Sophora moorcroftiana alkaloid extract and preparation method thereof
By using cellulose composite enzyme solution in the extraction of acetacid alkaloids for enzymatic and ultrasonic extraction, combined with heating reflux, the problems of low extraction rate and complex operation in the prior art were solved, and efficient and simple alkaloids were achieved Extraction significantly improves the extraction rate and purity.
Patent Information
- Application Number
- CN202411230864.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-04
- Publication Date
- 2024-12-10
AI Technical Summary
In the prior art, the extraction method of aceta alkaloid alkaloids has high limitations, complex operation and expensive operation, resulting in serious losses of active ingredients and low production capacity.
Enzymatic decomposition of cellulose complex enzyme solution and saccharomyces was performed, combined with ultrasonic extraction and heating reflux extraction, and multiple extractions were performed through alcohol solution to improve the extraction rate and purity of the alkaloids.
The alkaloid extraction rate has been increased to 90.15%, which is 31.8% higher than the traditional method. It is easy to operate and the recycling and utilization of active ingredients is more efficient.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of plant extraction, and particularly relates to a Sophora moracha alkaloid extract and a preparation method thereof. Background Art
[0002] Sophora moorcroftiana is a small shrub of the genus Sophora in the Leguminosae family, with many dense branches that are sometimes forked. It blooms from May to July, and fruits from July to October. Its young shoots, leaves and young buds can be used as wintering fodder for livestock, and its stems can be used as fuel. It is of great significance in windbreak and sand fixation, soil and water conservation, and ecological environment protection. In addition, the alkaloids contained in the seeds of Sophora moorcroftiana have detoxification and anti-inflammatory effects, and are widely used.
[0003] In the prior art, the extraction of Sophora moracifolia alkaloids mostly adopts solvent extraction, ultrasonic assisted extraction, acidic aqueous solution extraction, alkaline aqueous solution extraction and supercritical fluid extraction, but the above extraction methods are not only highly limited, complicated to operate and expensive, but also cause the serious loss of effective ingredients, which leads to low production capacity of Sophora moracifolia alkaloids. Therefore, how to improve the extraction rate of alkaloids in Sophora moracifolia, reduce the loss of effective ingredients, and realize industrial production is a problem to be solved in this field. Summary of the invention
[0004] The invention aims to provide a Sophora moracha alkaloid extract and a preparation method thereof, which is simple to operate and has a high extraction rate of alkaloids.
[0005] The present invention provides a method for preparing a Sophora moracha alkaloid extract, comprising the following steps:
[0006] Mixing Sophora japonica and cellulose complex enzyme solution and performing enzymolysis to obtain an enzymolysis mixture;
[0007] The enzymatic hydrolysis mixture is mixed with an alcohol solution and subjected to ultrasonic extraction to obtain an ultrasonically extracted mixture;
[0008] The ultrasonic mixture is subjected to heating reflux extraction to obtain a filtrate;
[0009] Concentrating and extracting the filtrate in sequence to obtain the alkaloid extract;
[0010] The cellulose complex enzyme includes cellulase and hemicellulase.
[0011] Preferably, the cellulose complex enzyme solution uses water as a solvent; the mass concentration of the cellulose complex enzyme solution is 4% to 12%;
[0012] The enzymatic activity of the cellulose complex enzyme is 20000-50000 U / g.
[0013] Preferably, the mass concentration of the Sophora japonica var. moorlandris in the cellulose complex enzyme solution is 6.25% to 12.5%.
[0014] Preferably, the enzymatic hydrolysis time is 2 to 3 hours; the enzymatic hydrolysis temperature is 40 to 50°C.
[0015] Preferably, the alcohol solution comprises an ethanol solution; the volume concentration of the ethanol solution is 60% to 80%;
[0016] The mass concentration of the morabella in the ethanol solution is 1.67% to 5%.
[0017] Preferably, the ultrasonic extraction time is 25 to 65 minutes, the temperature is 40 to 50° C., and the power is 100 to 300W.
[0018] Preferably, the heating reflux extraction is performed ≥ 3 times; the time of each heating reflux is 2 to 3 hours, and the temperature is 78 to 85°C.
[0019] Preferably, the concentration is accompanied by rotation; the rotation speed is 80-100 rpm and the temperature is 35-40°C.
[0020] Preferably, the morabella spp. comprises seeds of morabella spp.
[0021] The present invention provides an alkaloid extract obtained by the preparation method described in the above technical scheme.
[0022] Beneficial effects:
[0023] The present invention provides a method for preparing a Sophora moracha alkaloid extract. The present invention performs enzymolysis by mixing Sophora moracha with a cellulose complex enzyme solution including cellulase and hemicellulase, which is not only beneficial to keeping the effective components of the alkaloids from being destroyed, but also beneficial to the effective release of the alkaloids; ultrasonic extraction and heating reflux are beneficial to further improving the extraction rate and purity of the alkaloids. Therefore, an alkaloid extract with rich effective components and high extraction rate can be prepared. Experiments have shown that the alkaloid extract obtained by the technical solution provided by the present invention has an extraction rate of ≥90.15%, which is 31.8% higher than that of the traditional organic solvent method, and the operation process is simple, which is more conducive to the recovery and utilization of effective components. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.
[0025] Figure 1 It is the HPLC peak retention time diagram of four alkaloids in the present invention. DETAILED DESCRIPTION
[0026] The invention provides a method for preparing a Sophora morabica alkaloid extract, comprising the following steps: mixing Sophora morabica with a cellulose complex enzyme solution and then performing enzymolysis to obtain an enzymolysis mixture; mixing the enzymolysis mixture with an alcohol solution and performing ultrasonic extraction to obtain an ultrasonic mixture; heating the ultrasonic mixture for reflux extraction to obtain a filtrate; and sequentially concentrating and extracting the filtrate to obtain the alkaloid extract; the cellulose complex enzyme comprises cellulase and hemicellulase.
[0027] In the present invention, unless otherwise specified, the raw materials and reagents used are purchased conventionally.
[0028] In the present invention, the moraine Sophora japonica is preferably crushed to obtain crushed moraine Sophora japonica. In the present invention, the moraine Sophora japonica preferably includes moraine Sophora japonica seeds; the crushed moraine Sophora japonica is preferably sieved to obtain moraine Sophora japonica of 40 to 80 meshes, more preferably 40 meshes.
[0029] After obtaining the crushed sand-growing Sophora japonica, the present invention mixes the crushed sand-growing Sophora japonica with a cellulose complex enzyme solution for enzymolysis to obtain an enzymolysis mixture. In the present invention, the cellulose complex enzyme solution uses water as a solvent; the mass concentration of the cellulose complex enzyme solution is 4% to 12%, more preferably 10%; the cellulose complex enzyme preferably includes cellulase and hemicellulase; there is no special requirement for the added quality of cellulase and hemicellulase in the cellulose complex enzyme, and the cellulose complex enzyme with an enzyme activity of 20,000 to 50,000 U / g is preferably obtained, and more preferably 24,000 U / g. The present invention has no special limitation on the source of the cellulose complex enzyme, and the above composition and enzyme activity can be guaranteed. For example, in a specific embodiment of the present invention, the cellulose complex enzyme is purchased from Weifang Kangdian Biotechnology Co., Ltd., and the product model is Textile Enzyme DP3000.
[0030] In the present invention, when mixing, the mass volume ratio of the crushed Sophora japonica to the cellulose complex enzyme solution is preferably 1g:8-16mL, further preferably 1g:10-14mL, more preferably 1g:10mL, that is, the mass concentration of the crushed Sophora japonica in the cellulose complex enzyme solution is preferably 6.25%-12.5%, more preferably 10%; the enzymolysis time is preferably 2-3h, more preferably 2h; the enzymolysis temperature is preferably 40-50°C, more preferably 45°C. Enzymolysis is beneficial to the destruction of the seed cell wall to increase the release of intracellular substances.
[0031] After obtaining the enzymatic hydrolysis mixture, the present invention mixes the enzymatic hydrolysis mixture with an alcohol solution for ultrasonic extraction to obtain an ultrasonic mixture. In the present invention, the alcohol solution includes an ethanol solution; the volume concentration of the ethanol solution is preferably 60% to 80%, more preferably 75%; when performing ultrasonic extraction, the added volume of the ethanol solution is preferably based on the crushed sand Sophora japonica, that is, 20 to 60 mL of ethanol solution is required for each 1 g of crushed sand Sophora japonica to be mixed with the enzymatic hydrolysis mixture produced by it, and the mass volume ratio of the crushed sand Sophora japonica to the ethanol solution is further preferably 1g:20 to 60mL, more preferably 1g:40mL, that is, the mass concentration of sand Sophora japonica in the ethanol solution is 1.67% to 5%, more preferably 2.5%; the ultrasonic time is preferably 25 to 65min, more preferably 45min; the ultrasonic temperature is preferably 40 to 50°C, more preferably 45°C; the ultrasonic power is preferably 100 to 300W, further preferably 200 to 300W, more preferably 250w. Ultrasound is beneficial to the destruction of seed cell walls to increase the release of intracellular substances.
[0032] After obtaining the ultrasonic mixture, the present invention performs heating reflux extraction on the ultrasonic mixture to obtain a filtrate to be concentrated. The heating reflux extraction process preferably includes heating reflux and filtration, that is, the heating reflux device is connected to the suction filtration bottle, so that the refluxed liquid can be directly filtered to obtain a filtrate; the number of heating reflux extraction is preferably ≥3 times, more preferably 3 times; through the above process, it is beneficial to extract the intracellular substances of the seeds as much as possible.
[0033] The present invention preferably performs a first heating reflux extraction on the mixture after ultrasound to obtain a first filtrate and a first filter residue; the time of the first heating reflux extraction is preferably 2 to 3 hours, more preferably 2 hours; the temperature of the first heating reflux is preferably 78 to 85°C, more preferably 80°C.
[0034] After obtaining the first filter residue, the present invention preferably mixes the first filter residue with an alcohol solution and performs a second heating reflux extraction to obtain a second filtrate and a second filter residue. The type, volume concentration, and addition ratio of the alcohol solution are the same as those of the alcohol solution in the ultrasonic extraction process, and will not be described in detail here; the process of the second heating reflux extraction is the same as that of the first heating reflux extraction, and will not be described in detail here.
[0035] After obtaining the second filter residue, the present invention preferably mixes the second filter residue with an alcohol solution and performs a third heating reflux extraction to obtain a third filtrate and a third filter residue; the process of the third heating reflux extraction is the same as the process of the second heating reflux extraction, and will not be repeated here; when the heating reflux extraction is performed more than three times, the process of the heating reflux extraction is the same as the process of the second heating reflux extraction.
[0036] In the present invention, after the first filtrate, the second filtrate and the third filtrate are mixed, the filtrate to be concentrated is obtained, that is, the filtrate to be concentrated is the general term for the filtrate after multiple heating and reflux extraction.
[0037] After obtaining the filtrate to be concentrated, the present invention concentrates the filtrate to be concentrated to 100 mL to obtain a concentrated solution. In the present invention, the concentration is accompanied by rotation; the rotation speed is preferably 80 to 100 rpm, more preferably 90 rpm; the rotation temperature is preferably 35 to 40° C., more preferably 40° C.
[0038] After obtaining the concentrated solution, the present invention extracts the concentrated solution to obtain an extract; in the present invention, the extraction reagents are preferably petroleum ether and chloroform in sequence; there are no special requirements for the amount and method of use of the petroleum ether and chloroform, and the techniques well known in the art can be used.
[0039] After obtaining the extract, the present invention preferably re-concentrates the extract to obtain a paste-like substance, which is the alkaloid extract. In the present invention, the re-concentration is accompanied by rotation; the rotation speed is preferably 80 to 100 rpm, more preferably 90 rpm; the rotation temperature is preferably 35 to 40° C., more preferably 40° C.
[0040] The present invention also provides an alkaloid extract obtained by the preparation method described in the above technical solution, and the yield of the alkaloid extract is ≥3.604%.
[0041] Experiments have shown that the alkaloid extract obtained by the technical solution provided by the present invention not only has a high alkaloid yield, but also has a simple operation process and is more conducive to the recovery and utilization of effective ingredients.
[0042] In order to further illustrate the present invention, a Sophora japonica alkaloid extract and a preparation method thereof provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.
[0043] The cellulose complex enzyme used in the examples and comparative examples was purchased from Weifang Kangdien Biotechnology Co., Ltd. The product name is textile enzyme DP3000, which is composed of cellulase and hemicellulase; the enzyme activity of the complex enzyme is 24000 U / g.
[0044] The method for determining the alkaloid content in the extract of Sophora japonica seeds by HPLC is as follows:
[0045] The standard substances used are matrine, oxymatrine, sophoracarpine and oxymatrine alkaloids. The preparation methods of the standard curves of the four alkaloids are the same. Take the preparation process of the standard curve of matrine as an example: weigh 2 mg of matrine standard substance dried to constant mass at 105°C, dissolve it in 5 mL of methanol, and prepare a matrine standard solution with a mass concentration of 0.4 mg / mL. Accurately pipette 0, 0.05, 0.1, 0.25, 0.5, 0.75 and 1 mL of the matrine standard solution into a test tube, add 5, 4.95, 4.9, 4.75, 4.5, 4.25 and 4 mL of methanol solution respectively, shake well, pipette 1 mL respectively, use the corresponding reagent as the blank control, and measure its absorption peak area value at a wavelength of 210 nm. The standard curve was drawn with the peak area (Y) as the ordinate and the mass concentration of matrine (X) as the abscissa. The regression equation of the standard curve of matrine was Y=196.20711X+3.31635, and the correlation coefficient R 2 =0.9998. The standard curve regression equations corresponding to the remaining oxymatrine, sophocarpine and oxysophocarpine are Y=49.84393X+10.30458, Y=363.66922X+0.76102 and Y=87.00174X+1.09337, respectively. The HPLC peak retention time diagram of the four alkaloids is shown in Figure 1 As shown (in Figure 1 In the figure, 1 represents the HPLC peak retention time of oxymatrine, 2 represents the HPLC peak retention time of oxysophocarpine, 3 represents the HPLC peak retention time of matrine, and 4 represents the HPLC peak retention time of sophocarpine).
[0046] Calculation of yield: Calculate according to standard number: GB / T 22293-2008: dilute the extracted extract by a certain multiple, take 1 mL of the dilution, determine the peak area value by high performance liquid chromatography, calculate the yield of the four alkaloids according to the standard curve, and calculate the yield of the four alkaloids according to the formula. The sum of the yields of the four alkaloids is the content of alkaloids in Sophora moracha.
[0047] The total flavonoid yield was calculated according to the standard curve, and the total alkaloid yield was calculated according to formula (1).
[0048]
[0049] In the formula: r is the alkaloid yield, %; c is the alkaloid mass concentration, mg / mL; V is the total volume of the extraction solution to be tested, mL; G is the mass of the raw material, ω is the water content of the raw material, %; n is the dilution multiple.
[0050] Example 1
[0051] A method for preparing a Sophora moracha alkaloid extract (3 parallel repeated experiments), comprising the following steps:
[0052] 1) crushing the seeds of Sophora japonica with a pulverizer and passing through a 40-mesh sieve to obtain Sophora japonica seed powder;
[0053] 2) 5.000 g (accurate to 0.001) of Sophora morata seed powder was placed in a 250 mL round-bottom flask, and a 10% cellulose complex enzyme aqueous solution (i.e., 0.5 g of cellulose complex enzyme was dissolved in 50 mL of water) was added to the flask, and enzymolysis was performed at 45° C. for 2 h to obtain an enzymolysis mixture;
[0054] 3) adding 200 mL of 75% ethanol to the enzymatic hydrolysis mixture to make the mass concentration of Sophora moracha seed powder (placed in the ethanol solution) 2.5%, and ultrasonically treating at 45° C. and 250 W for 45 min to obtain an ultrasonically treated enzymatic hydrolysis mixture;
[0055] 4) The enzymatic hydrolysis mixture after ultrasonication was subjected to a first heating extraction for 2 h (the heating temperature was 80° C.), and was subjected to a first filtration to obtain a first filter residue and a first filtrate, and the first filtrate was transferred to a glass bottle with a cover;
[0056] 200 mL of 75% ethanol solution was added to the first filter residue again, and the mixture was heated and extracted for 2 h (the heating temperature was 80° C.), and filtered for the second time to obtain a second filter residue and a second filtrate, and the second filtrate was mixed with the first filtrate;
[0057] 200 mL of 75% ethanol solution was added to the second filter residue again, and the mixture was heated and extracted for 2 h (the heating temperature was 80° C.), and filtered for the third time to obtain a third filter residue and a third filtrate, and the third filtrate, the second filtrate and the first filtrate were mixed to obtain a mixed filtrate;
[0058] 5) The mixed filtrate was rotary evaporated at 40°C and 90rpm to 100mL, first extracted with petroleum ether to remove ester compounds, then extracted with chloroform, the alkaloids were dissolved in the chloroform layer, and then rotary evaporated at 40°C and 90rpm to obtain a paste-like substance, which is the alkaloid extract. The alkaloid extract was completely dissolved in anhydrous methanol and fixed to 100mL for later use.
[0059] Example 2
[0060] The method for extracting alkaloids from Sophora moracha seeds by enzyme treatment (3 parallel repeated experiments), the steps are:
[0061] 1) is the same as step 1) in Example 1;
[0062] 2) 5.000 g (accurate to 0.001) of Sophora morata seed powder was placed in a 250 mL round-bottom flask, 50 mL of a 10% cellulose complex enzyme solution was added to the flask, and enzymolysis was performed at 45° C. for 2 h to obtain an enzymolysis mixture;
[0063] 3) adding 200 mL of 75% by volume ethanol to the enzymatic hydrolysis mixture to adjust the material-liquid ratio (i.e., Sophora moracha seed powder and ethanol) to 1 g:40 mL to obtain a mixed solution;
[0064] 4) heating the mixed solution to reflux to obtain a mixed filtrate; the specific process of heating to reflux is the same as step 4) in Example 1;
[0065] 5) is the same as step 5) in Example 1.
[0066] Example 3
[0067] Ultrasonic extraction of alkaloids from Sophora moracha seeds (3 replicate experiments), the steps are:
[0068] 1) is the same as step 1) in Example 1;
[0069] 2) 5.000 g (accurate to 0.001) of Sophora morata seed powder was placed in a 250 mL round-bottom flask, 200 mL of 75% ethanol by volume was added to the flask to adjust the material-liquid ratio (i.e. Sophora morata seed powder and ethanol) to 1 g:40 mL, and ultrasonic treatment was performed at 45° C. for 45 min (ultrasonic power was 250 W) to obtain a mixed solution after ultrasonic treatment;
[0070] 3) heating the mixed solution to reflux to obtain a mixed filtrate; the specific process of heating to reflux is the same as step 4) in Example 1;
[0071] 4) is the same as step 5) in Example 1.
[0072] Comparative Example 1
[0073] The method for extracting alkaloids from Sophora moracha seeds by solvent method (3 parallel repeated experiments), the steps are:
[0074] 1) is the same as step 1) in Example 1;
[0075] 2) 5.000 g (accurate to 0.001) of Sophora morata seed powder was placed in a 250 mL round-bottom flask, and 200 mL of 75% ethanol was added to the flask to adjust the material-liquid ratio (i.e. Sophora morata seed powder and ethanol) to 1 g:40 mL to obtain a mixed solution;
[0076] 3) heating the mixed solution to reflux to obtain a mixed filtrate; the specific process of heating to reflux is the same as step 4) in Example 1;
[0077] 4) is the same as step 5) in Example 1.
[0078] The yields of the alkaloid extracts in Examples 1 to 3 and Comparative Example 1 were measured respectively. The results are shown in Table 1.
[0079] Table 1 Yield of Sophora japonica alkaloids from different extraction methods
[0080] deal with method Alkaloid extraction rate Example 1 Solvent extraction + ultrasound-coupled enzyme-assisted extraction 90.15％ Example 2 Solvent extraction + enzymatic extraction 79.69％ Example 3 Solvent extraction + ultrasonic extraction 75.79％ Comparative Example 1 Solvent Extraction 68.41％
[0081] It can be seen from Table 1 that the extraction rate of alkaloids in Example 1 is the highest, which is 90.15%, which is 31.8% higher than that of the traditional extraction method in Comparative Example 1. Therefore, the technical method provided by the present invention is the best extraction method for alkaloids from Sophora moracha seeds.
[0082] In summary, the technical solution provided by the present invention has a yield of more than 30% higher than that of traditional organic solvent extraction, more than 15% higher than that of ultrasonic-assisted extraction, and more than 10% higher than that of enzymatic-assisted extraction. It can be seen that the present invention provides a low-temperature, green and efficient extraction method, which solves the shortcomings of the extraction rate of effective components being limited by the equilibrium concentration inside and outside the cell, the long extraction time, the high solvent consumption, the low extraction efficiency, and the easy loss of effective components, and has a wide application prospect in the effective extraction of alkaloids.
[0083] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A method for preparing a Sophora japonica alkaloid extract, characterized in that: The steps include: Mixing Sophora japonica and cellulose complex enzyme solution and performing enzymolysis to obtain an enzymolysis mixture; The enzymatic hydrolysis mixture is mixed with an alcohol solution and subjected to ultrasonic extraction to obtain an ultrasonically extracted mixture; The ultrasonic mixture is subjected to heating reflux extraction to obtain a filtrate; Concentrating and extracting the filtrate in sequence to obtain the alkaloid extract; The cellulose complex enzyme includes cellulase and hemicellulase.
2. The preparation method according to claim 1, characterized in that: The cellulose complex enzyme solution uses water as a solvent; the mass concentration of the cellulose complex enzyme solution is 4% to 12%; The enzymatic activity of the cellulose complex enzyme is 20000-50000 U / g.
3. The preparation method according to claim 1 or 2, characterized in that: The mass concentration of the morabella sphaerocarpa in the cellulose complex enzyme solution is 6.25% to 12.5%.
4. The preparation method according to claim 1, characterized in that: The enzymolysis time is 2 to 3 hours; the enzymolysis temperature is 40 to 50°C.
5. The preparation method according to claim 1, characterized in that: The alcohol solution includes an ethanol solution; the volume concentration of the ethanol solution is 60% to 80%; The mass concentration of the morabella in the ethanol solution is 1.67% to 5%.
6. The preparation method according to claim 1, characterized in that: The ultrasonic extraction time is 25 to 65 minutes, the temperature is 40 to 50° C., and the power is 100 to 300W.
7. The preparation method according to claim 1, characterized in that: The heating reflux extraction is performed for ≥3 times; the time of each heating reflux is 2 to 3 hours, and the temperature is 78 to 85°C.
8. The preparation method according to claim 1, characterized in that: The concentration is accompanied by rotation; the rotation speed is 80-100 rpm, and the temperature is 35-40°C.
9. The preparation method according to any one of claims 1, 2 and 4 to 8, characterized in that: The morabella plant includes the seeds of morabella plant.
10. The alkaloid extract obtained by the preparation method according to any one of claims 1 to 9.