A kind of polygonatum stock solution and its preparation method and application

Abstract

The present invention proposes a polygonatum stock solution and a preparation method and application thereof, and belongs to the field of medical technology. The polygonatum rhizome is dried, added to an extracting solution for soaking and absorption, sterilized and enzyme-killed, steamed nine times and dried nine times, added to salt water for extraction, filtered, the filtrate is dialyzed, concentrated to obtain stock solution 1, solid fermentation, immobilized amino acid imidazolyl ionic liquid extraction, desorption, liquid concentration to obtain stock solution 2, mixed evenly with stock solution 1, filtered, sterilized, packaged, and the polygonatum stock solution is obtained. The polygonatum stock solution prepared by the present invention has a high concentration, simple raw materials, good effects of improving immunity, delaying aging, resisting fatigue, and lowering blood lipids and / or blood sugar, high content of active components, low cost, simple process operation, stable quality, and easy to achieve large-scale industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a polygonatum stock solution and a preparation method and application thereof. Background Art

[0002] Polygonatum cyrtonema Siberian Polygonum The dried rhizome of Astragalus membranaceus was first recorded in "Ming Yi Bie Lu", also known as "sun grass", which can nourish yin, strengthen spleen, moisten lungs and benefit kidneys, and is known as the "king of qi and blood supplement". Astragalus membranaceus The dried root of Bge. enters the spleen and lung meridians and contains effective ingredients such as astragalus polysaccharide, saponin, flavonoids, etc. Modern pharmacological research has shown that Polygonatum sibiricum has multiple pharmacological effects such as enhancing immunity, lowering blood lipids and blood sugar, and delaying aging.

[0003] 1. Anti-aging effect

[0004] Studies have shown that Polygonatum sibiricum extract can increase the activity of glutathione peroxidase and blood SOD in a dose-dependent manner; compared with the positive control group, the ability of Polygonatum sibiricum extract to inhibit the production of LPO in the heart and liver was significantly increased, also in a dose-dependent manner, indicating that Polygonatum sibiricum has anti-aging effects.

[0005] 2. Regulate immune function

[0006] Polygonatum sibiricum can improve the bone marrow hematopoietic function of mice treated with cyclophosphamide, increase the number of white blood cells and red blood cells, reduce the micronucleus rate of bone marrow polychromatic erythrocytes, and improve the phagocytic function of mouse peritoneal macrophages. Polygonatum sibiricum crude polysaccharide can significantly promote the increase of thymus and spleen weight in normal mice, significantly enhance the clearance rate of intravenously injected colloidal carbon particles in mice, and significantly activate and enhance the phagocytic function of the mouse reticuloendothelial system. Polygonatum sibiricum polysaccharide also has a significant protective effect on the hematopoietic function of radiation-damaged mice.

[0007] 3. Improve learning and memory

[0008] Polygonatum sibiricum polysaccharide can significantly improve the learning and memory and memory retrieval abilities of aged rats, reduce the number of errors, and significantly shorten the latency time of rats in maze tests.

[0009] 4. Anti-inflammatory and antiviral effects

[0010] The anti-inflammatory effect of Polygonatum sibiricum polysaccharide was investigated by using Polygonatum sibiricum polysaccharide eye drops in rabbit conjunctival and corneal inflammation models. The results showed that Polygonatum sibiricum polysaccharide eye drops could eliminate local symptoms such as conjunctival congestion, edema, increased secretions, corneal opacity, and ciliary congestion in the rabbit model; Polygonatum sibiricum polysaccharide could also significantly inhibit the swelling of the auricles of mice and the swelling of the toes of rats; reduce the weight of rat granulomas and reduce the exudation in granulomas; and have significant therapeutic effects on the primary lesions and secondary lesions of immune arthritis in rats, indicating that Polygonatum sibiricum polysaccharide has good anti-inflammatory effects. Polygonatum sibiricum polysaccharide preparation was used to treat experimental herpes simplex virus keratitis in rabbits, and the results showed that the efficacy of Polygonatum sibiricum polysaccharide preparation group was statistically significant compared with that of chloramphenicol group; Polygonatum sibiricum polysaccharide eye drops combined with Polygonatum sibiricum polysaccharide injection subconjunctival injection and Polygonatum sibiricum polysaccharide eye drops combined with Polygonatum sibiricum polysaccharide oral liquid were better than those of the acyclovir group.

[0011] 5. Regulate blood sugar and blood lipids

[0012] Polygonatum sibiricum polysaccharide significantly reduced the cAMP content in the liver of Ad model mice, so it is speculated that its hypoglycemic mechanism is to reduce the cAMP content in liver cells, thereby hindering the activation of phosphorylase and the inactivation of glycogen synthase, resulting in accelerated glycogen synthesis and slowed glycogen decomposition. A certain dose of Polygonatum sibiricum polysaccharide has the effect of reducing blood lipids in experimental animals with hyperlipidemia and inhibiting the formation of foam cells in the arterial intima.

[0013] 6. Anti-fatigue effect

[0014] Intraperitoneal injection of Polygonatum sibiricum solution can significantly improve the mice's ability to tolerate hypoxia. Oral administration of Polygonatum sibiricum decoction to mice can significantly prolong their swimming time.

[0015] 7. Anti-tumor effect

[0016] A certain dose of Polygonatum sibiricum polysaccharide can enhance the activity of natural killer cells in tumor-bearing mice and has a significant inhibitory effect on tumor growth.

[0017] So far, no high-quality, highly physiologically active Polygonatum sibiricum concentrate has been reported. Summary of the invention

[0018] The purpose of the present invention is to provide a polygonatum stock solution and a preparation method and application thereof, which has high concentration, simple raw materials, good effects of improving immunity, delaying aging, resisting fatigue, lowering blood lipids and / or blood sugar, high content of active components, low cost, simple process operation, stable quality, and is easy to realize large-scale industrial production.

[0019] The technical solution of the present invention is achieved in this way:

[0020] The invention provides a method for preparing a polygonatum stock solution. The method comprises the following steps: drying polygonatum rhizomes, adding them into an extracting solution for soaking and absorption, sterilizing the rhizomes, steaming and drying them for nine times, adding them into salt water for extraction, filtering, dialyzing the filtrate, and concentrating them to obtain a stock solution 1; solid fermentation, extraction with an immobilized amino acid imidazolyl ionic liquid, desorption, and liquid concentration to obtain a stock solution 2, uniformly mixing the stock solution with the stock solution 1, filtering, sterilizing, and packaging to obtain the polygonatum stock solution.

[0021] As a further improvement of the present invention, the following steps are included:

[0022] S1. Select fresh rhizomes of Polygonatum sibiricum, wash, dry and slice to prepare Polygonatum sibiricum slices;

[0023] S2. The Huangjing slices were soaked in the extract and absorbed, and the soaked Huangjing slices were air-dried to kill the enzyme;

[0024] S3. The enzyme-killing Huangjing slices were steamed and dried nine times, and air-dried after each steaming;

[0025] S4. The nine-processed Huangjing tablets were crushed, extracted with salt water, filtered, the solid residue was retained, the filtrate was dialyzed for desalination, and the non-permeated liquid was concentrated to obtain a stock solution 1;

[0026] S5. The solid residue is added to water, sterilized, inoculated with selenium-enriched yeast and Bacillus subtilis seed solution, fermented, filtered, and the filtrate is extracted by adding an immobilized amino acid imidazolyl ionic liquid, and the solid portion is filtered again after extraction and collected, and the solid portion is added to water and heated for desorption, filtered to obtain a solid and a liquid, the solid is separated, washed, recycled, and the liquid is concentrated to obtain a stock solution 2;

[0027] S6. Mix the stock solution 1 and the stock solution 2 evenly, filter, sterilize, and package to obtain the polygonatum stock solution.

[0028] As a further improvement of the present invention, the water content of the polygonatum sibiricum slices in step S1 is less than 1%; the extract in step S2 is a mixed solution containing snail enzyme and cellulase, the content of the snail enzyme is 2-4wt%, the content of the cellulase is 1-3wt%, the mass ratio of the polygonatum sibiricum slices to the extract is 1:1-1.5, the soaking and absorption time is 1-2h, and the method of killing and inactivating the enzyme is to use steam at 95-100°C to kill and inactivate the enzyme.

[0029] As a further improvement of the present invention, the brine in step S4 is a NaCl or KCl solution with a concentration of 2-5wt%, the extraction time is 0.5-1.5h, the pore size of the dialysis bag used for dialysis is 1k-2kDa, the dialysis time is 3-5h, and the density of the concentrated stock solution 1 relative to water is 1.15-1.17.

[0030] As a further improvement of the present invention, the bacterial content of the bacterial seed liquid in step S5 is 10 8 -10 9 cfu / mL, the inoculation amounts of the selenium-enriched yeast and Bacillus subtilis seed solutions are 1-3v / v% and 1.5-2.5v / v% respectively; the fermentation conditions are 25-30°C, 100-200r / min, and the fermentation is carried out for 24-36h; the addition amount of the immobilized amino acid imidazolyl ionic liquid is 3-5wt% of the total mass of the system, the heating temperature is 35-45°C, and the relative density of the concentrated stock solution 2 to water is 1.12-1.15.

[0031] As a further improvement of the present invention, the preparation method of the immobilized amino acid imidazolyl ionic liquid is as follows:

[0032] T1. Soaking silica gel in acid solution, filtering, washing and drying to obtain activated silica gel;

[0033] T2. Add activated silica gel to toluene, add silane coupling agent KH560 and pyridine under inert gas protection, heat to reflux and stir to react, filter, wash, and dry to obtain modified silica gel;

[0034] T3. 1-methylimidazole was added to ethanol, concentrated sulfuric acid was added dropwise, modified silica gel was added, the reaction was heated and stirred, filtered, washed, and dried to obtain imidazole-modified silica gel;

[0035] T4. Add lysine and aspartic acid into alkaline solution, add imidazole-modified silica gel, heat and stir to react, filter, wash and dry to obtain immobilized amino acid imidazole-based ionic liquid.

[0036] As a further improvement of the present invention, the acid solution in step T1 is 5-10wt% HCl or sulfuric acid solution, and the immersion time is 3-5h; the mass ratio of activated silica gel, silane coupling agent KH560 and pyridine in step T2 is 10:3-4:0.5-1, and the heating reflux stirring reaction time is 2-4h; the mass ratio of 1-methylimidazole, concentrated sulfuric acid and modified silica gel in step T3 is 2-3:1-2:10, the heating stirring reaction temperature is 60-70°C, and the time is 5-7h; the alkali solution in step T4 is 10-15wt% NaOH or KOH solution, the mass ratio of lysine, aspartic acid and imidazole-modified silica gel is 2-3:3-4:10, the heating stirring reaction temperature is 80-90°C, and the time is 3-5h.

[0037] As a further improvement of the present invention, the mass ratio of the stock solution 1 to the stock solution 2 in step S6 is 10:3-4.

[0038] The present invention further protects a polygonatum stock solution prepared by the above preparation method.

[0039] The present invention further protects the use of the above-mentioned polygonatum stock solution in the preparation of medicines for enhancing immunity, lowering blood lipids and / or blood sugar, delaying aging and resisting fatigue.

[0040] The present invention has the following beneficial effects:

[0041] The invention significantly improves the extraction rate of effective ingredients in polygonatum through the process of nine-steaming and nine-drying. The prepared polygonatum stock solution has stable quality and significant medicinal effect. The process operation is simple, which can not only improve the medicinal effect of polygonatum, but also ensure the stability of product quality, and is easy to realize large-scale production, and has good market application prospects.

[0042] The present invention soaks the dried polygonatum slices in an extracting solution containing snail enzyme and cellulase, promotes the breaking of plant cell walls and softens fibers during the soaking process, thereby allowing active components to break through the barrier and dissolve, and further adds the dried polygonatum slices to a hypertonic solution, and the active substances penetrate out along with the cell fluid, thereby obtaining a high-activity stock solution 1;

[0043] The present invention inoculates the solid residue with selenium-enriched yeast and Bacillus subtilis seed liquid for fermentation. The Chinese medicine residue contains a large amount of nutrients, bioactive substances and some unknown growth-promoting substances, and has a high starch content, which is suitable for use as a fermentation matrix. By selecting a suitable microorganism to ferment it, it can be converted into a high value-added product, realizing the transformation of waste into treasure. On the one hand, it can promote the proliferation of microorganisms, and on the other hand, the microorganisms themselves can also promote the breaking of plant cell walls, thereby promoting the dissolution of highly active substances, such as components such as polysaccharides, amino acids, proteins, flavonoids, triterpenes, and saponins. A part of the active components is converted into active substances such as short-chain fatty acids, antimicrobial peptides, etc. produced by microorganisms by microorganisms, and the other part enters the fermentation liquid, so that the obtained fermentation liquid contains rich active substances.

[0044] The present invention prepares an immobilized amino acid imidazolyl ionic liquid, using silica gel as a carrier. Silica gel, as a stable and low-cost inorganic porous material, has good performance. Amino acid ionic liquids are new functional ionic liquids with low toxicity and biodegradability. At the same time, they have good effects of separating and purifying proteins, amino acids, etc. The present invention uses lysine and aspartic acid as anions of the ionic liquid, and the separation effect has a synergistic effect, which significantly improves the separation and purification effect of amino acids, short peptides, protein peptides, and proteins.

[0045] The present invention uses selenium-enriched yeast and Bacillus subtilis for fermentation, and the fermentation liquid contains rich active substances such as selenium proteins and selenium amino acids, so that the protein content in the original solution 2 is relatively high, and has good effects of improving immunity, delaying aging and resisting fatigue.

[0046] The polygonatum sibiricum stock solution prepared by the invention has high concentration, simple raw materials, good effects of improving immunity, delaying aging, resisting fatigue, lowering blood lipids and / or blood sugar, high content of active components, low cost, simple process operation, stable quality, and is easy to realize large-scale industrial production. DETAILED DESCRIPTION

[0047] 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.

[0048] Snail enzyme, with a cell wall breaking rate of 90%, was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; cellulase, 10,000 U / g, was purchased from Guangdong Mingtong Biotechnology Co., Ltd.

[0049] Selenium-enriched yeast, 20 billion cfu / g, was purchased from Angel Yeast Co., Ltd.; Bacillus subtilis, 20 billion cfu / g, was purchased from Jiangsu Caiwei Biotechnology Co., Ltd.;

[0050] Preparation method of bacterial seed liquid: inoculate the bacterial strain into Gao's medium, activate and culture for 18-24h at 30℃ and 100r / min, and obtain a liquid containing 10 8 -10 9 cfu / mL of bacterial seed solution.

[0051] Silica gel, 100 mesh, was purchased from Shandong Tenghai Chemical Co., Ltd.

[0052] Preparation Example 1 Preparation of immobilized amino acid imidazolyl ionic liquid

[0053] Here’s how:

[0054] T1. 10 g of silica gel was added to 100 mL of 5 wt% HCl solution and soaked for 3 h, filtered, washed, and dried to obtain activated silica gel;

[0055] T2. 10 g of activated silica gel was added to 200 mL of toluene, and under nitrogen protection, 3 g of silane coupling agent KH560 and 0.5 g of pyridine were added, and the mixture was heated to reflux and stirred for 2 h, filtered, washed, and dried to obtain modified silica gel;

[0056] T3. Add 2 g of 1-methylimidazole to 200 mL of ethanol, add 1 g of concentrated sulfuric acid dropwise, add 10 g of modified silica gel, heat to 60 ° C, stir and react for 5 h, filter, wash, and dry to obtain imidazole-modified silica gel;

[0057] T4. Add 2 g of lysine and 3 g of aspartic acid to a 10 wt% NaOH solution, add 10 g of imidazole-modified silica gel, heat to 80°C, stir and react for 3 h, filter, wash, and dry to obtain an immobilized amino acid imidazole-based ionic liquid.

[0058] Preparation Example 2 Preparation of immobilized amino acid imidazolyl ionic liquid

[0059] Here’s how:

[0060] T1. 10 g of silica gel was added to 100 mL of a 10 wt% sulfuric acid solution and soaked for 5 h, filtered, washed, and dried to obtain activated silica gel;

[0061] T2. 10 g of activated silica gel was added to 200 mL of toluene, and under nitrogen protection, 4 g of silane coupling agent KH560 and 1 g of pyridine were added, and the mixture was heated to reflux and stirred for 4 h, filtered, washed, and dried to obtain modified silica gel;

[0062] T3. Add 3 g of 1-methylimidazole to 200 mL of ethanol, add 2 g of concentrated sulfuric acid dropwise, add 10 g of modified silica gel, heat to 70°C, stir and react for 7 hours, filter, wash, and dry to obtain imidazole-modified silica gel;

[0063] T4. Add 3 g of lysine and 4 g of aspartic acid to a 15 wt% KOH solution, add 10 g of imidazole-modified silica gel, heat to 90°C, stir and react for 5 h, filter, wash, and dry to obtain an immobilized amino acid imidazole-based ionic liquid.

[0064] Preparation Example 3 Preparation of immobilized amino acid imidazolyl ionic liquid

[0065] Here’s how:

[0066] T1. 10 g of silica gel was added to 100 mL of 7 wt % HCl solution and soaked for 4 h, filtered, washed, and dried to obtain activated silica gel;

[0067] T2. 10 g of activated silica gel was added to 200 mL of toluene, and 3.5 g of silane coupling agent KH560 and 0.7 g of pyridine were added under nitrogen protection, and the mixture was heated to reflux and stirred for 3 h, filtered, washed, and dried to obtain modified silica gel;

[0068] T3. Add 2.5 g of 1-methylimidazole to 200 mL of ethanol, add 1.5 g of concentrated sulfuric acid dropwise, add 10 g of modified silica gel, heat to 65°C, stir and react for 6 hours, filter, wash, and dry to obtain imidazole-modified silica gel;

[0069] T4. Add 2.5 g of lysine and 3.5 g of aspartic acid to a 12 wt% NaOH solution, add 10 g of imidazole-modified silica gel, heat to 85°C, stir and react for 4 h, filter, wash, and dry to obtain an immobilized amino acid imidazole-based ionic liquid.

[0070] Comparative Preparation Example 1

[0071] Compared with Preparation Example 3, the difference is that lysine is not added in step T4.

[0072] The details are as follows:

[0073] T1. 10 g of silica gel was added to 100 mL of 7 wt % HCl solution and soaked for 4 h, filtered, washed, and dried to obtain activated silica gel;

[0074] T2. 10 g of activated silica gel was added to 200 mL of toluene, and 3.5 g of silane coupling agent KH560 and 0.7 g of pyridine were added under nitrogen protection, and the mixture was heated to reflux and stirred for 3 h, filtered, washed, and dried to obtain modified silica gel;

[0075] T3. Add 2.5 g of 1-methylimidazole to 200 mL of ethanol, add 1.5 g of concentrated sulfuric acid dropwise, add 10 g of modified silica gel, heat to 65°C, stir and react for 6 hours, filter, wash, and dry to obtain imidazole-modified silica gel;

[0076] T4. Add 6 g of aspartic acid to a 12 wt% NaOH solution, add 10 g of imidazole-modified silica gel, heat to 85°C, stir and react for 4 h, filter, wash, and dry to obtain an immobilized amino acid imidazole-based ionic liquid.

[0077] Comparative Preparation Example 2

[0078] Compared with Preparation Example 3, the difference is that aspartic acid is not added in step T4.

[0079] The details are as follows:

[0080] T1. 10 g of silica gel was added to 100 mL of 7 wt % HCl solution and soaked for 4 h, filtered, washed, and dried to obtain activated silica gel;

[0081] T2. 10 g of activated silica gel was added to 200 mL of toluene, and 3.5 g of silane coupling agent KH560 and 0.7 g of pyridine were added under nitrogen protection, and the mixture was heated to reflux and stirred for 3 h, filtered, washed, and dried to obtain modified silica gel;

[0082] T3. Add 2.5 g of 1-methylimidazole to 200 mL of ethanol, add 1.5 g of concentrated sulfuric acid dropwise, add 10 g of modified silica gel, heat to 65°C, stir and react for 6 hours, filter, wash, and dry to obtain imidazole-modified silica gel;

[0083] T4. Add 6 g of lysine to a 12 wt% NaOH solution, add 10 g of imidazole-modified silica gel, heat to 85°C, stir and react for 4 h, filter, wash, and dry to obtain an immobilized amino acid imidazole ionic liquid.

[0084] Comparative Preparation Example 3

[0085] Compared with Preparation Example 3, the difference is that step T4 is not performed.

[0086] The details are as follows:

[0087] T1. 10 g of silica gel was added to 100 mL of 7 wt % HCl solution and soaked for 4 h, filtered, washed, and dried to obtain activated silica gel;

[0088] T2. 10 g of activated silica gel was added to 200 mL of toluene, and 3.5 g of silane coupling agent KH560 and 0.7 g of pyridine were added under nitrogen protection, and the mixture was heated to reflux and stirred for 3 h, filtered, washed, and dried to obtain modified silica gel;

[0089] T3. Add 2.5 g of 1-methylimidazole to 200 mL of ethanol, add 1.5 g of concentrated sulfuric acid dropwise, add 10 g of modified silica gel, heat to 65°C, stir and react for 6 hours, filter, wash and dry to obtain imidazole-modified silica gel, which is the immobilized amino acid imidazole ionic liquid.

[0090] Test Example 1

[0091] The specific surface area and total pore volume of the immobilized amino acid imidazolyl ionic liquids prepared in Preparation Examples 1-3 and Comparative Preparation Examples 1-3 were measured using a 3-FLEX 3500 multi-station high-throughput gas adsorption instrument. The results are shown in Table 1.

[0092] Table 1

[0093]

[0094] It can be seen from the above table that the immobilized amino acid imidazolyl ionic liquids prepared in Preparation Examples 1-3 of the present invention have higher specific surface areas and total pore volumes.

[0095] Example 1

[0096] This embodiment provides a preparation method of Polygonatum sibiricum stock solution, and the method comprises the following steps:

[0097] S1. Select fresh rhizomes of Polygonatum sibiricum, wash, dry and slice them to make Polygonatum sibiricum slices with a water content of <1%;

[0098] S2. 10 g of Polygonatum sibiricum tablets were added to 10 g of the extract and soaked for 1 hour, air-dried, and sterilized with 95°C steam for 10 minutes;

[0099] The extract is a mixed solution containing snail enzyme and cellulase, wherein the content of the snail enzyme is 2wt% and the content of the cellulase is 1wt%;

[0100] S3. The soaked Huangjing slices were steamed and dried nine times, and aired after each steaming;

[0101] S4. The nine-processed Huangjing tablets were crushed, extracted by adding 2wt% NaCl solution, filtered, the solid residue was retained, the filtrate was dialyzed for desalination using a dialysis bag with a pore size of 1kDa for 3h, the non-permeated solution was concentrated to obtain a stock solution 1, the relative water density was 1.15;

[0102] S5. Add 10g of solid residue to 200mL of water, sterilize, inoculate with selenium-enriched yeast and Bacillus subtilis seed liquid, the inoculation amount is 1v / v% and 1.5v / v%, respectively, 25°C, 100r / min, ferment and culture for 36h, filter, add the immobilized amino acid imidazolyl ionic liquid prepared in Preparation Example 1 to the filtrate for extraction, the immobilized amino acid imidazolyl ionic liquid is added in an amount of 3wt% of the total mass of the system, filter, add the solid to 100mL of water and heat to 35°C, desorb for 1h, filter to obtain solid and liquid, separate the solid, wash, recycle and reuse, concentrate the liquid to obtain stock solution 2, the density relative to water is 1.12;

[0103] S6. Stir and mix 10 g of solution 1 and 3 g of solution 2 for 15 min, filter, sterilize, and package to obtain polygonatum stock solution.

[0104] Example 2

[0105] This embodiment provides a preparation method of Polygonatum sibiricum stock solution, and the method comprises the following steps:

[0106] S1. Select fresh rhizomes of Polygonatum sibiricum, wash, dry and slice them to make Polygonatum sibiricum slices with a water content of <1%;

[0107] S2. 10g of Polygonatum sibiricum tablets were added to 15g of the extract and soaked for 2h, air-dried, and sterilized with steam at 100°C for 10min;

[0108] The extract is a mixed solution containing snail enzyme and cellulase, wherein the content of the snail enzyme is 4wt% and the content of the cellulase is 3wt%;

[0109] S3. The soaked Huangjing slices were steamed and dried nine times, and aired after each steaming;

[0110] S4. The nine-processed Huangjing tablets were crushed, extracted by adding 5wt% KCl solution, filtered, the solid residue was retained, the filtrate was dialyzed for desalination using a 2kDa pore size dialysis bag for 5h, the non-permeated solution was concentrated to obtain a stock solution 1, the relative water density was 1.17;

[0111] S5. Add 10g of solid residue to 200mL of water, sterilize, inoculate with selenium-enriched yeast and Bacillus subtilis seed liquid, the inoculation amount is 3v / v% and 2.5v / v%, respectively, 30°C, 200r / min, ferment and culture for 24h, filter, add the immobilized amino acid imidazolyl ionic liquid prepared in Preparation Example 2 to the filtrate for extraction, the immobilized amino acid imidazolyl ionic liquid is added in an amount of 5wt% of the total mass of the system, filter, add the solid to 100mL of water and heat to 45°C, desorb for 1h, filter, obtain solid and liquid, separate the solid, wash, recycle and reuse, concentrate the liquid to obtain stock solution 2, the density relative to water is 1.15;

[0112] S6. Stir and mix 10 g of solution 1 and 4 g of solution 2 for 15 min, filter, sterilize, and package to obtain polygonatum stock solution.

[0113] Example 3

[0114] This embodiment provides a preparation method of Polygonatum sibiricum stock solution, and the method comprises the following steps:

[0115] S1. Select fresh rhizomes of Polygonatum sibiricum, wash, dry and slice them to make Polygonatum sibiricum slices with a water content of <1%;

[0116] S2. 10 g of Polygonatum sibiricum tablets were added to 12 g of the extract and soaked for 1.5 h, air-dried, and sterilized with steam at 100 ° C for 10 min;

[0117] The extract is a mixed solution containing snail enzyme and cellulase, wherein the content of the snail enzyme is 3wt% and the content of the cellulase is 2wt%;

[0118] S3. The soaked Huangjing slices were steamed and dried nine times, and aired after each steaming;

[0119] S4. The nine-processed Huangjing tablets were crushed, extracted by adding 3.5wt% NaCl solution, filtered, the solid residue was retained, and the filtrate was dialyzed for desalination using a dialysis bag with a pore size of 1.5kDa for 4h, and the non-permeated liquid was concentrated to obtain a stock solution 1, with a relative water density of 1.16;

[0120] S5. Add 10g of solid residue to 200mL of water, sterilize, inoculate with selenium-enriched yeast and Bacillus subtilis seed liquid, the inoculation amount is 2v / v% and 2v / v%, respectively, 27°C, 150r / min, ferment and culture for 30h, filter, add the immobilized amino acid imidazolyl ionic liquid prepared in Preparation Example 3 to the filtrate for extraction, the immobilized amino acid imidazolyl ionic liquid is added in an amount of 4wt% of the total mass of the system, filter, add the solid to 100mL of water and heat to 40°C, desorb for 1h, filter, obtain solid and liquid, separate the solid, wash, recycle and reuse, concentrate the liquid to obtain stock solution 2, the relative density of water is 1.13;

[0121] S6. Stir and mix 10 g of solution 1 and 3.5 g of solution 2 for 15 min, filter, sterilize, and package to obtain polygonatum stock solution.

[0122] Example 4

[0123] Compared with Example 3, the difference is that the extract is an aqueous solution containing snail enzyme, and the content of the snail enzyme is 5wt%.

[0124] Example 5

[0125] Compared with Example 3, the difference is that the extract is an aqueous solution containing cellulase, and the content of the cellulase is 5wt%.

[0126] Comparative Example 1

[0127] Compared with Example 3, the difference is that the extracting liquid is replaced by water.

[0128] Comparative Example 2

[0129] Compared with Example 3, the difference is that the selenium-enriched yeast seed liquid is not inoculated in step S5.

[0130] The details are as follows:

[0131] S5. Add 10g of solid residue to 200mL of water, sterilize, inoculate with Bacillus subtilis seed liquid at an inoculation rate of 4v / v%, culture at 27°C, 150r / min, ferment for 30h, filter, add the immobilized amino acid imidazolyl ionic liquid prepared in Preparation Example 3 to the filtrate for extraction, wherein the immobilized amino acid imidazolyl ionic liquid is added in an amount of 4wt% of the total mass of the system, filter, add the solid to 100mL of water and heat to 40°C, desorb for 1h, filter to obtain solid and liquid, separate the solid, wash, recycle and reuse, and concentrate the liquid to obtain stock solution 2, whose density relative to water is 1.13.

[0132] Comparative Example 3

[0133] Compared with Example 3, the difference is that the Bacillus subtilis seed liquid is not inoculated in step S5.

[0134] The details are as follows:

[0135] S5. Add 10g of solid residue into 200mL of water, sterilize, inoculate with selenium-enriched yeast seed liquid at an inoculation rate of 4v / v%, culture at 27℃, 150r / min, ferment for 30h, filter, add the immobilized amino acid imidazolyl ionic liquid prepared in Preparation Example 3 to the filtrate for extraction, wherein the immobilized amino acid imidazolyl ionic liquid is added in an amount of 4wt% of the total mass of the system, filter, add the solid into 100mL of water and heat to 40℃, desorb for 1h, filter to obtain solid and liquid, separate the solid, wash, recycle and reuse, and concentrate the liquid to obtain stock solution 2, whose density relative to water is 1.13.

[0136] Comparative Example 4

[0137] Compared with Example 3, the difference is that in step S5, no selenium-enriched yeast and Bacillus subtilis seed liquid are inoculated.

[0138] The details are as follows:

[0139] S5. Add 10g of solid residue to 200mL of water, heat to 50°C, extract for 30h, filter, add the immobilized amino acid imidazolyl ionic liquid prepared in Preparation Example 3 to the filtrate for extraction, wherein the immobilized amino acid imidazolyl ionic liquid is added in an amount of 4wt% of the total mass of the system, filter, add the solid to 100mL of water, heat to 40°C, desorb for 1h, filter to obtain solid and liquid, separate the solid, wash, recycle and reuse, and concentrate the liquid to obtain stock solution 2, whose density relative to water is 1.13.

[0140] Comparative Example 5

[0141] Compared with Example 3, the difference is that the immobilized amino acid imidazolyl ionic liquid is prepared by Comparative Preparation Example 1.

[0142] Comparative Example 6

[0143] Compared with Example 3, the difference is that the immobilized amino acid imidazolyl ionic liquid is prepared by Comparative Preparation Example 2.

[0144] Comparative Example 7

[0145] Compared with Example 3, the difference is that the immobilized amino acid imidazolyl ionic liquid is prepared by Comparative Preparation Example 3.

[0146] Test Example 2

[0147] The protein content of the Polygonatum sibiricum stock solution prepared in Examples 1-5 and Comparative Examples 1-7 of the present invention was determined by referring to the method of "GB 5009.5-2016 National Food Safety Standard Determination of Protein in Food"; the polysaccharide content in the Polygonatum sibiricum stock solution was determined by referring to the method of "GB / T18672-2014 Lycium barbarum". The selenium content in the Polygonatum sibiricum stock solution was determined by the method of "GB5009.93-2017 National Food Safety Standard Determination of Selenium in Food". The results are shown in Table 2.

[0148] Table 2

[0149]

[0150] It can be seen from the above table that the polygonatum sibiricum stock solutions prepared in Examples 1-3 of the present invention are rich in protein, polysaccharides and selenium.

[0151] Test Example 3

[0152] The polygonatum stock solution prepared in Example 1-5 or Comparative Example 1-7 was diluted 100 times to prepare a sample solution.

[0153] Pipette 2 mL of sample solution or ascorbic acid solution (0.5 g / L), add 2 mL of DPPH· (0.2 mmol / L, prepared with anhydrous ethanol) solution into a test tube and mix evenly (use anhydrous ethanol instead of DPPH· as sample blank). Place at room temperature away from light for 30 minutes, then detect the absorbance at a wavelength of 517 nm. Each concentration should be measured 3 times.

[0154] Clearance rate (%) = [1-(A i -A j ) / A c ]×100%

[0155] In the formula, A i A is the absorbance of a mixture of 2 mL DPPH· solution and 2 mL of the sample solution to be determined; j A is the absorbance of a mixture of 2 mL of anhydrous ethanol and 2 mL of the sample solution to be measured; c It is the absorbance of a mixture of 2 mL of DPPH solution and 2 mL of anhydrous ethanol.

[0156] The results are shown in Table 3.

[0157] Table 3

[0158]

[0159] It can be seen from the above table that the Polygonatum sibiricum stock solutions prepared in Examples 1-3 of the present invention have good antioxidant properties.

[0160] Test Example 4

[0161] SPF female ICR mice with a body weight of 18-22g were selected and randomly divided into a negative control group, Example 1-5 groups, and Comparative Example 1-7 groups, with 10 mice in each group. The experimental group was given 0.5 mg / kg·d of the corresponding prepared Polygonatum sibiricum stock solution by oral gavage, and the negative control group was given an equal amount of normal saline. After continuous oral gavage for 30 days, 0.2 mL of 2% (V / V) sheep red blood cell solution was intraperitoneally injected into each mouse on the 31st day. After 4 days of immunization, the thickness of the left hind toe of the mouse was accurately measured, and 20 μL of 20% (V / V) sheep red blood cell solution was subcutaneously injected at the measuring site for re-sensitization. After 24 hours of re-immunization, the thickness of the left hind toe of the mouse was observed and measured again, and the difference in the thickness of the left hind toe of the mouse after the first sensitization and the second sensitization was calculated, that is, the degree of delayed-type hypersensitivity (DTH) was characterized by the swelling of the toes. The results are shown in Table 4.

[0162] Table 4

[0163]

[0164] Note: * Compared with the negative control group, P < 0.05.

[0165] It can be seen from the above table that the polygonatum stock solution prepared in Examples 1-3 of the present invention can significantly increase the difference in the degree of swelling of toes and significantly enhance the immune effect on mice.

[0166] 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 polygonatum stock solution, characterized in that: The following steps are involved: S1. Select fresh rhizomes of Polygonatum sibiricum, wash, dry and slice to prepare Polygonatum sibiricum slices; S2. The Huangjing slices are added to the extract and soaked and absorbed, and the soaked Huangjing slices are air-dried to kill the enzyme; wherein the extract is a mixture containing snail enzyme and cellulase, the snail enzyme content is 2-4wt%, the cellulase content is 1-3wt%; S3. The enzyme-killing Huangjing slices were steamed and dried nine times, and air-dried after each steaming; S4. The nine-processed Huangjing tablets were crushed, extracted with salt water, filtered, the solid residue was retained, the filtrate was dialyzed for desalination, and the non-permeated liquid was concentrated to obtain a stock solution 1; S5. The solid residue is added to water, sterilized, inoculated with selenium-enriched yeast and Bacillus subtilis seed solution, fermented and cultured, filtered, and the filtrate is added with immobilized amino acid imidazolyl ionic liquid for extraction. After extraction, the solid portion is filtered again and the solid portion is collected. The solid portion is added to water for heating and desorption, filtered to obtain a solid and a liquid, the solid is separated, washed, recycled and reused, and the liquid is concentrated to obtain a stock solution 2; wherein the inoculation amounts of the selenium-enriched yeast and Bacillus subtilis seed solution are 1-3v / v% and 1.5-2.5v / v%, respectively; the preparation method of the immobilized amino acid imidazolyl ionic liquid is as follows: T1. Soaking silica gel in acid solution, filtering, washing and drying to obtain activated silica gel; T2. Add activated silica gel to toluene, add silane coupling agent KH560 and pyridine under inert gas protection, heat to reflux and stir to react, filter, wash, and dry to obtain modified silica gel; T3. 1-methylimidazole was added to ethanol, concentrated sulfuric acid was added dropwise, modified silica gel was added, the reaction was heated and stirred, filtered, washed, and dried to obtain imidazole-modified silica gel; T4. Lysine and aspartic acid were added to an alkaline solution, imidazole-modified silica gel was added, the reaction was heated and stirred, filtered, washed, and dried to obtain an immobilized amino acid imidazole-based ionic liquid; S6. Mix the stock solution 1 and the stock solution 2 evenly, filter, sterilize, and package to obtain the polygonatum stock solution.

2. The preparation method according to claim 1, characterized in that: The water content of the polygonatum sibiricum slices in step S1 is less than 1%; the mass ratio of the polygonatum sibiricum slices to the extract in step S2 is 1:1-1.5, the soaking and absorption time is 1-2h, and the method of killing the enzyme is to use steam at 95-100°C to kill the enzyme.

3. The preparation method according to claim 1, characterized in that: The brine in step S4 is a NaCl or KCl solution with a concentration of 2-5wt%, the extraction time is 0.5-1.5h, the dialysis bag used for dialysis has a pore size of 1k-2kDa, the dialysis time is 3-5h, and the density of the concentrated stock solution 1 relative to water is 1.15-1.

17.

4. The preparation method according to claim 1, characterized in that: The bacterial content of the bacterial seed liquid in step S5 is 10 8 -10 9 cfu / mL, the fermentation culture conditions are 25-30°C, 100-200r / min, and the fermentation culture is 24-36h; the addition amount of the immobilized amino acid imidazolyl ionic liquid is 3-5wt% of the total mass of the system, the heating temperature is 35-45°C, and the density of the concentrated stock solution 2 relative to water is 1.12-1.

15.

5. The preparation method according to claim 1, characterized in that: The acid solution in step T1 is 5-10wt% HCl or sulfuric acid solution, and the immersion time is 3-5h; the mass ratio of activated silica gel, silane coupling agent KH560 and pyridine in step T2 is 10:3-4:0.5-1, and the heating reflux stirring reaction time is 2-4h; the mass ratio of 1-methylimidazole, concentrated sulfuric acid and modified silica gel in step T3 is 2-3:1-2:10, the heating stirring reaction temperature is 60-70°C, and the time is 5-7h; the alkali solution in step T4 is 10-15wt% NaOH or KOH solution, the mass ratio of lysine, aspartic acid and imidazole-modified silica gel is 2-3:3-4:10, the heating stirring reaction temperature is 80-90°C, and the time is 3-5h.

6. The preparation method according to claim 1, characterized in that: The mass ratio of the stock solution 1 to the stock solution 2 in step S6 is 10:3-4.

7. A polygonatum stock solution obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the polygonatum sibiricum stock solution as claimed in claim 7 in the preparation of medicines for enhancing immunity.

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