CN108261427B - Low-iron blood replenishing agent, black fungus melanin active ingredient F1 and application thereof - Google Patents

Low-iron blood replenishing agent, black fungus melanin active ingredient F1 and application thereof Download PDF

Info

Publication number
CN108261427B
CN108261427B CN201810038176.4A CN201810038176A CN108261427B CN 108261427 B CN108261427 B CN 108261427B CN 201810038176 A CN201810038176 A CN 201810038176A CN 108261427 B CN108261427 B CN 108261427B
Authority
CN
China
Prior art keywords
black fungus
active ingredient
fungus melanin
iron
group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201810038176.4A
Other languages
Chinese (zh)
Other versions
CN108261427A (en
Inventor
雷虹
曾伟民
万鹏
江伟
冯磊
程文
诸琳
王淼
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Heilongjiang University
Original Assignee
Heilongjiang University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Heilongjiang University filed Critical Heilongjiang University
Priority to CN201810038176.4A priority Critical patent/CN108261427B/en
Publication of CN108261427A publication Critical patent/CN108261427A/en
Application granted granted Critical
Publication of CN108261427B publication Critical patent/CN108261427B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/55—Liquid-liquid separation; Phase separation

Abstract

A low-iron blood replenishing agent, black fungus melanin active ingredient F1 and application thereof relate to the low-iron blood replenishing agent, the black fungus melanin active ingredient and the application thereof. The invention solves the problems that the existing iron supplement can cause diarrhea and constipation, intestinal flora imbalance, harm to gastrointestinal health, anaphylactic shock and other side effects, or has high price, limited effect of improving anemia and easy recurrence. The preparation method of the black fungus melanin active ingredient F1 comprises the following steps: 1. dissolving black fungus melanin in NaOH solution, and then loading the solution on a dextran gel column; 2. eluting with PBS buffer solution, collecting the eluting solution for 34-50 min, regulating pH value, standing, centrifuging, and vacuum freeze drying. The black fungus melanin active ingredient F1 can obviously improve the hemoglobin and the number of red blood cells of an IDA mouse model, improve hematopoietic factors, increase iron absorption and effectively improve iron deficiency anemia; and can not cause adverse symptoms such as nausea, abdominal pain, diarrhea, constipation, etc.

Description

Low-iron blood replenishing agent, black fungus melanin active ingredient F1 and application thereof
Technical Field
The invention relates to a low-iron blood replenishing agent, an active ingredient of black fungus melanin and application thereof.
Background
Iron deficiency anemia (Iron Deficiency Anemia, IDA) is anemia arising from depletion of iron reserves in humans resulting in reduced hemoglobin synthesis. Such anemia is characterized by a lack of dyeable iron in bone marrow, liver, spleen and other tissues, and by a decrease in serum iron, transferrin saturation and ferritin. The causes of iron deficiency anemia are numerous, and are generally summarized as: (1) Iron needs to be increased, and occurs frequently in infants and pregnant women; (2) The common reasons for the insufficient iron intake are insufficient iron content and partial food, and phosphate, phytic acid and tannic acid in vegetables, cereals and tea in the food can reduce the absorption of iron in gastrointestinal tracts; (3) Chronic bleeding is caused by a number of causes, and male patients are usually acute and chronic bleeding of the gastrointestinal tract, and female patients are often suffering from menorrhagia. The final result of iron deficiency anemia is that iron deficiency anemia occurs due to iron deficiency in the body, which is a requirement for iron by body metabolism, affecting heme synthesis in blood cells.
As one of the nutritional deficiency type diseases with high incidence rate worldwide, iron deficiency anemia affects about 22 hundred million people worldwide, the incidence rate of the disease in developing countries is obviously higher than that in developed countries, the incidence rate of iron deficiency anemia in all levels and age groups of China is about 20%, and the incidence rate of iron deficiency anemia in children in severe areas is as high as 70%. According to the survey, display: the annual incidence rate of iron deficiency in infants between 6 months and 2 years old is 75.0 percent to 82.5 percent, the proportion of iron deficiency in women over 3 months of gestation is 66.7 percent, the proportion of women of childbearing age is 43.3 percent, and the proportion of teenagers between 10 years old and 17 years old is 13.2 percent; the prevalence of Iron Deficiency Anemia (IDA) in the above population is 33.8% -45.7%, 19.3%,11.4%,9.8% respectively.
When iron deficiency anemia occurs, the body is initially changed to have iron deficiency in a plurality of tissues or organs, the iron deficiency is manifested by iron deficiency, enzyme activity and enzyme activity related to iron in the tissues are obviously reduced, people suffering from the iron deficiency anemia show physical strength, immunity, muscle endurance and the like to be reduced, and the reduction of hemoglobin and red blood cell quantity leads to the reduction of oxygen transporting capacity of blood, so that the tissue hypoxia can be caused, and the symptoms of mental fatigue, mental state reduction, chest distress, short breath, dizziness and the like of patients appear.
The diet of most of the world population is deficient in elemental iron, especially in countries where vegetable foods predominate. In the process of preventing and treating iron deficiency anemia, an oral iron agent is obviously most ideal, and an iron supplement or a nutrition enhancer is currently used as a main method for treating or preventing iron deficiency anemia, and the dietary iron supplement and the oral iron supplement medicine are widely used for preventing and treating iron deficiency anemia due to the simplicity, convenience and easiness in clinical application.
Up to now, there are about 30 kinds of hematinic agents allowed to be used by the world health organization for treating iron deficiency anemia, and these iron supplements are mainly classified into two major classes of inorganic iron and organic iron according to the presence of iron, and into two major classes of heme iron and non-heme iron according to the presence of iron in the human body. However, despite the long-term use and development of these iron supplements, there are some drawbacks and other side effects. Ferrous compounds are the iron supplements with the best iron supplementing effect, but can cause various side effects. It was found that oral iron supplements may increase free radicals generated in the colon leading to mucosal cell damage and may also increase lipid peroxidation in the rat colon, affecting intestinal epithelial cell integrity; it has also been found that iron deficiency and iron overload are both associated with impaired immune responses, as a result of the passage of large amounts of iron into the intestine, which is not absorbed by the human body, after administration of iron supplements. Side effects of long-term iron supplementation include epigastric pain, nausea, diarrhea, constipation, so that the patient has to stop the treatment. In order to treat iron deficiency anemia, patients suffer from high dosage level iron supplementation and a large amount of unabsorbed iron is utilized by intestinal flora along with food and gastrointestinal peristalsis to the large intestine, so that the balance of the intestinal flora is broken, and the intestinal health is adversely affected.
Disclosure of Invention
The invention aims to solve the problems that the existing iron supplement can cause nausea, abdominal pain, diarrhea, constipation, intestinal flora imbalance, harm to gastrointestinal health and the like, and simultaneously greatly reduce the iron content in the preparation while maintaining at least equal blood supplementing effect, and avoid the influence of iron overload on human immune response, thereby providing a low-iron blood supplementing agent, black fungus melanin active ingredient F1 and application thereof.
The black fungus melanin active ingredient F1 is prepared by the following steps:
1. dissolving black fungus melanin in NaOH solution at a concentration of 5mg/mL, and loading the solution on a Sephadex G-200 column;
2. eluting with PBS buffer solution with concentration of 50mmol/L, pH of 7.5, flowing at 1.2mL/min, collecting eluate of 34-50 min, adjusting pH to 2, standing, centrifuging at 4000r/min for 15min, washing precipitate with distilled water, and vacuum freeze-drying to obtain black fungus melanin active ingredient F1;
wherein the concentration of the NaOH solution in the first step is 0.2mol/L.
The preparation method of the black fungus melanin comprises the following steps:
drying Auricularia, pulverizing, and sieving with 60 mesh sieve. Accurately weighing 10g of black fungus powder sample, adding 250mL of hydrochloric acid with the concentration of 3mol/L, stirring and leaching for 1h at 70 ℃, centrifuging the mixture for 15min at 4000r/min, separating supernatant and sediment, collecting sediment, adjusting the pH value to 12 by sodium hydroxide, fully dissolving and stirring, centrifuging for 15min at 4000r/min, taking supernatant, adjusting the pH value to 2 by hydrochloric acid with the concentration of 3mol/L, standing, centrifuging for 15min at 4000r/min to obtain a melanin crude product, hydrolyzing the melanin crude product by HCl with the concentration of 7mol/L at 100 ℃ for 2h, centrifuging for 20min at 10000r/min, washing sediment by chloroform, ethyl acetate and ethanol sequentially, washing sediment by distilled water for 3-5 times, centrifuging again, taking sediment, and freeze-drying to obtain black solid powdered black fungus melanin.
The invention relates to an active ingredient of black fungus melaninThe infrared scanning spectrum of the component F1 is shown in figure 1, the structural characteristics of the component F1 are very similar to those of complex biological macromolecules, and the black fungus melanin active component F1 has a plurality of functional groups so as to enable the infrared scanning spectrum to have a plurality of wide and strong absorption peaks. In infrared spectrum scanning spectrum of black fungus melanin active component F1, 3200cm -1 The broad peak appearing nearby is formed by OH and NH 2 The group is caused by stretching vibration; 2950 cm to 2850cm -1 The absorption peak at this point indicates the presence of aliphatic C-H; 1650-1500 cm -1 Strong absorption at, associated with vibrations of aromatic c=c and COO; 1400-1200 cm -1 The absorbance peak at the position shows the presence of phenolic COH and indole and NH; 800-600 cm -1 The relatively small absorption peak of the range represents the presence of an aromatic ring.
In black fungus melanin active ingredient F1 1 In the H NMR spectrum, the aliphatic region is in the range of 0.8 to 1.0ppm, and the signal appearing is regarded as CH of the alkyl fragment 3 Radicals, e.g. CH 2 CH 3 ，CH(CH 3 ) 2 Possibly from residual proteins; the signal around 2.0ppm is a signal belonging to the methylene group or the ester group (-OCOCOCH) 3 ) Indicating the presence of a carboxylic acid structure; the peak around 4ppm was identified as methyl (N-CH) bound to indole or pyrrole ring 3 ) and-OCH bound to the first position on the ring 3 The method comprises the steps of carrying out a first treatment on the surface of the 6.5 and 7.3ppm of an aromatic hydrogen atom of an indole or pyrrole ring; the signal around 8.5ppm is a carboxyl proton on the indole or pyrrole ring. Wherein the strong signal at the position of 5ppm appears as a peak of H in NaOH in the solvent. Black fungus melanin 1 In the H NMR spectrum, the signal near 2.0ppm is stronger than that of the active ingredient F1 of the black fungus melanin, which indicates that the carboxylic acid structure proportion of the black fungus melanin is higher; the peak area around 4ppm is smaller than that of the black fungus melanin active ingredient F1, representing a smaller proportion of indole or pyrrole rings. It is shown that the black fungus melanin contains more protein, and the black fungus melanin active ingredient F1 contains more indole or pyrrole rings used for forming a framework.
And (3) spraying Au on the black fungus melanin active components F1 and the black fungus melanin, directly imaging by using an environment scanning electron microscope, and observing the microstructure of the black fungus melanin active components F1 and the black fungus melanin.
The black fungus melanin is composed of spherical monomers with irregular surfaces, and is in a layer-by-layer stacked structure, and the average particle size of the black fungus melanin monomers is about 1 μm (as shown in fig. 4) measured by software. The polymerization of a large amount of black fungus melanin monomers is shown in fig. 5, and the black fungus melanin monomers are in a large-volume irregular block shape, and the pores on the surface indicate that the combination of the black fungus melanin is not tight.
The active component F1 of the black fungus melanin has a more obvious lamellar structure, and the surface of the active component F is smoother than that of the black fungus melanin, so that the combination of the monomers is more compact (shown in figure 6). The active component F1 of black fungus melanin is polymerized in a large quantity as shown in fig. 7 to form an irregular block shape with a larger volume, but the surface is smoother and denser than the black fungus melanin, which indicates that the active component F1 of pure black fungus melanin is more tightly combined with the black fungus melanin, and the connection between the monomers is stronger.
The black fungus melanin active component F1 has better stability than black fungus melanin under the illumination condition and the room temperature condition.
The black fungus melanin active ingredient F1 can obviously improve the hemoglobin and the number of red blood cells of an IDA mouse model, improve hematopoietic factors, increase iron absorption and effectively improve iron deficiency anemia. Because the black fungus melanin active component F1 does not contain a large amount of iron elements, free radicals can not be generated in the colon to damage mucosal cells, lipid peroxidation in the colon can not be increased, and the integrity of intestinal epithelial cells is not affected. And the human immune response system is not damaged, and adverse symptoms such as nausea, abdominal pain, diarrhea, constipation and the like are not caused. The black fungus melanin active ingredient F1 can improve the intestinal flora structure of people with iron deficiency anemia and increase the abundance.
The low-iron blood replenishing agent mainly comprises an active ingredient F1 of black fungus melanin.
Wherein the low-iron blood replenishing agent also comprises black fungus and melanin.
The application of black fungus melanin active ingredient F1 as low-iron blood replenishing agent is provided.
The black fungus melanin is used as a low-iron blood replenishing agent.
The application of the black fungus melanin active ingredient F1 as an intestinal flora regulator for patients with iron deficiency anemia.
The application of black fungus melanin as an intestinal flora regulator for patients with iron deficiency anemia.
The black fungus melanin active ingredient F1 can effectively improve the quantity of red blood cells and the concentration of hemoglobin of iron-deficiency anemia mice, and the quantity of red blood cells and the concentration of hemoglobin of iron-deficiency anemia mice of black fungus melanin active ingredient F1 and black fungus melanin are obviously higher than those of IDA groups and are recovered to normal level by oral administration. The recovery effect of the black fungus melanin active ingredient F1 on iron deficiency anemia is not interfered by dietary ingredients.
Drawings
Fig. 1 is an infrared scan of the black fungus melanin active ingredient F1 of the present invention.
FIG. 2 shows the black fungus melanin active ingredient F1 of the present invention 1 H NMR scanning pattern.
FIG. 3 is a black fungus melanin 1 H NMR scanning pattern.
Fig. 4 is a scanning electron microscope image of black fungus melanin.
FIG. 5 is a scanning electron microscope image of a mass of monomeric polymerized black fungus melanin.
Fig. 6 is a scanning electron microscope image of the black fungus melanin active ingredient F1.
Fig. 7 is a scanning electron microscope image of a mass of the monomer-polymerized black fungus melanin active ingredient F1.
FIG. 8 is a graph showing the composition analysis of bacterial flora in the intestinal tract of mice based on total DNA from the feces of mice in experiment 2.
FIG. 9 is a graph showing the results of the preservation rate test of the active ingredient F1 of black fungus melanin and the natural light irradiation condition of black fungus melanin in experiment 3.
FIG. 10 is a graph showing the results of the storage rate test of the active ingredient F1 of black fungus melanin and black fungus melanin at room temperature in experiment 3.
Detailed Description
The technical scheme of the invention is not limited to the specific embodiments listed below, and also includes any combination of the specific embodiments.
The first embodiment is as follows: the black fungus melanin active ingredient F1 of the embodiment is prepared and obtained by the following steps:
1. dissolving black fungus melanin in NaOH solution at a concentration of 5mg/mL, and loading the solution on a Sephadex G-200 column;
2. eluting with PBS buffer solution with concentration of 50mmol/L, pH of 7.5, flowing at 1.2mL/min, collecting eluate of 34-50 min, adjusting pH to 2, standing, centrifuging at 4000r/min for 15min, washing precipitate with distilled water, and vacuum freeze-drying to obtain black fungus melanin active ingredient F1;
wherein the concentration of the NaOH solution in the first step is 0.2mol/L.
The element contents of the black fungus melanin and the black fungus melanin active ingredient F1 are shown in Table 1, and the content of N, C, H, S elements of the black fungus melanin is higher than that of the black fungus melanin active ingredient F1. Wherein, the N content of the black fungus melanin is 10.38 percent higher than that of the active component F1 of the black fungus melanin, which indicates that the black fungus melanin possibly contains more protein, and the result of the Fourier infrared scanning chart is mutually proved.
TABLE 1
Note that: in the table, O% = 100% -N% -C% -H% -S%
The black fungus used in this embodiment is provided by the eastern black fungus industry base, strain number: HDXZ-1.
Hepcidin (Hepcidin) is used as the most critical regulator in iron metabolism regulation (as insulin regulates blood sugar), regulates the metabolic absorption capacity of human body for iron, and the activity of Hepcidin has a negative correlation with the human body iron absorption capacity. The analysis proves that the black fungus melanin active ingredient F1 can act on hepcidin to improve the absorption and utilization of iron.
The low temperature is favorable for preserving the black fungus melanin active ingredient F1, and the preservation rate of the black fungus melanin active ingredient F1 can be reduced after long-time heating; the pH value can obviously influence the solubility of the black fungus melanin active ingredient F1, and the alkaline condition is more favorable for the dissolution of the black fungus melanin active ingredient F1; k (K) + 、Ca 2+ And Mg (magnesium) 2+ Can obviously reduce the absorbance value of the black fungus melanin active component F1, cu 2+ And Fe (Fe) 3+ The absorbance of the black fungus melanin active ingredient F1 is increased; the black fungus melanin active component F1 is easily oxidized by an oxidant, but the reducing agent has no obvious influence on the stability of the black fungus melanin active component F1; the four food additives of sodium benzoate, potassium sorbate, sodium glutamate and butylated hydroxyanisole have little influence on the stability of the black fungus melanin active ingredient F1.
The active ingredient F1 of black fungus melanin takes pyrrole rings and indole rings as skeletons, fatty carbon chains and carbonyl groups are connected to the skeletons, a large number of structures are polymerized to form irregular monomers, and the monomers are polymerized in a lamination mode to form the active ingredient F1 of the black fungus melanin with a smoother surface. The black fungus melanin active component F1 has a strong absorption peak at 207nm, and the content ratio of eumelanin to brownish melanin in the black fungus melanin active component F1 is 10.36:1.
The second embodiment is as follows: the present embodiment differs from the first embodiment in that: in the second step, the Sephadex G-200 is washed and stabilized by PBS buffer solution with the concentration of 50mmol/L, pH of 7.5 and then is sampled. Other steps and parameters are the same as in the first embodiment.
And a third specific embodiment: the second embodiment differs from the first embodiment in that the Sephadex G-200 has a diameter of 1.6cm and a length of 64cm. Other steps and parameters are the same as in one or more embodiments.
The specific embodiment IV is as follows: the low-iron blood replenishing agent of the embodiment mainly comprises an active ingredient F1 of black fungus melanin.
The low-iron blood replenishing agent of the embodiment can also comprise black fungus melanin; the mass ratio of the black fungus melanin to the black fungus melanin active component F1 is 1-100:1.
Fifth embodiment: the black fungus melanin active ingredient F1 of the embodiment is applied as a low-iron blood replenishing agent.
Specific embodiment six: the black fungus melanin of the embodiment is applied as a low-iron blood replenishing agent.
Seventh embodiment: the application of the black fungus melanin active ingredient F1 in the embodiment as the intestinal flora regulator for patients with iron deficiency anemia.
Eighth embodiment: the black fungus melanin of the embodiment is applied to an intestinal flora regulator for patients with iron deficiency anemia.
Experiment 1:
1.1 method for modeling iron deficiency anemia mice model
Referring to the method in the implementation manual of the technical specifications for testing and evaluating health food, the test adopts 24-day-old male mice of Kunming species with the weight of 14.0+/-1.7 g, 130 mice are randomly selected to start feeding low-iron feed after the mice are adapted for 3-5 days in a laboratory, deionized water is drunk, and a non-iron cage cover and a plastic cage body are adopted, so that iron pollution is strictly avoided. 10 mice are randomly selected after two weeks to collect blood from the tail, an automatic hemocytometer is adopted to measure the hemoglobin content of the mice, and if the mice are fed with low-iron feed to test that the average hemoglobin (Hb) of the mice is less than or equal to 100g/L, the molding is successful.
1.2 mouse rearing and grouping
Mice were kept in the animal culture room of the university of Heilongjiang student's life sciences laboratory. Laboratory facility conditions and daily animal management work are implemented according to the environmental and facility standards of the Ministry of health of the people's republic of China, the environmental temperature is 20+/-2 ℃, the relative humidity of the environment is 45-65%, and the daily replacement is automatically controlled for 12 hours.
After successful molding, the mold is randomly divided into 4 groups, 30 groups are respectively iron deficiency anemia group (IDA), iron deficiency anemia+black fungus melanin group (IDA+M), iron deficiency anemia+black fungus melanin active ingredient F1 group (IDA+F) 1 ) Iron deficiency anemia + ferrous sulfate group (IDA + Fe 2+ ) The method comprises the steps of carrying out a first treatment on the surface of the Another 30 normal healthy male mice of 29 days old were taken as normal Control group (Control).
Distilled water for gastric lavage of Control group and IDA group, and melanin for gastric lavage of IDA+M group with dosage of 200 mg/kg.d, IDA+F 1 Group lavage of melanin F 1 The administration dosage is 179.51 mg/kg.d, IDA+Fe 2+ The administration dosage of the group-lavage ferrous sulfate group is 5 mg/kg.d, and the stomach is continuously lavaged. The normal group is always fed with normal feed, and each iron-deficiency anemia group is always fed with low-iron feed. From the start of the experiment, all groups of mice were measured for body weight every 7d and recorded. On the day of stopping gastric lavage, mouse eyeball blood is taken for routine analysis of peripheral blood, and measurement of serum Erythropoietin (EPO), stem Cell Factor (SCF), serum specific transcription factor GATA-1 and Serum Iron (SI) content.
Experimental observation:
in the molding process, the mice begin to have phenomena of hair falling, sparse fur, pale foot paw and tail skin, reduced activity, wilting, aversion to cold and the like, and the phenomena are more obvious along with the extension of the test, and the growth is slow, so that the fur loses luster. As can be seen from the blood routine index, the hemoglobin (Hb) content, the red blood cell count (RBC), the Hematocrit (HCT) and the platelet count (PLT) of the mice after the molding were reduced by 47.5%, 36.89%, 37.58% and 64.46%, respectively, compared with the Control group, and the results showed statistically significant differences (p<0.01 Mean erythrocyte volume (MCV) was reduced by 16.15% compared to Control group, with significant differences (p<0.05). The average hemoglobin content (Hb) of IDA group mice is less than or equal to 100g/L, and the red blood cell number (RBC) is less than or equal to 7.7X10 12 The volume of red blood cells (HCT) is less than or equal to 36 percent, and the average volume of red blood cells (MCV) is less than or equal to 48 mu m 3 Platelet count (PLT) is less than or equal to 157×10 6 and/L. Indicating that the mice fed with the low-iron feed have obvious anemia, which indicates successful modeling.
TABLE 2 peripheral blood image of mice at the end of modeling
Hb(g/L) RBC(10 12 /L) HCT(％) MCV(μm 3 ) PLT(10 6 /L)
Normal control group 166.67±15.28 10.93±0.74 51.67±4.73 51.13±2.06 192.67±29.56
Model group 87.50±12.58** 6.90±0.63** 32.25±3.59** 42.88±2.90* 68.50±24.84**
Reference value 100～190 7.7～12.5 36～50 48～51 157～260
The normal group is always fed with normal feed, and each iron deficiency anemia group is always fed with the feedLow iron feed. The mice were filled with distilled water at 2 weeks prior to the experiment, the mice were in the rapid growth phase, the body weight of each iron deficiency anemia group and the normal feed group increased steadily, and there was no difference between the groups (p<0.05). The corresponding gastric lavage (IDA+M group, administration of gastric lavage melanin at 200 mg/kg.d, IDA+F) was started from week 3 1 Group lavage of melanin F 1 The administration dosage is 179.51 mg/kg.d, IDA+Fe 2+ The weight of each group of mice is steadily increased along with the time, but the increasing speeds are different, the weight of the Control group is fastest to increase, and the weight of the IDA+M group is inferior, and the weight of the IDA+F group is inferior 1 Group mice grew at a slightly slower rate than ida+m groups, ida+fe 2+ Group body weight gain rate was lower than ida+f 1 Group IDA group had lower body weight than the remaining groups, being the lowest of the groups.
Blood of each group of mice is conventionally shown in table 3. IDA+F 3 weeks after drug administration gastric lavage intervention 1 Group mice had an average increase in red blood cell count, hemoglobin amount, hematocrit, average red blood cell volume, average hemoglobin amount, average hemoglobin concentration, platelets by 43.56%, 94.34%, 60%, 6.31%, 14.33%, 5.42%, 21.81% (p)<0.05 At the same time no significant difference from the Control group (p>0.05 A normal level has been reached; the black fungus melanin active ingredient F1 can effectively improve iron deficiency anemia of mice. The black fungus melanin active ingredient F1 can effectively improve the quantity of red blood cells and the concentration of hemoglobin of iron-deficiency anemia mice, and the quantity of red blood cells and the concentration of hemoglobin of iron-deficiency anemia mice with the gastric-irrigation black fungus melanin active ingredient F1 and black fungus melanin are obviously higher than those of IDA groups and are recovered to the level of Control groups. The hemoglobin of mice in the IDA group still keeps a low level of red blood cells, and the mice are in an anemic state, so that the mice are continuously fed with low-iron feed during the test period, the anemia is not recovered due to normal diet recovery, and the effect of the black fungus melanin active ingredient F1 on iron-deficiency anemia is verified from the side surface, and the anemia is recovered due to no diet change.
The conventional detection data of the blood of the mice reflect that the black fungus melanin active ingredient F1 and the black fungus melanin have the effects of improving the hematocrit, the red cell volume, the average hemoglobin amount and the average hemoglobin concentration.
TABLE 3 Table 3
Control IDA IDA+Fe 2+ IDA+M IDA+F1
WBC(10 9 /L) 6.42±0.90a 1.82±0.50c 3.28±1.06b 6.45±1.20a 6.80±0.86a
RBC(10 12 /L) 10.10±0.82b 7.15±0.74c 10.88±0.92ab 11.38±1.37a 10.22±1.31b
Hb(g/L) 155.00±15.17bc 85.00±15.17d 156.67±8.16bc 171.67±7.53ab 171.67±11.69ab
HCT(％) 51.67±6.22a 38.83±4.75b 51.17±4.36a 54.50±8.02a 52.00±6.72a
MCV(fL) 51.08±2.69a 47.58±2.81b 47.05±2.52b 52.63±1.55a 50.30±3.10a
MCH(pg) 15.37±0.54ab 13.88±1.33c 14.43±0.58bc 15.90±0.47a 15.15±1.67ab
MCHC(g/L) 300.67±8.82cd 291.33±12.31d 307.17±18.03c 340.67±9.56a 314.50±14.27b
PLT(10 9 /L) 818.33±262.94ab 653.33±269.79b 1058.33±132.27a 885.00±227.93ab 806.17±109.43b
The low-iron feed is continuously fed during the test period, so that the factor that the anemia symptoms of the mice are recovered and interfered by dietary components is eliminated.
Experiment time 3 weeks, IDA+Fe 2+ The average red blood cell volume of mice in the group is obviously reduced compared with that of mice in the Control group, the serum stem cell factor is reduced by 3.86 percent, the concentration of serum erythropoietin is obviously reduced by 11.05 percent, and the concentration of serum specific green transfer factor GATA-1 is obviously reduced by 20.26 percent, which shows that ferrous sulfate is likely to damage the hematopoietic function of organisms except gastrointestinal discomfort symptoms when treating iron deficiency anemia, and has larger potential danger; and IDA+M group and IDA+F 1 None of the group mice developed the symptoms described above. The experimental time was 3 weeks, although ida+fe 2+ The group mice had a greater return in leukocyte count than the IDA group, but were still significantly lower than the Control group, IDA+M group and IDA+F group 1 The experiment was carried out with the time prolonged to 2 months, IDA+Fe 2+ The leukocyte count of the mice in the group was maintained at (3.46.+ -. 1.21). Times.10 9 L is not improved; and IDA+Fe 2+ Group mice also had diarrhea, constipation, dysphoria, etc., whereas IDA+M group and IDA+F group 1 None of the mice in the group showed similar symptoms.
The hematopoietic factor data for each group of mice is shown in table 4.
TABLE 4 Table 4
Control IDA IDA+Fe 2+ IDA+M IDA+F1
SCF 751.90±50.92bc 827.31±64.84b 610.48±98.13d 904.01±53.30a 889.91±52.19a
EPA 136.57±13.64c 137.62±11.62c 154.65±11.38b 182.28±17.70a 181.70±14.66a
GATA-1 11.35±2.12ab 10.72±1.02c 10.66±1.62c 13.00±0.42a 13.08±1.53a
SI 1.24±0.06b 0.75±0.04d 1.20±0.14c 1.44±0.06a 1.41±0.14a
Stem cell factor SCF is an important hematopoietic cytokine, and serum SCF is responsible for hematopoietic dysfunction and plays a key role in the adhesion of hematopoietic stem cells and early progenitor cells to the bone marrow hematopoietic microenvironment. IDA+M group and IDA+F group compared to IDA group 1 The concentration of the serum stem cell factor of the group is obviously increased by 12.89 percent and 10.21 percent respectively (p)<0.05 Ida+fe) 2+ There was no significant difference in the concentration of group serum stem cell factor (p>0.05). And compared with the Control group, IDA+M group and IDA+F group 1 The concentration of the serum stem cell factor of the group is obviously increased by 19.20 percent and 16.37 percent (p)<0.05 While the serum stem cell factor concentration of IDA group was slightly elevated, but not significant (p>0.05)。
Therefore, when iron deficiency anemia occurs in the organism, the stress reaction of the organism can cause the concentration of serum stem cell factors of the organism to be increased, but the increase is not obvious, and when iron deficiency anemia occurs, the concentration of serum stem cell factors of black fungus melanin and black fungus melanin active ingredients F1 can be obviously increased, which indicates that the black fungus melanin active ingredients F1 and black fungus melanin have better functions of promoting the production of serum stem cell factors.
Erythropoietin (EPO) is a glycoprotein hormone mainly produced by kidneys, is a main regulatory factor involved in erythroid hematopoiesis, and has the effects of promoting proliferation and differentiation of erythroid progenitor cells and maintaining erythrocyte and hemoglobin stability. IDA+M group and IDA+F group compared to IDA group 1 The serum erythropoietin concentration of the mice in the group is obviously improved by 28.48 percent and 26.25 percent (p)<0.05 Ida+fe), however 2+ The concentration of serum erythropoietin in group mice did not significantly differ from that in IDA group (p>0.05 A) is provided; when iron deficiency anemia occurs, the serum erythropoietin concentrate of the miceThe degree was not lowered, but there was no significant difference in Control group (p>0.05). Thus, when iron deficiency anemia occurs, administration of black fungus melanin active ingredient F1 and black fungus melanin increases serum erythropoietin concentration.
Serum Iron (SI) refers to iron bound to transferrin in blood, which is a complex formed by binding to transferrin, and is used to identify iron-deficiency as well as non-iron-deficiency anaemia. The binding and transport capacity of transferrin to iron is regulated by and inversely related to the regulatory factor hepcidin. IDA+F 1 There was no significant difference in serum iron concentration between group and Control and ida+m groups (p>0.05 All maintained at normal levels; IDA+M group and IDA+F group compared to IDA group mice 1 The serum iron concentration of group mice was significantly increased (p<0.05 87.53% and 84.68%, respectively; and IDA+Fe 2+ The serum iron concentration of group mice was not recovered, no significant difference from IDA group (p>0.05). Thus, for IDA mice, supplementation with black fungus melanin active ingredient F1 and black fungus melanin can result in recovery of serum iron concentration.
In conclusion, the black fungus melanin active ingredient F1 and black fungus melanin can improve the anemia condition, and because the existing iron supplementing and blood supplementing agents are added with a large amount of iron sources, the iron deficiency and anemia symptoms of the human body are improved through the supplement of a large amount of iron elements. Once the medicine is stopped, iron-deficiency anemia is easy to relapse, and the continuous taking of the medicine for a warehouse time adversely affects the hematopoietic function of a human body. The invention does not use iron supplement for increasing the iron source simplicity, and the active ingredients F1 of the black fungus melanin and the black fungus melanin can activate and improve the concentration of serum stem cell factors in the body, so that the effects of iron supplement and blood supplementing can be continuously and for a long time after the administration, and the invention has the advantages of long acting time, difficult recurrence of iron deficiency anemia and safety and effectiveness after long-time administration.
Experiment 2:
2.1 method for modeling iron deficiency anemia mice model
Referring to the method in the implementation manual of the technical specifications for testing and evaluating health food, the test adopts 24-day-old male mice of Kunming species with the weight of 14.0+/-1.7 g, 70 mice are randomly selected to start feeding low-iron feed after the mice are adapted for 3-5 days in a laboratory, deionized water is drunk, and a non-iron cage cover and a plastic cage body are adopted, so that iron pollution is strictly avoided. 10 mice are randomly selected after two weeks to collect blood from the tail, an automatic hemocytometer is adopted to measure the hemoglobin content of the mice, and if the mice are fed with low-iron feed to test that the average hemoglobin (Hb) of the mice is less than or equal to 100g/L, the molding is successful.
2.2 mouse rearing and grouping
Mice were kept in the animal culture room of the university of Heilongjiang student's life sciences laboratory. Laboratory facility conditions and daily animal management work are implemented according to the environmental and facility standards of the Ministry of health of the people's republic of China, the environmental temperature is 20+/-2 ℃, the relative humidity of the environment is 45-65%, and the daily replacement is automatically controlled for 12 hours.
After successful molding, the mold is randomly divided into 3 groups, 20 groups are respectively iron deficiency anemia group (IDA), iron deficiency anemia and black fungus melanin active ingredient F1 group (IDA+F) 1 ) Iron deficiency anemia + ferrous sulfate group (IDA + S); another 20 normal healthy male mice of 29 days old were taken as normal Control group (Control).
After 4 weeks of the experiment, normal groups are always fed with normal feed, and each iron-deficiency anemia group is always fed with low-iron feed. Control group and IDA group mice lavage distilled water, IDA+F 1 Group lavage of melanin F 1 The administration dosage is 179.51 mg/kg.d, and the administration dosage is 5 mg/kg.d for the IDA+S group of intragastric ferrous sulfate group.
2.3 extraction of total DNA from faeces of mice of each group
Experimental observation:
the composition of the bacterial community of the intestinal flora of each group of mice is analyzed and obtained according to the total DNA of the feces of each group of mice as shown in figure 8.
Bacteria of the S24-7_norank species are the most abundant flora in the intestinal tracts of mice of the normal group, the normal+ferrous sulfate group, the normal+black fungus melanin group and the iron deficiency anemia+black fungus melanin group, and the abundance values are 23.48%, 46.73%, 24.85% and 24.77% respectively; however, in the intestinal tracts of mice in the iron deficiency anemia group and iron deficiency anemia+ferrous sulfate group, the most abundant bacteria were bacteria of the genus allobaculom, with abundance values of 30.74% and 29.65%, respectively. The second abundance of bacteria is Alistipes in the normal group, and the abundance is 10.73%; the normal+ferrous sulfate group and the normal+black fungus melanin group are Ruminocarpeae_unctulted and Ruminocarpeae_unclassified respectively, and the abundance is 7.81 percent and 11.36 percent respectively; s24-7_norank is adopted in the iron deficiency anemia group and the iron deficiency anemia and ferrous sulfate group, and the abundance is respectively 20.90% and 17.21%; the iron deficiency anemia plus black fungus melanin group is Bactroides, and the abundance is 15.99%; the third group of bacteria with abundance is Alistipes in the normal+ferrous sulfate group and the iron deficiency anemia+ferrous sulfate group, and the abundance is 6.90% and 14.27% respectively; the abundance is 9.51% in the normal control group, 9.09% in the normal+auricularia melanin group; in the iron deficiency anemia group, bactroides, abundance was 9.60%; the iron deficiency anemia group is allobaculom, and the abundance is 9.53%.
The black fungus melanin active ingredient F1 can twist the abundance of 6 main species bacteria such as S24-7_normal, alistines, ruminococaceae_unctuted, blauthia, ruminococaceae_unctuted, prevoltellaceae_unctuted and the like in the intestinal canal of the iron-deficiency anemia mice. The black fungus melanin active ingredient F1 can reduce the abundance of Mycoplasma Albaculum and Parabacteriaceae Parabacteroides in the intestinal canal of mice with iron deficiency anemia.
When iron deficiency anemia occurs, the abundance of the main flora in the intestinal tract of the mice is obviously changed, and the black fungus melanin active ingredient F1 can play a role in regulating the abundance change of the main flora so as to recover the main flora; ferrous sulfate does not achieve the above-mentioned effects.
The black fungus melanin active ingredient F1 does not have obvious differential influence on the abundance of the species classification level of the normal mouse intestinal flora relative to ferrous sulfate, but has obvious differential influence on the abundance of the intestinal flora of the iron deficiency anemia mouse.
The overall difference analysis of the intestinal flora of the mice shows that the black fungus melanin active ingredient F1 can reduce the difference between the intestinal flora of the mice with iron deficiency anemia and the normal mice, and ferrous sulfate has no effect.
According to the analysis of the microbial diversity index of the intestinal flora of the mice, when iron deficiency anemia occurs, the abundance, uniformity and diversity index of the intestinal flora of the mice are reduced, and the abundance, uniformity and diversity of the intestinal flora of the mice are increased when the black fungus melanin active ingredient F1 is given to the iron deficiency anemia mice and normal mice; ferrous sulfate was given to iron-deficiency anaemia mice with only a slight increase in the abundance, uniformity and diversity index of the intestinal flora.
Analysis of intestinal metabolites in mice: when iron deficiency anemia occurs, the content of propionic acid, butyric acid and lactic acid in the intestinal tract of the mice is obviously reduced, and is lower than that of a normal control group, and the content of acetic acid is not obviously changed. The black fungus melanin active ingredient F1 can obviously improve the content of propionic acid, butyric acid and lactic acid in the intestinal tracts of mice with iron deficiency anemia, and the ferrous sulfate can obviously improve the content of lactic acid in the intestinal tracts of the mice, but the content of propionic acid and butyric acid in the intestinal tracts of the mice is obviously lower than that of the mice with iron deficiency anemia.
Experiments prove that
The black fungus melanin active ingredient F1 improves the abundance, uniformity and diversity index of intestinal flora of the iron-deficiency anemia mice, reduces the difference between the iron-deficiency anemia mice and the normal mice, and has a certain effect of adjusting the intestinal flora. The black fungus melanin active ingredient F1 can obviously improve the concentration of metabolites such as propionic acid, butyric acid, palmitic acid, stearic acid, oleic acid, amino acid and the like in the intestinal tract of the iron deficiency anemia mice, and can reduce the difference of intestinal metabolites of the anemia mice and normal mice as a whole, so that the abnormal changes of the intestinal metabolites of the anemia mice are recovered and gradually tend to be normal.
Experiment 3: performance comparative test of black fungus melanin active ingredient F1 and black fungus melanin:
3.1 illumination
The natural light irradiation was conducted at 4W (the preservation rate measurement result is shown in fig. 9), and the preservation rates of both the black fungus melanin active ingredient F1 and black fungus melanin showed a decrease trend, and the differences from the initial values were significant (p < 0.05). The damage of illumination to the black fungus melanin increases with time, probably because the illumination changes the structure of each component of the black fungus melanin to different degrees, so that the preservation rate of the black fungus melanin is reduced to different degrees. Therefore, the black fungus melanin is sensitive to illumination and needs to be preserved in dark. The comparison shows that the stability of the black fungus melanin active ingredient F1 is better than that of the black fungus melanin under the illumination condition, and the storage rate of the black fungus melanin active ingredient F1 and the black fungus melanin active ingredient F1 are obviously different from the storage rate of the black fungus melanin active ingredient F1 under the illumination condition and the 4W storage rate of the black fungus melanin active ingredient F is obviously different (p is more than 0.05). The stability of the black fungus melanin active component F1 to light irradiation is better than that of the black fungus melanin.
3.2 room temperature
The measurement results of the black fungus melanin active ingredient F1 and the black fungus melanin in the environment at 25 ℃ showed no significant difference in the preservation rate of the black fungus melanin active ingredient F1 (p > 0.05) with the lapse of time, and the decrease in the preservation rate of the black fungus melanin between the respective periods (p < 0.05) were shown in fig. 10. The black fungus melanin active ingredient F1 was demonstrated to have more excellent room temperature shelf life than black fungus melanin under room temperature conditions.

Claims (6)

1. The application of the black fungus melanin active ingredient F1 in preparing the medicine for treating iron deficiency anemia is characterized in that the black fungus melanin active ingredient F1 is an active ingredient in the medicine;
the black fungus melanin active ingredient F1 is prepared and obtained by the following steps:
1. dissolving black fungus melanin in NaOH solution at a concentration of 5mg/mL, and loading the solution on a Sephadex G-200 column;
2. eluting with PBS buffer solution with concentration of 50mmol/L, pH of 7.5, and flow rate of 1.2mL/min, collecting eluate 34-50 min, adjusting pH to 2, standing, centrifuging at 4000r/min for 15min, washing precipitate with distilled water, and vacuum freeze-drying to obtain black fungus melanin active ingredient F1;
wherein the concentration of the NaOH solution in the first step is 0.2mol/L.
2. The use of black fungus melanin active ingredient F1 in the preparation of a medicament for treating iron deficiency anemia according to claim 1, wherein in step two, sephadex G-200 is washed and stabilized with PBS buffer solution having a concentration of 50mmol/L, pH of 7.5 before loading.
3. The use of black fungus melanin active ingredient F1 in the preparation of a medicament for treating iron deficiency anemia according to claim 1, wherein in step two, sephadex G-200 has a diameter of 1.6cm and a length of 64cm.
4. The application of the black fungus melanin active ingredient F1 in preparing the medicine for regulating intestinal flora of patients with iron deficiency anemia is characterized in that the black fungus melanin active ingredient F1 is an active ingredient in the medicine;
the black fungus melanin active ingredient F1 is prepared and obtained by the following steps:
1. dissolving black fungus melanin in NaOH solution at a concentration of 5mg/mL, and loading the solution on a Sephadex G-200 column;
2. eluting with PBS buffer solution with concentration of 50mmol/L, pH of 7.5, and flow rate of 1.2mL/min, collecting eluate 34-50 min, adjusting pH to 2, standing, centrifuging at 4000r/min for 15min, washing precipitate with distilled water, and vacuum freeze-drying to obtain black fungus melanin active ingredient F1;
wherein the concentration of the NaOH solution in the first step is 0.2mol/L.
5. The use of black fungus melanin active ingredient F1 in preparing medicine for regulating intestinal flora of iron deficiency anemia patients according to claim 4, wherein in the second step, sephadex G-200 is washed and stabilized by PBS buffer with concentration of 50mmol/L, pH of 7.5, and then loaded.
6. The use of black fungus melanin active ingredient F1 in preparing medicine for regulating intestinal flora of iron deficiency anemia patients according to claim 4, wherein in the second step, sephadex G-200 has a diameter of 1.6cm and a length of 64cm.
CN201810038176.4A 2018-01-16 2018-01-16 Low-iron blood replenishing agent, black fungus melanin active ingredient F1 and application thereof Active CN108261427B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810038176.4A CN108261427B (en) 2018-01-16 2018-01-16 Low-iron blood replenishing agent, black fungus melanin active ingredient F1 and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810038176.4A CN108261427B (en) 2018-01-16 2018-01-16 Low-iron blood replenishing agent, black fungus melanin active ingredient F1 and application thereof

Publications (2)

Publication Number Publication Date
CN108261427A CN108261427A (en) 2018-07-10
CN108261427B true CN108261427B (en) 2023-09-15

Family

ID=62775529

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810038176.4A Active CN108261427B (en) 2018-01-16 2018-01-16 Low-iron blood replenishing agent, black fungus melanin active ingredient F1 and application thereof

Country Status (1)

Country Link
CN (1) CN108261427B (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012129047A1 (en) * 2011-03-18 2012-09-27 Albert Einstein College Of Medicine Of Yeshiva University Oral administration of melanin for protection against radiation
CN104382953A (en) * 2014-11-28 2015-03-04 哈尔滨墨医生物技术有限公司 Extraction method of melanin in black fungi
CN104672407A (en) * 2015-02-05 2015-06-03 西南林业大学 Preparation method of natural melanin-acrylic acid composite superabsorbent resin

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012129047A1 (en) * 2011-03-18 2012-09-27 Albert Einstein College Of Medicine Of Yeshiva University Oral administration of melanin for protection against radiation
CN104382953A (en) * 2014-11-28 2015-03-04 哈尔滨墨医生物技术有限公司 Extraction method of melanin in black fungi
CN104672407A (en) * 2015-02-05 2015-06-03 西南林业大学 Preparation method of natural melanin-acrylic acid composite superabsorbent resin

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
《Isolation and characterization of melanin pigment from Pleurotus cystidiosus (telomorph of Antromycopsis macrocarpa)》;P. Selvakumar等;《World Journal of Microbiology and Biotechnology volume》;20080320;第2125–2131页 *
《Production of natural edible melanin byAuricularia auricula and its physicochemical properties》;SUN,Shujing等;《FOOD CHEMISTRY》;20160430;第486-492页 *
《食用黑木耳治疗缺铁性贫血》;张传儒;《中国民间疗法》;19961228;第47页 *
《鱿鱼墨黑色素铁对大鼠缺铁性贫血的治疗作用》;雷敏等;《中国药科大学学报》;20071225;第539-543页 *
《黑木耳黑色素对细菌群体感应调控行为的抑制及其抗氧化活性的研究》;李斌;《中国博士学位论文全文数据库 农业科技辑》;20140215;摘要 *
《黑龙江省黑木耳加工进展与开发前景》;李家磊等;《中国食物与营养》;20140828;第33-35页 *
黑木耳黑色素组分分析及其抗氧化活性研究;邹宇等;《食品科学》;20130507(第23期);第138-141页 *

Also Published As

Publication number Publication date
CN108261427A (en) 2018-07-10

Similar Documents

Publication Publication Date Title
Gutyj et al. Morphological and biochemical indicators of blood of rats poisoned by carbon tetrachloride and subject to action of liposomal preparation
Bauchart-Thevret et al. Sulfur amino acid deficiency upregulates intestinal methionine cycle activity and suppresses epithelial growth in neonatal pigs
CN102885310B (en) Health care food composition capable of assisting in improving memory, and preparation method thereof
Srikantia et al. Megaloblastic anaemia of infancy and vitamin B12
Zhang et al. Effects of dietary xylooligosaccharide on growth performance, enzyme activity and immunity of juvenile grass carp, Ctenopharyngodon idellus
Janczyk et al. Evaluation of nutritional value and safety of the green microalgae Chlorella vulgaris treated with novel processing methods
EP1362587A1 (en) Use of glutamine for treating low arginine levels in the animal or human body
WO2019162425A1 (en) New process of preparation of glycan composition &amp; uses thereof
CN108261427B (en) Low-iron blood replenishing agent, black fungus melanin active ingredient F1 and application thereof
KR101388395B1 (en) Composition for improving nutritional status, reducing frequency of fever and/or increasing immunocompetence of elderly
El-Shenawy et al. Impact of carbohydrate to lipid ratio and bile salts supplementation on performance, body gain and body composition of Nile tilapia fish
CN115530380B (en) Small molecule composite peptide composition and preparation method and application thereof
CN115989873A (en) Nutritional composition for pregnant and lying-in women and preparation method and application thereof
CN1911103A (en) Health-care food for improving human body physical agility and endurance
CN112451651A (en) Application of chlorella pyrenoidosa peptide
CN102406174A (en) Functional food composite and application
CN102578499B (en) Nutritive health-care black soybean food formula and use thereof
RU2079307C1 (en) Antianemic agent
CN105341938B (en) The health food of rate of respiratory infection after a kind of reduction strenuous exercise
CN114868842B (en) Coated nutritional feed additive for preventing anemia and promoting immune function of piglets and preparation method and application thereof
Ahmed Effects of using L-arginine on production and physiological performance of rabbits
NL2032797B1 (en) Application of glycyrrhiza uralensis and lactic acid bacteria in preventing and/or alleviating damage of moldy feeds and additive thereof
RU2794424C1 (en) Method of application of prebiotic lactulose-containing feed additive in rabbit diets
CN112493487B (en) Composition for enhancing human immunity, preparation method thereof and health-care product
JPS6313407B2 (en)

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant