Combined biological agent and application thereof

Abstract

The invention discloses a combined biological agent and application thereof, and belongs to the technical field of biology. Amino acid and black fungus melanin are combined for intragastric administration to treat iron-deficiency anemia mice, a synergistic effect can be achieved, and the iron-deficiency anemia symptoms of the mice are remarkably improved. Compared with the prior art, the combined biological agent disclosed by the invention can be used for effectively improving blood indexes and intestinal tissue structures of mouse iron-deficiency anemia by regulating and controlling expression of iron transporter, so that the aim of improving symptoms of iron-deficiency anemia is fulfilled. The invention aims at finding a scientific, safe, small-toxic-side-effect and long-term-use iron-deficiency anemia treating agent which is expected to be specifically applied to treatment of iron-deficiency anemia and further replace a traditional iron supplementing agent to become a next-generation novel iron supplementing agent from raw materials with homology of medicine and food.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a combined biological preparation and application thereof. Background Art

[0002] Iron deficiency anemia (IDA) is one of the four major nutritional deficiencies announced by the World Health Organization (WHO). Studies have shown that IDA is a disease caused by reduced or lost iron intake and increased iron use, which leads to depletion of iron reserves in the body. The occurrence of the disease will affect the quality of human life, cause abnormal development and cognitive function, cause nervous system disorders, weakened digestive ability, reduced immune ability, disordered intestinal metabolites and imbalanced iron metabolism.

[0003] Currently, the effective measure for the treatment of IDA is iron supplementation, but the intake cycle is long, the effect is poor, and it is accompanied by side effects such as nausea, epigastric pain, diarrhea, and constipation. In addition to oral iron therapy, there are other alternative treatments for the treatment of IDA. Among them, Chinese herbal medicine has achieved satisfactory clinical results in the treatment of anemia, and oral iron therapy by increasing the intake of iron-rich foods is also a treatment for IDA. The natural product black bean seed coat extract can regulate iron metabolism by inhibiting the expression of hepcidin and promote erythropoiesis. Silkworm excrement extract also has certain anti-anemia, anti-inflammatory, and antioxidant biological activities. It shows that natural products may be a potential safe and effective new method to improve IDA.

[0004] Black fungus is an important medicinal and edible fungus in my country, and is also recognized as a health food in the world. It has pharmacological effects such as blood tonic, blood sugar lowering, blood lipid regulation and immune enhancement. Melanin, as one of the main biologically active ingredients of black fungus, has strong biological activities such as scavenging free radicals, anti-radiation, antibacterial and antiviral, improving liver damage, protecting DNA, etc. It is particularly interesting that the melanin in tiger-striped squid ink can improve inflammation and relieve oxidative stress damage. The melanin in squid ink has a significant therapeutic effect on the anemia symptoms of IDA rats.

[0005] Amino acids are a class of amphoteric organic compounds containing basic amino groups and acidic carboxyl groups, and are the basic building blocks of biological functional macromolecular proteins.

[0006] Amino acids can be divided into proteinogenic amino acids and non-proteinogenic amino acids. Among them, proteinogenic amino acids are also called standard amino acids, which are amino acids that directly participate in the synthesis of protein molecules. According to the different positions of the amino group on the carbon chain, amino acids can be divided into α-, β-, γ- and other amino acids, but the amino acids that make up natural proteins in the biological world are all α-amino acids, a total of 22 types, including 20 common amino acids and 2 uncommon amino acids. [2-3] Non-proteinogenic amino acids cannot directly participate in the synthesis of protein molecules and need to be modified before they can participate in protein synthesis, such as citrulline, ornithine and hydroxyproline.

[0007] Amino acids have different properties due to their different structures and R groups, such as isoelectric point and optical rotation. The detection of amino acids can also be screened and identified based on these different properties. Currently developed detection methods include spectrophotometry, liquid chromatography, gas chromatography, infrared detection and other methods. Amino acids are raw materials used in organisms to make antibody proteins, hemoglobin, enzyme proteins, hormone proteins, neurotransmitter substances, and can even be used to provide energy sources for organisms. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a combined biological preparation and its application which is "food and medicine of the same origin", can be taken for a long time, has high safety performance, good biocompatibility, low toxic and side effects, and can significantly improve iron deficiency anemia.

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0010] In one aspect, the present invention provides a combined biological preparation comprising black fungus melanin and amino acids.

[0011] Furthermore, the amino acid is tryptophan or tyrosine.

[0012] Furthermore, the usage ratio of black fungus melanin and amino acid is 1:4.5-6.

[0013] Furthermore, the intragastric dose of black fungus melanin is 100-300 mg / kg·d.

[0014] Furthermore, the method for preparing the black fungus melanin comprises:

[0015] (1) Crush the black fungus and extract the powder in concentrated hydrochloric acid;

[0016] (2) adding sodium hydroxide to step (1) to adjust the pH value, and centrifuging to obtain the supernatant;

[0017] (3) adding hydrochloric acid to step (2) to adjust the pH value, allowing to stand, and collecting the precipitate;

[0018] (4) The precipitate in step (3) is placed in concentrated hydrochloric acid, the precipitate is taken out in a water bath, and freeze-dried to obtain the precipitate.

[0019] Furthermore, in the step (1), 25 g of black fungus powder was dissolved in 500 mL of 3 mol / L concentrated hydrochloric acid and extracted in a water bath at 70° C. for 1.5 h.

[0020] Furthermore, in step (2), the pH value is adjusted to 12 with sodium hydroxide, and the mixture is centrifuged at 4000 rpm for 10 min; and in step (3), the pH value is adjusted to 2 with hydrochloric acid.

[0021] Furthermore, in step (4), the precipitate is placed in 150 mL of 7 mol / L concentrated hydrochloric acid, boiled in a boiling water bath for 2 h, centrifuged at 10,000 rpm for 20 min, and freeze-dried to obtain the product.

[0022] On the other hand, the present invention provides the use of the above-mentioned combined biological preparation, which is applied to medicines or health products for treating iron deficiency anemia.

[0023] Furthermore, the medicine or health product contains an effective therapeutic dose of black fungus melanin and amino acids.

[0024] The present invention has the following beneficial effects:

[0025] In the present invention, the mixed use of black fungus melanin and amino acids can produce a significant synergistic effect, improving the blood indicators of iron deficiency anemia SI, RBC, HCT, Hb and other blood indicators of mice; black fungus melanin plays a role in improving IDA by activating tryptophan metabolism; among them, cytological experiments found that when tryptophan participated in the incubation of iron-deficient cells with black fungus melanin, the expression effect of key genes in iron metabolism was most significant; the antibiotic cocktail was used to create a pseudo-sterile IDA mouse model, and black fungus melanin and tryptophan were gavaged, and transcriptomics research was carried out, which showed that tryptophan supplementation successfully activated the expression of AhR, thereby regulating the downstream HIFs signaling pathway; at the same time, tryptophan supplementation activated the IRP / IRE signaling pathway of iron metabolism in IDA mice, regulated the expression of intestinal iron transporter, and thus improved IDA; in short, in the present invention, tryptophan or tyrosine and black fungus melanin synergistically acted on each other for gavage treatment, which can significantly improve the symptoms of iron deficiency anemia in mice, and is expected to be targetedly used in the treatment of iron deficiency anemia and further replace traditional iron supplements to become the next generation of new iron supplements, providing a potential new strategy for the treatment of iron deficiency anemia patients in clinic. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the cytotoxicity experiment of the combined biological agent on iron-deficient HCT-116 cells in Example 1 of the present invention;

[0027] Figure 2The effect of the combined biological agent on the mRNA expression level of key genes in iron metabolism in Example 2 of the present invention is shown in FIG. express: P<0.05, P<0.01, P<0.001, Significance at the P < 0.0001 level;

[0028] Figure 3 Figure 3 is a diagram of establishing two experimental mouse animal models in Example 3 of the present invention;

[0029] Figure 4 This is a graph showing the determination of multiple blood indices of IDA mice in the IDA+ABX+M+Trp model group in Example 3 of the present invention;

[0030] Figure 5 This is a graph showing the improvement of colon lesions in IDA mice by the combination preparation of black fungus and melanin in the colon tissue pathology test of mice in Example 3 of the present invention;

[0031] Figure 6 This is the effect of the combined preparation of tryptophan and black fungus melanin in Example 4 of the present invention on the IRPs / IREs signaling pathway of IDA mice. DETAILED DESCRIPTION

[0032] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments. However, the present invention is by no means limited to these examples. The following is only a preferred embodiment of the present invention, which is only used to explain the present invention and cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that any modification, equivalent substitution and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0033] Unless otherwise specified, the components used in the present invention are all commercially available products. The experimental test conditions are not otherwise specified and are all conventional technical operations in the art. The tryptophan and tyrosine used in the present invention are purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0034] Black fungus was provided by Dongning Black Fungus Industrial Base, strain collection number: HDXZ-1.

[0035] The black fungus melanin used in the present invention is obtained by a method of hydrochloric acid hydrolysis-filtration-alkali dissolution-acid precipitation.

[0036] The specific preparation method is as follows: after the black fungus is crushed, it is passed through an 80-mesh sieve, 25 g of black fungus powder is taken in 500 mL of 3 mol / L concentrated hydrochloric acid, and extracted in a water bath at 70°C for 1.5 h, and then sodium hydroxide is added to adjust the pH value to 12, and the mixture is centrifuged at 4000 rpm for 10 min after stirring. 3 mol / L hydrochloric acid is added to the supernatant to adjust the pH value to 2, and after standing for a period of time, the mixture is centrifuged at 4000 rpm for 20 min, and the precipitate is obtained to obtain the crude melanin. The crude product is placed in 150 mL-7 mol / L concentrated hydrochloric acid, in a boiling water bath for 2 h, and centrifuged at 10000 rpm for 20 min. The obtained precipitate is washed twice with chloroform, ethyl acetate and ethanol, washed 7 times with deionized water, and the solid is freeze-dried to obtain purified black fungus melanin.

[0037] The combined biological preparation provided by the invention is prepared by mixing black fungus melanin and amino acids in a ratio of 1:4.5-6.

[0038] The present invention aims at the safety problems such as side effects during long-term use of conventional iron supplements in the prior art, and provides a combined biological preparation which is both medicinal and edible, has good safety performance and can significantly improve iron deficiency anemia, and its application.

[0039] Example 1 Cytotoxicity Experiment of Combined Biological Agents

[0040] This example explores the safe concentration range of melanin, amino acids, and a mixture of amino acids and black fungus melanin samples, and the specific steps are as follows:

[0041] HCT-116 cells in logarithmic growth phase were cultured at 1×10 5 3 mL of cell suspension was added to each well of a 6-well plate and incubated in a 5% CO 2 The cells were cultured in a cell culture incubator at 37°C for 24 h. When the cells adhered to the wall, the cell culture medium was discarded and 3 mL of cell culture medium containing deferoxamine was added to the 6-well plate for 24 h. After the modeling was successful, the cell culture medium containing deferoxamine in the plate was aspirated and digested with trypsin to prepare an iron-deficient cell suspension. The iron-deficient cell suspension was inoculated into a 96-well plate, 100 μL was added to each well, and the plate was incubated in 5% CO. 2The cells were cultured in a cell culture incubator at 37°C for 24 h. When the cells attached to the wall, the cell culture medium was discarded and different concentrations of black fungus melanin (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mg / mL), tryptophan (0, 5, 10, 20, 30, 40, 50 mmol / L), tyrosine (0, 5, 10, 20, 30, 40, 50 mmol / L), tryptophan (0, 5, 10, 20, 30, 40, 50 mmol / L) + black fungus melanin (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mg / mL), and tyrosine (0, 5, 10, 20, 30, 40, 50 mmol / L) were added to the 96-well plate. The cytotoxicity was detected after 24 h of incubation.

[0042] Cytotoxicity was detected by MTT method to detect cell growth rate. 100 μL cell suspension was added to each well of 96-well plate, and then 5 μL MTT solution was added to each well. After 4 h of culture, the MTT culture medium in the well was removed, and 200 μL DMSO was added to each well. The mixture was shaken for 10 min, and the absorbance was measured at 490 nm using a microplate reader. The absorbance values ​​of each gradient sample were compared with the blank sample, and the cell survival rate was below 90%, P < 0.05 as the significant index. The t test was performed, and there was a significant difference, indicating that the cells were inhibited by key intestinal metabolites; the cell survival rate was above 90%, and there was no significant difference, indicating that the key intestinal metabolites would not inhibit cell growth, that is, the safe concentration range of key intestinal metabolites for iron-deficient HCT-116 cells was obtained.

[0043] like Figure 1 As shown, melanin was not toxic to iron-deficient HCT-116 cells within the range of 0.8 mg / mL, tryptophan 20 mmol / L, tyrosine 20 mmol / L, tryptophan + black fungus melanin 10 mmol / L + 0.4 mg / mL, and tyrosine + black fungus melanin 10 mmol / L + 0.4 mg / mL, and this concentration was selected for subsequent genetic testing.

[0044] Example 2 Amino Acids Assist Black Fungus Melanin to Activate Cellular Iron Absorption and Transport

[0045] 1. HCT-116 colorectal cancer cell culture

[0046] Culture medium: high-glucose sterile DMEM culture medium, containing 10% fetal bovine serum and 1% double antibody. Culture bottle: 25 cm 2Cell culture flask. Culture conditions: 95% air; 5% carbon dioxide; 37°C. Culture method: Culture by subculture, change the cell culture medium every two days. Observe the cell growth status under a microscope to see if the cell density reaches about 80%~90%. If it reaches this level, the cells begin to be subcultured. Discard the waste liquid, rinse twice with PBS buffer, 2 mL each time, add 2 mL of trypsin to digest, observe the morphology of the cells after digestion under a microscope, add 2 mL of cell culture medium to stop digestion, blow the cells until they are suspended as a single cell, and inoculate and culture at a ratio of 1:2. Heat the purified water to 60-70 degrees to dissolve, prepare the glycyrrhizic acid mother solution, and slowly stir to dissolve.

[0047] 2. Cytotoxicity assay

[0048] MTT method to detect cell growth rate: 100 μL cell suspension in each well of 96-well plate, then add 5 μL MTT solution to each well, culture for 4 h, remove MTT culture medium in the well, add 200 μL DMSO to each well, shake for 10 min, and measure absorbance at 490 nm using a microplate reader. The absorbance values ​​of each gradient sample were compared with the blank sample, and P<0.05 was used as the significance index to perform a t test. If there was a significant difference, it meant that the cells were inhibited by DFO; if there was no significant difference, it meant that DFO would not inhibit cell growth, that is, the safe concentration range of deferoxamine for HCT-116 colorectal cancer cells was obtained, and when the cell growth rate of the test substance at different concentrations was 90% or above, it was non-toxic, and the cell growth rate formula was:

[0049] Cell growth rate = (OD value of experimental group / OD value of control group) × 100%;

[0050] 3. Establishment of HCT-116 colorectal cancer iron-deficient cell model

[0051] Within the toxicity range, the maximum concentration of deferoxamine solution was selected to establish the deferoxamine iron-deficient cell model. HCT-116 colorectal cancer cells in the logarithmic growth phase were cultured at a rate of 1×10 5 100 cells / mL were inoculated into a 6-well plate, and 3 mL of HCT-116 colorectal cancer cell suspension was added to each well. 2 The cells were cultured at 37°C in a cell culture incubator for 24 h. After the cells adhered to the wall, the cell culture medium was discarded and 3 ml of the deferoxamine solution with the highest concentration within the toxic range was added to the 6-well plate. The deferoxamine solution was incubated for 24 to 72 h. The cell iron content was measured every 24 h to determine the optimal time for successful modeling. If it was not successful after 72 h, the cells were continued to be cultured with a new deferoxamine culture medium of the same concentration, and the gradient was detected every 24 h.

[0052] The method for detecting cellular iron content was based on the instructions of the tissue iron content determination kit provided by Nanjing Jiancheng Bioengineering Research Institute, and the operation was performed strictly in accordance with the instructions.

[0053] 4. Real-time fluorescence quantitative PCR to detect key gene expression

[0054] The blank group consisted of HCT-116 colorectal cancer cells without any treatment, and the qRT-PCR method was used to detect the expression of HIF-1α, HIF-2α and Hepicdin in the cells at the mRNA level.

[0055] The results are as follows Figure 2 As shown in A, B, and C, the experiment found that when black fungus melanin was co-incubated with tryptophan and tyrosine, compared with the model group, HIF-1α (P < 0.0001), HIF-2α (P < 0.01, P < 0.0001), and Hepcidin (P < 0.0001) showed significant differences in mRNA levels.

[0056] Among them, when tryptophan participated in the incubation of black fungus melanin in iron-deficient cells, the expression of key genes of iron metabolism, HIF-1α, HIF-2α, and Hepicdin, was the most significant.

[0057] Example 3 Experiment on the improvement of IDA mouse model by tryptophan and black fungus melanin

[0058] 1 Establishment of mouse model

[0059] Male Kunming mice aged 24 days with a body weight range of 14.8 ± 1.62 g were randomly divided into groups with 12 mice in each group. Figure 3 As shown, they are iron deficiency anemia + antibiotic cocktail + black fungus melanin group (IDA+ABX+M) and iron deficiency anemia + antibiotic cocktail + black fungus melanin + amino acid group (IDA+ABX+M+Met).

[0060] The experiment started after one week of adaptive feeding. The mice in the IDA+ABX+M and IDA+ABX+M+Met groups were fed a low-iron diet and had free access to deionized water. The model was established for 2 weeks.

[0061] During the experimental treatment period, the IDA+ABX+M and IDA+ABX+M+Met groups were gavaged with antibiotic cocktail for 7 days for 3 weeks, and black fungus melanin was mixed with tryptophan or tyrosine to form a combined biological agent for gavage treatment (the mass concentration ratio of the two was 1:6, 1:5, and 1:4.5, respectively, and the dosage of black fungus melanin was 200 mg / kg·d).

[0062] During the experiment, the two groups of mice were fed low-iron feed and drank deionized water containing ABX. To avoid exogenous iron contamination, plastic cages and non-iron cage covers were used in the experiment. The mice were weighed every 7 days since the beginning of the experiment.

[0063] 2. Routine blood test of mice

[0064] Blood was collected from the eyeballs of the experimental model mice, and 20 μL of whole blood was added to the bullet containing the diluent. After mixing, various indicators were detected using a blood cell automatic analyzer, and the changes in the content of Hb, HCT, RBC and other indicators of the two model mice were recorded;

[0065] 3. Mouse colon histopathology

[0066] After the test period, the test mice were quickly dissected, the colon segments were cut, rinsed with cold saline, fixed in formaldehyde, prepared into paraffin blocks, cut and stained with hematoxylin-eosin (HE), and then the changes in colon tissue damage were observed under a microscope. Inflammation was determined by measuring the degree of inflammatory cell infiltration, epithelial damage and crypt loss. The specific steps are as follows:

[0067] The colon was fixed by immersion in neutral formalin solution for 5 h; dehydrated with 85% ethanol for 90 min, 95% ethanol for 40 min, and anhydrous ethanol for 15 min to remove water from the tissue block; the block was placed in xylene opacifier for 10 min to make it transparent. This step was repeated twice.

[0068] The transparent tissue block was placed in paraffin, immersed and embedded; the embedded block was placed in a cutter and cut into 4 μm thickness; the slices were baked at 70°C for 45 min.

[0069] Dewaxing with xylene for 10 min, repeated three times, and then rehydrated and washed with ethanol of different concentrations; Harris hematoxylin staining for 5 min, washing with hematoxylin stain in running water for 1 min; differentiation with 0.5% hydrochloric acid ethanol for 10 s, washing with running water for 15 min; reblueing with 50℃ hot water; restaining with eosin stain for 1 min, washing twice with distilled water; elution with 85% ethanol, 95% ethanol, and anhydrous ethanol for 1 min each; dehydration by washing with xylene for 2 min to make it transparent; sealing with neutral gum; observation and photography under a microscope.

[0070] The inventors explored the data on the effect of mixed intragastric administration of black fungus melanin and amino acids and found that when the black fungus melanin and amino acids were mixed intragastricly in a mass ratio of 1:5 (the dosage of black fungus melanin was 100 mg / kg·d), the effect of improving blood indicators was significant. The dosages of black fungus melanin and amino acids mixed intragastricly in the embodiments of the present technical solution were all the dosages in the optimal ratio; and the effect of the combined preparation of tryptophan and black fungus melanin was more significant than that of the combined preparation of tyrosine and black fungus melanin.

[0071] like Figure 4 As shown, when the black fungus melanin and tryptophan were supplemented simultaneously compared with the single gavage of black fungus melanin, the hemoglobin content in the IDA+ABX+M+Trp group was significantly increased by 81.25% (P < 0.001), the hematocrit was significantly increased by 76.29% (P < 0.001), the red blood cell content was significantly increased by 70.97% (P < 0.0001), and the serum iron content was significantly increased by 34.49% (P < 0.001), reaching normal levels.

[0072] like Figure 5 As shown in Figure 2, HE staining of the colon tissues of mice in the IDA+ABX+M+Trp group and the IDA+ABX+M group revealed that the colon tissue structure of mice in the IDA+ABX+M group was damaged, the crypt structure and goblet cells were distorted, the mucosal layer of the tissue was atrophied, the arrangement of mucosal epithelial cells was lost, and the upper mucosa and barrier were deformed (see Figure 2). Figure 5 A). In contrast, the colon epithelial cells of mice in the IDA+ABX+M+Trp group were arranged in a tight and orderly manner, with a large number of well-structured crypts and villi, which were long and clear, without atrophy of the mucosal layer or defective arrangement of epithelial cells, indicating that tryptophan participates in the improvement of black fungus melanin on the colon lesions of IDA mice (see Figure 5 B).

[0073] Example 4: Regulation of IRPs / IREs signaling pathway by tryptophan and black fungus melanin in IDA mice

[0074] The samples were selected from the animal model established in Example 3, and the livers of two groups of model mice were taken. The expression of key genes IRP1, IRP2, TfR1, and ferritin-L in the IRPs / IREs signaling pathway in the mouse liver at the mRNA level was detected by qRT-PCR.

[0075] like Figure 6 In the figure, A, B, C, and D represent iron regulatory protein 1 (IRP1), iron regulatory protein 2 (IRP2), light chain ferritin (ferritin-L), and transferrin receptor 1 (TfR1), respectively. express: P<0.05, P<0.01, P<0.001, Significance at the P<0.0001 level.

[0076] Figure 6 As shown in AC, compared with the IDA+ABX+M group, the mRNA levels of IRP1 (P<0.0001), IRP2 (P<0.001), and TfR1 (P<0.001) in the IDA+ABX+M+Trp group were significantly increased. Figure 6 D, ferritin-L (P < 0.001) was significantly decreased at the mRNA level.

[0077] The results showed that black fungus melanin, with the participation of tryptophan, increased IRPs, thereby regulating the expression of ferritin-L and TfR1, promoting the body's iron absorption and further improving the body's IDA.

[0078] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A combined biological preparation, characterized in that: The combined biological preparation includes black fungus melanin and amino acids.

2. The combined biological preparation according to claim 1, characterized in that: The amino acid is tryptophan or tyrosine.

3. The combined biological preparation according to claim 2, characterized in that: The mass ratio of black fungus melanin to amino acid is 1:4.5-6.

4. The combined biological preparation according to claim 2, characterized in that: The intragastric dose of black fungus melanin is 100-300 mg / kg·d.

5. The combined biological preparation according to claim 4, characterized in that: The preparation method of black fungus melanin comprises: (1) Crush the black fungus and extract the powder in concentrated hydrochloric acid; (2) adding sodium hydroxide to step (1) to adjust the pH value, and centrifuging to obtain the supernatant; (3) adding hydrochloric acid to step (2) to adjust the pH value, allowing to stand, and collecting the precipitate; (4) The precipitate in step (3) is placed in concentrated hydrochloric acid, the precipitate is taken out in a water bath, and freeze-dried to obtain the precipitate.

6. The combined biological preparation according to claim 5, characterized in that: In the step (1), 25 g of black fungus powder was dissolved in 500 mL of 3 mol / L concentrated hydrochloric acid and extracted in a water bath at 70° C. for 1.5 h.

7. The combined biological preparation according to claim 5, characterized in that: In the step (2), the pH value is adjusted to 12 with sodium hydroxide, and the mixture is centrifuged at 4000 rpm for 10 min. In the step (3), the pH value is adjusted to 2 with hydrochloric acid.

8. The combined biological preparation according to claim 5, characterized in that: In the step (4), the precipitate is placed in 150 mL of 7 mol / L concentrated hydrochloric acid, boiled in a boiling water bath for 2 h, centrifuged at 10,000 rpm for 20 min, and freeze-dried to obtain the precipitate.

9. An application of a combined biological agent, characterized in that: Used in medicines or health products for treating iron deficiency anemia.

10. The use of a combined biological agent according to claim 9, characterized in that: The medicine or health product contains black fungus melanin and amino acids in effective therapeutic doses.

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