helical antibacterial peptide has a random structure in aqueous solution and has no antibacterial activity. However, when the two were compounded with 25 μM/L DTAB and 50 μM/LAMP-18 compound system, and 100 μM/L DTAB and 5 μM/LAMP-18 compound system, they were effective against Escherichia coli (ATCC8739) and albino Trichozyme (ATCC10231) produced 98%-99% inhibitory effect.

The combination of DTAB and AMP-18 has a good bactericidal effect when the concentration ratio of the two is within the range of DTAB 1μM/L, AMP-185μM/L to DTAB 100μM/L, AMP-185μM/L.

Further experiments show that: when the concentration of AMP-18 is 1 μM/L, combined with DTAB of 75-100 μM/L, the bactericidal effect of more than 90% can be achieved. When the concentration of AMP-18 is 5 μM/L, combined with 1-100 μM/L DTAB, the bactericidal effect of more than 90% can be achieved. When the concentration of DTAB-18 is 50 μM/L, it can achieve a bactericidal effect of more than 90% when combined with 0.01-100 μM/L DTAB.

<Example 4>

1. Cosmetic emulsion formula 1: 1μM DTAB and 5μM AMP-18 compound system

Preparation steps:

(1) To prepare 100g of the product, try to calculate and weigh each component according to the formula in the table.

(2) Stir and heat phase A to 80-85°C, and keep stirring for more than 10 minutes.

(3) Stir and heat phase B to 85-90°C, and keep stirring for more than 10 minutes.

(4) Add phase A into phase B, stir for 3 minutes, and then stir for 1 minute.

(5) After cooling down to 50°C, add phase C, stir well and homogenize for 2 minutes.

(6) After cooling down to 45°C, add phase D and stir evenly.

2. After the preparation is completed, the antiseptic challenge test of the cosmetics is carried out. The test process is based on: "Chinese Pharmacopoeia" (CP) 2015. The formulation of this embodiment was able to pass the preservation challenge test.

<Embodiment 5>

1. Cosmetic emulsion formula 2:

Preparation steps:

(1) To prepare 100g of the product, try to calculate and weigh each component according to the formula in the table.

(2) Stir and heat phase A to 80-85°C, and keep stirring for more than 10 minutes.

(3) Stir and heat phase B to 85-90°C, and keep stirring for more than 10 minutes.

(4) Add phase A into phase B, stir for 3 minutes, and then stir for 1 minute.

(5) After cooling down to 50°C, add phase C, stir well and homogenize for 2 minutes.

(6) After cooling down to 45°C, add phase D and stir evenly.

2. After the preparation is completed, the antiseptic challenge test of the cosmetics is carried out. The test process is based on: "Chinese Pharmacopoeia" (CP) 2015. The formulation of this embodiment was able to pass the preservation challenge test.

<Example 6>

3. Cosmetic emulsion formula three:

Preparation steps:

(1) To prepare 100g of the product, try to calculate and weigh each component according to the formula in the table.

(2) Stir and heat phase A to 80-85°C, and keep stirring for more than 10 minutes.

(3) Stir and heat phase B to 85-90°C, and keep stirring for more than 10 minutes.

(4) Add phase A into phase B, stir for 3 minutes, and then stir for 1 minute.

(5) After cooling down to 50°C, add phase C, stir well and homogenize for 2 minutes.

(6) After cooling down to 45°C, add phase D and stir evenly.

2. After the preparation is completed, the antiseptic challenge test of the cosmetics is carried out. The test process is based on: "Chinese Pharmacopoeia" (CP) 2015. The formulation of this embodiment was able to pass the preservation challenge test.

<Embodiment 7>

1. Shampoo formula 1: 1μM DTAB and 5μM AMP-18 compound system.

The pigment, citric acid, essence and deionized water in the formula do not give specific mass percentages, and can be added in an appropriate amount according to the total mass of the product.

Preparation steps:

(1) Heat 16/18 alcohol, monoglyceride, and pearl flakes to 75°C to dissolve them completely, store at a constant temperature and record it as No. 1;

(2) After dispersing cationic guar gum, polyquaternium-7, and glycerol, add a solution of Carbopol U20 (pre-dispersed in advance), heat and dissolve it into a transparent and uniform solution, store it at a constant temperature of 60°C, and record for number 2;

(3) AES, 6501, BS-12, K12, and EDTA are heated to 75°C, stirred at a constant temperature at a slow speed to dissolve completely, and recorded as No. 3;

(4) Add No. 1 to No. 3, and at the same time add DC345 silicone oil and stir slowly until the mixture is complete, and keep the temperature at 75°C for 15 minutes. When the temperature drops to about 60°C, add No. 2 into the system together, and homogenize for 3 minutes while it is hot;

(5) When the temperature drops to 45°, add essence, preservatives (DTAB and AMP-18), and replenish the deionized water evaporated by heating, and adjust the pH value with citric acid.

2. After the preparation is completed, the antiseptic challenge test of the cosmetics is carried out. The test process is based on: "Chinese Pharmacopoeia" (CP) 2015. The formulation of this embodiment was able to pass the preservation challenge test.

<Embodiment Eight>

1. Shampoo formula 2: 1μM DTAB and 5μM AMP-18 compound system.

The pigment, citric acid, essence and deionized water in the formula do not give specific mass percentages, and can be added in an appropriate amount according to the total mass of the product.

Preparation steps:

(1) Heat 16/18 alcohol, monoglyceride, and pearl flakes to 75°C to dissolve them completely, store at a constant temperature and record it as No. 1;

(2) After dispersing cationic guar gum, polyquaternium-7, and glycerol, add a solution of Carbopol U20 (pre-dispersed in advance), heat and dissolve it into a transparent and uniform solution, store it at a constant temperature of 60°C, and record for number 2;

(3) AES, 6501, BS-12, K12, and EDTA are heated to 75°C, stirred at a constant temperature at a slow speed to dissolve completely, and recorded as No. 3;

(4) Add No. 1 to No. 3, and at the same time add DC345 silicone oil and stir slowly until the mixture is complete, and keep the temperature at 75°C for 15 minutes. When the temperature drops to about 60°C, add No. 2 into the system together, and homogenize for 3 minutes while it is hot;

(5) When the temperature drops to 45°, add essence, preservatives (DTAB and AMP-18), and replenish the deionized water evaporated by heating, and adjust the pH value with citric acid.

2. After the preparation is completed, the antiseptic challenge test of the cosmetics is carried out. The test process is based on: "Chinese Pharmacopoeia" (CP) 2015. The formulation of this embodiment was able to pass the preservation challenge test.

<Example Nine>

1. Shampoo formula three: 1μM DTAB and 5μM AMP-18 compound system.

The pigment, citric acid, essence and deionized water in the formulas of Examples 7 to 9 have no specific mass percentages, and can be added in an appropriate amount according to the total mass of the product. Colors and flavors can range from 0 to 1% by mass. Citric acid is used to adjust the pH, as long as the proper pH is achieved. Deionized water is enough to replenish the remaining percentage of the mass.

Preparation steps:

(1) Heat 16/18 alcohol, monoglyceride, and pearl flakes to 75°C to dissolve them completely, store at a constant temperature and record it as No. 1;

(2) After dispersing cationic guar gum, polyquaternium-7, and glycerol, add a solution of Carbopol U20 (pre-dispersed in advance), heat and dissolve it into a transparent and uniform solution, store it at a constant temperature of 60°C, and record for number 2;

(3) AES, 6501, BS-12, K12, and EDTA are heated to 75°C, stirred at a constant temperature at a slow speed to dissolve completely, and recorded as No. 3;

(4) Add No. 1 to No. 3, and at the same time add DC345 silicone oil and stir slowly until the mixture is complete, and keep the temperature at 75°C for 15 minutes. When the temperature drops to about 60°C, add No. 2 into the system together, and homogenize for 3 minutes while it is hot;

(5) When the temperature drops to 45°, add essence, preservatives (DTAB and AMP-18), and replenish the deionized water evaporated by heating, and adjust the pH value with citric acid.

2. After the preparation is completed, the antiseptic challenge test of the cosmetics is carried out. The test process is based on: "Chinese Pharmacopoeia" (CP) 2015. The formulation of this embodiment was able to pass the preservation challenge test.

In the above embodiments, only the application of the complex of α-helical antimicrobial peptide and surfactant in emulsion and shampoo is provided. In fact, it can be applied in a wider range of cosmetics to play a role of antiseptic.

Currently commonly used cosmetic preservatives, such as formaldehyde releasing body, bropol and paraben, have the risk of sensitization or even carcinogenicity; and the preservatives of the Kathon class have chronic toxicity to the skin; other preservatives such as anisic acid or Potassium sorbate and the like have higher requirements on the pH of the system, which limits the prospect of its application. Moreover, these preservatives are generally used in large amounts in the system, and it is usually necessary to add anti-allergic substances to the formula to reduce the probability of allergies, which essentially increases the burden on the skin. However, the α-helical antimicrobial peptide and surfactant complex of the present invention uses peptides as the antibacterial component, and the dosage is 1-2 orders of magnitude lower than that of traditional preservatives, so it has broad prospects in cosmetic antiseptic.

sequence listing

<110> Guangzhou Pharmaceutical University; Shanghai Nuoke Biotechnology Co., Ltd.

<120> Complex of α-helical antimicrobial peptide and surfactant and its application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> PRT

<213> artificial

<400> 1

Cys Trp Val Arg Leu Gly Arg Tyr Leu Leu Arg Arg Leu Lys Thr Pro

1 5 10 15

Phe Thr

Claims (10)

1. A-helical antimicrobial peptide and surfactant complex, is characterized in that, comprises: Anionic α-helical peptides and cationic surfactants. 2. α-helical antimicrobial peptide and surfactant complex as claimed in claim 1, is characterized in that: Wherein, the sequence of the anionic α-helical peptide is: Ac-CWVRLGRYLLRRLKTPFT- _NH2 . 3. α-helical antimicrobial peptide and surfactant complex as claimed in claim 1, is characterized in that: Wherein, the cationic surfactant is any one of dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide or cetyltrimethylammonium bromide. 4. α-helical antimicrobial peptide and surfactant complex as claimed in claim 1, is characterized in that: Wherein, the cationic surfactant is dodecyltrimethylammonium bromide. 5. α-helical antimicrobial peptide and surfactant complex as claimed in claim 1, is characterized in that: Wherein, the concentration ratio of the cationic surfactant to the anionic α-helical peptide is 1 μM/L:5 μM/L˜100 μM/L:5 μM/L. 6. α-helical antimicrobial peptide and surfactant complex as claimed in claim 1, is characterized in that: Wherein, the concentration ratio of the anionic α-helical peptide and the cationic surfactant is: 50 μM/L: 25 μM/L. 7. The application of a complex of anionic α-helical peptide and cationic surfactant in the preparation of antibacterial drugs. 8. The application according to claim 7, characterized in that: Wherein, the sequence of the anionic α-helical peptide is: Ac-CWVRLGRYLLRRLKTPFT- _NH2 . 9. The application according to claim 7, characterized in that: Wherein, the cationic surfactant is any one of dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide or cetyltrimethylammonium bromide. 10. The application of an anionic α-helical peptide and cationic surfactant complex in the preparation of cosmetics, characterized in that: the sequence of the anionic α-helical peptide is: Ac-CWVRLGRYLLRRLKTPFT- _NH2 .