Preparation method of anti-cancer drug based on pear fermentation

Abstract

The invention discloses a preparation method of an anticancer drug based on pear fermentation, which comprises the following steps: lactobacillus plantarum CGMCC 1.555 and saccharomyces cerevisiae CICC 1010 are selected as core strains, lactic acid bacteria and saccharomycetes are mixed according to a ratio of 2: 1 (v / v) or 3: 1 (v / v), and the saccharomycetes metabolizes to generate ethanol to promote the growth of the lactic acid bacteria, degrade pear pectin and release bound polyphenol at the same time. According to the preparation method of the anti-cancer drug based on pear fermentation, lactobacillus plantarum CGMCC 1.555 and saccharomyces cerevisiae CICC 1010 are selected and mixed according to the proportion of 2: 1 or 3: 1, ethanol generated by metabolism of saccharomycetes can promote growth of lactic acid bacteria, pear pectin is degraded, combined polyphenol is released, and compared with single strain fermentation, the release amount of active ingredients is increased by 30% or above. After the pear or snowflake pear is pulped, the sugar content is adjusted to 15%, the pH value is adjusted to 5.0, 0.15% of cysteine and 0.05% of EDTA are added to enhance oxidation resistance, and 50-100-mesh crushing particle size control is matched, so that the viscosity of the fruit pulp is smaller than or equal to 350 mPa.s, polyphenol oxidation is effectively prevented, and the integrity of active ingredients of the raw materials is maintained.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a method for preparing an anticancer drug based on pear fermentation. Background Art

[0002] Pears are rich in potential anti-cancer compounds such as polyphenols, flavonoids, and polysaccharides, but their natural bioavailability is low. Existing technologies have limited anti-cancer effects from directly extracting pear ingredients, and traditional extraction processes are inefficient and costly.

[0003] Microbial fermentation can degrade plant cell walls, release active substances, and produce new metabolites (such as short-chain fatty acids, bacteriocins, etc.), but the current process still has shortcomings in fermentation efficiency and safety assessment.

[0004] Existing pear component extraction processes are inefficient and costly. The anti-cancer components such as polyphenols and flavonoids in natural pears are bound by the cell wall structure, with a bioavailability of less than 15%. Traditional extraction processes have problems such as high solvent consumption and complicated steps, resulting in high production costs. The anti-cancer effect of a single component is limited and the mechanism of action is single. Although natural pear extracts contain multiple active substances, when not fermented and converted, the inhibition rate on the proliferation of liver cancer cells HepG2 is less than 30%, and it cannot simultaneously act on multiple links such as tumor cell proliferation, apoptosis, and metastasis. Microbial fermentation process parameters lack precise control. In traditional fermentation processes, due to the lack of real-time monitoring methods for pH and metabolites, excessive fermentation or untimely termination often occurs, resulting in degradation of active ingredients, and the total phenol content fluctuates by as much as ±20%. The active ingredient purification process is prone to loss and lacks purity. Conventional purification methods have a low retention rate for polyphenol-polysaccharide complexes with a molecular weight of 800-1500Da, and the purity of flavonoid components is generally less than 70%, which cannot meet the quality requirements of pharmaceutical preparations. Summary of the Invention

[0005] (1) Technical problems solved

[0006] In response to the shortcomings of the existing technology, the present invention provides a method for preparing anti-cancer drugs based on pear fermentation, and provides a method for efficiently extracting ingredients with significant anti-cancer activity by co-fermenting pears with specific strains of bacteria, thereby achieving process optimization and cost reduction, and solving the above-mentioned problems.

[0007] (2) Technical solution

[0008] To achieve the above object, the present invention provides the following technical solution: a method for preparing an anticancer drug based on pear fermentation, comprising:

[0009] 1.1. Lactobacillus plantarum CGMCC 1.555 and Saccharomyces cerevisiae CICC 1010 were selected as core strains. The lactic acid bacteria and yeast were mixed in a ratio of 2:1 (v / v) or 3:1 (v / v). The yeast metabolized ethanol to promote the growth of lactic acid bacteria and degraded pear pectin to release bound polyphenols.

[0010] 1.2. Select Yali pear or Xuehua pear as raw material, beat it and adjust the sugar content to 15%, pH value to 5.0, and add 0.15% cysteine ​​+ 0.05% EDTA to enhance antioxidant. The Xuehua pear pulp is crushed to 50-100 mesh;

[0011] 1.3. Mix the treated pear raw material with the core strain and perform anaerobically fermentation at 37°C for 48 hours, then switch to aerobic fermentation at 28°C for 24 hours; or perform anaerobically fermentation at 28°C for 5-7 days. Terminate the fermentation when the pH of the fermentation system drops to 3.5-3.8 and the total phenol content reaches 7.5-8 mg GAE / g.

[0012] 1.4. The fermentation broth was centrifuged at 8000 rpm for 15 min to obtain a supernatant, which was concentrated by nanofiltration (100 kDa) and vacuum concentration at low temperature. The inlet air temperature of the spray dryer was 150°C and the outlet air temperature was 80°C to obtain a crude extract.

[0013] 1.5. The crude extract was subjected to graded purification, using ultrafiltration (molecular weight cutoff 100 kDa) to obtain the anticancer polysaccharide component FPS-1. After extraction, the ethyl acetate was removed by rotary evaporation, and the residue was dissolved in propylene glycol to obtain the component FPE-2. The flavonoid complex FPF-3 was obtained by purification using macroporous resin AB-8 and elution with 60% ethanol.

[0014] Alternatively, a 100kDa nanofiltration membrane was used to concentrate the polysaccharide, and immobilized β-glucosidase was used to catalyze the improvement of flavonoid purity.

[0015] Preferably, the fermentation raw material is Yali pear or Xuehua pear, and after beating, the sugar content is adjusted to 15%, the pH is 5.0-5.5, and 0.15% cysteine ​​+ 0.05% EDTA is added to enhance antioxidant properties;

[0016] The sugar content of the duck pear or snow pear is adjusted by adding sucrose or glucose and satisfies the formula:

[0017] Brix=S+0.25×(A-0.65)

[0018] Where S is the mass percentage of added sugar (%), A is the initial soluble solid content of the raw material (%), the crushing particle size is controlled to be 50–100 mesh, and the viscosity of the pulp after crushing is ≤350 mPa·s.

[0019] Preferably, the fermentation termination conditions are pH 3.5-3.8 and total phenol content ≥7.5 mg GAE / g, or determined by MMP-2 / -9 inhibitory activity index;

[0020] The fermentation termination conditions are dynamically monitored by real-time pH sensor combined with near-infrared spectroscopy when the following conditions are met simultaneously:

[0021] It automatically terminates when pH ≤ 3.8 and total phenols ≥ 7.5 mg GAE / g;

[0022] Alternatively, an MMP-2 / -9 inhibition rate ≥ 65% can be used as an auxiliary judgment indicator.

[0023] Preferably, the active ingredient extraction comprises precipitating the polysaccharide with 70% ethanol, removing the ethyl acetate by rotary evaporation after extraction, dissolving the residue in propylene glycol, purifying the flavonoids with macroporous resin AB-8, or concentrating the polyphenol-polysaccharide complex with a molecular weight of 800-1500 Da through a 100 kDa nanofiltration membrane;

[0024] In the fractionated purification:

[0025] a) The ethanol precipitation step requires controlling the final ethanol concentration to 68–72%, the temperature to 4±1°C, and the standing time to 10–14 hours (Example 1, Step 10);

[0026] b) extraction with ethyl acetate using an acidified aqueous phase at pH 2.5-3.0, with the number of extractions ≥ 3 times (Example 1, step 11);

[0027] c) Membrane flux of nanofiltration membrane during concentration ≥ 25L / (m 2 h), molecular weight cut-off 800–1500 Da (Example 2, step 23).

[0028] Preferably, the polysaccharide FPS-1 (molecular weight 1.2×10 5 Daβ-glucan), phenolic acid FPE-2 (combination of chlorogenic acid and arbutin), flavonoid FPF-3 (quercetin-3-O-glucoside content ≥85%), and polyphenol-polysaccharide complex;

[0029] Contains the following core components in the following weight proportions:

[0030] FPS-1:FPE-2:FPF-3=(40–60):(20–30):(15–25);

[0031] The proportion of quercetin-3-O-glucoside in the polyphenol-polysaccharide complex is ≥18%.

[0032] Preferably, the polyphenol-polysaccharide complex has a molecular weight of 800-1500 Da and can reduce MMP-2 / -9 expression by inhibiting the PI3K / AKT pathway;

[0033] The molecular weight distribution of the polyphenol-polysaccharide complex is determined by HPSEC-MALS coupling technology, and the fitting formula is:

[0034] Mw=Σ(ciMi) / Σci

[0035] Where ci is the concentration of component i, and Mi is the molecular weight of component i.

[0036] Preferably, the drug is used to inhibit the proliferation of liver cancer cells (such as HepG2), apoptosis of breast cancer cells (such as MCF-7), or metastasis of non-small cell lung cancer (NSCLC).

[0037] Preferably, the drug blocks tumor cell invasion by inhibiting the EMT (epithelial-mesenchymal transition) process, thereby reducing the number of lung metastatic nodules by ≥60%.

[0038] Preferably, a toxicological validation step is also included, including a single oral gavage MTD of ≥ 2000 mg / kg in mice, no liver and kidney damage in rats after repeated administration for 90 days (ALT, Cr indicators p>0.05), and no mutagenicity in the Ames test.

[0039] A pear fermented anticancer drug preparation, characterized in that: it comprises the active ingredient according to claim 5 and a liposome carrier, wherein the nanoliposome particle size is 100-150 nm and the encapsulation efficiency is ≥85%;

[0040] The liposome carrier was composed of phosphatidylcholine: cholesterol = 5:1 (w / w), with a hydration volume of 5–10 mL / g lipid;

[0041] Particle size control formula:

[0042] D=k·(P -0.5 )·t 0.3

[0043] Where D is the particle size (nm), P is the ultrasonic power (W), t is the time (min), and k is the material constant.

[0044] (3) Beneficial effects

[0045] Compared with the prior art, the present invention provides a method for preparing an anticancer drug based on pear fermentation, which has the following beneficial effects:

[0046] This pear fermentation-based anticancer drug preparation method utilizes a mixture of Lactobacillus plantarum CGMCC 1.555 and Saccharomyces cerevisiae CICC 1010 in a 2:1 or 3:1 ratio. The ethanol produced by yeast metabolism promotes the growth of lactic acid bacteria while simultaneously degrading pear pectin, releasing bound polyphenols. Compared to single-strain fermentation, the release of active ingredients is increased by over 30%. After pulping, the sugar content of Yali or Xuehua pears is adjusted to 15% and the pH to 5.0. 0.15% cysteine ​​and 0.05% EDTA are added to enhance antioxidant activity. The pulp is then ground to a 50-100 mesh particle size to maintain a viscosity of ≤350 mPa·s, effectively preventing polyphenol oxidation and preserving the integrity of the active ingredients. By anaerobic fermentation at 37°C for 48 hours followed by aerobic fermentation at 28°C for 24 hours, or anaerobic fermentation at 28°C for 5-7 days, combined with dynamic termination conditions of pH ≤ 3.8 and total phenolics ≥ 7.5 mg GAE / g, the targeted accumulation of anti-cancer polysaccharides, phenolic acids, flavonoids, and other components can be achieved, with the total phenolic content increased by 25%-30% compared to traditional fermentation processes. Using 70% ethanol precipitation, ethyl acetate extraction, and macroporous resin AB-8 purification, the flavonoid complex FPF-3 with a purity ≥ 85% was obtained, along with the phenolic acid component FPE-2 containing chlorogenic acid and arbutin, and a molecular weight of 1.2×10 5 Da's β-glucan FPS-1 has a polysaccharide yield of over 6.5%, which is 40% higher than that of traditional extraction methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a schematic diagram of the preparation process of a broad-spectrum anticancer active ingredient in a method for preparing an anticancer drug based on pear fermentation proposed in the present invention;

[0048] Figure 2 This is a schematic diagram of the preparation process of a polyphenol-polysaccharide complex for preventing lung cancer metastasis, according to a method for preparing an anti-cancer drug based on pear fermentation proposed in the present invention;

[0049] Figure 3 This is a schematic diagram of the preparation process of a nanoliposome drug-loaded preparation of an anticancer drug preparation method based on pear fermentation proposed in the present invention;

[0050] Figure 4 This is a schematic diagram of the toxicological safety verification process of the method for preparing an anticancer drug based on pear fermentation proposed in the present invention; DETAILED DESCRIPTION

[0051] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0052] The present invention provides a technical solution: a method for preparing an anticancer drug based on pear fermentation, comprising:

[0053] Fermentation strain combination

[0054] Core strains: Lactobacillus plantarum CGMCC 1.555 + Saccharomyces cerevisiae CICC 1010.

[0055] Ratio: Lactic acid bacteria: yeast = 2:1 (v / v), which can be adjusted to 3:1 (v / v) as needed to optimize the accumulation of lung cancer metastasis inhibitory components.

[0056] Synergistic effect: Yeast metabolism produces ethanol to promote the growth of lactic acid bacteria, while degrading pear pectin and releasing bound polyphenols.

[0057] 2. Fermentation process

[0058] Raw material processing: Select duck pear or snow pear, adjust the sugar content to 15% and pH 5.0 after pulping, add 0.15% cysteine ​​+ 0.05% EDTA to enhance antioxidant; the snow pear pulp can be crushed to 50-100 mesh.

[0059] Fermentation conditions: anaerobic fermentation at 37°C for 48 hours, then switch to aerobic fermentation at 28°C for 24 hours; or anaerobic fermentation at 28°C for 5-7 days (focusing on lung cancer metastasis inhibitory components).

[0060] Termination conditions: terminate when the pH drops to 3.5-3.8 and the total phenol content reaches 7.5-8 mg GAE / g.

[0061] 3. Extraction and purification of active ingredients

[0062] Preparation of crude extract: fermentation broth centrifugation (8000 rpm, 15 min) → supernatant concentration by nanofiltration membrane (100 kDa) + low-temperature vacuum concentration (inlet air 150°C, outlet air 80°C).

[0063] Fractional purification:

[0064] The polysaccharide was precipitated with 70% ethanol to obtain the anticancer polysaccharide component FPS-1 (molecular weight 1.2×10 5 Da of β-glucan).

[0065] After extraction, the ethyl acetate was removed by rotary evaporation, and the residue was dissolved in propylene glycol to obtain fraction FPE-2 (containing chlorogenic acid and arbutin). The flavonoid complex FPF-3 (quercetin-3-O-glucoside content ≥85%) was purified using macroporous resin AB-8 and eluted with 60% ethanol. Alternatively, the polysaccharide was concentrated using a 100 kDa nanofiltration membrane, and the flavonoid purity was increased to 93% using immobilized β-glucosidase (immobilized on magnetic Fe3O4 nanoparticles).

[0066] IV. Active Ingredients and Anticancer Applications

[0067] Core ingredients: polysaccharide FPS-1, phenolic acid FPE-2, flavonoid FPF-3, and polyphenol-polysaccharide complex with a molecular weight of 800-1500Da.

[0068] Anticancer mechanism: FPS-1 (200 μg / mL) inhibited HepG2 liver cancer cells by 72.3%. FPE-2 (50 μg / mL) induced apoptosis in MCF-7 breast cancer cells (early apoptosis rate 38.7%).

[0069] The ethyl acetate extract can reduce the expression level of MMP-2 / -9 in A549 cells (inhibition rate 68.3±2.1%), block tumor cell invasion by inhibiting the PI3K / AKT pathway, and reduce the number of lung metastatic nodules in Lewis lung cancer mice by 62.5%.

[0070] 5. Toxicology Verification System

[0071] Short-term toxicity: MTD of single oral administration in mice is ≥2000 mg / kg.

[0072] Long-term toxicity: After repeated administration of 50 mg / kg / d for 90 days to SD rats, there was no significant change in liver and kidney function indicators (ALT, Cr) (p>0.05).

[0073] Genotoxicity: Ames test (TA98 strain) showed no mutagenicity.

[0074] Solvent system:

[0075] Use propylene glycol + water (60:40) as the base fluid:

[0076] Propylene glycol can dissolve flavonoids, phenolic acids and polysaccharide complexes and has antibacterial properties.

[0077] Add 0.15% cysteine ​​+ 0.05% EDTA to enhance antioxidant properties.

[0078] Anti-corrosion system:

[0079] Add 0.1% potassium sorbate

[0080] Example 1

[0081] For large-scale preparation of broad-spectrum anticancer active ingredients, please refer to Figure 1

[0082] Raw material pretreatment

[0083] Take 20 kg of fresh pears, wash them with running water, peel and core them, and crush them into 80 mesh pulp using a beater;

[0084] 3% sucrose (osmotic pressure adjusted to 15° Brix), 0.15% cysteine ​​+ 0.05% EDTA were added to enhance antioxidant activity, and the pH was adjusted to 5.0 with citric acid.

[0085] Strain inoculation and fermentation control

[0086] Inoculate Lactobacillus plantarum (CGMCC 1.555, bacterial concentration 1×10 8 CFU / mL) and 40 mL of Saccharomyces cerevisiae (CICC1010, bacterial concentration 1×10 8 CFU / mL) 20mL, mix well;

[0087] Anaerobic fermentation was carried out at 37°C for 48 hours (the tank was sealed and the pressure was maintained at 0.1 MPa). When the pH dropped to 4.0, it was switched to aerobic fermentation at 28°C (with aeration volume of 0.5 vvm) for 24 hours, and the fermentation liquid pH was stabilized at 3.5 and the total phenol content reached 8.2 mg GAE / g.

[0088] Active ingredient fractionation extraction

[0089] Centrifugation: The fermentation broth was centrifuged at 8000 rpm for 15 minutes and the supernatant was collected;

[0090] Polysaccharide precipitation: 3 volumes of 70% ethanol were added to the supernatant, and the mixture was allowed to stand at 4°C for 12 hours. The precipitate was collected by centrifugation (10,000 rpm, 20 minutes) and freeze-dried to obtain FPS-1 (β-glucan, molecular weight 1.2×10 5 Da, yield 6.5%);

[0091] Phenolic acid extraction: The supernatant after centrifugation was extracted three times with an equal volume of ethyl acetate, the organic phases were combined and concentrated by rotary evaporation to obtain FPE-2 (chlorogenic acid and arbutin combination, purity ≥90% by HPLC);

[0092] Flavonoid purification: The aqueous phase was passed through a macroporous resin AB-8 column (column diameter, 10 cm × 50 cm), first rinsed with distilled water until colorless, then eluted with 60% ethanol (flow rate, 2 BV / h), and the propylene glycol / water mixed solvent was collected and freeze-dried to obtain FPF-3 (quercetin-3-O-glucoside content, 85.6%).

[0093] Activity verification;

[0094] In vitro experiments: FPS-1 (200 μg / mL) inhibited HepG2 cells by 72.3%, FPE-2

[0095] (50 μg / mL) induced an early apoptosis rate of 38.7% in MCF-7 cells;

[0096] In vivo experiments: FPF-3 (20 mg / kg / d) inhibited the tumor volume of S180 sarcoma mice by 64%.

[0097] (p<0.01).

[0098] Example 2

[0099] Directed preparation of polyphenol-polysaccharide complexes for anti-lung cancer metastasis Figure 2 ,

[0100] Raw material pretreatment

[0101] Take 15 kg of snow pear, clean it and crush it into 50 mesh, add 2% glucose to adjust the sugar content to 15%, and adjust the pH to 5.5 with NaOH.

[0102] Optimize fermentation process;

[0103] Inoculate 60 mL of Lactobacillus plantarum (CGMCC 1.555) and 20 mL of Saccharomyces cerevisiae (CICC 1010)

[0104] (Bacteria agent ratio 3:1 v / v);

[0105] Anaerobic fermentation at 28℃ for 7 days (tank protected from light, humidity controlled at 70%), pH at the end of fermentation

[0106] 3.6, the content of MMP-2 / -9 inhibition-related metabolites reached a peak.

[0107] Highly efficient purification process;

[0108] The fermentation broth was concentrated by 100 kDa nanofiltration membrane (membrane flux 25 L / (m 2 h)), the retentate was chromatographed on a macroporous resin AB-8 column, and eluted with a 60% ethanol gradient (10%→60%, 5 BV per gradient);

[0109] Elution fractions with molecular weights between 800 and 1500 Da were collected, desalted by ultrafiltration, and then freeze-dried to obtain polyphenol-polysaccharide complexes (yield 12.3%, HPLC characteristic peaks positively correlated with lung cancer metastasis inhibitory activity). Anti-metastasis mechanism verification;

[0110] In vitro Transwell assay: 100 μg / mL of the complex reduced the number of H1299 cell migration to 29% of the control group (p<0.01), and the cell healing rate in the scratch test decreased by 58% after 48 hours;

[0111] In vivo experiments: In the Lewis lung cancer mouse model, oral administration of 50 mg / kg / d reduced the number of lung metastatic nodules by 62.5% (n=10, p<0.001), and Western blot showed that the phosphorylation level of PI3K / AKT pathway proteins decreased by 47%.

[0112] Example 3

[0113] For the preparation of nanoliposome drug-loaded formulations, please refer to Figure 3 ,

[0114] Construction of drug delivery system

[0115] FPF-3 (100 mg), phosphatidylcholine (500 mg), and cholesterol (100 mg) were dissolved in chloroform, and the solvent was removed by rotary evaporation to form a lipid film;

[0116] 5 mL of PBS buffer (pH 7.4) was added, and ultrasonic oscillation (power 300 W, time 15 min) was performed to form a nanoliposome suspension;

[0117] The particles were sterilized by 0.22 μm filter membrane and the particle size distribution was determined by dynamic light scattering (average particle size 120 ± 10 nm, PDI < 0.2).

[0118] Formulation characterization and activity

[0119] Encapsulation efficiency: HPLC test showed that the FPF-3 encapsulation efficiency reached 89.5%;

[0120] Sustained release properties: 48-hour cumulative release rate 72% (PBS, 37°C);

[0121] In vivo efficacy: The nanoliposome preparation (10 mg / kg) increased the tumor inhibition rate of S180 sarcoma by 18% (p<0.05) compared with free FPF-3, and prolonged the plasma half-life by 2.3 times.

[0122] Example 4:

[0123] Toxicological safety verification; acute toxicity test, please refer to Figure 4 ,

[0124] ICR mice (n=50, body weight 20±2g) were divided into 5 groups and given FPE-2 by single oral gavage.

[0125] (dose gradient 500, 1000, 2000, 4000, 5000 mg / kg), observation for 14 days;

[0126] Results: There was no death in the 5000 mg / kg dose group, MTD>2000 mg / kg, and pathological examination showed no liver and kidney damage (ALT and Cr indicators showed no significant difference from the control group, p>0.05).

[0127] Genotoxicity test

[0128] Ames test: TA98 strain was used, FPE-2 concentration was 0.1-0.5 mg / dish, and co-cultured with the strain for 48 hours;

[0129] Results: The number of reverse mutant colonies in each dose group did not exceed 2 times of the negative control, confirming that it had no mutagenicity.

[0130] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations, an element defined by the phrase "comprising a" does not exclude the presence of additional identical elements in the process, method, article, or device comprising the element.

[0131] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing an anticancer drug based on pear fermentation, characterized in that: include: 1.

1. Lactobacillus plantarum CGMCC 1.555 and Saccharomyces cerevisiae CICC 1010 were selected as core strains. The lactic acid bacteria and yeast were mixed in a ratio of 2:1 (v / v) or 3:1 (v / v). The yeast metabolized ethanol to promote the growth of lactic acid bacteria and degraded pear pectin to release bound polyphenols. 1.

2. Select Yali pear or Xuehua pear as raw material, beat it and adjust the sugar content to 15%, pH value to 5.0, and add 0.15% cysteine ​​+ 0.05% EDTA to enhance antioxidant. The Xuehua pear pulp is crushed to 50-100 mesh; 1.

3. Mix the treated pear raw material with the core strain and perform anaerobically fermentation at 37°C for 48 hours, then switch to aerobic fermentation at 28°C for 24 hours; or perform anaerobically fermentation at 28°C for 5-7 days. Terminate the fermentation when the pH of the fermentation system drops to 3.5-3.8 and the total phenol content reaches 7.5-8 mg GAE / g. 1.

4. The fermentation broth was centrifuged at 8000 rpm for 15 min to obtain a supernatant, which was concentrated by nanofiltration (100 kDa) and vacuum concentration at low temperature. The inlet air temperature of the spray dryer was 150°C and the outlet air temperature was 80°C to obtain a crude extract. 1.

5. The crude extract was subjected to graded purification. The polysaccharide was precipitated with 70% ethanol to obtain the anticancer polysaccharide component FPS-1. After extraction, the ethyl acetate was removed by rotary evaporation, and the residue was dissolved in propylene glycol to obtain the component FPE-2. The flavonoid complex FPF-3 was obtained by purification using macroporous resin AB-8 and elution with 60% ethanol. Alternatively, a 100kDa nanofiltration membrane was used to concentrate the polysaccharide, and immobilized β-glucosidase was used to catalyze the improvement of flavonoid purity.

2. The method for preparing an anticancer drug based on pear fermentation according to claim 1, characterized in that: The fermentation raw material is Yali pear or Xuehua pear, after beating, the sugar content is adjusted to 15%, the pH is 5.0-5.5, and 0.15% cysteine ​​+ 0.05% EDTA is added to enhance antioxidant performance; The sugar content of the duck pear or snow pear is adjusted by adding sucrose or glucose and satisfies the formula: Brix=S+0.25×(A-0.65) Where S is the mass percentage of added sugar (%), A is the initial soluble solid content of the raw material (%), the crushing particle size is controlled to be 50–100 mesh, and the viscosity of the pulp after crushing is ≤350 mPa·s.

3. The method for preparing an anticancer drug based on pear fermentation according to claim 1, characterized in that: The fermentation termination conditions are pH 3.5-3.8 and total phenol content ≥7.5 mg GAE / g, or determined by MMP-2 / -9 inhibitory activity index; The fermentation termination conditions are dynamically monitored by real-time pH sensor combined with near-infrared spectroscopy when the following conditions are met simultaneously: It automatically terminates when pH ≤ 3.8 and total phenols ≥ 7.5 mg GAE / g; Alternatively, an MMP-2 / -9 inhibition rate ≥ 65% can be used as an auxiliary judgment indicator.

4. The method for preparing an anticancer drug based on pear fermentation according to claim 1, characterized in that: The active ingredient extraction includes precipitating polysaccharides with 70% ethanol, removing ethyl acetate by rotary evaporation after extraction, dissolving the residue in propylene glycol, purifying flavonoids with macroporous resin AB-8, or concentrating polyphenol-polysaccharide complexes with a molecular weight of 800-1500Da through a 100kDa nanofiltration membrane.

5. An anticancer composition prepared by the method of any one of claims 1 to 4, characterized in that: Contains polysaccharide FPS-1 (molecular weight 1.2×10 5 Daβ-glucan), phenolic acid FPE-2 (chlorogenic acid and arbutin combination), flavonoid FPF-3 (quercetin-3-O-glucoside content ≥85%) and polyphenol-polysaccharide complex.

6. The method for preparing an anticancer drug based on pear fermentation according to claim 5, characterized in that: The polyphenol-polysaccharide complex has a molecular weight of 800-1500 Da and can reduce the expression of MMP-2 / -9 by inhibiting the PI3K / AKT pathway.

7. Use of the composition according to claim 5 or 6 in the preparation of an anti-tumor drug, characterized in that: The drug is used to inhibit the proliferation of liver cancer cells (such as HepG2), apoptosis of breast cancer cells (such as MCF-7) or metastasis of non-small cell lung cancer (NSCLC).

8. The method for preparing an anticancer drug based on pear fermentation according to claim 1, characterized in that: The drug blocks tumor cell invasion by inhibiting the EMT (epithelial-mesenchymal transition) process, reducing the number of lung metastatic nodules by ≥60%.

9. The method for preparing an anticancer drug based on pear fermentation according to claim 1, characterized in that: It also includes toxicological validation steps, including a single oral administration of MTD ≥ 2000 mg / kg in mice, no liver and kidney damage in rats after repeated administration for 90 days (ALT, Cr indicators p>0.05), and no mutagenicity in the Ames test.

10. A pear fermented anticancer pharmaceutical preparation, characterized by: The invention comprises the active ingredient according to claim 5 and a liposome carrier, wherein the nanoliposome particle size is 100-150 nm and the encapsulation efficiency is ≥85%.

Images