A large white pile polypeptide-ferric chelate and its preparation method and application

Abstract

The present application provides a kind of big white pile mushroom polypeptide-ferrous chelate and its preparation method and application, belong to the technical field of peptide iron chelate.The present application first provides a kind of preparation method of big white pile mushroom polypeptide-ferrous chelate, the big white pile mushroom polypeptide-ferrous chelate obtained by the preparation method of the present application has in vitro antioxidant effect and bacteriostatic effect, high stability, and has certain inhibitory effect on liver cancer and rectal cancer, and has certain improvement effect on the iron deficiency anemia disease caused by it, the big white pile mushroom polypeptide-ferrous chelate provided in the present application can be used as a new potential drug for preventing, intervening, auxiliary treatment of gastrointestinal malignant tumor liver cancer, rectal cancer and related anemia.

Description

Technical Field

[0001] This invention belongs to the field of peptide iron chelate technology, and particularly relates to a polypeptide-ferrous chelate of *Pleurotus ostreatus*, its preparation method and application. Background Technology

[0002] Cancer-related anemia (CRA) is a common comorbidity of malignant tumors. It refers to a decrease in the number of red blood cells per unit volume or a reduction in hemoglobin concentration in peripheral blood during the development and treatment of cancer, resulting in insufficient oxygen supply to surrounding tissue cells. The causes of CRA mainly include tumor-related factors (such as blood loss, hemolysis, and bone marrow invasion) and factors related to cancer treatment (such as the bone marrow suppression effects of chemotherapy and radiotherapy). CRA has a low treatment rate, can cause various clinical symptoms, severely reduces patients' quality of life, and shortens the survival time of CRA patients. It is a risk factor affecting the survival of cancer patients independently of radiotherapy and chemotherapy. Among cancers, the incidence of anemia is higher in gastrointestinal tumors than in other systems.

[0003] Liver cancer and colon cancer are among the most common malignant digestive system tumors in my country, and the treatment of accompanying anemia has a significant impact on patient prognosis and quality of life. Current treatments for anemia associated with liver and colon cancer mainly include blood transfusions, iron supplementation, and erythropoietin (EPO) therapy. While these treatments effectively alleviate anemia, they also have significant adverse reactions. EPO can lead to thrombosis, and transfusions can cause infection and iron overload. Furthermore, the impact of these treatments on patient survival remains highly controversial. Therefore, iron supplementation alone is crucial for treating chronic liver cancer (CRA), and there is an urgent need to develop novel iron supplements to address this issue. Peptide iron chelates are an excellent iron supplement and one of the best iron supplements for alleviating iron deficiency. They are odorless, have high bioavailability, and exhibit significant preventative and therapeutic effects against iron deficiency. Most importantly, in addition to their good iron supplementation effect, peptide iron chelates also possess antioxidant, antibacterial, and immunomodulatory activities.

[0004] In our daily diet, protein-rich foods include meat, dairy products, eggs, legumes, and nuts. Edible fungi can also serve as an important source of protein, with a protein content ranging from 7.7% to 49.9%. Many varieties contain higher levels of protein than animal-based, plant-based, and egg-based foods. Furthermore, the amino acid profiles of enoki mushrooms and king oyster mushrooms are similar to those of egg protein, and the essential amino acid content of cordyceps militaris exceeds 50% of that of milk, making it extremely nutritious and a good natural protein source for preparing bioactive peptides.

[0005] Previous research by our group found that the protein content of the fruiting body of *Mucuna macrocarpa* is 49.12 g / 100 g (dry weight), which is much higher than that of other types of edible fungi, such as morel (28.43 g / 100 g) and animal, plant, and egg sources. The total amount of 17 free amino acids is 33.95 g / 100 g, of which essential amino acids account for 31.41% (excluding tryptophan). It is not only rich in umami substances aspartic acid and glutamic acid, but also has an amino acid score (35.26) that is higher than 35% of the FAO / WHO pattern spectrum, giving *Mucuna macrocarpa* a better taste and flavor. Therefore, *Mucuna macrocarpa* can be used as a good natural protein source for the preparation of bioactive peptides. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a *Pleurotus ostreatus* polypeptide-ferrous chelate, its preparation method and application. The *Pleurotus ostreatus* polypeptide-ferrous chelate provided by this invention has antioxidant and antibacterial effects, and has significant therapeutic effects on iron deficiency anemia, malignant digestive tract tumors such as liver cancer and rectal cancer and their related anemia.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for preparing *Pleurotus ostreatus* polypeptide-ferrous chelate, comprising the following steps: using liquid fermentation product of *Pleurotus ostreatus* strain as raw material, obtaining *Pleurotus ostreatus* protein by alkali dissolution and acid precipitation, hydrolyzing *Pleurotus ostreatus* protein with protease, heating the hydrolysis product after hydrolysis to inactivate the enzyme, collecting the *Pleurotus ostreatus* polypeptide fraction less than 3 kDa from the supernatant, adding ascorbic acid and ferrous chloride to react and obtain *Pleurotus ostreatus* polypeptide-ferrous chelate.

[0009] Preferably, the liquid culture medium for liquid fermentation comprises: 16-35 g / L carbon source, 4-6 g / L nitrogen source, 0.4-0.6 g / L potassium dihydrogen phosphate, 0.2-0.3 g / L magnesium sulfate, and 8-12 mg / L vitamin B1, wherein the pH value of the liquid culture medium is 4-6.

[0010] Preferably, the protease includes at least one of alkaline protease, neutral protease, trypsin, pepsin, and papain.

[0011] Preferably, the enzyme content of the protease is 6000-8000 U / mL.

[0012] Preferably, the pH value of the hydrolysis is 6.0-7.0, the hydrolysis temperature is 30-50℃, and the hydrolysis time is 4.0-7.0h.

[0013] Preferably, the amount of ascorbic acid added is 0.075-0.15%, the concentration of the *Pleurotus ostreatus* polypeptide is 2-6%, and the mass ratio of the *Pleurotus ostreatus* polypeptide to ferrous chloride is 3:1-6:1.

[0014] Preferably, the reaction temperature is 10-50℃, the pH value is 4-7, and the time is 10-50 min.

[0015] The present invention also provides the *Pleurotus ostreatus* polypeptide-ferrous chelate prepared by the above preparation method.

[0016] This invention also provides the application of the above-mentioned *Pleurotus ostreatus* polypeptide-ferrous chelate in the preparation of drugs for the prevention and treatment of liver cancer and / or rectal cancer.

[0017] This invention also provides the application of the above-mentioned *Pleurotus ostreatus* polypeptide-ferrous chelate in the preparation of drugs for the prevention and treatment of anemia associated with liver cancer and / or rectal cancer.

[0018] This invention also provides the application of the above-mentioned *Pleurotus ostreatus* polypeptide-ferrous chelate in the preparation of drugs for the prevention and treatment of iron deficiency anemia.

[0019] The beneficial effects of this invention are:

[0020] This invention provides a method for preparing a *Pleurotus ostreatus* polypeptide-ferrous chelate. The *Pleurotus ostreatus* polypeptide-ferrous chelate prepared by this method exhibits in vitro antioxidant and antibacterial effects, high stability, and a certain inhibitory effect on liver cancer and rectal cancer. In addition, it has a certain effect on improving iron deficiency anemia. The *Pleurotus ostreatus* polypeptide-ferrous chelate provided by this invention can serve as a novel potential drug for the prevention, intervention, and treatment of liver cancer and related anemia. Attached Figure Description

[0021] Figures 1-6 The effects of different carbon sources, nitrogen sources, carbon-nitrogen ratios, pH values, fermentation temperatures, and fermentation times on the soluble protein content in fermentation products during liquid fermentation were investigated.

[0022] Figures 7-11 The effects of different protease types, enzyme dosages, pH values, temperatures, and times on peptide content during hydrolysis were investigated.

[0023] Figures 12-17 The effects of peptide-iron mass ratio, peptide concentration, ascorbic acid addition, chelation time, pH value, and chelation temperature on the chelation rate during the chelation reaction were investigated.

[0024] Figure 18 The scavenging effects of different groups on DPPH free radicals;

[0025] Figure 19 The antibacterial activity of different groups;

[0026] Figure 20 The results show the body weight of rats in different groups;

[0027] Figure 21 Results of serum iron content in rats from different groups;

[0028] Figure 22 Results of serum ferritin levels in rats from different groups;

[0029] Figure 23 The inhibition rate of different groups on rectal cancer Caco2 cells;

[0030] Figure 24 The inhibition rate of different groups on HepG2 liver cancer cells;

[0031] Figure 25 Results of red blood cell counts for different groups;

[0032] Figure 26 Results of hemoglobin levels in different groups. Detailed Implementation

[0033] This invention provides a method for preparing *Pleurotus ostreatus* polypeptide-ferrous chelate, comprising the following steps: using liquid fermentation product of *Pleurotus ostreatus* strain as raw material, obtaining *Pleurotus ostreatus* protein by alkali dissolution and acid precipitation, hydrolyzing *Pleurotus ostreatus* protein with protease, heating the hydrolysis product after hydrolysis to inactivate the enzyme, collecting the *Pleurotus ostreatus* polypeptide fraction less than 3 kDa from the supernatant, adding ascorbic acid and ferrous chloride to react and obtain *Pleurotus ostreatus* polypeptide-ferrous chelate.

[0034] In this invention, the preferred strain of *Mammillaria pubescens* is *Mammillaria pubescens* collected at an altitude of 1943.00m, latitude of 38.69°, and longitude of 111.81°. In this invention, when liquid fermenting the *Pleurotus ostreatus* strain, the preferred liquid culture medium includes: 16-35 g / L carbon source, 4-6 g / L nitrogen source, 0.4-0.6 g / L potassium dihydrogen phosphate, 0.2-0.3 g / L magnesium sulfate, and 8-12 mg / L vitamin B1. The pH of the liquid culture medium is 4-6. The preferred carbon source is glucose, trehalose, and sucrose, with glucose being more preferred. The preferred nitrogen source is peptone and beef extract, with peptone being more preferred. The preferred carbon-to-nitrogen ratio is 5:1-30:1, more preferably 5:1-20:1, and most preferably 10:1. Combining the most preferred carbon source being glucose and the most preferred nitrogen source being peptone, the most preferred glucose:peptone ratio is 4:1. The more preferred fermentation pH is 5. The preferred fermentation temperature is 20-30℃, and most preferably 25℃. The preferred fermentation time is 12-16 days, and most preferably 14 days. This invention does not have any special limitations on the specific raw materials and methods for the alkaline dissolution and acid precipitation method; the conventional alkaline dissolution and acid precipitation method for separating proteins in the art can be used.

[0035] In this invention, the protease preferably includes at least one of alkaline protease, neutral protease, trypsin, pepsin, and papain, more preferably a complex enzyme using both alkaline protease and papain, wherein the mass ratio of alkaline protease to papain in the complex enzyme is 1:1. This invention does not specifically limit the source of any of the above enzymes. In this invention, the enzyme content of the protease is preferably 6000-8000 U / mL. When hydrolyzing *Pleurotus ostreatus* protein with protease, the pH value of the hydrolysis is preferably 6.0-7.0, more preferably 6.5; the hydrolysis temperature is preferably 30-50℃, more preferably 35-45℃, and most preferably 40℃; the hydrolysis time is preferably 4-7 h, more preferably 5-6 h, and most preferably 5 h.

[0036] In this invention, the mass ratio of *Pleurotus ostreatus* polypeptide to ferrous chloride is preferably 3:1-6:1, more preferably 3:1-5:1, and most preferably 3:1; the concentration of *Pleurotus ostreatus* polypeptide is preferably 2-6%, more preferably 3-5%, and most preferably 3%; the amount of ascorbic acid added is preferably 0.075-0.15%, more preferably 0.1-0.15%, and most preferably 0.1%, where the percentage refers to the percentage ratio of *Pleurotus ostreatus* polypeptide solution; the pH value of the chelation reaction is preferably 4-7, more preferably 5-6, and most preferably 6; the chelation reaction time is preferably 10-50 min, more preferably 15-40 min, and most preferably 20 min; the chelation reaction temperature is preferably 10-50℃, more preferably 10-40℃, and most preferably 10℃. This invention also provides the *Pleurotus ostreatus* polypeptide-ferrous chloride chelate prepared by the above method.

[0037] This invention also provides the application of the above-mentioned *Pleurotus ostreatus* polypeptide-ferrous chelate in the preparation of drugs for the prevention and treatment of liver cancer and / or rectal cancer, drugs for the prevention and treatment of liver cancer-related anemia, and drugs for the prevention and treatment of iron deficiency anemia.

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Unless otherwise specified, the following embodiments are all conventional methods.

[0040] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0041] Example 1

[0042] The *Pleurotus ostreatus* cultivar was subjected to liquid fermentation. The liquid fermentation method was as follows: the liquid culture medium contained 16 g / L carbon source, 4 g / L nitrogen source, 0.4 g / L potassium dihydrogen phosphate, 0.2 g / L magnesium sulfate, and 0.8 mg / L vitamin B1. The pH of the liquid culture medium was 5, the fermentation temperature was 25℃, and the fermentation time was 14 days, yielding the fermented product. Using this fermented product as raw material, *Pleurotus ostreatus* protein was prepared by alkali dissolution and acid precipitation. The protein was hydrolyzed using an alkaline protease with an enzyme concentration of 6000 U / mL at a pH of 7.2, a temperature of 54℃, and a hydrolysis time of 3.0 h. After hydrolysis, the hydrolysate was heated to 100℃ and held for 10 min to inactivate the enzyme. The supernatant was obtained by centrifugation at 3500 r / min for 30 min at room temperature. The fraction of the hydrolysate smaller than 3 kDa was collected using an ultrafiltration membrane (3 kDa). After freeze-drying, the obtained LGP (Large White Mushroom Peptide) was stored at -20℃ for later use.

[0043] Weigh a certain amount of *Lactobacillus davidii* polypeptide freeze-dried powder, add 3% polypeptide concentration to ultrapure water and mix well, add 0.1% ascorbic acid and mix well, add ferrous chloride at a peptide-iron mass ratio of 3:1 and adjust the pH of the solution to 6, seal with a film, react at 10℃ for 20 min, centrifuge at 3800 r / min for 10 min and collect the supernatant, add 6 times the volume of 95% anhydrous ethanol and let stand for 1 h, centrifuge at 8000 r / min for 20 min, collect the precipitate, wash the precipitate 3 times with 95% anhydrous ethanol and centrifuge, collect the precipitate, which is *Lactobacillus davidii* polypeptide-ferrous chelate (LGP-Fe). 2+ The product was freeze-dried into powder in a freeze dryer at -80℃, and the freeze-dried powder was collected and stored at -20℃ for later use.

[0044] Example 2

[0045] Screening of *Pleurotus ostreatus* strains with high soluble protein content

[0046] The protein content of five species of white mushrooms collected from different regions (see Table 1 for sampling location information) was tested, and the results are shown in Table 1.

[0047] Table 1 Screening of *Dalbergia odorifera* strains

[0048] 1 1723.50 36.60° 112.08° 36.17 25.58 2 2283.80 38.76° 111.64° 38.98 26.13 3 2223.30 39.05° 113.44° 38.22 24.41 4 1943.00 38.69° 111.81° 40.12 26.35 5 2280.80 38.97° 112.13° 35.39 21.96

[0049] As shown in Table 1, the seed bodies of *Pleurotus ostreatus*, which originated from an altitude of 1943.00m, latitude of 38.69°, and longitude of 111.81°, had the highest protein and soluble protein content.

[0050] Example 3

[0051] Using the soluble protein content in the liquid fermentation broth and mycelium as the evaluation index, single-factor experiments were conducted on the specific liquid fermentation method in Example 1. Specifically, six variables were selected for single-factor experiments: carbon source (glucose, trehalose, sucrose, maltose, lactose), nitrogen source (peptone, beef extract, yeast powder, ammonium sulfate, wheat bran), carbon-nitrogen ratio (5:1, 10:1, 20:1, 30:1, 40:1), fermentation pH (4, 5, 6, 7, 8), fermentation temperature / ℃ (10, 15, 20, 25, 30℃), and fermentation time / d (8, 10, 12, 14, 16). The effects of each factor on the soluble protein content in the liquid fermentation broth and mycelium of *Millettia divaricata* were investigated. The results are as follows: Figures 1-6 As shown.

[0052] The results showed that the optimal conditions for enriching soluble protein in *Pleurotus ostreatus* were: carbon source, glucose, trehalose, and sucrose, with glucose being the most preferred; nitrogen source options were peptone and beef extract, with peptone being the most preferred; the carbon-to-nitrogen ratio was 5:1-30:1, preferably 5:1-20:1, and most preferably 10:1. Combining the optimal carbon source of glucose and the optimal nitrogen source of peptone, the optimal carbon-to-nitrogen ratio, glucose:peptone, was 4:1; the fermentation pH was 4-8, preferably 4-6, and most preferably 5; the fermentation temperature was 20-30℃, most preferably 25℃; and the fermentation time was 12-16 days, most preferably 14 days. Under these optimal conditions, the soluble protein content in the liquid fermentation broth and fermentation mycelium was 42.08%.

[0053] Example 4

[0054] Using peptide content as the evaluation index, single-factor experiments were conducted on the enzymatic hydrolysis conditions in Example 1. Specifically, five variables were selected for single-factor experiments: enzyme type (alkaline protease, neutral protease, trypsin, pepsin, papain, and two compound enzymes), enzyme dosage (2000, 4000, 6000, 8000, 10000 U / mL), hydrolysis pH (5, 6, 6.5, 7, 7.5, 8), hydrolysis temperature / ℃ (30, 35, 40, 45, 50, 55, 60℃), and hydrolysis time / h (2, 3, 4, 5, 6, 7). The effects of each factor on peptide content were investigated. The results are as follows. Figures 7-11 As shown.

[0055] The results showed that the optimal conditions for preparing the polypeptide were as follows: an enzyme mixture of alkaline protease and papain in a 1:1 ratio; an enzyme concentration of 5000-10000 U / mL, preferably 6000-8000 U / mL, and most preferably 6000 U / mL; a hydrolysis pH of 6.0-8.0, preferably 6.0-7.0, and most preferably 6.5; a hydrolysis temperature of 30-50℃, preferably 35-45℃, and most preferably 40℃; and a hydrolysis time of 4-7 h, preferably 5-6 h, and most preferably 5 h. Under these optimal conditions, the polypeptide yield was 12.65%.

[0056] Example 5

[0057] Using the ferrous chelation rate (excluding ascorbic acid addition amount, the precipitation content was used as the evaluation index) as the evaluation index, single-factor experiments were conducted on the ferrous chelation conditions in Example 1. Specifically, six variables were selected for single-factor experiments: peptide-iron mass ratio (2:1, 3:1, 4:1, 5:1, 6:1), peptide concentration (2, 3, 4, 5, 6%), ascorbic acid addition amount (0.05, 0.075, 0.1, 0.125, 0.15%), pH (3, 4, 5, 6, 7), time (10, 20, 30, 40, 50 min), and temperature (20, 30, 40, 50, 60℃). The effects of each factor on the ferrous chelation rate were investigated. The results are as follows. Figures 12-17 As shown.

[0058] The results showed that the optimal conditions for ferrous chelation preparation were: a peptide-to-iron mass ratio of 3:1-6:1, preferably 3:1-5:1, and most preferably 3:1; a peptide concentration of 2-6%, preferably 3-5%, and most preferably 3%; an ascorbic acid addition of 0.075-0.15%, preferably 0.1-0.15%, and most preferably 0.1%; a pH of 4-7, preferably 5-6, and most preferably 6; a time of 10-50 min, preferably 15-40 min, and most preferably 20 min; and a temperature of 10-50℃, preferably 10-40℃, and most preferably 10℃. Under these optimal conditions, the ferrous chelation rate was 75.32%.

[0059] Example 6

[0060] Study on the antioxidant and antibacterial activities of the *Pleurotus ostreatus* polypeptide-ferrous chelate prepared in Example 1

[0061] In vitro antioxidant activity assay: determination of DPPH free radical scavenging ability

[0062] The LGP-Fe3+ polypeptide-ferrous chelate prepared in Example 1 was used. 2+ The experimental group consisted of Vitamin C and the LGP (unchelated polypeptide) prepared in Example 1, which served as the control group. The antioxidant activities of the different groups were measured. DPPH solution: 5.0 mg of DPPH was weighed, dissolved in an appropriate amount of anhydrous ethanol, and sonicated in the dark to ensure complete dissolution. The solution was then diluted to 100.0 mL with anhydrous ethanol to prepare a solution of 50.0 μg / mL. -1 The DPPH solution should be prepared fresh for immediate use. Accurately measure 3.0 mL of the DPPH solution and add 1.0 mL of the test solution. Mix thoroughly and incubate at room temperature in the dark for 30 min. Measure the absorbance using a UV-Vis spectrophotometer at a wavelength of 517 nm (zeroing is performed using the sample solvent). Results are as follows: Figure 18As shown, the chelated peptide-iron chelate consistently exhibited higher DPPH radical scavenging ability than the unchelated peptide at the same concentration. At a concentration of 0.8 g / L, the DPPH scavenging rate of the peptide-iron chelate reached 91.35%, which was not significantly different from that of vitamin C at the same concentration.

[0063] Determination of antibacterial activity

[0064] The LGP-Fe3+ polypeptide-ferrous chelate prepared in Example 1 was used. 2+ The experimental group consisted of Vitamin C and the unchelated *Lysimachia christinae* polypeptide (LGP) prepared in Example 1, serving as the control group. Antibacterial activities (Staphylococcus aureus, Bacillus subtilis, and *Escherichia coli*) of different groups were measured. The antibacterial effect was determined using the paper disc diffusion method. First, bacteria were inoculated onto agar plates using a coating method (to form a uniform bacterial film). Then, filter paper discs (0.5 mm × 0.5 mm) soaked in 20 μL of 100 mg / L Cordyceps polypeptide solution (<3 kDa) were placed on the plates. Within a certain range, the polypeptide concentration inhibited bacterial growth, forming a transparent zone (inhibition zone) where no bacteria grew. The larger the inhibition zone, the better the antibacterial effect. Cefixime was used as a positive control. The plates with the added samples were incubated at 37°C for 24 hours. After incubation, the size of the inhibition zone was measured. The experiment was repeated three times, and the average value was taken. The results are as follows: Figure 19 As shown in the figure, after chelation, the antibacterial effect of the polypeptide against Staphylococcus aureus, Bacillus subtilis, and Escherichia coli was significantly higher than that of the unchelated polypeptide, and slightly lower than that of VC, but the difference was not statistically significant.

[0065] The above results indicate that the *Pleurotus ostreatus* polypeptide-ferrous chelate prepared in this invention has significant antibacterial and antioxidant effects.

[0066] Example 7

[0067] Example 1: Study on the iron supplementation effect of the prepared *Gnaphalium affine* polypeptide-ferrous chelate on iron-deficiency anemia mice.

[0068] Establishment of a rat model of iron deficiency anemia

[0069] Thirty male SD rats, 3 weeks old and weighing 40-50g, were used. The temperature was controlled between 20-26℃ and the humidity between 40-70%, with 12-hour alternating light and dark exposure. They had free access to water and food. After 3 days of acclimatization, the 30 rats were randomly divided into 6 groups (n=5 / group). One group was randomly selected as the normal control group, fed a standard diet throughout the experiment. The remaining 25 rats were fed an iron-deficient diet to establish the IDA rat model. After 28 days, blood was collected from the orbital sinus of each of the 25 model rats for routine blood tests. Rats with hemoglobin (Hb) <100g / L were considered to have successfully established the model.

[0070] The groups were divided into four groups: normal control group (CN, fed standard diet + sterile water), iron-deficient control group (CD, fed iron-deficient diet + sterile water), low-dose peptide iron chelate group (CL, fed iron-deficient diet + 0.5 mg / kg peptide iron chelate), medium-dose peptide iron chelate group (CM, fed iron-deficient diet + 1 mg / kg peptide iron chelate), high-dose peptide iron chelate group (CH, fed iron-deficient diet + 2 mg / kg peptide iron chelate), and FeCl2 group (CF, fed iron-deficient diet + 1 mg / kg FeCl2). The corresponding diets were fed daily for 24 days. Body weight, serum iron content, and serum ferritin content were measured in each group. Results are as follows: Figure 20-22 As shown.

[0071] The results showed that before the experiment, the body weight of rats with iron deficiency anemia was significantly lower than that of the normal control group (CN). At the end of the experiment, except for the iron deficiency control group (CD), there was no significant difference in body weight between the other five groups and the normal control group (CN) (p>0.05). The body weight of each intervention group was significantly different from that of the CD group. There was also no significant difference among the low, medium and high doses of Da Bai Zhuang Mushroom Peptide Iron Chelate intervention groups. This indicates that iron deficiency anemia significantly affects the growth and development of rats, and the intervention of Da Bai Zhuang Mushroom Peptide Iron Chelate can supplement the iron requirements of rats with iron deficiency anemia and restore the body weight of rats to normal levels.

[0072] Serum iron and serum ferritin levels are relevant indicators for diagnosing iron deficiency anemia. Iron deficiency anemia significantly reduces serum iron and serum ferritin levels in rats. Compared with the iron-deficient control group (CD), the serum iron and serum ferritin levels in all intervention groups were significantly increased. The levels of peptide iron chelate were positively correlated with the peptide iron chelate concentration in all rats, and the serum iron and serum ferritin levels in rats treated with high concentrations of peptide iron chelate were significantly higher than those in the high concentration CF group, indicating that ferrous chelate was more effective than FeCl2.

[0073] Example 8

[0074] Effects of the prepared *Pleurotus ostreatus* polypeptide-ferrous chelate on the growth of rectal cancer Caco2 cells and liver cancer HepG2 cells in Example 1

[0075] The effects of different concentrations of *Gnaphalium affine* polypeptide, peptide-iron chelate, and a positive control group (5-fluorouracil injection) on the growth of rectal cancer Caco2 cells and liver cancer HepG2 cells were studied for 48 hours. The results are as follows: Figure 23-24 As shown, different concentrations of *Pleurotus ostreatus* polypeptide, peptide-iron chelate, and the positive control group all inhibited the growth of rectal cancer Caco2 cells and liver cancer HepG2 cells. However, the inhibitory effect of peptide-iron chelate was significantly higher than that of polypeptide, and its inhibition rate was significantly positively correlated with the concentration of peptide-iron chelate. At high concentrations, the inhibition rate on the growth of both cell types was higher than that of the positive control.

[0076] Example 9

[0077] The effect of improving cancer-related anemia

[0078] Using tumor-bearing H22 mice (n=20), they were randomly divided into two groups: the model group (CM, fed with standard diet + saline) and the treatment group (PM, fed with 0.5 mg / kg of *Gnaphalium affine* polypeptide iron chelate prepared in Example 1 + saline). Healthy mice served as the blank control group (CK, fed with standard diet + saline). Administration began on day 2, once daily for 10 consecutive days. On days 1, 5, and 10, 0.5 mL of blood was collected from the eyeballs of each group of mice, placed in anticoagulant tubes, and the number of red blood cells and hemoglobin content in peripheral blood were measured.

[0079] The results are as follows Figure 25 , 26 As shown, with the extension of time, the red blood cell count in the model group was significantly reduced compared to the blank control group, indicating the possible presence of cancer-related anemia (cancer-related anemia). The red blood cell count in the group treated with *Gnaphalium affine* polypeptide iron chelate was increased compared to the model group. Compared to the blank control group, the hemoglobin content in the model group was significantly reduced, also indicating the possible presence of cancer-related anemia. The hemoglobin content in the group treated with *Gnaphalium affine* polypeptide iron chelate gradually increased, suggesting that *Gnaphalium affine* polypeptide iron chelate has a certain hematopoietic effect on tumor-bearing mice.

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a *Pleurotus ostreatus* polypeptide-ferrous chelate in the preparation of a drug for treating rectal cancer, characterized in that, The preparation method of the *Pleurotus ostreatus* polypeptide-ferrous chelate includes the following steps: using liquid fermentation product of *Pleurotus ostreatus* strain as raw material, *Pleurotus ostreatus* protein is obtained by alkali dissolution and acid precipitation method, *Pleurotus ostreatus* protein is hydrolyzed by protease, after hydrolysis, the hydrolysis product is heated to inactivate the enzyme, the supernatant is taken to collect the *Pleurotus ostreatus* polypeptide fraction less than 3kDa in the hydrolysis product, ascorbic acid and ferrous chloride are added to react and the *Pleurotus ostreatus* polypeptide-ferrous chelate is obtained.

2. The application according to claim 1, characterized in that, The liquid culture medium for liquid fermentation includes: 16-35 g / L carbon source, 4-6 g / L nitrogen source, 0.4-0.6 g / L potassium dihydrogen phosphate, 0.2-0.3 g / L magnesium sulfate, and 8-12 mg / L vitamin B1. The pH value of the liquid culture medium is 4-6.

3. The application according to claim 1, characterized in that, The protease is at least one of alkaline protease, neutral protease, trypsin, pepsin, and papain.

4. The application according to claim 1, characterized in that, The enzyme content of the protease is 6000-8000 U / mL.

5. The application according to claim 1, characterized in that, The hydrolysis is performed at a pH of 6.0-7.0, at a temperature of 30-50°C, and for a time of 4.0-7.0 hours.

6. The application according to claim 1, characterized in that, The amount of ascorbic acid added is 0.075-0.15%, the concentration of the *Pleurotus ostreatus* polypeptide is 2-6%, and the mass ratio of the *Pleurotus ostreatus* polypeptide to ferrous chloride is 3:1-6:

1.

7. The application according to claim 1, characterized in that, The reaction is carried out at a temperature of 10-50℃, a pH of 4-7, and a time of 10-50 minutes.

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