Preparation of an antibacterial drug based on agaricus blazei lectin and enteromorpha polysaccharide and application thereof

Abstract

This invention relates to the application of Agaricus blazei lectin and Ulva prolifera polysaccharide in the preparation of antibacterial drugs, specifically using a mixture of Agaricus blazei lectin and Ulva prolifera polysaccharide to inhibit Penicillium sclerosis. A pharmaceutical composition, which is a gel, contains the following components per 100 ml of gel: 1.2–1.8 g Agaricus blazei lectin, 1.0–1.8 g Ulva prolifera polysaccharide, 0.3–0.6 g Bletilla striata gum, 1–1.2 g sodium chloride, a pH buffer in a pharmaceutically acceptable amount, a stabilizer in a pharmaceutically acceptable amount, and a preservative in a pharmaceutically acceptable amount. The advantages of this invention are: the discovery of the role of Agaricus blazei lectin in inhibiting Penicillium sclerosis, and the discovery that adding Ulva prolifera polysaccharide to Agaricus blazei lectin can enhance the inhibitory effect on Penicillium sclerosis, providing a theoretical basis for the development of drugs to inhibit Penicillium sclerosis.

Description

Technical Field

[0001] This invention relates to the field of antibacterial drugs, and in particular to a method for preparing antibacterial drugs based on Agaricus blazei lectin and Ulva prolifera polysaccharide and their application. Background Technology

[0002] Lectins are a class of proteins with specific sugar-binding activity, possessing one or more non-catalytic domains that can specifically and reversibly bind to monosaccharides or oligosaccharides. Lectins have important applications in biology and medicine, primarily in blood typing and microbial identification.

[0003] Currently, the main application of Agaricus blazei lectin is in the field of anticancer drugs, and its application in other fields is still in the exploratory stage. Similarly, the application of Ulva prolifera polysaccharide is also currently in the field of anticancer drugs. Although some studies have shown that Ulva prolifera polysaccharide has certain antibacterial effects, the results are not yet ideal. Summary of the Invention

[0004] The purpose of this invention is to provide an application of Agaricus blazei lectin and Ulva prolifera polysaccharide in the preparation of antibacterial drugs. It has been discovered that adding Ulva prolifera polysaccharide to Agaricus blazei lectin can enhance the inhibitory effect on Penicillium circulatory mildew, providing a theoretical basis for the development of drugs that inhibit Penicillium circulatory mildew.

[0005] An application of Agaricus blazei lectin and Ulva prolifera polysaccharide in the preparation of antibacterial drugs, wherein the mixture of Agaricus blazei lectin and Ulva prolifera polysaccharide is used to inhibit Penicillium circulatory spores.

[0006] A pharmaceutical composition, which is a gel, wherein each 100 ml of gel contains the following components: 1.2-1.8 g of Agaricus blazei lectin, 1.0-1.8 g of Ulva prolifera polysaccharide, 0.3-0.6 g of Bletilla striata gum, 1-1.2 g of sodium chloride, a pH buffer in a pharmaceutically acceptable amount, a stabilizer in a pharmaceutically acceptable amount, and a preservative in a pharmaceutically acceptable amount.

[0007] The pH buffer is an acetate-sodium acetate buffer solution with a pH of 4.6-4.7. The stabilizer is PEG6000. The preservative is potassium sorbate.

[0008] The advantages of this invention patent are: it discovers the role of Agaricus blazei lectin in inhibiting Penicillium circulatory mildew, and it discovers that adding Ulva prolifera polysaccharide to Agaricus blazei lectin can enhance the inhibitory effect on Penicillium circulatory mildew, providing a theoretical basis for the development of drugs to inhibit Penicillium circulatory mildew. Attached Figure Description

[0009] Figure 1 This is a schematic diagram of Penicillium arcuate hyphae on a culture medium.

[0010] Figure 2 For comparison, see the experimental diagram. Detailed Implementation

[0011] Example 1

[0012] The Agaricus blazei lectin of the present invention is prepared by the following method, which includes the following steps:

[0013] Step 1: The fruiting bodies of Agaricus blazei were added to phosphate buffer at a mass / volume ratio of 1:5, and then extracted at 4-6℃ for 24 hours; wherein the concentration of phosphate buffer was 0.04mol / L.

[0014] Step 2: The phosphate buffer containing fruiting bodies from Step 1 was subjected to cryogenic centrifugation, and the supernatant was collected for later use. The cryogenic centrifugation conditions were: 4-6℃, 8000 r / min, and 40 min. Subsequently, the samples were screened using various hollow fiber membranes based on their molecular weight.

[0015] Step 3: Precipitate the supernatant obtained in Step 2 by ammonium sulfate precipitation; collect the precipitate with 40% to 60% ammonium sulfate saturation.

[0016] Step 4: Redissolve the precipitates obtained in Step 3 in a small amount of PBS buffer, then place them in a dialysis bag for desalting, and dialyze until no SO4 is visible in the external dialysate. 2- Upon detection, crude Agaricus blazei lectin was obtained by freeze-drying. The concentration of PBS buffer used was 0.04 mol / L.

[0017] Step 5: Load the crude Agaricus blazei agglutinin obtained in Step 4 onto a DEAE-Sepharose FastFlow column, elute with the appropriate buffer solution and collect in separate tubes, detect at a wavelength of 280 nm, detect the coagulation activity of each peak with rabbit red blood cells, and collect the active peaks.

[0018] The active peaks of Agaricus blazei lectin were then further purified by Sephadex G-100 column chromatography. The active peaks of Agaricus blazei lectin were purified by CM-Sepharose FastFlow chromatography, and the active peaks were collected, desalted, and freeze-dried to obtain pure Agaricus blazei lectin.

[0019] Example 2

[0020] The *Ulva prolifera* polysaccharide of the present invention is prepared by the following method, which includes the following steps:

[0021] Fresh seaweed was rinsed with tap water and then washed with distilled water. One kilogram of fresh seaweed was crushed in a plant crusher at 16000 rpm for two minutes, then centrifuged at 5000 rpm for 10 minutes in a 90°C water bath. The supernatant was collected. Protein was removed using the Sevag method. The lower clear layer was collected by centrifugation at 5000 rpm for 10 minutes. The mixture was concentrated and then three times its volume of anhydrous ethanol was added. Alcohol precipitation was carried out overnight (at least 12 hours). Ethanol and water were removed, and the mixture was freeze-dried. The precipitate was collected by centrifugation at 5000 rpm for 10 minutes. The precipitate was washed with ethanol, decolorized, and passed through a hollow fiber membrane at various molecular weights. Purification was performed using a high-performance liquid chromatography (HPLC). The crude polysaccharide extraction rate was 31.3%, the total sugar content was 39.62%, the reducing sugar content was 3.2%, and the polysaccharide content was 36.42%.

[0022] The crude polysaccharide was then detected by high performance liquid chromatography and was roughly divided into 5 components. The peak times were 5.148, 7.563, 8.638, 9.919, and 11.144, respectively, and the proportions of each component were 6.54%, 44.23%, 16.33%, 31.06%, and 1.84%, respectively.

[0023] Then, polysaccharide purification was performed: 0.2g of polysaccharide powder was dissolved in 40ml of ultrapure water at 70°C for 1 hour, filtered through a 0.45µm microporous membrane, and the column packing was washed with ultrapure water until no alcohol odor was detected. The column was then filled with 3 times its volume of ultrapure water, sonicated for 5 minutes to remove air bubbles, and packed into a column. Elution was performed sequentially with water, 0.2MnAcl, 0.4MnAcl, 0.6MnAcl, and 0.8MnAcl. Dialysis was performed using a dialysis bag for 48 hours, with water changed every 6 hours. After concentration in an evaporating dish, precipitation was carried out with 3 times its volume of anhydrous ethanol, and the product was dried to obtain the *Ulva prolifera* polysaccharide.

[0024] Example 3

[0025] A drug composition of Agaricus blazei lectin and Ulva prolifera polysaccharide, which is a gelling agent, contains the following components per 100 ml of gel: 1.6 g Agaricus blazei lectin, 1.2 g Ulva prolifera polysaccharide, 0.3 g Bletilla striata gum, 1 g sodium chloride, a pH buffer of 4.6 acetate-sodium acetate buffer, a stabilizer of 1.6 g PEG6000, and a preservative of 0.2 g potassium sorbate.

[0026] Example 4

[0027] like Figure 1 As shown: Take the preserved Penicillium arcus culture, inoculate it on Bengal red agar medium, and incubate at 30°C until the mycelium covers the entire plate.

[0028] like Figure 2 As shown: On a petri dish, pick up a round mycelium about 10 mm in size and inoculate it in the center of a Bengal red agar plate. Add 100 μL of four sets of solvents around the mycelium.

[0029] The four solvents are respectively

[0030] Control group 1: sterile PBS buffer (pH 7.2);

[0031] Control group 2: A gel containing the same concentration of *Ulva prolifera* polysaccharide as in Example 3;

[0032] Control group 3: A gel containing the same concentration of Agaricus blazei lectin as in Example 3;

[0033] Experimental group: The gelling agent disclosed in Example 3;

[0034] The above four groups were incubated at 30℃ for 18 hours, and each treatment was repeated 3 times.

[0035] Mycelial growth inhibition rate / % [1 - (treatment group growth diameter - mycelial cake diameter) ÷ (control group growth diameter - mycelial cake diameter)] × 100.

[0036] from Figure 2 As can be seen, the mycelium grew completely in the environment of control group 1, while its growth was somewhat inhibited in the environments of controls 2 and 3; and in the experimental group environment of this invention, the growth of the mycelium was clearly strongly inhibited.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. The use of a pharmaceutical composition comprising Agaricus blazei lectin and Ulva prolifera polysaccharide in the preparation of a drug for inhibiting Penicillium circulatory spp., characterized in that: The pharmaceutical composition is a gel, wherein each 100 ml of gel contains the following components: 1.6 g Agaricus blazei lectin, 1.2 g Ulva prolifera polysaccharide, 0.3 g Bletilla striata gum, 1 g sodium chloride, a pH buffer of 4.6 acetate-sodium acetate buffer, a stabilizer of PEG6000 (1.6 g by weight), and a preservative of potassium sorbate (0.2 g by weight).

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