WO2023065491A1

WO2023065491A1 - Antimicrobial peptide drug from marine organisms, and preparation method therefor

Info

Publication number: WO2023065491A1
Application number: PCT/CN2021/137377
Authority: WO
Inventors: 郭红星; 赵子方; 周尽学
Original assignee: 海南三元星生物科技股份有限公司
Priority date: 2021-10-20
Filing date: 2021-12-13
Publication date: 2023-04-27
Also published as: CN113786473A

Abstract

Provided in the present invention is an antimicrobial peptide drug from marine organisms, which drug comprises an antimicrobial peptide, an oil phase matrix, an aqueous phase matrix, a preservative and a medicinal material. Further provide in the present invention is a method for preparing the antimicrobial peptide drug from marine organisms.

Description

A kind of marine biological antimicrobial peptide drug and preparation method thereof

This application claims the priority of the Chinese patent application submitted to the China Patent Office on October 20, 2021 with the application number 202111221474.5 and the title of the invention "An antimicrobial peptide drug from marine organisms and its preparation method", the entire contents of which are incorporated by reference in this application.

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to a marine biological antibacterial peptide drug and a preparation method thereof.

Background technique

The prior art discloses the functions and preparation methods of various antimicrobial peptides as drugs. When treating scalded patients, coating the drug has the functions of repairing the skin, antibacterial and other effects, but the effect of the drugs in the prior art is relatively poor, and it takes one day It is very troublesome to apply it many times.

Contents of the invention

In view of this, the object of the present invention is to provide a marine organism antimicrobial peptide drug and a preparation method thereof. The marine organism antimicrobial peptide drug provided by the present invention has a better therapeutic effect on scalded patients.

The present invention provides a marine biological antimicrobial peptide medicine, comprising:

Antimicrobial peptides, oil phase base, water phase base, preservatives and medicinal materials.

Preferably, the mass ratio of the antimicrobial peptide, oil phase matrix, water phase matrix, preservative and medicinal material is (10-30): (5-15): (50-70): (0.5-1.5): (5 ~15).

Preferably, the oil phase base comprises:

Glyceryl monostearate, liquid paraffin, paraffin wax, sorbitan oleate, petrolatum and beeswax.

Preferably, the aqueous base comprises:

polysorbate and water.

Preferably, the medicinal materials include:

Rhubarb, Scutellaria, Phellodendron, Elephant Peel, Borneol, Frankincense, Wormwood and Peppermint.

The invention provides a preparation method of marine biological antibacterial peptide medicine, comprising:

The oil phase base, the water phase base, the preservative and medicinal materials are mixed with the antimicrobial peptide after decocting to obtain the marine biological antimicrobial peptide drug.

Preferably, the preparation method of the antimicrobial peptide comprises:

The large krill and the buffer solution are stirred, filtered and centrifugally precipitated to obtain the antimicrobial peptide.

Preferably, after said mixing also includes:

The obtained mixture is filtered, concentrated, dried and pulverized to obtain the marine biological antimicrobial peptide drug.

Preferably, the fresh-keeping method of the giant krill is a 1-MCP fresh-keeping method.

Preferably, the mixing temperature is 80-100°C.

In the present invention, traditional Chinese medicine and western medicine are used in combination. In the prior art, when treating scalded patients with medicine, paste-like medicine is mostly used to coat the injured part of the patient, which needs to be coated many times a day, which is cumbersome to operate, and the The effects of sterilization, skin repair, and wound healing are slow. The present invention combines the extracted antibacterial peptide with traditional Chinese medicine, and adds antipruritic medicine and peppermint extract. It also has a good bactericidal effect, and it is in the form of a traditional Chinese medicine plaster. It does not need to be applied multiple times a day, just stick the plaster on the burnt area, and it will be accompanied by itching and other problems during skin repair. The patient's experience is made more comfortable thanks to the antipruritic drug included to reduce itching and the formula contains peppermint extract.

Further, the present invention uses 1-MCP to keep fresh and sterilize the raw materials. The current selection of antimicrobial peptide extracts is wide, and the extracts are easily infected by bacteria during transportation, resulting in poor quality of the used extracts. The quality of the antimicrobial peptide extraction is affected. The present invention selects large krill as the extract, which is rich in resources. The extract is thawed and 1-MCP is used to keep the extract fresh to ensure that the quality of the extract remains unchanged during transportation. And it can sterilize the extract to prevent contamination.

Description of drawings

Fig. 1 is a process flow diagram for preparing marine biological antimicrobial peptide drugs according to an embodiment of the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other examples improved or modified by those skilled in the art belong to the protection scope of the present invention. It should be understood that the embodiments of the present invention are only used to illustrate the technical effect of the present invention, and are not used to limit the protection scope of the present invention. In the examples, the methods used are conventional methods unless otherwise specified.

In the present invention, the mass ratio of the antimicrobial peptide, oil phase matrix, water phase matrix, preservatives and medicinal materials is preferably (10-30): (5-15): (50-70): (0.5-1.5) : (5-15), more preferably (15-25): (8-12): (55-65): (0.8-1.2): (8-10), most preferably 20:10:60:1 :9.

In the present invention, the oil phase base preferably includes:

In the present invention, the mass ratio of glyceryl monostearate, liquid paraffin, solid paraffin, sorbitan oleate, vaseline and beeswax is preferably (6～7):(8～12):(1～3) : (1～2): (1.8～2.2): (0.7～1.1), more preferably (6.3～6.7): (9～11): (1.5～2.5): (1.3～1.7): (1.9～2.1 ):(0.8～1), most preferably 6.35:10:2:1.65:2.1:0.9.

In the present invention, the aqueous matrix preferably includes:

polysorbate and water.

In the present invention, the water is preferably distilled water.

In the present invention, the mass ratio of polysorbate to water is preferably (65-75): (65-70), more preferably (68-72): (66-69), most preferably 70:68 .

In the present invention, the preservative is preferably selected from ethylparaben.

In the present invention, the medicinal materials preferably include:

In the present invention, the cork cork is preferably a dry paste of cork cortex.

In the present invention, the elephant skin is preferably elephant skin powder.

In the present invention, the mint is preferably mint liquid.

In the present invention, the mass ratio of the total mass of rhubarb, scutellaria baicalensis and Phellodendron phellodendron, elephant skin, borneol, frankincense, wormwood and peppermint is preferably (8.5～9.5):(4～4.5):(6～7): (1～2): (2～6): (2～4), more preferably (8.6～9): (4.1～4.4): (6.2～6.8): (1.2～1.8): (3～5) : (2.5-3.5), most preferably 8.7:4.2:6.3:1.7:4:3.

In the present invention, the mass ratio of rhubarb, scutellaria baicalensis and Phellodendron Phellodendri is preferably 1:(0.8-1.2):(0.8-1.2), more preferably 1:1:1.

The present invention provides a preparation method of the marine biological antimicrobial peptide medicine described in the above technical scheme, comprising:

The process flow diagram of the preparation of marine biological antimicrobial peptide drugs according to the embodiment of the present invention is shown in Fig. 1 .

In the present invention, the oil phase base, the water phase base, the preservative and medicinal materials are used as the raw materials of the ointment.

In the present invention, it is preferred to soak and clean the oil phase matrix, water phase matrix, preservatives and medicinal materials; the soaking time is preferably 10 to 15 hours, more preferably 11 to 14 hours, and most preferably 12 to 13 hours. Hour.

In the present invention, the amount of water used in the soaking process is preferably 2 to 4 times that of the ointment raw material, more preferably 3 times.

In the present invention, in the decocting process, it is preferable to add oil phase matrix, water phase matrix, preservatives and medicinal materials in order to decoct; it is more preferred to add glycerol monostearate, liquid paraffin, solid paraffin, oleic acid Decoction with Tan, Vaseline, Beeswax, Polysorbate, Distilled Water, Ethyl Paraben, Rhubarb, Scutellaria, Phellodendron Dry Extract, Elephant Peel Powder, Borneol, Frankincense, Wormwood and Mint.

In the present invention, the decocting temperature is preferably 80-100°C, more preferably 85-95°C, most preferably 90°C.

In the present invention, large krill is preferably used as the raw material for antimicrobial peptide extraction.

In the present invention, the 1-MCP fresh-keeping method is preferably used to preserve and transport the giant krill during transportation after thawing.

In the present invention, the preparation method of the antimicrobial peptide preferably comprises:

The antimicrobial peptide is obtained by mixing the large krill and the buffer solution, stirring, filtering and centrifuging to precipitate.

In the present invention, it is preferable to wash the large krill.

In the present invention, the buffer is preferably a NaCl buffer; the pH of the buffer is preferably 7.5 to 8.5, more preferably 7.8 to 8.2, and most preferably 8; the concentration of the buffer is preferably 70 to 8.5. 80mmol/L, more preferably 73-77mmol/L, most preferably 75mmol/L.

In the present invention, the stirring is preferably carried out in a blender; during the stirring process, the homogenization is preferably 3 to 5 times, more preferably 4 times; the homogenization is preferably 15 to 25 s each time, more preferably 18 to 22 s , most preferably 20s.

In the present invention, the filtering is preferably filtering and discarding the supernatant.

In the present invention, the rotational speed in the centrifugal precipitation process is preferably 4000-5000r/min, more preferably 4300-4700r/min, most preferably 4500r/min; the temperature is preferably 3-7°C, more preferably 4- 6°C, most preferably 5°C; the time is preferably 10-20 minutes, more preferably 13-17 minutes, most preferably 15 minutes.

In the present invention, after the centrifugal precipitation, it is preferred to further include:

The supernatant was discarded, the obtained precipitate was dissolved in water, and the second centrifugation was carried out again.

In the present invention, the water is preferably ultrapure water.

In the present invention, the rotational speed in the secondary centrifugal precipitation process is preferably 6500-7500r/min, more preferably 6800-7200r/min, most preferably 7000r/min; the temperature is preferably 3-7°C, more preferably 4-6°C, most preferably 5°C; the heating time is preferably 10-20 minutes, more preferably 13-17 minutes, most preferably 15 minutes.

In the present invention, after the second centrifugation, preferably also include:

The obtained precipitate was mixed with sulfuric acid and centrifuged again three times.

In the present invention, the sulfuric acid is preferably a sulfuric acid solution, more preferably a dilute sulfuric acid solution, most preferably a hot dilute sulfuric acid solution.

In the present invention, the rotational speed in the three centrifugal precipitation processes is preferably 8000-12000r/min, more preferably 9000-11000r/min, most preferably 10000r/min; the temperature is preferably 3-5°C, more preferably 3.5 ~4.5°C, most preferably 4°C; the time is preferably 15-25 minutes, more preferably 18-22 minutes, most preferably 20 minutes.

In the present invention, after the centrifugation for three times, preferably also include:

The resulting supernatants were filtered and the filtrates were combined.

In the present invention, the combined filtrate preferably also includes:

Concentrate the combined solution, mix it with ethanol, place it, centrifuge again, wash, wait, and save it for future use.

In the present invention, the concentration is preferably distillation concentration.

In the present invention, the standing time is preferably 20 to 30 hours, more preferably 22 to 28 hours, most preferably 24 to 26 hours; the standing temperature is preferably 4 to 6°C, more preferably 4.5 to 26 hours. 5.5°C, most preferably 5°C.

In the present invention, the washing is preferably performed with acetone and ether respectively.

In the present invention, the waiting time is preferably 4-8 hours, more preferably 5-7 hours, most preferably 6 hours, so as to volatilize the organic matter.

In the present invention, the storage temperature is preferably 15-20°C, more preferably 16-18°C.

In the present invention, the mixing temperature is preferably 80 to 100°C, more preferably 85 to 95°C, and most preferably 90°C; the mixing is preferably:

The oil phase base, the water phase base, the preservative and the medicinal materials are decocted until the temperature reaches 90° C., and the antimicrobial peptides are added for mixing.

In the present invention, preferably after the mixing, it also includes:

The resulting mixture is repeatedly decocted.

In the present invention, the number of repeated decoctions is preferably 2 to 4 times, more preferably 3 times; the time for each decoction is preferably 0.5 to 1.5 hours, more preferably 0.8 to 1.2 hours, most preferably 1 hour.

In the present invention, after repeated decocting, it is preferred to further include:

The obtained medicinal solution was left to stand and then filtered.

In the present invention, the standing time is preferably 2-4 hours, more preferably 2.5-3.5 hours, most preferably 3 hours.

In the present invention, after the mixing is completed, it is preferred to further include:

In the present invention, the concentration is preferably concentrated by taking the filtered filtrate; the method of concentration is preferably heating and concentration.

In the present invention, the method of heating and concentrating preferably includes:

Heat the obtained filtrate to boiling, remove the froth, lower the temperature, stir and concentrate to paste.

In the present invention, the heating is preferably carried out in an evaporation pot.

In the present invention, after said concentration, preferably also include:

The concentrated plaster is left to stand and then pulverized to obtain a powdery plaster.

In the present invention, the standing time is preferably 1 to 3 hours, more preferably 2 hours.

In the present invention, the drying method is preferably sun drying.

In the present invention, preferably after the pulverization, it also includes:

The pulverized material is heated and extruded into thin sheets to stick on the medicine paper.

In the present invention, the heating is preferably performed under the condition of stirring.

In the present invention, it is preferred to heat the pulverized material, mix it with peppermint, antipruritic powder and anti-inflammatory water, and then stir until the ointment turns black, take it out, extrude the ointment into thin sheets and stick it on the medicine paper.

In the embodiment of the present invention, the process flow for the preparation of marine biological antimicrobial peptide drugs is shown in FIG. 1 .

The present invention adopts 1-MCP method to keep fresh and sterilize raw materials. The prior art has a wide range of selection directions for antimicrobial peptide extracts, and the extracts are easily infected by bacteria during transportation, resulting in poor quality of the used extracts, which directly affects the antibacterial peptides. The quality of the peptide extraction, the present invention selects large krill as the extract, and the large krill is rich in resources, thaws the extract and uses 1-MCP to keep the extract fresh, so as to ensure that the quality of the extract remains unchanged during transportation, and can The extract is sterilized to prevent contamination.

In the present invention, traditional Chinese medicine and western medicine are used in combination. Most of the existing medicines for scalded patients use paste-like medicines to coat the injured parts of the patients, which requires multiple coatings a day, which is cumbersome to operate, and the sterilization of medicines, skin The effect of repairing and wound healing is slow. The present invention combines the extracted antibacterial peptide with traditional Chinese medicine for treating scalds, and adds antipruritic medicine and peppermint extract. It grows and repairs, and has a good bactericidal effect at the same time. At the same time, it is in the form of a traditional Chinese medicine plaster. It does not need to be applied multiple times a day. It is only necessary to stick the plaster on the burnt area. It will be accompanied by itching and other problems during skin repair. The present invention contains antipruritics to reduce itching, and peppermint extract to make patients experience more comfortable.

Example 1

The formula of the marine biological antimicrobial peptide medicine provided by the present embodiment is:

Antimicrobial peptides, oil phase matrix, water phase matrix, preservatives and medicinal materials;

The mass ratio of antimicrobial peptide, oil phase matrix, water phase matrix, preservative and medicinal material is:

20%: 10%: 60%: 1%: 9%;

The oil phase base includes: 6.35g of glyceryl monostearate, 10g of liquid paraffin, 2g of solid paraffin, 1.65g of sorbitan oleate, 2.1g of petrolatum, and 0.9g of beeswax;

The aqueous base includes: polysorbate 70g, distilled water 68g;

Preservatives include: ethylparaben 2.3g;

The medicinal materials include: rhubarb 2.9g, scutellaria baicalensis 2.9g, phellodendron dry paste 2.9g (8.7g in total), elephant skin powder 4.2g, borneol 6.3g, frankincense 1.7g, wormwood 4g and mint liquid 3g.

The preparation method is:

Krill is used as an antimicrobial peptide extract. After the krill is thawed, the 1-MCP fresh-keeping method is used to keep the krill fresh and transported. After the krill is received, it is cleaned, and 300mL of 75mmol/L NaCl buffer solution with a pH value of 8 is added. Homogenize in a mixer for 4 times, each time for 20s, filter, then centrifuge at 4500r/min, 5°C for 15min, discard the supernatant, add ultrapure water to dissolve the obtained precipitate, and then carry out 7000r/min, 5°C Centrifuge for 15min to get the precipitate, heat the dilute sulfuric acid solution, centrifuge at 10000r/min, 4°C for 20min, take the supernatant, filter and combine the filtrate, concentrate the supernatant by distillation under reduced pressure, add ethanol, place for 24h, place at 4°C ~6°C, take out the precipitate by centrifugation again, wash with 10mL acetone and 10mL ether respectively, wait for 6h for the organic matter to volatilize, and store it for later use. The storage temperature is 15°C-20°C to obtain the antimicrobial peptide.

Add the oil phase matrix, water phase matrix, preservatives and medicinal materials to 3 times of clean water and soak for 12 hours, then add glyceryl monostearate, liquid paraffin, solid paraffin, sorbitan oleate, vaseline, beeswax, poly Sorbitate, distilled water, ethylparaben, rhubarb, baicalin, Phellodendron dry paste, elephant skin powder, borneol, frankincense, wormwood and mint are added to the pot for decoction, wait for the temperature to reach 90°C, add antimicrobial peptides, and decoct repeatedly 3 times, decoct for 1 hour each time, filter after standing for 3 hours, take out the filtrate and put it in an evaporating pot, heat it to boiling, remove the froth, lower the temperature and continuously stir and concentrate until it becomes a paste, take it out and let it stand for 2 hours to dissolve the plaster Crush, screen and set aside, take out the powdered ointment and heat it again, add mint, anti-itch powder and anti-inflammatory water and keep stirring until the ointment turns black, take out the ointment, squeeze the ointment into thin slices and stick it on the medicine paper, and get the marine biological antibacterial peptide ointment .

As can be seen from the above examples, the present invention combines the extracted antibacterial peptides with traditional Chinese medicines for treating scalds, and adds antipruritic medicines and peppermint extracts. The traditional Chinese medicines are less irritating to the skin of patients, and adding antimicrobial peptides can promote the growth of the skin And repair, at the same time play a good bactericidal effect, and at the same time in the form of a traditional Chinese medicine plaster, no need to apply it multiple times a day, just stick the plaster on the burnt position, it will be accompanied by itching and other problems during skin repair, because it contains Antipruritics reduce itching and contain peppermint extract for a more comfortable experience. The present invention selects giant krill as the extract, which is rich in resources, thaws the extract and uses 1-MCP to keep the extract fresh, so as to ensure that the quality of the extract remains unchanged during transportation, and the extract can be sterilized, To prevent contamination, it has obvious antibacterial activity against Staphylococcus aureus through enzymatic hydrolysis of polypeptides. The product provided by the invention is in the form of a paste, so it does not need to be replaced several times a day during use, and can be replaced after 24 hours according to the situation, which is convenient to use and has good effect, and the medicine can perfectly contact the patient's skin.

It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is a relationship between these entities or operations. There is no such actual relationship or order between them. Furthermore, the term "comprises", "comprises" or any other variation thereof is intended to cover a non-exclusive inclusion such that a process, method, article, or apparatus comprising a set of elements includes not only those elements, but also includes elements not expressly listed. other elements of or also include elements inherent in such a process, method, article, or device. Without further limitations, an element defined by the phrase "comprising a referenced structure" does not exclude the presence of additional identical elements in the process, method, article or device comprising said element.

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications and substitutions can be made to these embodiments without departing from the principle and spirit of the present invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims

A marine biological antimicrobial peptide medicine, comprising:

Antimicrobial peptides, oil phase base, water phase base, preservatives and medicinal materials.
The antimicrobial peptide drug of marine organisms according to claim 1, wherein the mass ratio of the antimicrobial peptide, oil phase matrix, water phase matrix, preservative and medicinal material is (10～30):(5～15): (50-70): (0.5-1.5): (5-15).
The marine organism antimicrobial peptide drug according to claim 1, wherein the oil phase matrix comprises:

Glyceryl monostearate, liquid paraffin, paraffin wax, sorbitan oleate, petrolatum and beeswax.
The marine biological antimicrobial peptide drug according to claim 1, wherein the aqueous matrix comprises:

polysorbate and water.
The marine organism antimicrobial peptide drug according to claim 1, wherein the medicinal material comprises:

Rhubarb, Scutellaria, Phellodendron, Elephant Peel, Borneol, Frankincense, Wormwood and Peppermint.
A preparation method of marine biological antimicrobial peptide medicine, comprising:

The oil phase base, the water phase base, the preservative and medicinal materials are mixed with the antimicrobial peptide after decocting to obtain the marine biological antimicrobial peptide drug.
method according to claim 6, is characterized in that, the preparation method of described antimicrobial peptide comprises:

The large krill and the buffer solution are stirred, filtered and centrifugally precipitated to obtain the antimicrobial peptide.
method according to claim 6, is characterized in that, after described mixing also comprises:

The obtained mixture is filtered, concentrated, dried and pulverized to obtain the marine biological antimicrobial peptide drug.
The method according to claim 7, characterized in that, the fresh-keeping method of the giant krill is a 1-MCP fresh-keeping method.
The method according to claim 6, characterized in that the mixing temperature is 80-100°C.