Rana sylvaticum antimicrobial peptide preparation against Helicobacter pylori and preparation method thereof

Abstract

The present application provides a wood frog antimicrobial peptide preparation for Helicobacter pylori and a preparation method thereof, wherein the wood frog antimicrobial peptide preparation comprises a polysaccharide-modified wood frog antimicrobial peptide concentrate. The wood frog antimicrobial peptide preparation of the present application can maintain the antibacterial effect and antioxidant activity for a long time in a low-pH gastric fluid environment, and is expected to be used as an oral drug to kill Helicobacter pylori in the gastric environment, providing a new class of drugs for the treatment of Helicobacter pylori infection.

Description

Technical Field

[0001] The present application belongs to the field of antibacterial drugs or gastric disease treatment. Specifically, the present application provides a Rana sylvatica antibacterial peptide preparation against Helicobacter pylori and a preparation method thereof. Background Art

[0002] The Northeast Forest Frog (Ranadybowskii) is an important amphibian with both medicinal and edible uses unique to my country. It has a long history of medicinal use and has many effects, including nourishing the lungs and yin, tonifying the kidneys and replenishing essence, improving immunity, and anti-aging. Through ultrasonic extraction, acid hydrolysis, enzymatic hydrolysis and other processing methods, many scientific research institutions and enterprises have isolated a variety of peptides or peptide mixtures with antibacterial and antiviral functions from the trunk or skin of the forest frog. These peptides or peptide mixtures have the effect of resisting a variety of bacteria, HPV, new coronavirus, and Helicobacter pylori.

[0003] Helicobacter pylori is one of the main pathogenic factors of gastritis, gastric ulcer and even gastric cancer. Because it lives in an environment filled with highly acidic gastric juice, most antibacterial drugs are not effective against it. Clinically, the so-called antibiotic quadruple therapy, which has great side effects, is currently the main treatment for Helicobacter pylori infection.

[0004] Antimicrobial peptide drugs are a potential method for treating Helicobacter pylori infection. The applicant has previously discovered the efficacy of forest frog antimicrobial peptides in inhibiting Helicobacter pylori, and based on this, prepared an oral spray against Helicobacter pylori for prevention. Currently, the main obstacle to developing forest frog antimicrobial peptides as a therapeutic drug for Helicobacter pylori is that the effect of the antimicrobial peptides will rapidly decrease in a low pH environment. Summary of the invention

[0005] In view of the above situation, the applicant further developed a novel Rana sylvaticum antimicrobial peptide preparation that stabilizes Rana sylvaticum antimicrobial peptide at low pH.

[0006] In one aspect, the present application provides a rana antimicrobial peptide preparation against Helicobacter pylori, wherein the rana antimicrobial peptide preparation comprises a rana antimicrobial peptide concentrate.

[0007] Furthermore, the Rana sylvaticum antimicrobial peptide concentrate is a polysaccharide-modified Rana sylvaticum antimicrobial peptide concentrate.

[0008] Furthermore, the Rana sylvatica antimicrobial peptide concentrate is prepared by the following method:

[0009] (1) taking dried frog as raw material;

[0010] (2) adding the Rana sylvatica stem body to an 8 wt % acetic acid aqueous solution having a weight of 10 times that of the Rana sylvatica stem body, and swelling the solution for 2 hours to obtain the swollen Rana sylvatica stem body and the swelling solution;

[0011] (3) subjecting the swollen Rana sylvatica stem and the swelling solution obtained in step (2) to ultrasonic-assisted extraction twice, each time for 30 minutes, with an interval of 10 minutes, the ultrasonic power being 200 W and the frequency being 20 kHz; filtering to obtain an extract;

[0012] (4) centrifuging the extract obtained in step (3) at 5000 prm for 15 min, discarding the precipitate, and collecting the supernatant; (5) treating the supernatant obtained in step (4) at 40° C. for 15 min, cooling the temperature to 5-10° C., and filtering; concentrating the filtrate to a specific gravity of 1.1-1.2 to obtain a concentrated solution of Rana sylvatica antimicrobial peptide.

[0013] Furthermore, the polysaccharide modification method is:

[0014] (A) preparing the polysaccharide into a 200 mg / L aqueous solution or suspension;

[0015] (B) mixing the polysaccharide aqueous solution with a polysaccharide aqueous solution or suspension having a volume twice that of the concentrated solution of Rana tenuissima antimicrobial peptide;

[0016] (C) placing the mixture obtained in step (B) in a 60° C. water bath for 120 minutes, and then placing it in a 85° C. water bath for 30 minutes;

[0017] (D) Cooling, polysaccharide-modified Rana sylvatica antimicrobial peptide concentrate.

[0018] Furthermore, the polysaccharide is gellan gum or xanthan gum.

[0019] Furthermore, the polysaccharide is gellan gum.

[0020] On the other hand, the present application provides the use of the above-mentioned Rana sylvatica antimicrobial peptide preparation in the preparation of a drug or preparation for inhibiting Helicobacter pylori.

[0021] Furthermore, the medicine or preparation is an oral medicine or preparation, such as an oral spray.

[0022] Furthermore, the medicine or preparation is an external medicine or preparation, such as an external spray.

[0023] Furthermore, the drug or preparation is an oral drug or preparation.

[0024] Furthermore, the medicine is in the form of an oral liquid.

[0025] Furthermore, the medicine also contains an antioxidant and a buffer.

[0026] On the other hand, the present application provides the use of the above-mentioned Rana sylvatica antimicrobial peptide preparation in the preparation of a health product having an auxiliary protective effect on gastric mucosal damage.

[0027] Through glycosylation modification, the stability of the forest frog antimicrobial peptide concentrate in the low pH gastric fluid environment is significantly improved, and it is expected to form an oral drug for the treatment of Helicobacter pylori infection and a health product for protecting the gastric mucosa with the cooperation of suitable excipients. Compared with the forest frog antimicrobial peptide oral spray previously applied by the applicant, the use of the forest frog antimicrobial peptide concentrate has been expanded. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the effect of the concentrated antimicrobial peptides of Rana sylvatica modified with different polysaccharides against artificial gastric juice treatment.

[0029] Figure 2 The figure shows the effect of the Rana sylvatica antimicrobial peptide concentrate treated with different ratios of concentrate and polysaccharide on resistance to artificial gastric juice treatment (the ratio described in the legend is the volume ratio of polysaccharide solution / suspension to antimicrobial peptide concentrate). DETAILED DESCRIPTION

[0030] Materials and methods

[0031] The Rana sylvatica antimicrobial peptide concentrate was prepared by the applicant, and the same batch of products was used in the experiment.

[0032] Artificial gastric juice was homemade: 1 w / v% saline was prepared, the pH was adjusted to 2.5 with hydrochloric acid, and 1% pepsin was added after sterilization; when the antimicrobial peptide concentrate was treated with artificial gastric juice, the antimicrobial peptide concentrate was mixed with 3 times the volume of artificial gastric juice, treated at 37°C for a certain period of time, and then used in subsequent experiments.

[0033] Helicobacter pylori standard strain: ATCC 43504.

[0034] Xanthan gum, locust bean gum, cassia gum, guar gum, gellan gum and soluble soybean polysaccharides are all domestic food grade.

[0035] Detection and calculation of sterilization rate

[0036] Add about 1×10 Helicobacter pylori in logarithmic growth phase into artificial gastric juice. 6 CFU / mL, add 1 / 10 volume of antimicrobial peptide concentrate (or antimicrobial peptide concentrate treated with an equal volume of antimicrobial peptide concentrate) and distilled water, mix for 5 minutes, take 10uL and dilute it 10 times to apply Columbia medium, count the colonies after 48 hours of culture, and calculate the bactericidal rate.

[0037] DPPH free radical scavenging rate determination:

[0038] The concentrated solution of Rana sylvatica antimicrobial peptide was diluted 10 times as the sample to be tested (or the sugar-modified sample was appropriately diluted so that the concentration of the concentrated solution of Rana sylvatica antimicrobial peptide in the sample to be tested was 1 / 10 of the original concentration); 2.5 mL of the liquid sample was taken and mixed with 2.5 mL of 0.1 mM DPPH anhydrous ethanol solution (reaction) or 2.5 mL of anhydrous ethanol (control), and reacted at 25°C in the dark for 30 minutes; the absorbance of the sample reaction solution, the control reaction solution and the reference (2.5 mL of anhydrous ethanol plus 2.5 mL of 0.1 mM DPPH anhydrous ethanol solution) at 519 nm was measured by a spectrophotometer;

[0039] DPPH free radical scavenging rate = (1-(A 反应 -A 对照 ) / A 参比 )×100%.

[0040] Example 1 Preparation of Rana sylvatica Antimicrobial Peptide Concentrate

[0041] The preparation of Rana sylvatica antimicrobial peptide concentrate basically follows the existing method:

[0042] (1) taking dried frog as raw material;

[0043] (2) adding the dried body of Rana sylvatica to an 8 wt % acetic acid aqueous solution with a weight of 10 times the dried body weight of Rana sylvatica and swelling it for 2 hours;

[0044] (3) subjecting the swollen Rana sylvatica stem obtained in step (2) to ultrasonic-assisted extraction twice in an ultrasonic generator with a power of 200 W and a frequency of 20 kHz, each time for 30 minutes with an interval of 10 minutes; filtering to obtain an extract;

[0045] (4) centrifuging the extract obtained in step (3) at 5000 prm for 15 min, discarding the precipitate, and collecting the supernatant; (5) treating the supernatant obtained in step (4) at 40° C. for 15 min, cooling the temperature to 5-10° C., and filtering; concentrating the filtrate to a specific gravity of 1.1-1.2 to obtain a concentrated solution of Rana sylvatica antimicrobial peptide.

[0046] The Rana sylvatica antimicrobial peptide concentrate was stored at 4°C.

[0047] Example 2 Effect of gastric acid treatment on the effect of Rana sylvatica antimicrobial peptides

[0048] The anti-Helicobacter pylori effect of the Rana sylvatica antimicrobial peptide concentrate in Example 1 was detected using the method in the "Materials and Methods" section. The results are shown in Table 1:

[0049] Table 1 Bactericidal rate of Rana sylvatica antimicrobial peptide concentrate against Helicobacter pylori

[0050]

[0051] The results showed that the forest frog antimicrobial peptide concentrate had a significant anti-Helicobacter pylori effect, but this effect was only obvious within 30 minutes. If it existed in gastric juice for a long time, its anti-Helicobacter pylori effect would be significantly reduced. Therefore, the antimicrobial peptide concentrate is not suitable for directly preparing drugs for the treatment of Helicobacter pylori infection.

[0052] Example 3 Glycosylation modification of Rana sylvatica antimicrobial peptide concentrate

[0053] On the one hand, the applicant attempted to embed the Rana sylvatica antimicrobial peptide concentrate or prepare it into a solid coating dosage form; on the other hand, the applicant used PEG modification and polysaccharide modification to improve the stability of the Rana sylvatica antimicrobial peptide concentrate in the low pH gastric fluid environment.

[0054] Basic methods of glycosylation modification:

[0055] The polysaccharide was prepared into a 200 mg / L aqueous solution or suspension, the polysaccharide aqueous solution was mixed with an equal volume of the Rana sylvatica antimicrobial peptide concentrate, the mixture was placed in a 60° C. water bath for 120 minutes, and then placed in a 85° C. water bath for 30 minutes.

[0056] The antibacterial effect of the polysaccharide-modified Rana sylvatica antimicrobial peptide concentrate after treatment in a low pH gastric fluid environment was detected using the methods in the “Materials and Methods” section.

[0057] The results showed that gellan gum and xanthan gum underwent Maillard reaction with the polypeptide / protein components in the Rana sylvatica antimicrobial peptide concentrate (the color was darker than the original concentrate), and the stability of the product at low pH was significantly improved. Taking 90% in WS / T650-2019 (5.2.1) as the standard, both products could still maintain effective bactericidal effects after 30-60 minutes of treatment.

[0058] In addition, the data of DPPH free radical scavenging rate showed that, except for xanthan gum (about 49%), the modification of various polysaccharides basically did not change the antioxidant properties of the Rana sylvatica polypeptide concentrate (all remained at a level of about 60%), and other health functions of the Rana sylvatica polypeptide concentrate were expected to be unaffected by sugar modification.

[0059] Example 4 Optimization of the ratio of glycosylation modification of Rana sylvatica antimicrobial peptide concentrate

[0060] The ratio of concentrate to polysaccharide was further optimized by selecting gellan gum with better effect.

[0061] The results are as follows Figure 2As shown, the stability is further improved at a ratio of 1:2 polysaccharide solution / suspension: antimicrobial peptide concentrate. Taking 90% in WS / T650-2019 (5.2.1) as the standard, the antibacterial effect after 120 minutes can be basically regarded as qualified (89.7%). Combined with the residence time of the drug / food in the stomach, such an effect can basically maintain the full effect of the drug in the stomach.

Claims

1. A Rana sylvaticum antimicrobial peptide preparation against Helicobacter pylori, characterized in that: The Rana sylvaticum antimicrobial peptide preparation comprises a polysaccharide-modified Rana sylvaticum antimicrobial peptide concentrate; The Rana sylvatica antimicrobial peptide concentrate is prepared by the following method: (1) taking a trunk of a Chinese frog; (2) adding the trunk of a Chinese frog to an 8 wt % acetic acid aqueous solution having a weight of 10 times that of the trunk of the Chinese frog, swelling the trunk for 2 hours, and obtaining a swollen trunk of a Chinese frog and a swelling solution; (3) subjecting the swollen trunk of a Chinese frog and the swelling solution obtained in step (2) to ultrasonic-assisted extraction twice, each time for 30 minutes, with an interval of 10 minutes, an ultrasonic power of 200 W, and a frequency of 20 kHz; filtering to obtain an extract; (4) centrifuging the extract obtained in step (3) at 5000 prm for 15 minutes, discarding the precipitate, and collecting the supernatant; (5) treating the supernatant obtained in step (4) at 40° C. for 15 minutes, reducing the temperature to 5-10° C., and filtering; concentrating the filtrate to a specific gravity of 1.1-1.2, and obtaining a Chinese frog antimicrobial peptide concentrate; The polysaccharide-modified Rana sylvatica antimicrobial peptide concentrate is prepared by the following method: (A) preparing gellan gum into a 200 mg / L aqueous solution; (B) mixing the gellan gum aqueous solution with 2 times the volume of the forest frog antimicrobial peptide concentrate to obtain a mixed solution; (C) placing the mixed solution obtained in step (B) in a 60° C. water bath for 120 minutes, and then placing it in a 85° C. water bath for 30 minutes; (D) cooling to obtain a polysaccharide-modified forest frog antimicrobial peptide concentrate.

2. Use of the Rana sylvatica antimicrobial peptide preparation according to claim 1 in the preparation of a drug for inhibiting Helicobacter pylori.

3. The use according to claim 2, wherein the medicine is an external medicine.

4. The use according to claim 3, wherein the drug is an oral drug.

5. Use of the Rana sylvatica antimicrobial peptide preparation according to claim 1 in the preparation of a health product having an auxiliary protective effect on gastric mucosal damage.

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