Composition for treating anemia and preparation method thereof

Abstract

The present invention proposes a composition for treating anemia and a preparation method thereof, and belongs to the field of medical technology. Millettia reticulata, Angelica sinensis, and Panax notoginseng are extracted and precipitated with water to obtain a composite polysaccharide, and the solid residue is fermented by iron-rich yeast and Lactobacillus plantarum to separate the fermented bacteria and the fermented product, and the fermented bacteria are hydrolyzed with snail enzyme and lysozyme to obtain an iron-rich lysozyme product, and succinic acid is activated and coupled with lactoferrin and vitamin C to obtain lactoferrin succinic acid / vitamin C iron, which is mixed with the fermented product and the iron-rich lysozyme product, and embedded in a carboxylated composite polysaccharide and sodium alginate microspheres to obtain a composition for treating anemia. The present invention has high bioavailability, and has substantially no irritation to the gastrointestinal mucosa, is highly safe, has obvious effects on rapid iron supplementation for anemia in pregnant women and promotes bone marrow blood regeneration, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a composition for treating anemia and a preparation method thereof. Background Art

[0002] Iron deficiency anemia during pregnancy is a common complication during pregnancy, which is related to poor eating habits and unbalanced nutrient intake. Iron is an important element to meet the physiological needs of fetal growth and development. The occurrence of iron deficiency anemia during pregnancy will not only affect the patient's immunity, but also increase the risk of adverse maternal and infant outcomes such as fetal distress and low birth weight, which needs to be taken seriously.

[0003] Currently, folic acid, iron preparations, etc. are mostly used in the clinical treatment of iron deficiency anemia during pregnancy. Among them, ferrous succinate tablets are oral iron supplements, which mainly play an iron-replenishing role and can effectively correct iron deficiency anemia. However, the effect of a single medication is limited, and long-term use will cause excessive deposition of iron in the body and increase adverse reactions. In order to improve the patient's condition, it is necessary to focus on combination medication.

[0004] Therefore, developing safe, effective and low-cost treatments for anemia during pregnancy has good clinical application prospects. Summary of the invention

[0005] The purpose of the present invention is to provide a composition for treating anemia and a preparation method thereof, which has no metallic odor, is not in a free state, has relatively stable water solubility, can directly transport iron ions into the body, and then rapidly increase the ferritin level, is conducive to the normal development of the fetus, and reduces the occurrence of adverse pregnancy outcomes. The iron element contained in the composition exists in molecular form and will not be decomposed during absorption. Therefore, when passing through the digestive tract, the effective ingredients in the drug are not affected by gastric acid and will not be released into divalent iron. The composition has high bioavailability and has basically no irritation to the gastrointestinal mucosa. The composition has high safety, has obvious effects on rapid iron supplementation for anemia in pregnant women and promoting bone marrow blood regeneration, and has broad application prospects.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The invention provides a preparation method of a composition for treating anemia. The method comprises the following steps: extracting millettia spatholobi, angelica sinensis and panax notoginseng with water and subjecting them to alcohol precipitation to obtain a composite polysaccharide; fermenting solid residue with iron-rich yeast and lactobacillus plantarum to separate the fermented bacteria and fermented product; hydrolyzing the fermented bacteria with snail enzyme and lysozyme to obtain an iron-rich lysozyme product; activating succinic acid and coupling it with lactoferrin and vitamin C to chelate iron to obtain lactoferrin succinic acid / vitamin C iron; mixing the lactoferrin succinic acid / vitamin C iron with the fermented product and the iron-rich lysozyme product, and embedding the lactoferrin succinic acid / vitamin C iron in carboxyl-modified composite polysaccharide and sodium alginate microspheres to obtain the composition for treating anemia.

[0008] As a further improvement of the present invention, the following steps are included:

[0009] S1. Millettia reticulata, Angelica sinensis, Panax notoginseng were washed, crushed, dried, added to water, heated to boiling for extraction, filtered, the solid residue was retained, ethanol was added to the filtrate for precipitation, the solid was washed, and dried to obtain a complex polysaccharide;

[0010] S2. adding the composite polysaccharide to water, adding α-ketoglutaric acid, stirring to react, then adding sodium borohydride, continuing the reaction, then adding ethanol to precipitate, filtering, washing the solid, and drying, adding the dried product and sodium hydroxide to water, adding chloroacetic acid, heating and stirring to react, adding ethanol to precipitate, filtering, washing the solid, and drying to obtain a carboxylated modified composite polysaccharide;

[0011] S3. The solid residue in step S1 is added to water, sterilized, inoculated with iron-rich yeast and Lactobacillus plantarum seed solution, fermented, filtered, solid and filtrate obtained, the solid is washed, the bacterial solution is collected, freeze-dried, fermented bacteria are obtained, and the filtrate is freeze-dried to obtain a fermentation product;

[0012] S4. The fermentation bacteria were added to water, snail enzyme and lysozyme were added, enzymatic hydrolysis was performed, the enzyme was inactivated, the filtrate was filtered, and the filtrate was freeze-dried to obtain an iron-rich lysozyme;

[0013] S5. The succinic acid was added to water, NHS and EDC were added, and the mixture was stirred for activation. Lactoferrin and vitamin C were added, iron salt was added, the reaction was stirred, dialyzed, and freeze-dried to obtain lactoferrin succinic acid / vitamin C iron;

[0014] S6. Add the fermented product, iron-rich lysing yeast and lactoferrin succinic acid / vitamin C iron into water, add carboxylated modified complex polysaccharide, lecithin and sodium alginate, stir and mix evenly, add dropwise into fish oil, emulsify, add dropwise calcium chloride solution, solidify at room temperature, centrifuge, wash and dry to obtain a composition for treating anemia.

[0015] As a further improvement of the present invention, the mass ratio of Millettia spatholobi, Angelica sinensis and Panax notoginseng in step S1 is 3-5:7-10:4-6, and the heating and boiling extraction time is 3-5h.

[0016] As a further improvement of the present invention, the mass ratio of the complex polysaccharide, α-ketoglutaric acid, sodium borohydride, sodium hydroxide and chloroacetic acid in step S2 is 10:3-3.5:0.7-0.9:2.7-3:0.2-0.25, the stirring reaction temperature is room temperature, the time is 20-24h, the continued reaction time is 8-10h, and the heating stirring reaction temperature is 55-65°C, and the time is 8-12h.

[0017] As a further improvement of the present invention, the solid-liquid ratio of the solid residue to water in step S3 is 1:5-7 g / mL, the inoculation amounts of the iron-rich yeast and plant lactobacillus seed solutions are 2-4 v / v% and 1-3 v / v%, respectively, and the bacterial content of the seed solution is 10 8 -10 9 cfu / mL, the fermentation culture conditions are 30-35°C, 100-200r / min, and the fermentation culture is 24-36h.

[0018] As a further improvement of the present invention, the mass ratio of the fermentation bacteria, snail enzyme and lysozyme in step S4 is 10:0.5-1:0.2-0.3, and the enzymolysis conditions are 35-45°C and the time is 2-4h.

[0019] As a further improvement of the present invention, the mass ratio of succinic acid, NHS, EDC, lactoferrin, vitamin C and iron salt in step S5 is 2-3:0.5-1:0.5-1:5-7:1-2:3-4, the iron salt is selected from at least one of ferric chloride, ferric sulfate and ferric nitrate, the dialysis bag pore size used in the dialysis is 5-7kDa, the dialysis time is 36-48h, the stirring reaction time is 10-12h, and the stirring activation time is 15-20min.

[0020] As a further improvement of the present invention, the mass ratio of the fermentation product, iron-rich lysing yeast, lactoferrin succinic acid / vitamin C iron, carboxylated modified complex polysaccharide, lecithin and sodium alginate in step S6 is 5-7:10:4-6:12-15:0.5-1:7-10, and the room temperature curing time is 20-30 min.

[0021] The present invention further protects a composition for treating anemia obtained by the above preparation method.

[0022] The present invention further protects the use of the above-mentioned composition for treating anemia in preparing a medicine for treating anemia during pregnancy.

[0023] The present invention has the following beneficial effects:

[0024] Millettia reticulata is bitter, sweet and warm in nature. It enters the liver and kidney meridians and has the effect of "removing blood stasis and generating new blood". It is known as the "holy medicine for blood". Modern pharmacological research has found that its chemical composition is complex, mainly polysaccharides, anthraquinones, sterols, flavonoids, etc., which have the effects of improving hematopoietic function, lowering blood lipids, protecting the liver, anti-inflammatory, and regulating immunity. Angelica sinensis is sweet, spicy and warm in nature. It enters the heart, spleen, and liver meridians. It has the effects of increasing the number of peripheral blood cells, enhancing immunity, delaying the aging of hematopoietic stem cells, anti-inflammatory, anti-tumor, regulating menstruation and relieving pain, and promoting the proliferation and differentiation of hematopoietic stem cells and hematopoietic progenitor cells. Angelica sinensis polysaccharide is one of the main active substances in hematopoiesis. Panax notoginseng is warm in nature, sweet and slightly bitter. It enters the liver and stomach meridians. It has the effects of nourishing blood and anti-inflammatory, and can be anti-oxidant and anti-aging. Panax notoginseng can dilate blood vessels, reduce coronary resistance, increase coronary flow, strengthen and improve coronary microcirculation, increase nutritive myocardial blood flow, and can also lower arterial pressure and slightly reduce heart rate, thereby significantly reducing myocardial oxygen consumption. It contains rich active saponins and polysaccharides.

[0025] The present invention adopts the method of water extraction and alcohol precipitation to obtain the composite polysaccharide of Millettia reticulata, Angelica sinensis and Panax notoginseng. The composite polysaccharide itself has good effects of promoting blood regeneration and hematopoiesis. At the same time, the polysaccharide contains amino groups and hydroxyls. After being carboxylated and modified, the carboxylated modified composite polysaccharide is obtained. The composite polysaccharide is mixed with sodium alginate to embed fermentation products, iron-rich lysozyme products, and lactoferrin succinic acid / vitamin C iron. The composite polysaccharide can well resist the acidity of gastric acid, is relatively stable in a gastric acid environment, protects the contents from being destroyed and dissolved, does not release free iron ions to damage the gastric mucosa, reduces the iron smell, and has less irritation. After entering the intestine, due to the increase in pH value, the capsule structure swells and is destroyed, the contents are dissolved, the trivalent iron ions are converted into divalent iron ions, and the absorption and utilization of iron by the intestine can be promoted.

[0026] After the solid residue of traditional Chinese medicine is fermented by iron-rich yeast and plant lactobacillus, on the one hand, it greatly promotes the breaking of plant cell walls, the dissolution of active components, and a large amount of anthraquinones, sterols, and flavonoids enter the fermentation liquid. At the same time, the fermentation of iron-rich yeast greatly increases the iron content of the fermented species. After the fermented bacteria are treated with snail enzyme and lysozyme to break the wall, iron-rich probiotic polysaccharides and iron-rich probiotic proteins are obtained. These polysaccharides and proteins form complexes with iron, which can supply iron to the body in the form of molecules after oral administration. The absorption rate is not affected by the reduction of gastric acid, food ingredients, etc., and the bioavailability is high. It has multiple functions such as enhancing the body's immunity and antiviral.

[0027] Succinate can increase the body's ability to absorb Fe 2+ Vitamin C can enhance the intestinal absorption of non-heme iron, and it can also reduce the Fe in heme iron. 3+ , turning it into easily absorbed Fe 2+Lactoferrin is an iron-binding glycoprotein with immune effects. It can promote the body's iron absorption by regulating the body's hepcidin and using whey protein as a carrier. Lactoferrin can promote the body's iron absorption metabolism faster and correct anemia symptoms. After carboxyl and amino dehydration, lactoferrin and succinic acid are coupled and compounded with vitamin C, which greatly increases the iron complexation capacity and, at the same time, can improve the bioavailability of iron.

[0028] The composition for treating anemia prepared by the present invention has no metallic odor, is not in a free state, and has relatively stable water solubility. It can directly transport iron ions into the body, thereby rapidly increasing the ferritin level, contributing to the normal development of the fetus, and reducing the occurrence of adverse pregnancy outcomes. The iron element contained in the composition exists in molecular form and will not be decomposed during absorption. Therefore, when passing through the digestive tract, the effective ingredients in the medicine are not affected by gastric acid and will not be released into divalent iron. The composition has high bioavailability and has basically no irritation to the gastrointestinal mucosa. It is highly safe, has obvious effects on rapid iron supplementation for anemia in pregnant women and promoting bone marrow blood regeneration, and has broad application prospects. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Iron-rich yeast, 20 billion cfu / g, was purchased from Jinan Shenghe Chemical Co., Ltd.; Lactobacillus plantarum, 10 billion cfu / g, was purchased from Xi'an Meihe Biotechnology Co., Ltd.

[0031] Preparation method of bacterial seed liquid: inoculate the bacterial strain into Gao's medium, activate and culture for 18-24 hours at 28-35℃ and 150r / min, and obtain a liquid containing 10 8 -10 9 cfu / mL of bacterial seed solution.

[0032] Snail enzyme, cell wall breaking rate 90%. Lysozyme, 200,000 U / g.

[0033] NHS, N-hydroxysuccinimide; EDC, 1-ethyl-(3-dimethylaminopropyl)carbodiimide. Example 1

[0034] This embodiment provides a method for preparing a composition for treating anemia, comprising the following steps:

[0035] S1. 3g Millettia reticulata, 7g Angelica sinensis, 4g Panax notoginseng were washed, crushed, dried, added to 200mL of water, heated to boiling and extracted for 3h, filtered, the solid residue was retained, ethanol was added to the filtrate until the ethanol content of the system was 75wt%, precipitated for 1h, the solid was washed and dried to obtain a composite polysaccharide;

[0036] S2. Add 10g of the complex polysaccharide to 200mL of water, add 3g of α-ketoglutaric acid, stir and react at room temperature for 20h, then add 0.7g of sodium borohydride, continue to react for 8h, then add ethanol until the ethanol content of the system is 75wt%, precipitate for 1h, filter, wash the solid, dry, add the dried product and 2.7g of sodium hydroxide to 200mL of water, add 0.2g of chloroacetic acid, heat to 55°C, stir and react for 8h, add ethanol until the ethanol content of the system is 75wt%, precipitate for 1h, filter, wash the solid, dry, and obtain a carboxyl-modified complex polysaccharide;

[0037] S3. Add 10 g of the solid residue in step S1 to 50 mL of water, sterilize, inoculate iron-rich yeast and Lactobacillus plantarum seed solution, the inoculation amount is 2 v / v% and 1 v / v%, respectively, at 30 ° C, 100 r / min, ferment and culture for 24 h, filter to obtain a solid and a filtrate, wash the solid, collect the bacterial solution, freeze-dry to obtain a fermented bacterium, freeze-dry the filtrate to obtain a fermentation product;

[0038] S4. Add 10 g of fermentation bacteria to 200 mL of water, add 0.5 g of snail enzyme and 0.2 g of lysozyme, enzymolyze at 35 ° C for 2 h, inactivate the enzyme, filter, and freeze-dry the filtrate to obtain an iron-rich lysozyme;

[0039] S5. 2 g of succinic acid was added to 200 mL of water, 0.5 g of NHS and 0.5 g of EDC were added, and the mixture was stirred for activation for 15 min, 5 g of lactoferrin and 1 g of vitamin C were added, 3 g of ferric chloride was added, and the reaction was stirred for 10 h, and the mixture was dialyzed for 36 h using a dialysis bag having a pore size of 5 kDa, and the non-permeated liquid was freeze-dried to obtain lactoferrin succinic acid / vitamin C iron;

[0040] S6. Add 5g of fermentation product, 10g of iron-rich lysing yeast and 4g of lactoferrin succinic acid / vitamin C iron into 200mL of water, add 12g of carboxylated modified complex polysaccharide, 0.5g of lecithin and 7g of sodium alginate, stir and mix for 15min, add dropwise into 500mL of fish oil, emulsify at 8000r / min for 15min, add dropwise 20mL of 2wt% calcium chloride solution, solidify at room temperature for 20min, centrifuge, wash and dry to obtain a composition for treating anemia. Example 2

[0041] This embodiment provides a method for preparing a composition for treating anemia, comprising the following steps:

[0042] S1. 5g Millettia reticulata, 10g Angelica sinensis, 6g Panax notoginseng were washed, crushed, dried, added to 200mL of water, heated to boiling and extracted for 5h, filtered, the solid residue was retained, ethanol was added to the filtrate until the ethanol content of the system was 75wt%, precipitated for 1h, the solid was washed and dried to obtain a composite polysaccharide;

[0043] S2. Add 10g of the complex polysaccharide to 200mL of water, add 3.5g of α-ketoglutaric acid, stir and react at room temperature for 24h, then add 0.9g of sodium borohydride, continue to react for 10h, then add ethanol until the ethanol content of the system is 75wt%, precipitate for 1h, filter, wash the solid, dry, add the dried product and 3g of sodium hydroxide to 200mL of water, add 0.25g of chloroacetic acid, heat to 65°C, stir and react for 12h, add ethanol until the ethanol content of the system is 75wt%, precipitate for 1h, filter, wash the solid, dry, and obtain a carboxyl-modified complex polysaccharide;

[0044] S3. Add 10 g of the solid residue in step S1 to 70 mL of water, sterilize, inoculate iron-rich yeast and Lactobacillus plantarum seed solution, the inoculation amount is 4 v / v% and 3 v / v%, respectively, at 35 ° C, 200 r / min, ferment and culture for 36 h, filter to obtain a solid and a filtrate, wash the solid, collect the bacterial solution, freeze-dry, obtain a fermented bacterium, freeze-dry the filtrate to obtain a fermentation product;

[0045] S4. Add 10 g of fermentation bacteria to 200 mL of water, add 1 g of snail enzyme and 0.3 g of lysozyme, hydrolyze at 45 ° C for 4 h, inactivate the enzyme, filter, and freeze-dry the filtrate to obtain an iron-rich lysozyme;

[0046] S5. 3 g of succinic acid was added to 200 mL of water, 1 g of NHS and 1 g of EDC were added, and the mixture was stirred for activation for 20 min, 7 g of lactoferrin and 2 g of vitamin C were added, 4 g of ferrous sulfate was added, and the reaction was stirred for 12 h, and the mixture was dialyzed for 48 h using a dialysis bag having a pore size of 7 kDa, and the non-permeated solution was freeze-dried to obtain lactoferrin succinic acid / vitamin C iron;

[0047] S6. Add 7g of fermentation product, 10g of iron-rich lysing yeast and 6g of lactoferrin succinic acid / vitamin C iron into 200mL of water, add 15g of carboxylated modified complex polysaccharide, 1g of lecithin and 10g of sodium alginate, stir and mix for 15min, add dropwise into 500mL of fish oil, emulsify at 8000r / min for 15min, add dropwise 20mL of 2wt% calcium chloride solution, solidify at room temperature for 30min, centrifuge, wash and dry to obtain a composition for treating anemia. Example 3

[0048] This embodiment provides a method for preparing a composition for treating anemia, comprising the following steps:

[0049] S1. 4g Millettia reticulata, 8g Angelica sinensis, 5g Panax notoginseng were washed, crushed, dried, added to 200mL of water, heated to boiling and extracted for 4h, filtered, the solid residue was retained, ethanol was added to the filtrate until the ethanol content of the system was 75wt%, precipitated for 1h, the solid was washed and dried to obtain a complex polysaccharide;

[0050] S2. Add 10g of the complex polysaccharide to 200mL of water, add 3.2g of α-ketoglutaric acid, stir and react at room temperature for 22h, then add 0.8g of sodium borohydride, continue to react for 9h, then add ethanol until the ethanol content of the system is 75wt%, precipitate for 1h, filter, wash the solid, dry, add the dried product and 2.9g of sodium hydroxide to 200mL of water, add 0.22g of chloroacetic acid, heat to 60°C, stir and react for 10h, add ethanol until the ethanol content of the system is 75wt%, precipitate for 1h, filter, wash the solid, dry, and obtain a carboxyl-modified complex polysaccharide;

[0051] S3. Add 10 g of the solid residue in step S1 to 60 mL of water, sterilize, inoculate iron-rich yeast and Lactobacillus plantarum seed solution, the inoculation amount is 3 v / v% and 2 v / v%, respectively, at 32°C, 150 r / min, ferment and culture for 30 h, filter to obtain a solid and a filtrate, wash the solid, collect the bacterial solution, freeze-dry to obtain a fermented bacterium, freeze-dry the filtrate to obtain a fermentation product;

[0052] S4. 10 g of fermentation bacteria was added to 200 mL of water, 0.7 g of snail enzyme and 0.25 g of lysozyme were added, and the mixture was hydrolyzed at 40 ° C for 3 h, the enzyme was inactivated, filtered, and the filtrate was freeze-dried to obtain an iron-rich lysozyme;

[0053] S5. 2.5 g of succinic acid was added to 200 mL of water, 0.7 g of NHS and 0.8 g of EDC were added, and the mixture was stirred for activation for 17 min, 6 g of lactoferrin and 1.5 g of vitamin C were added, 3.5 g of ferric nitrate was added, and the reaction was stirred for 11 h, and the mixture was dialyzed for 42 h using a dialysis bag having a pore size of 6 kDa, and the non-permeated liquid was freeze-dried to obtain lactoferrin succinic acid / vitamin C iron;

[0054] S6. Add 6g of fermentation product, 10g of iron-rich lysing yeast and 5g of lactoferrin succinic acid / vitamin C iron into 200mL of water, add 13g of carboxylated modified complex polysaccharide, 0.7g of lecithin and 8g of sodium alginate, stir and mix for 15min, add dropwise into 500mL of fish oil, emulsify at 8000r / min for 15min, add dropwise 20mL of 2wt% calcium chloride solution, solidify at room temperature for 25min, centrifuge, wash and dry to obtain a composition for treating anemia.

[0055] Comparative Example 1

[0056] Compared with Example 3, the difference is that step S2 is not performed.

[0057] The details are as follows:

[0058] S1. 4g Millettia reticulata, 8g Angelica sinensis, 5g Panax notoginseng were washed, crushed, dried, added to 200mL of water, heated to boiling and extracted for 4h, filtered, the solid residue was retained, ethanol was added to the filtrate until the ethanol content of the system was 75wt%, precipitated for 1h, the solid was washed and dried to obtain a complex polysaccharide;

[0059] S2. Add 10 g of the solid residue in step S1 to 60 mL of water, sterilize, inoculate iron-rich yeast and Lactobacillus plantarum seed solution, the inoculation amount is 3 v / v% and 2 v / v%, respectively, at 32 ° C, 150 r / min, ferment and culture for 30 h, filter to obtain a solid and a filtrate, wash the solid, collect the bacterial solution, freeze-dry, obtain a fermented bacterium, freeze-dry the filtrate to obtain a fermentation product;

[0060] S3. 10 g of fermentation bacteria was added to 200 mL of water, 0.7 g of snail enzyme and 0.25 g of lysozyme were added, and the mixture was hydrolyzed at 40 ° C for 3 h, the enzyme was inactivated, filtered, and the filtrate was freeze-dried to obtain an iron-rich lysozyme;

[0061] S4. 2.5 g of succinic acid was added to 200 mL of water, 0.7 g of NHS and 0.8 g of EDC were added, and the mixture was stirred for activation for 17 min, 6 g of lactoferrin and 1.5 g of vitamin C were added, 3.5 g of ferric nitrate was added, and the mixture was stirred for reaction for 11 h, and the mixture was dialyzed for 42 h using a dialysis bag having a pore size of 6 kDa, and the non-permeated liquid was freeze-dried to obtain lactoferrin succinic acid / vitamin C iron;

[0062] S5. Add 6g of fermentation product, 10g of iron-rich lysing yeast and 5g of lactoferrin succinic acid / vitamin C iron into 200mL of water, add 13g of complex polysaccharide, 0.7g of lecithin and 8g of sodium alginate, stir and mix for 15min, add dropwise into 500mL of fish oil, emulsify at 8000r / min for 15min, add dropwise 20mL of 2wt% calcium chloride solution, solidify at room temperature for 25min, centrifuge, wash and dry to obtain a composition for treating anemia.

[0063] Comparative Example 2

[0064] Compared with Example 3, the difference is that the iron-rich yeast seed liquid is not inoculated in step S3.

[0065] The details are as follows:

[0066] S3. Add 10 g of the solid residue in step S1 to 60 mL of water, sterilize, inoculate with Lactobacillus plantarum seed solution at an inoculation rate of 5 v / v%, culture at 32°C, 150 r / min, ferment for 30 h, filter to obtain a solid and a filtrate, wash the solid, collect the bacterial solution, freeze-dry to obtain a fermented bacterium, and freeze-dry the filtrate to obtain a fermentation product.

[0067] Comparative Example 3

[0068] Compared with Example 3, the difference is that the Lactobacillus plantarum seed liquid is not inoculated in step S3.

[0069] The details are as follows:

[0070] S3. Add 10 g of the solid residue in step S1 to 60 mL of water, sterilize, inoculate with iron-rich yeast seed solution at an inoculation rate of 5 v / v%, culture at 32°C, 150 r / min, ferment for 30 h, filter to obtain a solid and a filtrate, wash the solid, collect the bacterial solution, freeze-dry to obtain a fermented bacterium, and freeze-dry the filtrate to obtain a fermented product.

[0071] Comparative Example 4

[0072] Compared with Example 3, the difference is that no helicase is added in step S4.

[0073] The details are as follows:

[0074] S4. Add 10g of fermentation bacteria to 200mL of water, add 0.95g of lysozyme, hydrolyze at 40℃ for 3h, inactivate the enzyme, filter, and freeze-dry the filtrate to obtain iron-rich lysozyme.

[0075] Comparative Example 5

[0076] Compared with Example 3, the difference is that lysozyme is not added in step S4.

[0077] The details are as follows:

[0078] S4. Add 10g of fermentation bacteria to 200mL of water, add 0.95g of snail enzyme, enzymolyze at 40℃ for 3h, inactivate the enzyme, filter, and freeze-dry the filtrate to obtain iron-rich lysozyme.

[0079] Comparative Example 6

[0080] Compared with Example 3, the difference is that vitamin C is not added in step S5.

[0081] The details are as follows:

[0082] S5. Add 2.5 g of succinic acid to 200 mL of water, add 0.7 g of NHS and 0.8 g of EDC, stir and activate for 17 min, add 7.5 g of lactoferrin, add 3.5 g of ferric nitrate, stir and react for 11 h, dialyze using a dialysis bag with a pore size of 6 kDa for 42 h, freeze-dry the non-permeated liquid, and obtain lactoferrin ferric succinate.

[0083] Comparative Example 7

[0084] Compared with Example 3, the difference is that lactoferrin is not added in step S5.

[0085] The details are as follows:

[0086] S5. Add 2.5 g of succinic acid to 200 mL of water, add 0.7 g of NHS and 0.8 g of EDC, stir and activate for 17 min, add 7.5 g of vitamin C, add 3.5 g of ferric nitrate, stir and react for 11 h, dialyze using a dialysis bag with a pore size of 6 kDa for 42 h, freeze-dry the non-permeated liquid to obtain succinic acid / vitamin C iron.

[0087] Comparative Example 8

[0088] Compared with Example 3, the difference is that no fermentation product is added in step S6.

[0089] The details are as follows:

[0090] S6. Add 10g of iron-rich lysogeny yeast and 5g of lactoferrin succinic acid / vitamin C iron into 200mL of water, add 13g of carboxylated modified complex polysaccharide, 0.7g of lecithin and 8g of sodium alginate, stir and mix for 15min, add dropwise into 500mL of fish oil, emulsify at 8000r / min for 15min, add dropwise 20mL of 2wt% calcium chloride solution, solidify at room temperature for 25min, centrifuge, wash and dry to obtain a composition for treating anemia.

[0091] Comparative Example 9

[0092] Compared with Example 3, the difference is that no iron-rich lysing yeast is added in step S6.

[0093] The details are as follows:

[0094] S6. Add 6g of fermentation product and 5g of lactoferrin succinic acid / vitamin C iron into 200mL of water, add 13g of carboxylated modified complex polysaccharide, 0.7g of lecithin and 8g of sodium alginate, stir and mix for 15min, add dropwise into 500mL of fish oil, emulsify at 8000r / min for 15min, add dropwise 20mL of 2wt% calcium chloride solution, solidify at room temperature for 25min, centrifuge, wash and dry to obtain a composition for treating anemia.

[0095] Comparative Example 10

[0096] Compared with Example 3, the difference is that lactoferrin succinate / vitamin C iron is not added in step S6.

[0097] The details are as follows:

[0098] S6. Add 6g of fermentation product and 10g of iron-rich lysing yeast into 200mL of water, add 13g of carboxylated modified complex polysaccharide, 0.7g of lecithin and 8g of sodium alginate, stir and mix for 15min, add dropwise into 500mL of fish oil, emulsify at 8000r / min for 15min, add dropwise 20mL of 2wt% calcium chloride solution, solidify at room temperature for 25min, centrifuge, wash and dry to obtain a composition for treating anemia.

[0099] Comparative Example 11

[0100] Compared with Example 3, the difference is that no carboxyl-modified complex polysaccharide is added in step S6.

[0101] The details are as follows:

[0102] S6. Add 6g of fermentation product, 10g of iron-rich lysing yeast and 5g of lactoferrin succinic acid / vitamin C iron into 200mL of water, add 0.7g of lecithin and 21g of sodium alginate, stir and mix for 15min, add dropwise into 500mL of fish oil, emulsify at 8000r / min for 15min, add dropwise 20mL of 2wt% calcium chloride solution, solidify at room temperature for 25min, centrifuge, wash and dry to obtain a composition for treating anemia.

[0103] Test Example 1: Sustained-release test

[0104] 1 g of the composition for treating anemia prepared in Examples 1-3 of the present invention and Comparative Examples 1 and 11 was added to 9 mL of artificial simulated gastric juice and 9 mL of artificial simulated intestinal juice, respectively, and reacted at 37° C. and 70 r / min for 2 h and 3 h, respectively. In addition, an equal amount of the composition for treating anemia was added to 9 mL of artificial simulated gastric juice, first placed in a shaker, reacted at 37° C. and 70 r / min for 2 h, centrifuged, and then 9 mL of artificial simulated intestinal juice was added to continue the reaction for 3 h. After the reaction, the probiotic cell count was performed.

[0105] The release rate was calculated according to the following formula:

[0106] Release rate (%) = (W0-W t ) / W0×100%

[0107] Where W0 is the initial weight of the sample; W t It is the weight of the sample after incubation in vitro for a certain period of time.

[0108] The results are shown in Table 1.

[0109] Table 1

[0110]

[0111] As can be seen from the above table, the compositions for treating anemia prepared in Examples 1-3 of the present invention can maintain good integrity in artificial simulated gastric juice, release a large amount of active substances after being transferred to artificial simulated intestinal juice, have the characteristics of pH responsiveness and resistance to simulated gastric juice, and release a large amount of embedded active substances after entering the intestine, and have a good effect of targeted delivery and controlled release of active components in the intestine.

[0112] Test Example 2

[0113] 96 healthy female SD rats and 48 healthy male SD rats aged 8 weeks were selected. After 7 days of adaptive feeding, the 96 female SD rats were randomly divided into 16 groups according to body weight, including a normal group (Fe=181.6 mg / kg), an iron deficiency group (Fe=24.2 mg / kg), Example 1-3 and Comparative Example 1-11 group (Fe=24.2 mg / kg + 100 mg / kg of the corresponding composition for treating anemia). There were 6 rats in each group. Female rats were fed with feeds of each group respectively, and male rats were fed with standard feeds (feeds meeting the growth and reproduction period (G type) in GB 14924.3-2010). After 8 weeks, male and female rats were caged in a 1:1 ratio, and vaginal plugs were checked at 8 am the next day. If vaginal plugs were present, the day was determined to be the pregnancy day (GD0).

[0114] At GD18d, pregnant mice were anesthetized by intraperitoneal injection of 2% sodium pentobarbital solution, and blood was collected from the abdominal aorta. Blood samples were collected and organs were separated. The blood samples were coagulated, centrifuged, and serum was collected. Serum iron (SI) was detected using a biochemical kit; serum ferritin (SF) was detected using ELISA. Mindray's fully automatic blood cell analyzer BC-2800Vet was used to detect hemoglobin (Hb), number of red blood cells (RBC), and mean red blood cell volume (MCV).

[0115] The results are shown in Table 2.

[0116] Table 2

[0117]

[0118] Note: * compared with the normal group, P < 0.05; # compared with the iron deficiency group, P < 0.05.

[0119] It can be seen from the above table that the compositions for treating anemia prepared in Examples 1-3 of the present invention can significantly improve the anemia indicators of mice.

[0120] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a composition for treating anemia, characterized in that: The following steps are involved: S1. Wash, crush and dry Millettia reticulata, Angelica sinensis and Panax notoginseng, add them to water, heat and boil to extract, filter, retain the solid residue, add ethanol to the filtrate for precipitation, wash the solid, and dry to obtain a composite polysaccharide; the mass ratio of Millettia reticulata, Angelica sinensis and Panax notoginseng is 3-5:7-10:4-6; S2. Add the composite polysaccharide to water, add α-ketoglutaric acid, stir to react, then add sodium borohydride, continue to react, then add ethanol to precipitate, filter, wash the solid, dry, add the dried product and sodium hydroxide to water, add chloroacetic acid, heat and stir to react, add ethanol to precipitate, filter, wash the solid, dry to obtain a carboxylated modified composite polysaccharide; the mass ratio of the composite polysaccharide, α-ketoglutaric acid, sodium borohydride, sodium hydroxide, and chloroacetic acid is 10:3-3.5:0.7-0.9:2.7-3:0.2-0.25; S3. Add the solid residue in step S1 to water, sterilize, inoculate iron-rich yeast and Lactobacillus plantarum seed solution, ferment and culture, filter to obtain solid and filtrate, wash the solid, collect the bacterial solution, freeze-dry to obtain fermented bacteria, freeze-dry the filtrate to obtain a fermentation product; the solid-liquid ratio of the solid residue to water is 1:5-7g / mL, the inoculation amounts of the iron-rich yeast and Lactobacillus plantarum seed solution are 2-4v / v% and 1-3v / v%, respectively, and the bacterial content of the bacterial seed solution is 10 8 -10 9 cfu / mL; S4. The fermentation bacteria was added to water, snail enzyme and lysozyme were added, enzymolysis was performed, the enzyme was inactivated, the filtrate was filtered, and the filtrate was freeze-dried to obtain an iron-rich lysozyme; the mass ratio of the fermentation bacteria, snail enzyme and lysozyme was 10:0.5-1:0.2-0.3; S5. The succinic acid was added to water, NHS and EDC were added, stirred for activation, lactoferrin and vitamin C were added, iron salt was added, stirred for reaction, dialyzed, and freeze-dried to obtain lactoferrin succinic acid / vitamin C iron; the mass ratio of succinic acid, NHS, EDC, lactoferrin, vitamin C and iron salt was 2-3:0.5-1:0.5-1:5-7:1-2:3-4; S6. Add the fermented product, iron-rich bacteriolytic yeast and lactoferrin succinic acid / vitamin C iron into water, add carboxylated modified complex polysaccharide, lecithin and sodium alginate, stir and mix evenly, add dropwise into fish oil, emulsify, add calcium chloride solution dropwise, solidify at room temperature, centrifuge, wash and dry to obtain a composition for treating anemia; the mass ratio of the fermented product, iron-rich bacteriolytic yeast, lactoferrin succinic acid / vitamin C iron, carboxylated modified complex polysaccharide, lecithin and sodium alginate is 5-7:10:4-6:12-15:0.5-1:7-10.

2. The preparation method according to claim 1, characterized in that: The time of the heating and boiling extraction in step S1 is 3-5 hours.

3. The preparation method according to claim 1, characterized in that: The temperature of the stirring reaction in step S2 is room temperature, the time is 20-24 hours, the time of the continued reaction is 8-10 hours, and the temperature of the heated stirring reaction is 55-65°C, and the time is 8-12 hours.

4. The preparation method according to claim 1, characterized in that: The fermentation conditions in step S3 are 30-35°C, 100-200r / min, and fermentation for 24-36h.

5. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis conditions in step S4 are 35-45° C. and the time is 2-4 h.

6. The preparation method according to claim 1, characterized in that: In step S5, the iron salt is selected from at least one of ferric chloride, ferric sulfate, and ferric nitrate. The dialysis bag used for dialysis has a pore size of 5-7 kDa, the dialysis time is 36-48 hours, the stirring reaction time is 10-12 hours, and the stirring activation time is 15-20 minutes.

7. The preparation method according to claim 1, characterized in that: The time for room temperature curing in step S6 is 20-30 minutes.

8. A composition for treating anemia obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the composition for treating anemia as claimed in claim 8 in the preparation of a medicament for treating iron deficiency anemia during pregnancy.

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