Method for extracting osthole from Cnidium monnieri
By optimizing the enzymatic decomposition and extraction process, using a combination of cellulase, hemicellulase, calcium phosphate, sodium dihydrogen phosphate and sucrose esters, combined with β-cyclodextrin and sucrose esters, the problems of low extraction rate and sucrose esters in the prior art are solved, and efficient and environmentally friendly serpin extraction is achieved.
Patent Information
- Application Number
- CN202311402804.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-27
- Publication Date
- 2025-08-12
- Estimated Expiration
- 2043-10-27
AI Technical Summary
The existing methods of extracting snailoxin from snails have problems such as low extraction rate, long enzymatic decomposition time, and easy to damage the efficacy, which is difficult to meet the needs of industrial production.
The combination of cellulase, hemicellulase, calcium phosphate, sodium dihydrogen phosphate and sucrose esters is adopted, combined with the use of β-cyclodextrin and sucrose esters, and the enzymatic lysis and extraction process are used to optimize the enzymatic lysis conditions and stir the stirring speed to improve the enzymatic lysis efficiency and extraction rate.
The extraction rate of snailoxin was significantly improved to more than 98.8%, shortened the enzymatic decomposition time to 30-50 minutes, protected the efficacy of snailoxin, and reduced energy consumption and solvent use.
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Figure BDA0004515760360000101
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of osthole extraction, and more particularly to a method for extracting osthole from Cnidium monnieri. Background Art
[0002] Cnidium monnieri is the dried mature fruit of the Apiaceae plant Cnidium monnieri. The fruit is harvested in summer and autumn when ripe, removed of impurities, and sun-dried. Cnidium monnieri is a double-hanging fruit, oval in shape, 2 to 4 mm long, and approximately 2 mm in diameter. Its surface is grayish-yellow or grayish-brown, with two outward-curving plinths at the top and an occasional thin stalk at the base. The back of the schizocarp has five thin, raised longitudinal ridges, while the joint surface is flat and has two slightly raised brown longitudinal ridges. The peel is crisp and easily falls off when rubbed. The seeds are small, grayish-brown, and oily. It has a fragrant aroma and a spicy, cooling flavor that can leave a numbing sensation on the tongue.
[0003] Cnidium monnieri contains a variety of chemical components, including coumarins, phenolic glycosides, and volatile oils. Osthole is a quality-control component of Cnidium monnieri. In vivo pharmacological studies have shown that osthole has anti-inflammatory, neuroprotective, and hypoglycemic activities. Existing methods for extracting osthole from Cnidium monnieri mainly include ethanol reflux, ultrasound, microwave, and bio-enzymatic methods. While the ethanol reflux and reflux methods offer good extraction efficiency, they require energy to recover large amounts of ethanol. Ultrasonic equipment is expensive. Microwaves, due to their direct impact on the molecular structure, can easily damage the osthole structure, resulting in a loss of efficacy. Existing bio-enzymatic methods, while mild in extraction conditions, require long enzymatic hydrolysis times, resulting in low osthole extraction yields. Therefore, there is still much room for improvement in osthole extraction yield, efficiency, and efficacy retention to meet the needs of industrial production. Summary of the Invention
[0004] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.
[0005] In order to achieve these objects and other advantages according to the present invention, a method for extracting osthole from Cnidium monnieri is provided, comprising the following steps:
[0006] Step 1: Grind the Cnidium monnieri into Cnidium monnieri powder;
[0007] Step 2: Add appropriate amount of water to the Cnidium monnieri powder, mix, then add cellulase, hemicellulase, calcium phosphate, sodium dihydrogen phosphate and sucrose ester, mix, and stir at 40-55° C. to obtain an enzymatic hydrolysis mixture;
[0008] Step 3: dilute the enzymatic hydrolysis mixture with water, add β-cyclodextrin and sucrose ester, and stir and extract at 50-60°C;
[0009] Step 4: dilute with water, add β-cyclodextrin and sucrose ester, and continue to stir and extract at 50-60°C;
[0010] Step 5: Filter and collect the filtrate, and dry it to obtain an extract containing osthole.
[0011] Preferably, based on the weight of Cnidium monnieri, the added amounts of cellulase and hemicellulase are 800-1000 U / g and 500-800 U / g, respectively;
[0012] Based on the weight of Cnidium monnieri, the addition amounts of water, calcium phosphate, sodium dihydrogen phosphate, and sucrose ester in step 2 are 1-2 g / g, 30-50 mg / g, 50-100 mg / g, and 10-30 mg / g, respectively, and the stirring enzymolysis time is 30-50 min.
[0013] Preferably, based on the weight of Cnidium monnieri, the added amounts of water, β-cyclodextrin and sucrose ester in step 3 are 1-2 g / g, 100-300 mg / g and 5-10 mg / g respectively.
[0014] Preferably, based on the weight of Cnidium monnieri, the added amounts of water, β-cyclodextrin and sucrose ester in step 4 are 1-5 g / g, 200-500 mg / g and 5-10 mg / g respectively.
[0015] Preferably, the particle size of the Cnidium monnieri powder in step 1 is less than 50 mesh.
[0016] Preferably, the specific method of drying in step 5 is vacuum low-temperature drying.
[0017] Preferably, the stirring speed of the enzymatic hydrolysis in step 2 is 40 to 80 rpm.
[0018] Preferably, the stirring speed of the stirring extraction in step 3 and step 4 is 100-300 rpm.
[0019] Preferably, the stirring extraction time in step 3 and step 4 is 20 to 30 minutes and 20 to 40 minutes, respectively.
[0020] The present invention has at least the following beneficial effects:
[0021] First, by adding auxiliary agents such as calcium phosphate, sodium dihydrogen phosphate, and sucrose esters during the stirring enzymatic hydrolysis process, the enzymatic hydrolysis efficiency (the traditional enzymatic hydrolysis method requires more than 2 hours, while the present application only requires 30 to 50 minutes) and the osthole extraction rate can be significantly improved (the traditional enzymatic hydrolysis method has a maximum extraction rate of about 95%, while the present application reaches 98.8% and above).
[0022] Second, by adding auxiliary agents sucrose ester and β-cyclodextrin simultaneously during the stirring extraction process, the extraction rate of osthole can be significantly improved.
[0023] Third, by controlling the stirring speed within the range of 100-300 rpm during the stirring extraction process, the extraction rate of osthole can be significantly improved.
[0024] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. DETAILED DESCRIPTION
[0025] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.
[0026] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.
[0027] <Example 1>
[0028] The method for extracting osthole from Cnidium monnieri comprises the following steps:
[0029] Step 1: Weigh 1000g of Cnidium monnieri and grind it into less than 50 mesh using a grinder to obtain Cnidium monnieri powder.
[0030] Step 2: Measure 1000mL of water and add it to the Cnidium monnieri powder, then add cellulase, hemicellulase, calcium phosphate, sodium dihydrogen phosphate and sucrose ester, mix, and then stir and enzymolyze in a constant temperature water bath at 40°C for 30 minutes to obtain an enzymolysis mixture; wherein, based on the weight of Cnidium monnieri, the addition amounts of cellulase and hemicellulase are 800U / g and 500U / g, respectively, and the addition amounts of calcium phosphate, sodium dihydrogen phosphate, and sucrose ester are 30mg / g, 50mg / g, and 10mg / g, respectively. The stirring speed of the stirring enzymolysis is 40rpm;
[0031] Step 3: Measure 1000 mL of water and add it to the enzymatic hydrolysis mixture in step 2, then add β-cyclodextrin and sucrose ester, and stir and extract in a constant temperature water bath at 50°C for 20 minutes; wherein, based on the weight of Cnidium monnieri, the addition amounts of β-cyclodextrin and sucrose ester in step 3 are 100 mg / g and 5 mg / g, respectively, and the stirring speed of the stirring extraction is 100 rpm.
[0032] Step 4: Measure 1000 mL of water and add it to step 3 for dilution, and add β-cyclodextrin and sucrose ester, and continue stirring and extracting in a constant temperature water bath at 50°C for 20 minutes; wherein, based on the weight of Cnidium monnieri, the amount of β-cyclodextrin and sucrose ester added in step 4 is 200 mg / g and 5 mg / g, respectively, and the stirring speed of the stirring extraction is 100 rpm.
[0033] Step 5: Filter and collect the filtrate, and vacuum-dry the filtrate at 60° C. to obtain an extract containing osthole.
[0034] <Example 2>
[0035] The method for extracting osthole from Cnidium monnieri comprises the following steps:
[0036] Step 1: Weigh 1000g of Cnidium monnieri and grind it into less than 50 mesh using a grinder to obtain Cnidium monnieri powder.
[0037] Step 2: Measure 2000mL of water and add it to the Cnidium monnieri powder, then add cellulase, hemicellulase, calcium phosphate, sodium dihydrogen phosphate and sucrose ester, mix, and then stir and enzymolyze in a constant temperature water bath at 55°C for 50 minutes to obtain an enzymolysis mixture; wherein, based on the weight of Cnidium monnieri, the addition amounts of cellulase and hemicellulase are 1000U / g and 800U / g, respectively, and the addition amounts of calcium phosphate, sodium dihydrogen phosphate, and sucrose ester are 50mg / g, 100mg / g, and 30mg / g, respectively. The stirring speed of the stirring enzymolysis is 80rpm;
[0038] Step 3: Measure 2000 mL of water and add it to the enzymatic hydrolysis mixture in step 2, then add β-cyclodextrin and sucrose ester, and stir and extract for 30 minutes in a constant temperature water bath at 60°C; wherein, based on the weight of Cnidium monnieri, the addition amounts of β-cyclodextrin and sucrose ester in step 3 are 300 mg / g and 10 mg / g, respectively, and the stirring speed of the stirring extraction is 300 rpm.
[0039] Step 4: Measure 5000 mL of water and add it to the dilution in step 3, and add β-cyclodextrin and sucrose ester, and continue to stir and extract in a constant temperature water bath at 60°C for 40 minutes; wherein, based on the weight of Cnidium monnieri, the amount of β-cyclodextrin and sucrose ester added in step 4 is 500 mg / g and 10 mg / g, respectively, and the stirring speed of the stirring extraction is 300 rpm.
[0040] Step 5: Filter and collect the filtrate, and vacuum-dry the filtrate at 60° C. to obtain an extract containing osthole.
[0041] <Example 3>
[0042] The method for extracting osthole from Cnidium monnieri comprises the following steps:
[0043] Step 1: Weigh 1000g of Cnidium monnieri and grind it into less than 50 mesh using a grinder to obtain Cnidium monnieri powder.
[0044] Step 2: Measure 1500mL of water and add it to the Cnidium monnieri powder, then add cellulase, hemicellulase, calcium phosphate, sodium dihydrogen phosphate and sucrose ester, mix, and then stir and enzymolyze in a constant temperature water bath at 50°C for 40 minutes to obtain an enzymolysis mixture; wherein, based on the weight of Cnidium monnieri, the addition amounts of cellulase and hemicellulase are 800U / g and 800U / g, respectively, and the addition amounts of calcium phosphate, sodium dihydrogen phosphate, and sucrose ester are 40mg / g, 80mg / g, and 20mg / g, respectively. The stirring speed of the stirring enzymolysis is 60rpm;
[0045] Step three, measure 1500mL of water and add it to the enzymatic hydrolysis mixture in step two, then add β-cyclodextrin and sucrose ester, and stir and extract in a constant temperature water bath at 55°C for 20min; wherein, based on the weight of Cnidium monnieri, the addition amounts of β-cyclodextrin and sucrose ester in step three are 200mg / g and 8mg / g, respectively, and the stirring speed of the stirring extraction is 200rpm.
[0046] Step 4: Measure 3000 mL of water and add it to the dilution in step 3, and add β-cyclodextrin and sucrose ester, and continue stirring and extracting in a constant temperature water bath at 55°C for 30 minutes; wherein, based on the weight of Cnidium monnieri, the amount of β-cyclodextrin and sucrose ester added in step 4 is 300 mg / g and 8 mg / g, respectively, and the stirring speed of the stirring extraction is 200 rpm.
[0047] Step 5: Filter and collect the filtrate, and vacuum-dry the filtrate at 60° C. to obtain an extract containing osthole.
[0048] <Standard extraction method>
[0049] The osthole extract was extracted using the Chinese Pharmacopoeia standard method, specifically: Cnidium monnieri was crushed, passed through a No. 3 sieve, 0.1 g was weighed, placed in a stoppered conical flask, 25 mL of anhydrous ethanol was added, sealed, weighed, left for 2 hours, ultrasonically treated for 30 minutes with an ultrasonic power of 300 W and a frequency of 50 kHz, cooled, weighed again, and the lost weight was supplemented with anhydrous ethanol. The mixture was shaken well, 5 mL of the supernatant was accurately measured and placed in a 10 mL volumetric flask, anhydrous ethanol was added to the scale, and shaken well to obtain an extract containing osthole.
[0050] Detection:
[0051] The content of osthole was detected by high performance liquid chromatography method according to Chinese Pharmacopoeia (General Chapter 0512).
[0052] The test results are shown in Table 1:
[0053] Table 1 Extraction rate of osthole in Examples 1 to 3
[0054] Group Osthole content mg / g Extraction rate % Example 1 0.1539 98.8 Example 2 0.1550 99.5 Example 3 0.1544 99.1 Standard extraction method 0.1558 ——
[0055] Note: relative to the content of dry Cnidium monnieri powder.
[0056] As can be seen from Table 1, the extraction rate of osthole by the methods of Examples 1 to 3 is as high as 98.8% or above. The methods of Examples 1 to 3 are mild and can effectively protect the structure of osthole from being damaged by high temperature. They do not require the use of a large amount of ethanol reagent, thus eliminating the need to spend excessive costs on solvent recovery. The enzymatic hydrolysis time is 30 to 50 minutes, which significantly reduces the enzymatic hydrolysis time and improves the enzymatic hydrolysis efficiency.
[0057] Influencing factors:
[0058] The effects of extraction adjuvants (calcium phosphate, sodium dihydrogen phosphate, sucrose ester, β-cyclodextrin) and process parameters (stirring speed) on the extraction rate were studied, as shown in Comparative Examples 1 to 9:
[0059] Comparative Example 1
[0060] The method for extracting osthole from Cnidium monnieri comprises the following steps:
[0061] Step 1: Weigh 1000g of Cnidium monnieri and grind it into less than 50 mesh using a grinder to obtain Cnidium monnieri powder.
[0062] Step 2: Measure 1500 mL of water and add it to the Cnidium monnieri powder. Then, add cellulase and hemicellulase, mix, and then stir and hydrolyze in a constant temperature water bath at 50°C for 40 minutes to obtain an enzymatic hydrolysis mixture. The amount of cellulase and hemicellulase added is 800 U / g and 800 U / g, respectively, based on the weight of the Cnidium monnieri. The stirring speed of the stirring enzymatic hydrolysis is 60 rpm.
[0063] Step three, measure 1500mL of water and add it to the enzymatic hydrolysis mixture in step two, then add β-cyclodextrin and sucrose ester, and stir and extract in a constant temperature water bath at 55°C for 20min; wherein, based on the weight of Cnidium monnieri, the addition amounts of β-cyclodextrin and sucrose ester in step three are 200mg / g and 8mg / g, respectively, and the stirring speed of the stirring extraction is 200rpm.
[0064] Step 4: Measure 3000 mL of water and add it to the dilution in step 3, and add β-cyclodextrin and sucrose ester, and continue stirring and extracting in a constant temperature water bath at 55°C for 30 minutes; wherein, based on the weight of Cnidium monnieri, the amount of β-cyclodextrin and sucrose ester added in step 4 is 300 mg / g and 8 mg / g, respectively, and the stirring speed of the stirring extraction is 200 rpm.
[0065] Step 5: Filter and collect the filtrate, and vacuum-dry the filtrate at 60° C. to obtain an extract containing osthole.
[0066] Comparative Example 2
[0067] The method for extracting osthole from Cnidium monnieri comprises the following steps:
[0068] Step 1: Weigh 1000g of Cnidium monnieri and grind it into less than 50 mesh using a grinder to obtain Cnidium monnieri powder.
[0069] Step 2: Measure 1500 mL of water and add it to the Cnidium monnieri powder. Then, add cellulase, hemicellulase, and calcium phosphate, mix, and then stir and hydrolyze in a constant temperature water bath at 50°C for 40 minutes to obtain an enzymatic hydrolysis mixture. The amount of cellulase and hemicellulase added is 800 U / g and 800 U / g, respectively, based on the weight of Cnidium monnieri, and the amount of calcium phosphate added is 40 mg / g. The stirring speed of the stirring enzymatic hydrolysis is 60 rpm.
[0070] Step three, measure 1500mL of water and add it to the enzymatic hydrolysis mixture in step two, then add β-cyclodextrin and sucrose ester, and stir and extract in a constant temperature water bath at 55°C for 20min; wherein, based on the weight of Cnidium monnieri, the addition amounts of β-cyclodextrin and sucrose ester in step three are 200mg / g and 8mg / g, respectively, and the stirring speed of the stirring extraction is 200rpm.
[0071] Step 4: Measure 3000 mL of water and add it to the dilution in step 3, and add β-cyclodextrin and sucrose ester, and continue stirring and extracting in a constant temperature water bath at 55°C for 30 minutes; wherein, based on the weight of Cnidium monnieri, the amount of β-cyclodextrin and sucrose ester added in step 4 is 300 mg / g and 8 mg / g, respectively, and the stirring speed of the stirring extraction is 200 rpm.
[0072] Step 5: Filter and collect the filtrate, and vacuum-dry the filtrate at 60° C. to obtain an extract containing osthole.
[0073] Comparative Example 3
[0074] The method for extracting osthole from Cnidium monnieri comprises the following steps:
[0075] Step 1: Weigh 1000g of Cnidium monnieri and grind it into less than 50 mesh using a grinder to obtain Cnidium monnieri powder.
[0076] Step 2: Measure 1500 mL of water and add it to the Cnidium monnieri powder. Then, add cellulase, hemicellulase, and sodium dihydrogen phosphate, mix, and then stir and hydrolyze in a constant temperature water bath at 50°C for 40 minutes to obtain an enzymatic hydrolysis mixture. The amount of cellulase and hemicellulase added is 800 U / g and 800 U / g, respectively, based on the weight of the Cnidium monnieri, and the amount of sodium dihydrogen phosphate added is 80 mg / g. The stirring speed of the stirring enzymatic hydrolysis is 60 rpm.
[0077] Step three, measure 1500mL of water and add it to the enzymatic hydrolysis mixture in step two, then add β-cyclodextrin and sucrose ester, and stir and extract in a constant temperature water bath at 55°C for 20min; wherein, based on the weight of Cnidium monnieri, the addition amounts of β-cyclodextrin and sucrose ester in step three are 200mg / g and 8mg / g, respectively, and the stirring speed of the stirring extraction is 200rpm.
[0078] Step 4: Measure 3000 mL of water and add it to the dilution in step 3, and add β-cyclodextrin and sucrose ester, and continue stirring and extracting in a constant temperature water bath at 55°C for 30 minutes; wherein, based on the weight of Cnidium monnieri, the amount of β-cyclodextrin and sucrose ester added in step 4 is 300 mg / g and 8 mg / g, respectively, and the stirring speed of the stirring extraction is 200 rpm.
[0079] Step 5: Filter and collect the filtrate, and vacuum-dry the filtrate at 60° C. to obtain an extract containing osthole.
[0080] Comparative Example 4
[0081] The method for extracting osthole from Cnidium monnieri comprises the following steps:
[0082] Step 1: Weigh 1000g of Cnidium monnieri and grind it into less than 50 mesh using a grinder to obtain Cnidium monnieri powder.
[0083] Step 2: Measure 1500 mL of water and add it to the Cnidium monnieri powder. Then, add cellulase, hemicellulase, and sucrose ester, mix, and then stir and enzymolyze in a constant temperature water bath at 50°C for 40 minutes to obtain an enzymatic hydrolysis mixture. The amount of cellulase and hemicellulase added is 800 U / g and 800 U / g, respectively, based on the weight of Cnidium monnieri, and the amount of sucrose ester added is 20 mg / g. The stirring speed of the stirring enzymatic hydrolysis is 60 rpm.
[0084] Step three, measure 1500mL of water and add it to the enzymatic hydrolysis mixture in step two, then add β-cyclodextrin and sucrose ester, and stir and extract in a constant temperature water bath at 55°C for 20min; wherein, based on the weight of Cnidium monnieri, the addition amounts of β-cyclodextrin and sucrose ester in step three are 200mg / g and 8mg / g, respectively, and the stirring speed of the stirring extraction is 200rpm.
[0085] Step 4: Measure 3000 mL of water and add it to the dilution in step 3, and add β-cyclodextrin and sucrose ester, and continue stirring and extracting in a constant temperature water bath at 55°C for 30 minutes; wherein, based on the weight of Cnidium monnieri, the amount of β-cyclodextrin and sucrose ester added in step 4 is 300 mg / g and 8 mg / g, respectively, and the stirring speed of the stirring extraction is 200 rpm.
[0086] Step 5: Filter and collect the filtrate, and vacuum-dry the filtrate at 60° C. to obtain an extract containing osthole.
[0087] Comparative Example 5
[0088] The method for extracting osthole from Cnidium monnieri comprises the following steps:
[0089] Step 1: Weigh 1000g of Cnidium monnieri and grind it into less than 50 mesh using a grinder to obtain Cnidium monnieri powder.
[0090] Step 2: Measure 1500mL of water and add it to the Cnidium monnieri powder, then add cellulase, hemicellulase, calcium phosphate, sodium dihydrogen phosphate and sucrose ester, mix, and then stir and enzymolyze in a constant temperature water bath at 50°C for 40 minutes to obtain an enzymolysis mixture; wherein, based on the weight of Cnidium monnieri, the addition amounts of cellulase and hemicellulase are 800U / g and 800U / g, respectively, and the addition amounts of calcium phosphate, sodium dihydrogen phosphate, and sucrose ester are 40mg / g, 80mg / g, and 20mg / g, respectively. The stirring speed of the stirring enzymolysis is 60rpm;
[0091] Step 3: 1500 mL of water was added to the enzymatic hydrolysis mixture in step 2, and then stirred and extracted in a constant temperature water bath at 55° C. for 20 min; the stirring speed of the stirring extraction was 200 rpm.
[0092] Step 4: Measure 3000 mL of water and add it to dilute the solution in step 3, and continue to stir and extract in a constant temperature water bath at 55° C. for 30 min; wherein, the stirring speed of the stirring extraction is 200 rpm.
[0093] Step 5: Filter and collect the filtrate, and vacuum-dry the filtrate at 60° C. to obtain an extract containing osthole.
[0094] Comparative Example 6
[0095] The method for extracting osthole from Cnidium monnieri comprises the following steps:
[0096] Step 1: Weigh 1000g of Cnidium monnieri and grind it into less than 50 mesh using a grinder to obtain Cnidium monnieri powder.
[0097] Step 2: Measure 1500mL of water and add it to the Cnidium monnieri powder, then add cellulase, hemicellulase, calcium phosphate, sodium dihydrogen phosphate and sucrose ester, mix, and then stir and enzymolyze in a constant temperature water bath at 50°C for 40 minutes to obtain an enzymolysis mixture; wherein, based on the weight of Cnidium monnieri, the addition amounts of cellulase and hemicellulase are 800U / g and 800U / g, respectively, and the addition amounts of calcium phosphate, sodium dihydrogen phosphate, and sucrose ester are 40mg / g, 80mg / g, and 20mg / g, respectively. The stirring speed of the stirring enzymolysis is 60rpm;
[0098] Step 3: Measure 1500 mL of water and add it to the enzymatic hydrolysis mixture in step 2, then add β-cyclodextrin, and stir and extract in a constant temperature water bath at 55°C for 20 minutes; wherein, based on the weight of Cnidium monnieri, the amount of β-cyclodextrin added in step 3 is 200 mg / g, and the stirring speed of the stirring extraction is 200 rpm.
[0099] Step 4: Measure 3000 mL of water and add it to the dilution in step 3, and add β-cyclodextrin, and continue to stir and extract in a constant temperature water bath at 55°C for 30 minutes; wherein, based on the weight of Cnidium monnieri, the amount of β-cyclodextrin added in step 4 is 300 mg / g, and the stirring speed of the stirring extraction is 200 rpm.
[0100] Step 5: Filter and collect the filtrate, and vacuum-dry the filtrate at 60° C. to obtain an extract containing osthole.
[0101] Comparative Example 7
[0102] The method for extracting osthole from Cnidium monnieri comprises the following steps:
[0103] Step 1: Weigh 1000g of Cnidium monnieri and grind it into less than 50 mesh using a grinder to obtain Cnidium monnieri powder.
[0104] Step 2: Measure 1500mL of water and add it to the Cnidium monnieri powder, then add cellulase, hemicellulase, calcium phosphate, sodium dihydrogen phosphate and sucrose ester, mix, and then stir and enzymolyze in a constant temperature water bath at 50°C for 40 minutes to obtain an enzymolysis mixture; wherein, based on the weight of Cnidium monnieri, the addition amounts of cellulase and hemicellulase are 800U / g and 800U / g, respectively, and the addition amounts of calcium phosphate, sodium dihydrogen phosphate, and sucrose ester are 40mg / g, 80mg / g, and 20mg / g, respectively. The stirring speed of the stirring enzymolysis is 60rpm;
[0105] Step three, measure 1500mL of water and add it to the enzymatic hydrolysis mixture in step two, then add sucrose ester, and stir and extract in a constant temperature water bath at 55°C for 20min; wherein, based on the weight of Cnidium monnieri, the amount of sucrose ester added in step three is 8mg / g, and the stirring speed of the stirring extraction is 200rpm.
[0106] Step 4: Measure 3000 mL of water and add it to the dilution in step 3, and add sucrose ester, and continue to stir and extract in a constant temperature water bath at 55°C for 30 minutes; wherein, based on the weight of Cnidium monnieri, the amount of sucrose ester added in step 4 is 8 mg / g, and the stirring speed of the stirring extraction is 200 rpm.
[0107] Step 5: Filter and collect the filtrate, and vacuum-dry the filtrate at 60° C. to obtain an extract containing osthole.
[0108] Comparative Example 8
[0109] The method for extracting osthole from Cnidium monnieri comprises the following steps:
[0110] Step 1: Weigh 1000g of Cnidium monnieri and grind it into less than 50 mesh using a grinder to obtain Cnidium monnieri powder.
[0111] Step 2: Measure 1500mL of water and add it to the Cnidium monnieri powder, then add cellulase, hemicellulase, calcium phosphate, sodium dihydrogen phosphate and sucrose ester, mix, and then stir and enzymolyze in a constant temperature water bath at 50°C for 40 minutes to obtain an enzymolysis mixture; wherein, based on the weight of Cnidium monnieri, the addition amounts of cellulase and hemicellulase are 800U / g and 800U / g, respectively, and the addition amounts of calcium phosphate, sodium dihydrogen phosphate, and sucrose ester are 40mg / g, 80mg / g, and 20mg / g, respectively. The stirring speed of the stirring enzymolysis is 60rpm;
[0112] Step three, measure 4500mL of water and add it to the enzymatic hydrolysis mixture in step two, then add β-cyclodextrin and sucrose ester, and stir and extract for 70min in a constant temperature water bath at 55°C; wherein, based on the weight of Cnidium monnieri, the addition amounts of β-cyclodextrin and sucrose ester in step three are 500mg / g and 16mg / g, respectively, and the stirring speed of the stirring extraction is 200rpm.
[0113] Step 4: Filter and collect the filtrate, and vacuum dry the filtrate at 60° C. to obtain an extract containing osthole.
[0114] Comparative Example 9
[0115] The method for extracting osthole from Cnidium monnieri comprises the following steps:
[0116] Step 1: Weigh 1000g of Cnidium monnieri and grind it into less than 50 mesh using a grinder to obtain Cnidium monnieri powder.
[0117] Step 2: Measure 1500mL of water and add it to the Cnidium monnieri powder, then add cellulase, hemicellulase, calcium phosphate, sodium dihydrogen phosphate and sucrose ester, mix, and then stir and enzymolyze in a constant temperature water bath at 50°C for 40 minutes to obtain an enzymolysis mixture; wherein, based on the weight of Cnidium monnieri, the addition amounts of cellulase and hemicellulase are 800U / g and 800U / g, respectively, and the addition amounts of calcium phosphate, sodium dihydrogen phosphate, and sucrose ester are 40mg / g, 80mg / g, and 20mg / g, respectively. The stirring speed of the stirring enzymolysis is 60rpm;
[0118] Step 3: Measure 1500 mL of water and add it to the enzymatic hydrolysis mixture in step 2, then add β-cyclodextrin and sucrose ester, and stir and extract in a constant temperature water bath at 55°C for 20 minutes; wherein, based on the weight of Cnidium monnieri, the addition amounts of β-cyclodextrin and sucrose ester in step 3 are 200 mg / g and 8 mg / g, respectively, and the stirring speed of the stirring extraction is 80 rpm.
[0119] Step 4: Measure 3000 mL of water and add it to the dilution in step 3, and add β-cyclodextrin and sucrose ester, and continue stirring and extracting in a constant temperature water bath at 55°C for 30 minutes; wherein, based on the weight of Cnidium monnieri, the amount of β-cyclodextrin and sucrose ester added in step 4 is 300 mg / g and 8 mg / g, respectively, and the stirring speed of the stirring extraction is 80 rpm.
[0120] Step 5: Filter and collect the filtrate, and vacuum-dry the filtrate at 60° C. to obtain an extract containing osthole.
[0121] Detection:
[0122] The content of osthole was detected by high performance liquid chromatography method according to Chinese Pharmacopoeia (General Chapter 0512).
[0123] The test results are shown in Table 2:
[0124] Table 2 Extraction rate of osthole in comparative examples 1 to 9
[0125]
[0126] Note: relative to the content of dry Cnidium monnieri powder.
[0127] As can be seen from Table 2, in comparative examples 1 to 4, three auxiliary agents, calcium phosphate, sodium dihydrogen phosphate, and sucrose ester, act together during the stirring enzymolysis process, thereby significantly improving the osthole extraction rate. The applicant analyzed the reason as follows: calcium phosphate and sodium dihydrogen phosphate not only stabilize the pH of the entire enzymatic system, but also calcium ions are used to enhance the stability of the activity of cellulase and hemicellulose, avoiding the weakening of activity as the enzymatic hydrolysis time increases, and calcium ions can also bind to the pectin after the enzymatic hydrolysis of the osthole cell wall and then dissolve in the solution (originally calcium phosphate and pectin are both weakly soluble in water. After binding, they are easily soluble in water with hydrogen ions), thereby facilitating the opening of channels inside and outside the cell wall, facilitating the extraction of osthole, and sodium ions are used to balance the osmotic pressure inside and outside the cell wall to stabilize the effect of calcium ions. Since osthole is insoluble in water, sucrose ester is used as a medium carrier, or in other words, it plays a guiding role, drawing osthole out from around the cell wall, and continuously taking osthole away from around the cell wall in combination with the stirring effect.
[0128] As shown in Table 2, in Comparative Examples 5-7 and 9, the two additives, β-cyclodextrin and sucrose ester, and the stirring speed work together to significantly improve the osthole extraction yield during the stirring extraction process. The applicants analyze this as follows: β-cyclodextrin's molecular structure consists of a hydrophobic or non-polar cavity in the center, surrounded by a hydrophilic or polar ring. Due to the unique structure of osthole itself, β-cyclodextrin alone makes it difficult for osthole to dissolve within this cavity. However, with the combined assistance of sucrose ester and stirring speed, osthole should first bind to the sucrose ester and then rapidly transfer to the β-cyclodextrin cavity, forming a relatively stable equilibrium state, thereby improving the osthole extraction yield.
[0129] Comparative Example 8 explored the step-by-step stirring extraction method. The dilution factor in one step was too large, which affected the extraction rate of osthole. The applicant analyzed that the reason was that both β-cyclodextrin and sucrose ester were not easily soluble in water and had a slow dissolution rate. Therefore, when the amount of water was too large, they could not quickly and fully contact with osthole, thereby affecting the extraction rate.
[0130] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.
Claims
1. A method for extracting osthole from Cnidium monnieri, characterized in that: The following steps are involved: Step 1: Grind the Cnidium monnieri into Cnidium monnieri powder; Step 2: Add appropriate amount of water to the Cnidium monnieri powder, mix, then add cellulase, hemicellulase, calcium phosphate, sodium dihydrogen phosphate and sucrose ester, mix, and stir at 40-55° C. to obtain an enzymatic hydrolysis mixture; Step 3: dilute the enzymatic hydrolysis mixture with water, add β-cyclodextrin and sucrose ester, and stir and extract at 50-60°C; Step 4: dilute with water, add β-cyclodextrin and sucrose ester, and continue to stir and extract at 50-60°C; Step 5, filtering the filtrate and drying it to obtain an extract containing osthole; The stirring speed of the stirring enzymolysis in step 2 is 40-80 rpm; the stirring speed of the stirring extraction in steps 3 and 4 is 100-300 rpm.
2. The method for extracting osthole from Cnidium monnieri according to claim 1, wherein: Based on the weight of Cnidium monnieri, the addition amounts of cellulase and hemicellulase were 800-1000 U / g and 500-800 U / g, respectively; Based on the weight of Cnidium monnieri, the added amounts of water, calcium phosphate, sodium dihydrogen phosphate, and sucrose ester in step 2 are 1-2 g / g, 30-50 mg / g, 50-100 mg / g, and 10-30 mg / g, respectively, and the stirring enzymatic hydrolysis time is 30-50 min.
3. The method for extracting osthole from Cnidium monnieri according to claim 1, wherein: Based on the weight of Cnidium monnieri, the added amounts of water, β-cyclodextrin, and sucrose ester in step 3 are 1-2 g / g, 100-300 mg / g, and 5-10 mg / g, respectively.
4. The method for extracting osthole from Cnidium monnieri according to claim 1, wherein: Based on the weight of Cnidium monnieri, the added amounts of water, β-cyclodextrin, and sucrose ester in step 4 are 1-5 g / g, 200-500 mg / g, and 5-10 mg / g, respectively.
5. The method for extracting osthole from Cnidium monnieri according to claim 1, wherein: The particle size of the Cnidium monnieri powder in step 1 is less than 50 mesh.
6. The method for extracting osthole from Cnidium monnieri according to claim 1, wherein: The specific method of drying in step 5 is vacuum low-temperature drying.
7. The method for extracting osthole from Cnidium monnieri according to claim 1, wherein: The stirring extraction time in step 3 and step 4 is 20-30 min and 20-40 min, respectively.
Citation Information
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