A method for extracting active ingredients from antler velvet by combining deep eutectic

Abstract

The present invention discloses a method for extracting the effective components of antlers by combining deep eutectic multiple extractions, belonging to the technical field of drug extraction. This method sequentially performs antler pretreatment, deep eutectic solvent preparation, deep eutectic extraction, alcohol extraction, water extraction and enzymatic hydrolysis to extract the effective components of antlers. The present invention utilizes choline chloride and glycerol in a molar ratio of 1:3 to prepare a deep eutectic solvent, optimizes the comprehensive extraction process, and obtains a protein content of 0.230~0.310 mg / kg, an amino acid content of 325~350.23 mg / kg, a polysaccharide yield of 9.0~11.61%, and a total extract yield of 20.99%. The present invention is non-toxic and pollution-free, has high extraction efficiency, simple extraction operation, and is more environmentally friendly for component extraction; at the same time, the extraction process and detection technology are optimized to shorten the detection time and make the detection results more accurate.

Description

Technical Field

[0001] The invention belongs to the technical field of drug extraction, and particularly relates to a method for extracting effective components of pilose antler by combining deep eutectic multiple extractions. Background Art

[0002] Deer antler, the unossified antlers from the heads of male sika deer and red deer (Cervidae), is a traditional and valuable Chinese medicinal ingredient. As one of the most important animal medicines, it boasts benefits such as aphrodisiac properties, nourishing essence and blood, strengthening tendons and bones, and treating sores. Deer antler is rich in nutrients such as amino acids, effective proteins, and phospholipids. However, research on the combined extraction of active ingredients from deer antler is relatively limited, with extraction methods limited to a single extraction site, resulting in suboptimal extraction yield and purity, leading to waste of raw materials.

[0003] Deep eutectics are a new type of green extraction solvent, comprising hydrogen bond donors and hydrogen bond acceptors. They offer advantages such as high extraction efficiency, low toxicity, rapid degradation, and minimal environmental impact, making them widely used for the extraction of bioactive compounds. However, due to their low vapor pressure, the isolation of target compounds dissolved in deep eutectics is often difficult, hindering their application in industrial production. To obtain target compounds from deep eutectics, it is necessary to investigate appropriate conditions that denature the deep eutectics and disrupt hydrogen bonds to reduce the solubility of the target compound in the deep eutectics.

[0004] In addition, the existing extraction of effective ingredients from deer antlers still has problems such as complicated extraction process, long extraction time, low effective content in the extract, poor environmental friendliness, and long detection time. Therefore, it is urgent to develop a green, environmentally friendly, and low-cost extraction process. Summary of the Invention

[0005] Technical problems solved: In response to the above technical problems, the present invention provides a method for extracting the effective ingredients of antlers by combining deep eutectics, which has the characteristics of being green, environmentally friendly, efficient, low cost, high utilization rate of antler resources, short detection time and high extraction rate.

[0006] Technical solution: A method for extracting active ingredients from antler velvet by combining deep eutectic, comprising the following steps:

[0007] S1. Pretreatment of bloody antlers: drying the bloody antlers and then crushing them to obtain antler powder;

[0008] S2. Prepare a deep eutectic solvent: Mix choline chloride as a hydrogen bond acceptor and glycerol as a hydrogen bond donor in a molar ratio of 1:3, heat to 75-95°C, and stir at this temperature until the white solid dissolves into a colorless viscous liquid to obtain a deep eutectic solvent.

[0009] S3. Deep eutectic extraction: Weigh deer antler powder and add it to the deep eutectic solvent at a solid-liquid ratio of 1 g: (5-20) mL. After mechanical stirring, centrifuge the suspension and collect the supernatant. Set aside the supernatant. Wash the lower residue with water, dry it, and weigh it.

[0010] S4. Alcohol extraction: Add anhydrous ethanol to the residue after deep eutectic extraction at a solid-liquid ratio of 1 g:25 mL. Seal and weigh the mixture, then reflux under boiling conditions. After cooling, seal and weigh the mixture, centrifuge, and remove the supernatant for later use. Wash the lower residue with water, dry, and weigh it.

[0011] S5. Water extraction: Add deionized water to the residue after alcohol extraction at a material-liquid ratio of 1g:(20-40)mL. Extract at 60-100°C, weigh, centrifuge, and reserve the supernatant. Dry and weigh the residue below.

[0012] S6. Enzymatic hydrolysis: Add deionized water to the residue after water extraction at a solid-liquid ratio of 1g:10mL to prepare it as a substrate. Adjust the pH to 8.0 and add trypsin. After enzymatic hydrolysis, inactivate the enzyme, cool, and centrifuge in sequence. Take the supernatant for later use.

[0013] Preferably, in step S1, the antler velvet is crushed and sieved through a 65-85 mesh sieve to obtain antler powder.

[0014] Preferably, in step S2, the constant temperature stirring time is 0.5 to 2 hours.

[0015] Preferably, in step S3, the rotation speed of the mechanical stirring is 300-1500 rpm, and the stirring time is 2-6 hours.

[0016] Preferably, in step S3, the suspension is centrifuged at a speed of 10,000 rpm and for a time of 10 min.

[0017] Preferably, in step S5, the extraction time is 2 to 6 hours.

[0018] Preferably, in step S6, the enzyme accounts for 5-7 wt.% of the substrate.

[0019] Preferably, in step S6, the pH is adjusted by 1M NaOH.

[0020] Preferably, in step S6, the enzymatic hydrolysis temperature is 50° C., and the enzymatic hydrolysis time is 11 h; the enzyme inactivation temperature is 100° C., and the enzyme inactivation time is 15 min.

[0021] Preferably, the water washing temperature is 50-60°C.

[0022] Beneficial effects: The present invention extracts effective ingredients such as amino acids, effective proteins and polysaccharides from velvet antlers through deep eutectic extraction, alcohol extraction, water extraction and enzymatic hydrolysis in sequence, and has the characteristics of being green, environmentally friendly, efficient, non-toxic, low-cost, high in velvet antler resource utilization, short in detection time and high in extraction rate.

[0023] The present invention utilizes deep eutectic extraction combined with alcohol extraction, hydrolysis and enzymatic hydrolysis to determine the extraction sequence and extraction parameters, which can achieve a higher total extract yield of 20.99%. The total extract yield of the combination of alcohol extraction, hydrolysis and enzymatic hydrolysis is 17.37%.

[0024] The present invention uses choline chloride and glycerol in a 1:3 molar ratio to form a deep eutectic solvent. The optimized comprehensive extraction process results in a protein content of 0.230-0.310 mg / kg, an amino acid content of 325-350.23 mg / kg, and a polysaccharide yield of 9.0-11.61% in velvet antler. In the absence of a deep eutectic solvent, the protein content of 0.08-0.2 mg / kg, the amino acid content of 202.28-304.21 mg / kg, and the polysaccharide yield of 6.24%-10.56% can be extracted from velvet antler. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : This is a graph showing the effects of different parameters for preparing deep eutectic solvents on the extraction rate, where A shows the effect of the molar ratio of hydrogen bond donor to hydrogen bond acceptor on the extraction rate of antler protein; B shows the effect of stirring temperature on the extraction rate of antler protein; and C shows the effect of stirring time on the extraction rate of antler protein.

[0026] Figure 2 Figure 1 is a graph showing the effects of different parameters of deep eutectic extraction on the extraction rate, where A is the effect of the solid-liquid ratio of antler powder to deep eutectic solvent on the extraction rate of antler protein; B is the effect of stirring rate on the extraction rate of antler protein; and C is the effect of stirring time on the extraction rate of antler protein.

[0027] Figure 3 Figure 1 is the effect of different water extraction parameters on the extraction rate, where A is the effect of the solid-liquid ratio of antler powder to water on the polysaccharide yield; B is the effect of extraction time on the antler polysaccharide yield; C is the effect of extraction temperature on the antler polysaccharide yield;

[0028] Figure 4 This is a graph showing the effect of the solid-liquid ratio of antler powder and anhydrous ethanol on the total amount of amino acids in alcohol extraction;

[0029] Figure 5 This is a schematic diagram comparing the total amount of amino acids extracted by anhydrous ethanol and trypsin;

[0030] Figure 6 This is a schematic diagram comparing the total extract yields of different extraction methods. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0032] Example 1

[0033] A method for extracting active ingredients from antler velvet by combining deep eutectic multiple extractions comprises the following steps:

[0034] S1. Pretreatment of bloody antlers: Deer antlers were purchased from Changbai Mountain in Changchun City, Jilin Province. The bloody antlers were evenly cut and air-dried at room temperature for several days. The antlers were then crushed through an 80-mesh sieve to obtain antler powder, which was sealed and stored at room temperature until needed.

[0035] S2. Preparation of deep eutectic solvent: Place a hydrogen bond acceptor and a hydrogen bond donor in a 100-mL round-bottom flask at a molar ratio of 1:3. The hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is glycerol. Slowly raise the temperature to 85°C and stir at this constant temperature for 1 h until the white solid in the flask gradually dissolves and turns into a colorless viscous liquid, thereby obtaining a deep eutectic solvent.

[0036] S3. Deep eutectic extraction: Add 1.00 g of antler antler powder to a 100 mL round-bottom flask containing 10.3 g of deep eutectic solution and mechanically stir at 900 rpm for 4 h. After extraction, centrifuge the suspension at 10,000 rpm for 20 min, collect the supernatant, and dry the lower residue at 50°C and weigh it.

[0037] Remove 10 mL of the reserved supernatant, add 50 mL of deionized water, and pour into a 100 mL beaker. Store at 4°C for 18 h. Pour the suspended solids and precipitate into a centrifuge tube and centrifuge at 10,000 rpm for 10 min. Remove the protein layer and wash with 50 mL of water at 60°C for 2 h to remove the deep eutectic solvent.

[0038] S4. Alcohol extraction: Place the weighed powder residue and anhydrous ethanol in a 250 mL round-bottom flask at a solid-liquid ratio of 1g:25mL. Seal and weigh, connect a condenser reflux tube, heat to a slight boil for 1 hour, remove after cooling, seal and weigh, make up for the lost water with water, centrifuge, take the supernatant and set aside, dry the lower residue at 50℃ and weigh it.

[0039] S5. Water extraction: Add deionized water to the antler residue after alcohol extraction at a material-liquid ratio of 1 g:35 mL, soak at 90°C for 4 h, weigh it, make up the lost weight with water, centrifuge and weigh the supernatant for later use, dry the lower layer of residue at 50°C and weigh it.

[0040] S6. Enzymatic Hydrolysis: Add deionized water to the residue after aqueous extraction at a solid-to-liquid ratio of 1:10 to prepare the substrate. Adjust the pH to 8 (1 M NaOH). Add 0.05 g of trypsin (enzyme / substrate = 5 wt.%) and perform enzymatic hydrolysis at 50°C for 11 h. Heat at 100°C for 15 min (to inactivate the enzyme), cool, and allow to stand. Centrifuge at 6000 rpm for 10 min. Filter, remove the supernatant, and store in a refrigerator at 4°C until ready for use.

[0041] Example 2

[0042] The extraction process was optimized based on single-factor experimental design. The single-factor experiments in deep eutectic extraction included deep eutectic molar ratio, solid-liquid ratio, and extraction time; the single-factor experiments in alcohol extraction and water extraction included solid-liquid ratio, extraction time, and extraction temperature.

[0043] Preparation optimization of deep eutectic system:

[0044] The deep eutectic system is extracted according to the molar ratio of 1:3, the stirring temperature is 85℃, and the stirring time is 2 h. Under these conditions, the protein extraction efficiency can be guaranteed to be the highest. Figure 1 As shown, when HBA and HBD are mixed in a ratio of 1:3, glycerol and choline chloride form a uniform and transparent solution because glycerol has more hydroxyl groups, which helps to form more hydrogen bonds with proteins. When the ratio of choline chloride to glycerol changes from 1:1 to 1:3, the extraction efficiency of the protein increases, but when the ratio reaches 1:4, the extraction efficiency decreases. This is because initially as the glycerol content increases, the viscosity of the deep eutectic solvent decreases and the internal hydrogen bonds of choline chloride weaken, which increases the mass transfer rate. In addition, glycerol interacts with the hydrophobic functional groups of the protein, acting as an amphiphilic interface between the hydrophobic surface and choline chloride, and also acts as a stabilizer. However, the hydrogen bond interaction between choline chloride and protein is stronger than that between glycerol and protein, and excessive glycerol content will reduce the proportion of choline chloride in the entire system.

[0045] Extraction method optimization:

[0046] For deep eutectic extraction, the solid-liquid ratio was 1:5~1:13, the stirring rate was 300~1200 rpm, and the extraction time was 3~6 h. The extraction rate was tested, such as Figure 2 As shown in Figure A, as the solid-liquid ratio changes, the protein extraction rate first increases, stabilizes, and then slowly decreases. Insufficient solvent will lead to insufficient extraction, while excessive solvent will cause waste of extractant, and a small amount of powder will move to the top of the flask during stirring, resulting in insufficient extraction. Figure 2As shown in Figure B, as the stirring rate increases, the higher the rate, the higher the extraction rate. However, when the rate is too high, the fluid jet loses too much on the cup wall, resulting in a decrease in the extraction rate. Therefore, 900 rpm is selected as the optimal rotation rate. Figure 2 As shown in C, the extraction rate increases with time. In order to save time, 4 h was selected as the optimal extraction time.

[0047] The solid-liquid ratio of polysaccharide measured by water extraction method is 1:20~1:40, such as Figure 3 As shown in Figure A, the polysaccharide yield is highest when the solid-liquid ratio is 1:35. This may be because under the same dissolution conditions, as the solvent increases, the solute solubility also increases. 1g of antler powder in 35mL of water may have basically dissolved the polysaccharide, and the amount of solute is basically constant at this time. Therefore, from the perspective of saving cost and manpower, a solid-liquid ratio of 1:35 is more appropriate; Figure 3 As shown in B, the extraction time was selected to be 2~6 h, the extraction temperature was 80℃, and the extraction was carried out to measure the polysaccharide yield. As can be seen from the figure, the antler polysaccharide increased with the increase of extraction time. This is because the polysaccharide is more fully dissolved within a certain period of time. When the extraction time exceeds 4 h, the polysaccharide yield decreases, which may be because the stability of the polysaccharide gradually decreases under high temperature conditions. Therefore, the extraction time is preferably 4 h. The material-liquid ratio is 1:35, the extraction temperature is selected to be 60~100℃, and the extraction time is 4 h. Figure 3 As shown in Figure C, as the temperature increases, the polysaccharide yield increases from 7.93% to 10.16%. This may be because the thermal effect accelerates the molecular movement speed. At the same time, the thermal effect increases the damage to the antler cell wall, making the polysaccharide easier to extract. However, the continued increase in temperature may cause polysaccharide instability. In order to reduce polysaccharide loss, the extraction temperature should be 80℃.

[0048] In the alcohol extraction method, Figure 4 It can be seen that the total amount of amino acids is the highest when the solid-liquid ratio of antler powder to anhydrous ethanol is 1g:20mL.

[0049] In the alcohol extraction and enzymatic hydrolysis methods, Figure 5 It can be seen that trypsin extracts the highest amount of amino acids in the enzymatic hydrolysis method. The reason for this is that under suitable conditions, the enzyme decomposes macromolecular substances into small molecular amino acids. Figure 6 It can be seen that the total extract yield obtained by deep eutectic extraction, alcohol extraction, water extraction and enzymatic hydrolysis is much greater than the yield of each method alone.

Claims

1. A method for extracting active ingredients from antler velvet by combining deep eutectic, characterized in that: The steps are as follows: S1. Pretreatment of bloody antlers: drying the bloody antlers and then crushing them to obtain antler powder; S2. Prepare a deep eutectic solvent: Mix choline chloride as a hydrogen bond acceptor and glycerol as a hydrogen bond donor in a molar ratio of 1:3, heat to 75-95°C, and stir at this temperature until the white solid dissolves into a colorless viscous liquid to obtain a deep eutectic solvent. S3. Deep eutectic extraction: Weigh deer antler powder and add it to the deep eutectic solvent at a solid-liquid ratio of 1 g: (5-20) mL. After mechanical stirring, centrifuge the suspension and collect the supernatant. Set aside the supernatant. Wash the lower residue with water, dry it, and weigh it. S4. Alcohol extraction: Add anhydrous ethanol to the residue after deep eutectic extraction at a solid-liquid ratio of 1 g:25 mL. Seal and weigh the mixture, then reflux under boiling conditions. After cooling, seal and weigh the mixture, centrifuge, and remove the supernatant for later use. Wash the lower residue with water, dry, and weigh it. S5. Water extraction: Add deionized water to the residue after alcohol extraction at a material-liquid ratio of 1g:(20-40)mL. Extract at 60-100°C, weigh, centrifuge, and reserve the supernatant. Dry and weigh the residue below. S6. Enzymatic hydrolysis: Add deionized water to the residue after water extraction at a solid-liquid ratio of 1g:10 mL to prepare it as a substrate. Adjust the pH to 8.0 and add trypsin. After enzymatic hydrolysis, inactivate the enzyme, cool, and centrifuge in sequence. Collect the supernatant for later use.

2. The method for extracting active ingredients from velvet antlers by combining deep eutectic according to claim 1, characterized in that: In the step S1, the antler velvet is crushed and sieved with 65-85 mesh to obtain velvet velvet powder.

3. The method for extracting active ingredients from velvet antlers by combining deep eutectic according to claim 1, characterized in that: In step S2, the constant temperature stirring time is 0.5 to 2 hours.

4. The method for extracting active ingredients from velvet antlers by combining deep eutectic according to claim 1, characterized in that: In step S3, the rotation speed of the mechanical stirring is 300-1500 rpm, and the stirring time is 2-6 hours.

5. The method for extracting active ingredients from velvet antlers by combining deep eutectic according to claim 1, characterized in that: In step S3, the suspension is centrifuged at a speed of 10,000 rpm for 10 minutes.

6. The method for extracting active ingredients from velvet antlers by combining deep eutectic according to claim 1, characterized in that: In step S5, the extraction time is 2 to 6 hours.

7. The method for extracting active ingredients from velvet antlers by combining deep eutectic according to claim 1, characterized in that: In step S6, the enzyme accounts for 5-7 wt.% of the substrate.

8. The method for extracting active ingredients from velvet antlers by combining deep eutectic according to claim 1, characterized in that: In step S6, the pH is adjusted using 1 M NaOH.

9. The method for extracting active ingredients from velvet antlers by combining deep eutectic according to claim 1, characterized in that: In step S6, the enzymatic hydrolysis temperature is 50° C., and the enzymatic hydrolysis time is 11 h; the enzyme inactivation temperature is 100° C., and the enzyme inactivation time is 15 min.

10. The method for extracting active ingredients from velvet antlers by combining deep eutectic according to claim 1, characterized in that: The water washing temperature is 50-60°C.

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