CN108383885B - Process for extracting sophoricoside from fructus Sophorae and its application in anti-inflammation - Google Patents
Process for extracting sophoricoside from fructus Sophorae and its application in anti-inflammation Download PDFInfo
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- ISQRJFLLIDGZEP-CMWLGVBASA-N Sophoricoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C(C=2C(C3=C(O)C=C(O)C=C3OC=2)=O)C=C1 ISQRJFLLIDGZEP-CMWLGVBASA-N 0.000 title claims abstract description 31
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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Abstract
The invention belongs to the technical field of plant extraction, and discloses a process for extracting sophoricoside from sophora fruits, which comprises the following steps: crushing → high voltage → electric field treatment → shearing → enzyme reaction → microwave alcohol extraction → evaporation → drying → adsorption → elution → evaporation → drying → dissolution → crystallization. The invention has simple and feasible process and high extraction rate and purity of the total flavone and the sophoricoside.
Description
Technical Field
The invention belongs to the technical field of plant extraction, and particularly relates to a process for extracting sophoricoside from sophora fruits and application of sophoricoside in anti-inflammation.
Background
Fructus Sophorae is fruit of Sophora japonica L.of Leguminosae, and is a common Chinese medicinal material collected in pharmacopoeia, and the medicinal fructus Sophorae usually adopts mature pod harvested in winter. Dried pod of fructus Sophorae picked after winter solstice is cylindrical and sometimes curved, and its seeds are contracted into continuous bead shape, and after wrapping, its seeds are brown or tawny, and its seeds are brownish black; the theory of the traditional Chinese medicine considers that the fructus sophorae is cold in nature and bitter in taste, enters liver and large intestine channels, has the functions of clearing heat and purging fire, and cooling blood and stopping bleeding, and is suitable for intestinal heat hematochezia, hemorrhoid bleeding, liver heat headache, dizziness and conjunctival congestion.
Chemical research on sophora fruits has been reported in the prior art, researchers have separated several flavonoids, alkaloids, triterpenes, amino acids, saccharides and other components from sophora fruits, wherein the content of the flavonoids is higher than that of flavonoids in other plants. In the pod of sophorae, the total flavonoids determined at present comprise more than 20, and the total flavonoids mainly comprise quercetin, kaempferol, isorhamnetin and genistein isoflavone, and preferably comprise a large amount of sophoricoside. Isoflavones represented by genistein have good cancer preventing and resisting and female hormone-like activity, and can be used for preventing and treating cancer, cardiovascular diseases, osteoporosis and female climacteric syndrome. Rutin is the highest content of quercetin glycoside; sophoricoside is the main component of genistein glycoside. At present, a plurality of processes for extracting the sophorae fructus flavone mostly adopt chemical reagents for extraction, and the conventional extraction method has unsafe factors such as organic solvent residues and the like and has potential threats to human health, and the prior art improves the extraction method, for example, the process optimization of extracting the total flavone in the sophorae fructus by an enzyme method, the extraction of the sophorae fructus flavone by the enzyme method in 2012 of food process science has the advantages of no toxicity, mild reaction conditions and the like, but the method has the defects of large enzyme dosage, low extraction rate and the like, and needs to be further improved. The invention has been improved on the extraction process of the general flavone in the prior work, and has already reported patent technology, on the basis of which, the inventor continues to separate the effective components of the general flavone.
Disclosure of Invention
On the basis of the previous results of the applicant, the invention continuously separates the effective cost of the total flavonoids of the sophora fruits, and provides a process for extracting sophoricoside from the sophora fruits and the application of the sophoricoside in resisting inflammation.
The invention is realized by the following technical scheme:
the process for extracting sophoricoside from sophora fruits comprises the following steps:
crushing → high voltage → electric field treatment → shearing → enzyme reaction → microwave alcohol extraction → evaporation → drying → adsorption → elution → evaporation → drying → dissolution → crystallization.
Specifically, the extraction process comprises the following steps:
pulverizing fructus Sophorae, sieving with 100-200 mesh sieve to obtain fructus Sophorae powder, placing in a reactor, controlling pressure in the reactor at 2-3MPa, maintaining the pressure for 30-40s, and performing pulsed electric field treatment; adding 3-5 times of water, shearing with a high-speed shearing machine, adding cellulase for enzyme reaction, adding 70% ethanol with the same volume, heating to 60 deg.C, and extracting with microwave; filtering, and collecting the extract; then entering a rotary evaporator, evaporating under reduced pressure to remove ethanol, then freeze-drying under vacuum to obtain a total flavone crude extract, adsorbing with macroporous adsorption resin, eluting with 70% ethanol, collecting eluate, entering the rotary evaporator again, evaporating under reduced pressure to remove ethanol, and finally freeze-drying under vacuum to obtain the total flavone extract; adding ethyl acetate five times the weight of the total flavone extract, stirring, filtering, collecting insoluble substance, adding 85% ethanol dropwise into the insoluble substance while heating, stirring at 100rpm until the insoluble substance is completely dissolved, standing at 0 deg.C for 30min, filtering, and collecting crystal.
Preferably, the pulsed electric field has a field strength of 10kv/cm, a total treatment time of 200. mu.s, a pulse width of 4. mu.s, and a pulse frequency of 400 pps.
Preferably, the first and second electrodes are formed of a metal,
the shearing time of the high-speed shearing machine is 3min, and the shearing speed is 5000 rpm.
Preferably, the first and second electrodes are formed of a metal,
the enzyme reaction conditions are as follows: adding cellulase according to the proportion of 1 ten thousand to 2 ten thousand U to 100g of sophora fruit powder, controlling the enzyme reaction temperature to be 35 ℃, and controlling the reaction time to be 60 to 90 min.
Preferably, the first and second electrodes are formed of a metal,
the microwave extraction conditions are that the power is 400- & lt600 & gt W, and the extraction time is 30-50 min.
The invention also claims sophoricoside prepared by the extraction process.
The sophoricoside provided by the invention is used for resisting inflammation.
The technology of the invention adopts the process, and the advantages mainly comprise but are not limited to the following aspects:
the pagoda tree pod has a compact structure because cell walls are rich in cellulose and hemicellulose, and active ingredients are difficult to fully dissolve out by direct ultrasonic or microwave extraction, the cell walls are deformed or partially broken by high-pressure treatment, then pulsed electric field treatment is carried out, perforation can be carried out on the cell walls, and enzymolysis treatment is carried out on the cellulose of the cell walls by cellulase;
the conventional extraction method has unsafe factors such as organic solvent residue and the like, and has potential threat to human health. The invention reduces the adoption of chemical organic preparations, and improves the safety performance of the extract;
the invention adopts various process technologies, organically combines high voltage, pulse electric field, high-speed mechanical force shearing, enzyme method and microwave alcohol extraction method together, combines the methods, realizes advantage complementation, makes up the disadvantages of a single process, and has the characteristics of low energy consumption, high efficiency, no damage to effective components and the like, thereby obtaining higher extraction efficiency and purity;
the invention adopts ethyl acetate to dissolve other types of soluble flavonoids, and then utilizes the different solubility of the sophoricoside in cold and hot ethanol to separate out the sophoricoside crystal, has high yield and purity, and can be applied to anti-inflammatory drugs.
Drawings
FIG. 1: the influence of microwave power and time on the yield of total flavonoids.
Detailed Description
Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.
Example 1
The process for extracting sophoricoside from sophora fruits comprises the following steps:
crushing → high voltage → electric field treatment → shearing → enzyme reaction → microwave alcohol extraction → evaporation → drying → adsorption → elution → evaporation → drying → dissolution → crystallization.
The method specifically comprises the following steps:
crushing fructus Sophorae, sieving with 100 mesh sieve to obtain fructus Sophorae powder, placing in a reactor, controlling pressure in the reactor at 3MPa, maintaining pressure for 30s, performing pulsed electric field treatment with electric field intensity of 10kv/cm, total treatment time of 200 μ s, pulse width of 4 μ s, and pulse frequency of 400 pps; adding 5 times of water by weight, shearing for 3min by a high-speed shearing machine at the shearing speed of 5000rpm, adding cellulase according to the proportion of 2 ten thousand U to 100g of locust bean powder, controlling the enzyme reaction temperature to be 35 ℃ and the reaction time to be 60min, then adding 70% ethanol with the same volume, heating to 60 ℃, extracting by microwave, controlling the microwave power to be 600W and the extraction time to be 30 min; filtering, and collecting the extract; then entering a rotary evaporator, evaporating under reduced pressure to remove ethanol, then freeze-drying under vacuum to obtain a total flavone crude extract, adsorbing with AB-8 type macroporous adsorbent resin, eluting with 2BV of 70% ethanol at an elution speed of 0.5BV/h, collecting the eluate, entering the rotary evaporator again, evaporating under reduced pressure to remove ethanol, and finally freeze-drying under vacuum to obtain the total flavone extract; adding ethyl acetate five times the weight of the total flavone extract, stirring, filtering, collecting insoluble substance, adding 85% ethanol dropwise into the insoluble substance while heating, stirring at 100rpm until the insoluble substance is completely dissolved, standing at 0 deg.C for 30min, filtering, and collecting crystal. Detecting the crystal by using a high performance liquid chromatography system (the operation process can refer to 'simultaneously determining rutin and sophoricoside in sophora fruit by HPLC', 2010 in Huaxi J medicament), weighing the weight of the crystal, and analyzing the content and purity of sophoricoside, wherein the content of sophoricoside in sophora fruit powder is 4.71% calculated by 1000g of sophora fruit powder, the collected crystal is 45.8g, the purity of sophoricoside in the crystal is 95.1%, and the yield of sophoricoside is 92.5%.
Example 2
The process for extracting sophoricoside from sophora fruits comprises the following steps:
crushing → high voltage → electric field treatment → shearing → enzyme reaction → microwave alcohol extraction → evaporation → drying → adsorption → elution → evaporation → drying → dissolution → crystallization.
The method specifically comprises the following steps:
crushing fructus Sophorae, sieving with 200 mesh sieve to obtain fructus Sophorae powder, placing in a reactor, controlling the pressure in the reactor to 2MPa, maintaining the pressure for 40s, and performing pulsed electric field treatment with electric field strength of 10kv/cm, total treatment time of 200 μ s, pulse width of 4 μ s, and pulse frequency of 400 pps; adding 4 times of water by weight, shearing for 3min by a high-speed shearing machine at the shearing speed of 5000rpm, adding cellulase according to the proportion of 1 ten thousand U to 100g of locust bean powder, controlling the enzyme reaction temperature to be 35 ℃ and the reaction time to be 90min, then adding 70% ethanol with the same volume, heating to 60 ℃, extracting by microwave, controlling the microwave power to be 500W and the extraction time to be 60 min; filtering, and collecting the extract; then entering a rotary evaporator, evaporating under reduced pressure to remove ethanol, then freeze-drying under vacuum to obtain a total flavone crude extract, adsorbing with HPD450 type macroporous adsorbent resin, eluting with 3BV of 70% ethanol at an elution speed of 1BV/h, collecting the eluate, entering the rotary evaporator again, evaporating under reduced pressure to remove ethanol, and finally freeze-drying under vacuum to obtain a total flavone extract; adding ethyl acetate five times the weight of the total flavone extract, stirring, filtering, collecting insoluble substance, adding 85% ethanol dropwise into the insoluble substance while heating, stirring at 100rpm until the insoluble substance is completely dissolved, standing at 0 deg.C for 30min, filtering, and collecting crystal. The detection proves that the purity of the sophoricoside in the crystal is 94.7 percent, and the yield of the sophoricoside is 93.1 percent.
Comparative example 1
The process for extracting sophoricoside from sophora fruits comprises the following steps:
pulverizing fructus Sophorae, sieving with 100 mesh sieve to obtain fructus Sophorae powder, placing in a reactor, and performing pulsed electric field treatment with electric field intensity of 10kv/cm, total treatment time of 200 μ s, pulse width of 4 μ s, and pulse frequency of 400 pps; adding 5 times of water by weight, shearing for 3min by a high-speed shearing machine at the shearing speed of 5000rpm, adding cellulase according to the proportion of 2 ten thousand U to 100g of locust bean powder, controlling the enzyme reaction temperature to be 35 ℃ and the reaction time to be 60min, then adding 70% ethanol with the same volume, heating to 60 ℃, extracting by microwave, controlling the microwave power to be 500W and the extraction time to be 60 min; filtering, and collecting the extract; then entering a rotary evaporator, evaporating under reduced pressure to remove ethanol, then freeze-drying under vacuum to obtain a total flavone crude extract, adsorbing with AB-8 type macroporous adsorbent resin, eluting with 2BV of 70% ethanol at an elution speed of 0.5BV/h, collecting the eluate, entering the rotary evaporator again, evaporating under reduced pressure to remove ethanol, and finally freeze-drying under vacuum to obtain the total flavone extract; adding ethyl acetate five times the weight of the total flavone extract, stirring, filtering, collecting insoluble substance, adding 85% ethanol dropwise into the insoluble substance while heating, stirring at 100rpm until the insoluble substance is completely dissolved, standing at 0 deg.C for 30min, filtering, and collecting crystal.
Comparative example 2
The process for extracting sophoricoside from sophora fruits comprises the following steps:
crushing fructus Sophorae, sieving with 100 mesh sieve to obtain fructus Sophorae powder, placing in a reactor, controlling the pressure in the reactor at 3MPa, maintaining the pressure for 30s, adding 5 times of water, shearing with a high speed shearing machine for 3min at a shearing speed of 5000rpm, adding cellulase at a ratio of 2 ten thousand U to 100g fructus Sophorae powder, controlling the enzyme reaction temperature at 35 deg.C and the reaction time at 60min, adding 70% ethanol with the same volume, heating to 60 deg.C, extracting with microwave at 400W and 60 min; filtering, and collecting the extract; then entering a rotary evaporator, evaporating under reduced pressure to remove ethanol, then freeze-drying under vacuum to obtain a total flavone crude extract, adsorbing with AB-8 type macroporous adsorbent resin, eluting with 2BV of 70% ethanol at an elution speed of 0.5BV/h, collecting the eluate, entering the rotary evaporator again, evaporating under reduced pressure to remove ethanol, and finally freeze-drying under vacuum to obtain the total flavone extract; adding ethyl acetate five times the weight of the total flavone extract, stirring, filtering, collecting insoluble substance, adding 85% ethanol dropwise into the insoluble substance while heating, stirring at 100rpm until the insoluble substance is completely dissolved, standing at 0 deg.C for 30min, filtering, and collecting crystal.
Comparative example 3
The process for extracting sophoricoside from sophora fruits comprises the following steps:
crushing fructus Sophorae, sieving with 100 mesh sieve to obtain fructus Sophorae powder, placing in a reactor, controlling pressure in the reactor at 3MPa, maintaining pressure for 30s, performing pulsed electric field treatment with electric field intensity of 10kv/cm, total treatment time of 200 μ s, pulse width of 4 μ s, and pulse frequency of 400 pps; adding 5 times of water, shearing with a high speed shearing machine at 5000rpm for 3min, adding 70% ethanol with the same volume, heating to 60 deg.C, extracting with microwave power of 500W for 50 min; filtering, and collecting the extract; then entering a rotary evaporator, evaporating under reduced pressure to remove ethanol, then freeze-drying under vacuum to obtain a total flavone crude extract, adsorbing with AB-8 type macroporous adsorbent resin, eluting with 2BV of 70% ethanol at an elution speed of 0.5BV/h, collecting the eluate, entering the rotary evaporator again, evaporating under reduced pressure to remove ethanol, and finally freeze-drying under vacuum to obtain the total flavone extract; adding ethyl acetate five times the weight of the total flavone extract, stirring, filtering, collecting insoluble substance, adding 85% ethanol dropwise into the insoluble substance while heating, stirring at 100rpm until the insoluble substance is completely dissolved, standing at 0 deg.C for 30min, filtering, and collecting crystal.
Comparative example 4
The process for extracting sophoricoside from sophora fruits comprises the following steps:
crushing fructus Sophorae, sieving with 100 mesh sieve to obtain fructus Sophorae powder, placing in a reactor, controlling pressure in the reactor at 3MPa, maintaining pressure for 30s, performing pulsed electric field treatment with electric field intensity of 10kv/cm, total treatment time of 200 μ s, pulse width of 4 μ s, and pulse frequency of 400 pps; then adding 5 times of water by weight, adding cellulase according to the proportion of 2 ten thousand U to 100g of sophora fruit powder, controlling the enzyme reaction temperature to be 35 ℃ and the reaction time to be 60min, then adding 70 percent ethanol with the same volume, heating to 60 ℃, adopting microwave extraction, controlling the microwave power to be 500W and the extraction time to be 60 min; filtering, and collecting the extract; then entering a rotary evaporator, evaporating under reduced pressure to remove ethanol, then freeze-drying under vacuum to obtain a total flavone crude extract, adsorbing with AB-8 type macroporous adsorbent resin, eluting with 2BV of 70% ethanol at an elution speed of 0.5BV/h, collecting the eluate, entering the rotary evaporator again, evaporating under reduced pressure to remove ethanol, and finally freeze-drying under vacuum to obtain the total flavone extract; adding ethyl acetate five times the weight of the total flavone extract, stirring, filtering, collecting insoluble substance, adding 85% ethanol dropwise into the insoluble substance while heating, stirring at 100rpm until the insoluble substance is completely dissolved, standing at 0 deg.C for 30min, filtering, and collecting crystal.
Example 3
And (3) total flavone determination: taking the sophora fruit in the Lunan area as an example, 1000g of the sophora fruit is adopted in the experiment, the content of the total flavone in the sophora fruit powder is determined to be 19.7% by an HPLC method, and the experiment group selects the example 1 and the comparative examples 1 to 4 respectively. Weighing to obtain the weight of the total flavone extract, measuring the content of the total flavone in the extract by an HPLC method, and calculating the yield of the total flavone by the following calculation formula: the yield is ((mass x content of extract)/(mass x content of sophora fruit)) × 100%. Specific results are shown in table 1:
TABLE 1
| Group of | Weight of Sophora fruit powder (g) | Total Flavonoids extract (g) | The total flavone content in the extract% | Yield% |
| Example 1 | 1000 | 245.9 | 75.8 | 94.6 |
| Comparative example 1 | 1000 | 234.7 | 70.4 | 83.9 |
| Comparative example 2 | 1000 | 221.8 | 68.1 | 76.7 |
| Comparative example 3 | 1000 | 218.3 | 62.6 | 69.4 |
| Comparative example 4 | 1000 | 247.6 | 72.9 | 91.6 |
The invention also examined the weight of the crystals of example 1 and comparative examples 1-4 and the content of sophoricoside in the crystals, as shown in table 2:
TABLE 2
| Group of | Sophora japonica powderWeight (g) | Weight of crystals (g) | Content% |
| Example 1 | 1000 | 45.8 | 92.5 |
| Comparative example 1 | 1000 | 41.6 | 91.4 |
| Comparative example 2 | 1000 | 42.9 | 90.6 |
| Comparative example 3 | 1000 | 38.7 | 91.7 |
| Comparative example 4 | 1000 | 44.8 | 92.1 |
As shown in the above tables 1-2, the yield and purity of total flavonoids were higher in the example 1 group than in the comparative examples 1-4, and the weight of the crystals and the content of sophoricoside in the example 1 group were the highest in each group; the invention adopts various process technologies, organically combines high voltage, pulse electric field, high-speed mechanical force shearing, enzyme method and microwave alcohol extraction method together, combines the methods, realizes advantage complementation, makes up the disadvantages of single process, and has the characteristics of low energy consumption, high efficiency, no damage to effective components and the like, thereby obtaining higher extraction efficiency and purity.
Example 4
The microwave power and the microwave time have the influence on the yield, and are respectively set to be 200w, 400w, 600w, 800w and 1000w, and respectively set to be 10 min, 20 min, 30min, 40 min, 50min, 60min, 70 min and 80 min. As shown in FIG. 1, the yield of total flavonoids can be improved by increasing the microwave power or increasing the microwave treatment time, but when the microwave power is increased to 600w, the yield is not increased any more, but is reduced, and probably the power is too large, so that flavonoids are damaged; the microwave time is within 50min, the yield is obviously improved, and the yield is not obviously changed along with the increase of the time. Therefore, the microwave power is selected to be 400-600w, and the microwave time is 30-50 min.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (2)
1. The process for extracting sophoricoside from sophora fruits is characterized by comprising the following steps:
pulverizing fructus Sophorae, sieving with 100-200 mesh sieve to obtain fructus Sophorae powder, placing in a reactor, controlling pressure in the reactor at 2-3MPa, maintaining the pressure for 30-40s, and performing pulsed electric field treatment; adding 3-5 times of water, shearing with a high-speed shearing machine, adding cellulase for enzyme reaction, adding 70% ethanol with the same volume, heating to 60 deg.C, and extracting with microwave; filtering, and collecting the extract; then entering a rotary evaporator, evaporating under reduced pressure to remove ethanol, then freeze-drying under vacuum to obtain a total flavone crude extract, adsorbing with macroporous adsorption resin, eluting with 70% ethanol, collecting eluate, entering the rotary evaporator again, evaporating under reduced pressure to remove ethanol, and freeze-drying under vacuum to obtain a total flavone extract; adding ethyl acetate five times the weight of the total flavone extract, stirring well, filtering, collecting insoluble substance, adding 85% ethanol dropwise into the insoluble substance while heating, stirring at 100rpm until the insoluble substance is completely dissolved, standing at 0 deg.C for 30min, filtering, and collecting crystal;
the field intensity of the pulse electric field is 10kv/cm, the total processing time is 200 mus, the pulse width is 4 mus, and the pulse frequency is 400 pps;
the enzyme reaction conditions are as follows: adding cellulase according to the proportion of 1 ten thousand to 2 ten thousand U to 100g of sophora fruit powder, controlling the enzyme reaction temperature to be 35 ℃, and controlling the reaction time to be 60 to 90 min; the microwave extraction conditions are that the power is 400- & lt600 & gt W, and the extraction time is 30-50 min.
2. The extraction process according to claim 1, wherein the high speed shearing machine has a shearing time of 3min and a shearing speed of 5000 rpm.
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