Anti-tumor grifola frondosa water-soluble protein complex preparation and preparation method thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to an anti-tumor grifola frondosa water-soluble protein complex preparation and a preparation method thereof.The preparation raw materials of the complex preparation include the following components in parts by weight: grifola frondosa water-soluble protein 2-3 parts, fermentation preparation 0.8-1.2 parts, rosemary acid 0.1-0.2 parts.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an antitumor water-soluble protein compound preparation of *Lactobacillus thunbergii* and its preparation method. Background Technology

[0002] Hepatocellular carcinoma, a common malignant tumor, is currently mainly treated with surgery combined with chemotherapy. However, this approach has drawbacks, including a high recurrence rate and significant toxic side effects. Therefore, finding safe, effective, and low-toxicity anti-cancer active ingredients from natural products is urgently needed. *Mushroom arvensis*, a representative species of *Mushroom arvensis*, is a fungus used in both food and medicine. It is delicious, tender, and nutritious. Containing polysaccharides, caliciferine, and other bioactive substances, it possesses various biological functions, including scavenging excess free radicals, enhancing immune function, and exhibiting anti-tumor activity. It can also treat symptoms such as measles and restlessness in children. Current research has shown that *Mushroom arvensis* possesses various activities, such as antioxidant, anti-tumor, and antibacterial effects. However, research on the anti-tumor activity of *Mushroom arvensis* water-soluble proteins is rarely reported. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention proposes an anti-tumor water-soluble protein compound preparation of *Lactobacillus thunbergii* and its preparation method.

[0004] This invention is achieved through the following technical solution:

[0005] A water-soluble protein compound preparation of antitumor *Pleurotus ostreatus*, the raw materials for preparation include the following components in parts by weight: 2-3 parts water-soluble protein of *Pleurotus ostreatus*, 0.8-1.2 parts fermentation preparation, and 0.1-0.2 parts rosmarinic acid.

[0006] Furthermore, the method for preparing the water-soluble protein from *Pleurotus ostreatus* includes the following steps:

[0007] L1. Dry the large white mushroom to constant weight, crush it and pass it through an 80-mesh sieve. Add it to a phosphate buffer solution with pH 7.8 0.2 mol / L at a material-to-liquid ratio of 1 g: 20-30 mL. Stir at 400-500 rpm for 4 h, centrifuge at 10000 r / min for 15-20 min, and take the supernatant.

[0008] L2. Take the supernatant obtained in step L1, add ammonium sulfate to saturation, let stand overnight at 4°C, centrifuge, place the precipitate in a 3500 Da dialysis bag and dialyze in deionized water for 24 h, freeze-dry the dialysate to obtain water-soluble protein from Mushroom sphaerocephala.

[0009] Furthermore, the method for preparing the fermentation agent includes the following steps:

[0010] V1. Dry the large white mushrooms to constant weight, pulverize them through an 80-mesh sieve, add them to distilled water at a material-to-liquid ratio of 1 g: 10-15 mL, sterilize at 121℃ for 20 min, and after cooling, use 10... 7 Inoculate activated Lactobacillus paracasei at an inoculum size of CFU / mL, ferment at 37℃ for 24 h, and centrifuge at 8000 r / min for 10-15 min.

[0011] V2. Take the supernatant after centrifugation in step V1, filter it through a 0.22μm filter membrane, add 4 times the volume of ethanol while stirring, let it stand overnight at 4℃, centrifuge at 8000 rpm for 10-15 min, wash the precipitate with anhydrous ethanol, reconstitute it with distilled water, and freeze dry it under vacuum to obtain the fermentation preparation.

[0012] Furthermore, the present invention also provides a method for preparing the antitumor *Pleurotus ostreatus* water-soluble protein complex preparation, comprising the following steps:

[0013] S1. Add the water-soluble protein of *Pleurotus ostreatus* to PBS (0.01M) buffer at pH 6.8, stir overnight at 4°C, adjust the pH to 9 with 1 mol / L NaOH, add rosmarinic acid and stir for 24 h, dialyze in deionized water for 48 h, freeze dry the dialysate to obtain RA-water-soluble protein;

[0014] S2. Mix the RA-water-soluble protein obtained in step S1 with the fermentation agent evenly to obtain the anti-tumor white mushroom water-soluble protein complex preparation.

[0015] Further, in step S1, the mass concentration of the water-soluble protein of *Pleurotus ostreatus* in PBS buffer is 20 mg / mL.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention provides an anti-tumor *Pleurotus ostreatus* water-soluble protein complex formulation. Through the scientific formulation and preparation process of specific components, the components synergistically enhance their effects, exhibiting significant anti-tumor activity. This invention extracts water-soluble proteins from *Pleurotus ostreatus*, which possess certain anti-tumor activity, but their effectiveness is limited when used alone. This invention covalently complexes rosmarinic acid with *Pleurotus ostreatus* water-soluble proteins under alkaline conditions. Rosmarinic acid binds to the water-soluble proteins through covalent bonding, forming an RA-water-soluble protein complex. This improves the bioavailability and stability of rosmarinic acid, alters the conformation of the water-soluble proteins, or generates new active sites, thereby enhancing the inhibition rate of HepG2 cell proliferation. This invention uses *Lactobacillus paracasei* to ferment *Pleurotus ostreatus*, degrading the large polysaccharides in *Pleurotus ostreatus* into smaller molecules that are more easily absorbed and have higher activity, improving bioavailability and biological activity, and exerting a synergistic anti-tumor effect with the RA-water-soluble protein. The water-soluble protein of *Pleurotus ostreatus* contains amino acid residues with antioxidant capabilities, and rosmarinic acid is a potent natural antioxidant. The fermented preparation may be rich in antioxidants produced by the microorganisms and their metabolism. The components work synergistically to form a complex antioxidant system, which helps to reduce the level of oxidative stress in the tumor microenvironment, directly inhibit the proliferation of tumor cells, enhance the function of the body's immune cells, and exert an anti-tumor effect. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only for this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The HepG2 cell inhibitory effect of the compound preparations described in Examples 1-3 and Comparative Examples 1-3 of this invention;

[0020] Figure 2 This describes the free radical scavenging effect of the composite formulations described in Examples 1-3 and Comparative Examples 1-3 of the present invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. However, this invention is not limited to the following embodiments. It should be noted that, unless otherwise specified, all chemical reagents involved in this invention are purchased through commercial channels.

[0022] Example 1: A water-soluble protein compound preparation of anti-tumor mushroom, the raw materials for preparation include the following components in parts by weight: 3 parts water-soluble protein of mushroom, 1.2 parts fermentation preparation, and 0.2 parts rosmarinic acid.

[0023] The preparation method of water-soluble protein from *Pleurotus ostreatus* includes the following steps:

[0024] L1. Dry the large white mushroom to constant weight, crush it through an 80-mesh sieve, add it to a 0.2 mol / L phosphate buffer solution at a material-to-liquid ratio of 1 g:30 mL, stir at 500 rpm for 4 h, centrifuge at 10000 r / min for 20 min, and take the supernatant.

[0025] L2. Take the supernatant obtained in step L1, add ammonium sulfate to saturation, let stand overnight at 4°C, centrifuge, place the precipitate in a 3500 Da dialysis bag and dialyze in deionized water for 24 h, freeze-dry the dialysate to obtain water-soluble protein from Mushroom sphaerocephala.

[0026] The preparation method of the fermentation agent includes the following steps:

[0027] V1. Dry the large white mushrooms to constant weight, pulverize them through an 80-mesh sieve, add them to distilled water at a material-to-liquid ratio of 1 g: 15 mL, sterilize at 121℃ for 20 min, and after cooling, use 10... 7 Inoculate activated Lactobacillus paracasei at an inoculum size of CFU / mL, ferment at 37℃ for 24 h, and centrifuge at 8000 r / min for 15 min.

[0028] V2. Take the supernatant after centrifugation in step V1, filter it through a 0.22 μm filter membrane, add 4 times the volume of ethanol while stirring, let it stand overnight at 4℃, centrifuge at 8000 rpm for 15 min, wash the precipitate with anhydrous ethanol, reconstitute it with distilled water, and freeze dry it under vacuum to obtain the fermentation preparation.

[0029] This embodiment also provides a method for preparing the antitumor *Pleurotus ostreatus* water-soluble protein complex preparation, including the following steps:

[0030] S1. Add 3 g of water-soluble protein from *Pleurotus ostreatus* to 150 mL of 0.01 M PBS buffer at pH 6.8, stir overnight at 4°C, adjust the pH to 9 with 1 mol / L NaOH, add 0.2 g of rosmarinic acid, stir for 24 h, dialyze in deionized water for 48 h, freeze-dry the dialysate to obtain RA-water-soluble protein;

[0031] S2. Mix the RA-water-soluble protein obtained in step S1 with 1.2 g of fermentation agent to obtain an anti-tumor white mushroom water-soluble protein complex preparation.

[0032] Example 2: A water-soluble protein compound preparation of anti-tumor mushroom, the raw materials for preparation include the following components in parts by weight: 2 parts water-soluble protein of mushroom, 0.8 parts fermentation preparation, and 0.1 parts rosmarinic acid.

[0033] The preparation method of water-soluble protein from *Pleurotus ostreatus* includes the following steps:

[0034] L1. Dry the large white mushroom to constant weight, crush it through an 80-mesh sieve, add it to a 0.2 mol / L phosphate buffer solution at a ratio of 1 g: 20 mL, stir at 400 rpm for 4 h, centrifuge at 10000 r / min for 15 min, and take the supernatant.

[0035] L2. Take the supernatant obtained in step L1, add ammonium sulfate to saturation, let stand overnight at 4°C, centrifuge, place the precipitate in a 3500 Da dialysis bag and dialyze in deionized water for 24 h, freeze-dry the dialysate to obtain water-soluble protein from Mushroom sphaerocephala.

[0036] The preparation method of the fermentation agent includes the following steps:

[0037] V1. Dry the large white mushrooms to constant weight, pulverize them through an 80-mesh sieve, add them to distilled water at a material-to-liquid ratio of 1 g: 10 mL, sterilize at 121℃ for 20 min, and after cooling, use 10... 7 Inoculate activated Lactobacillus paracasei at an inoculum size of CFU / mL, ferment at 37℃ for 24 h, and centrifuge at 8000 r / min for 10 min.

[0038] V2. Take the supernatant after centrifugation in step V1, filter it through a 0.22 μm filter membrane, add 4 times the volume of ethanol while stirring, let it stand overnight at 4℃, centrifuge at 8000 rpm for 10 min, wash the precipitate with anhydrous ethanol, reconstitute it with distilled water, and freeze dry it under vacuum to obtain the fermentation preparation.

[0039] This embodiment also provides a method for preparing the antitumor *Pleurotus ostreatus* water-soluble protein complex preparation, including the following steps:

[0040] S1. Add 2 g of water-soluble protein from *Pleurotus ostreatus* to 100 mL of PBS (0.01 M) buffer at pH 6.8, stir overnight at 4°C, adjust the pH to 9 with 1 mol / L NaOH, add 0.1 g of rosmarinic acid and stir for 24 h, dialyze in deionized water for 48 h, freeze-dry the dialysate to obtain RA-water-soluble protein;

[0041] S2. Mix the RA-water-soluble protein obtained in step S1 with 0.8 g of the fermentation agent to obtain an anti-tumor white mushroom water-soluble protein complex preparation.

[0042] Example 3: A water-soluble protein compound preparation of anti-tumor mushroom, the raw materials for preparation include the following components in parts by weight: 2.5 parts of water-soluble protein of mushroom, 1 part of fermentation preparation, and 0.15 parts of rosmarinic acid.

[0043] The preparation method of water-soluble protein from *Pleurotus ostreatus* includes the following steps:

[0044] L1. Dry the large white mushroom to constant weight, crush it through an 80-mesh sieve, add it to a 0.2 mol / L phosphate buffer solution at a material-to-liquid ratio of 1 g: 25 mL, stir at 450 rpm for 4 h, centrifuge at 10000 r / min for 18 min, and take the supernatant.

[0045] L2. Take the supernatant obtained in step L1, add ammonium sulfate to saturation, let stand overnight at 4°C, centrifuge, place the precipitate in a 3500 Da dialysis bag and dialyze in deionized water for 24 h, freeze-dry the dialysate to obtain water-soluble protein from Mushroom sphaerocephala.

[0046] The preparation method of the fermentation agent includes the following steps:

[0047] V1. Dry the large white mushrooms to constant weight, pulverize them through an 80-mesh sieve, add them to distilled water at a material-to-liquid ratio of 1 g: 12 mL, sterilize at 121℃ for 20 min, and after cooling, use 10... 7 Inoculate activated Lactobacillus paracasei at an inoculum size of CFU / mL, ferment at 37℃ for 24 h, and centrifuge at 8000 r / min for 12 min.

[0048] V2. Take the supernatant after centrifugation in step V1, filter it through a 0.22 μm filter membrane, add 4 times the volume of ethanol while stirring, let it stand overnight at 4℃, centrifuge at 8000 rpm for 12 min, wash the precipitate with anhydrous ethanol, reconstitute it with distilled water, and freeze dry it under vacuum to obtain the fermentation preparation.

[0049] This embodiment also provides a method for preparing the antitumor *Pleurotus ostreatus* water-soluble protein complex preparation, including the following steps:

[0050] S1. Add 2.5 g of the water-soluble protein of *Pleurotus ostreatus* to 125 mL of PBS (0.01 M) buffer at pH 6.8, stir overnight at 4°C, adjust the pH to 9 with 1 mol / L NaOH, add 0.15 g of rosmarinic acid and stir for 24 h, dialyze in deionized water for 48 h, freeze-dry the dialysate to obtain RA-water-soluble protein;

[0051] S2. Mix the RA-water-soluble protein obtained in step S1 with 1 g of fermentation agent to obtain an anti-tumor white mushroom water-soluble protein complex preparation.

[0052] The only difference between Comparative Example 1 and Example 1 is that rosmarinic acid is not added, and RA-water-soluble protein is replaced with Pleurotus ostreatus water-soluble protein.

[0053] The only difference between Comparative Example 2 and Example 1 is that the large white mushrooms were dried to constant weight, crushed and passed through an 80-mesh sieve, added to distilled water at a material-to-liquid ratio of 1 g: 15 mL, heated at 80°C for 2 h, filtered, and 4 times the volume of ethanol was added to the filtrate while stirring. The mixture was allowed to stand overnight at 4°C, centrifuged at 8000 rpm for 15 min, the precipitate was washed with anhydrous ethanol, reconstituted with distilled water, and freeze-dried under vacuum. The resulting product was used to replace the fermentation agent.

[0054] The only difference between Comparative Example 3 and Example 1 is that the water-soluble protein of *Pleurotus ostreatus*, the fermentation preparation and the physical mixture of rosmarinic acid are used instead of the antitumor *Pleurotus ostreatus* water-soluble protein compound preparation.

[0055] Experimental Example 1: HepG2 cells were routinely cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin antibiotics at 37°C in a 5% CO2 incubator. HepG2 cells in the logarithmic growth phase were then introduced at a rate of 1×10⁻⁶ cells / cells. 5 100 μL of MTT solution was seeded into each well of a 96-well plate at a concentration of / mL. After 24 h of incubation, the cells were divided into two groups: Examples 1-3 and Comparative Examples 1-3. The old culture medium was discarded, and each group was replaced with 100 μL of DMEM medium containing 50 mg / L of the anti-tumor *Gnaphalium affine* water-soluble protein complex prepared in Examples 1-3 and Comparative Examples 1-3, respectively. A control group without the complex preparation was also included. After 48 h of incubation at 37°C, 20 μL of 5 mg / mL MTT solution was added to each well, and the cells were incubated for another 4 h. The culture medium was then discarded, and 150 μL of DMSO was added to each well. The cells were incubated with shaking for 10 min, and the absorbance was measured at 570 nm using a microplate reader. The cell mortality rate in the experimental groups was calculated with the cell viability of the control group as 100%. The cell proliferation inhibition rate (%) was calculated as [(OD control group - OD experimental group) / OD control group] × 100%. The results are as follows: Figure 1 As shown.

[0056] Figure 1 The results showed that the water-soluble protein compound preparations of *Lactobacillus paracasei* in Examples 1-3 and Comparative Examples 1-3 could effectively inhibit the proliferation of HepG2 cells. The cell proliferation inhibition rate of HepG2 cells in Examples 1-3 was significantly better than that in Comparative Examples 1-3. In Comparative Example 1, no rosmarinic acid was added, and the cell proliferation inhibition rate decreased. In Comparative Example 2, no *Lactobacillus paracasei* fermentation was performed, and the cell proliferation inhibition rate decreased. In Comparative Example 3, no rosmarinic acid was combined with the water-soluble protein of *Lactobacillus paracasei*, and only physical blending was performed, resulting in a decrease in the cell proliferation inhibition rate.

[0057] Experimental Example 2: A 7 mmol / L ABTS solution and a 2.45 mmol / L K₂S₂O₈ solution were prepared and mixed in a 1:1 ratio. The mixture was then incubated in the dark for 12 h to obtain the ABTS working solution. 50 μL of the composite formulation solution (100 μg / mL) prepared in Examples 1-3 and Comparative Examples 1-3, and 150 μL of the ABTS working solution were added to a 96-well plate. The plate was incubated at room temperature for 6 min, and the absorbance value (A1) was measured at 734 nm. Pure water was used instead of the ABTS working solution and sample, representing A2 and A0 respectively. The ABTS scavenging rate (%) was calculated as 100% - [(A1 - A2) / A0]. The results are as follows: Figure 2 As shown.

[0058] Figure 2 The results showed that the water-soluble protein compound preparations of *Lactobacillus paracasei* in Examples 1-3 and Comparative Examples 1-3 could effectively scavenge ABTS free radicals. The free radical scavenging rate of Examples 1-3 was significantly better than that of Comparative Examples 1-3. Comparative Example 1 did not add rosmarinic acid, so its antioxidant effect was reduced. Comparative Example 2 did not undergo *Lactobacillus paracasei* fermentation, so its free radical scavenging effect was reduced. Comparative Example 3 did not combine rosmarinic acid with water-soluble protein of *Lactobacillus paracasei*, but only performed physical blending, so its free radical scavenging effect was reduced.

[0059] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

Claims

1. A water-soluble protein compound preparation of *Pleurotus ostreatus* with antitumor properties, characterized in that, The raw materials for preparation include the following components in parts by weight: 2-3 parts of water-soluble protein from *Pleurotus ostreatus*, 0.8-1.2 parts of fermentation agent, and 0.1-0.2 parts of rosmarinic acid; The method for preparing the fermentation agent includes the following steps: V1. Dry and crush the large white mushroom, add it to distilled water, sterilize, cool, inoculate with activated Lactobacillus paracasei, ferment, and centrifuge; V2. Take the supernatant after centrifugation in step V1, filter, add ethanol, let stand at 4°C, centrifuge, wash the precipitate, reconstitute with distilled water, freeze dry, and obtain the fermentation preparation; The preparation method of the antitumor *Pleurotus ostreatus* water-soluble protein complex preparation includes the following steps: S1. Add the water-soluble protein of *Pleurotus ostreatus* to PBS buffer, stir overnight, adjust the pH to 9, add rosmarinic acid, stir, dialyze, freeze dry, and obtain RA-water-soluble protein; S2. Mix the RA-water-soluble protein obtained in step S1 with the fermentation agent evenly to obtain the anti-tumor white mushroom water-soluble protein complex preparation; The method for preparing the water-soluble protein of the mushroom *Pleurotus ostreatus* includes the following steps: L1. Dry and crush the large white mushroom, add it to phosphate buffer, stir, centrifuge, and take the supernatant; L2. Take the supernatant obtained in step L1, add ammonium sulfate to saturation, let stand at 4°C, centrifuge, place the precipitate in a 3500 Da dialysis bag and dialyze in deionized water, freeze dry to obtain water-soluble protein from Mushroom sphaerocephala.

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