CN112716991A - Method for extracting and purifying total flavonoids of senecio dianthus - Google Patents
Method for extracting and purifying total flavonoids of senecio dianthus Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
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- A—HUMAN NECESSITIES
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
Abstract
The invention discloses a method for extracting and purifying total flavonoids of senecio dianthus, which comprises the following steps: pulverizing herba Senecionis Scandentis, and sieving; adding 10-40 parts by weight of ethanol solution into 1 part by weight of senecio dianthus powder, performing reflux extraction at 60-80 ℃ for 1-2h, and filtering to obtain a first filtrate and a first filter residue; adding 3-5 parts by weight of citric acid solution into the first filter residue, stirring at normal temperature for 1-2h, and then performing freeze-thaw cycle treatment; adding 5-25 parts by weight of ethanol solution into the unfrozen mixed solution, performing ultrasonic extraction at 50-70 ℃ for 15-25min, and filtering to obtain a second filtrate and a second filter residue; mixing the first filtrate and the second filtrate, adding yeast powder 0.2-0.3 weight parts, stirring at 25-35 deg.C for 22-30min, centrifuging, collecting supernatant, and concentrating under reduced pressure. The method of the invention can obviously improve the yield and the purity of the total flavone.
Description
Technical Field
The invention relates to the field of natural product extraction. More specifically, the invention relates to a method for extracting and purifying total flavonoids of senecio dianthus.
Background
The Tibetan medicine, radix seu herba Senecionis Scandentis (Senecio dianthus Franch), is a plant of Senecio genus of Compositae family, and has effects of clearing away heat and toxic materials, dispelling pathogenic wind and removing dampness, promoting blood circulation and dredging collaterals. The senecio scandens contains a large amount of flavonoid components, and in order to deeply research the medicinal value of the senecio scandens, the flavonoid components need to be extracted and separated. The existing extraction method of the senecio dianthus total flavonoids is mainly ethanol reflux extraction, and research is focused on optimizing the ethanol reflux extraction process, but the yield of the total flavonoids is still low and needs to be improved.
Disclosure of Invention
The invention aims to provide a method for extracting and purifying total flavonoids of senecio scandens so as to improve the yield and the purity of the total flavonoids.
To achieve the objects and other advantages in accordance with the present invention, there is provided a method for extracting and purifying senecio dianthus total flavonoids, comprising the steps of:
step one, crushing the groundsel herb, and sieving the ground groundsel herb with a 60-mesh sieve to obtain groundsel herb powder;
step two, adding 10-40 parts by weight of 60-80% ethanol solution in volume concentration into 1 part by weight of senecio scandens powder, performing reflux extraction at 60-80 ℃ for 1-2h, and filtering to obtain a first filtrate and a first filter residue;
adding 3-5 parts by weight of citric acid solution with the mass concentration of 10-15% into the first filter residue, stirring at normal temperature for 1-2h, and then performing freeze-thaw cycle treatment;
step four, adding 5-25 parts by weight of 60-80% ethanol solution in volume concentration into the unfrozen mixed solution, performing ultrasonic extraction at 50-70 ℃ for 15-25min, and filtering to obtain a second filtrate and a second filter residue;
and step five, combining the first filtrate and the second filtrate, adding 0.2-0.3 part by weight of yeast powder, stirring for 22-30min at 25-35 ℃, centrifuging, taking supernatant, and concentrating under reduced pressure to obtain the finished product.
Preferably, in the second step, the volume concentration of the ethanol solution is 70%, the addition amount is 30 parts by weight, the reflux extraction temperature is 70 ℃, and the extraction time is 1.5 h.
Preferably, in the method for extracting and purifying the senecio dianthus total flavonoids, in the third step, the citric acid solution is 13% in mass concentration, 5 parts by weight in addition, the stirring speed is 200r/min, and the stirring time is 1 h.
Preferably, in the method for extracting and purifying the senecio dianthus total flavonoids, in the third step, the freeze-thaw cycle treatment specifically comprises the following steps: the mixed solution is firstly transferred to be frozen for 8h at minus 18 ℃, then transferred to be unfrozen at 30 ℃, after unfreezing, the mixed solution passes through a magnetizer with the magnetic field intensity of 600 and 800mT at the flow rate of 120ml/s, and then the magnetized mixed solution is transferred to be minus 18 ℃ for next freeze-thaw cycle treatment, wherein the freeze-thaw cycle treatment is carried out for at least 3 times.
Preferably, in the fourth step, the volume concentration of the ethanol solution is 70%, the addition amount is 20 parts by weight, the ultrasonic extraction temperature is 60 ℃, the ultrasonic power is 600w, the frequency is 30KHz, and the extraction time is 20 min.
Preferably, in the method for extracting and purifying the senecio dianthus total flavonoids, in the fifth step, the yeast powder is activated before adding the filtrate, specifically, 0.2-0.3 part by weight of the yeast powder is added into 5 parts by weight of a sucrose solution with the mass concentration of 4%, water bath oscillation is carried out for 30min at 30 ℃, then 3 parts by weight of the first filtrate is added, water bath oscillation is carried out for 15min, centrifugation is carried out, and precipitates are taken and washed.
Preferably, in the fifth step, the addition amount of the yeast powder is 0.22 part by weight, the stirring temperature is 30 ℃, the stirring speed is 50r/min, and the stirring time is 25 min.
The invention at least comprises the following beneficial effects:
firstly, after the senecio scandens powder is subjected to primary ethanol reflux extraction, the obtained filter residue is sequentially subjected to soaking in a citric acid solution, freeze thawing treatment, ultrasonic extraction and yeast fermentation, so that the yield and the purity of the total flavonoids of the senecio scandens can be effectively improved, and compared with the conventional ethanol reflux extraction method, the yield of the total flavonoids is improved by 35.9-60.9%.
Secondly, the freeze-thaw cycle treatment is combined with the magnetization of the magnetizer, the magnetization treatment improves the activity of water molecules in the mixed solution, and is beneficial to the cell wall pores formed when the solvent passes through capillaries or the freeze-thaw treatment to enter the cell wall, on one hand, flavonoid components in the cell can be dissolved, on the other hand, the damage degree to the cell wall in the next freeze-thaw treatment can be increased, and the synergy of the two promotes the dissolution of the total flavonoids, thereby further improving the yield of the total flavonoids.
Thirdly, by carrying out proper yeast fermentation treatment on the filtrate, nutrient substances in the filtrate can be consumed in the yeast fermentation process, the content of impurities such as soluble protein, saccharides and the like in the filtrate is reduced, and further the purity of the total flavone in the freeze-dried powder is improved.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It is to be noted that the experimental procedures described in the following examples are conventional ones unless otherwise specified, and the reagents and materials described therein are commercially available without otherwise specified.
Example 1:
the method for extracting and purifying the senecio dianthus total flavonoids comprises the following steps:
step one, crushing the groundsel herb, and sieving the ground groundsel herb with a 60-mesh sieve to obtain groundsel herb powder;
step two, adding 1000ml of 80% ethanol solution in volume concentration into 100g of senecio scandens powder, performing reflux extraction at 60 ℃ for 1h, and filtering to obtain a first filtrate and a first filter residue;
adding 300ml of citric acid solution with the mass concentration of 15% into the first filter residue, stirring for 1h at normal temperature at 200r/min, then transferring the mixed solution to-18 ℃ for freezing for 8h, then transferring the mixed solution to 30 ℃ for unfreezing, transferring the unfrozen mixed solution to-18 ℃ for next freeze-thaw cycle treatment, and performing the freeze-thaw cycle treatment for 3 times;
step four, adding 500ml of 80% ethanol solution into the unfrozen mixed solution, performing ultrasonic extraction at 50 ℃ for 15min, wherein the ultrasonic power is 600w, the frequency is 30KHz, and filtering to obtain a second filtrate and a second filter residue;
and step five, combining the first filtrate and the second filtrate, adding 20g of yeast powder, stirring at 25 ℃ at 50r/min for 30min, centrifuging at 6000r/min for 8min, taking supernatant, and concentrating under reduced pressure to 1000ml to obtain the compound.
Example 2:
the method for extracting and purifying the senecio dianthus total flavonoids comprises the following steps:
step one, crushing the groundsel herb, and sieving the ground groundsel herb with a 60-mesh sieve to obtain groundsel herb powder;
step two, 4000ml of 60% ethanol solution with volume concentration is added into 100g of senecio scandens powder, reflux extraction is carried out for 2 hours at 80 ℃, and filtration is carried out to obtain first filtrate and first filter residue;
adding 500ml of citric acid solution with the mass concentration of 10% into the first filter residue, stirring for 2 hours at normal temperature at 200r/min, then transferring the mixed solution to-18 ℃ for freezing for 8 hours, then transferring the mixed solution to 30 ℃ for unfreezing, transferring the unfrozen mixed solution to-18 ℃ for next freeze-thaw cycle treatment, and performing the freeze-thaw cycle treatment for 3 times;
adding 2500ml of 60% ethanol solution into the unfrozen mixed solution, performing ultrasonic extraction at 70 ℃ for 25min, wherein the ultrasonic power is 600w, the frequency is 30KHz, and filtering to obtain a second filtrate and a second filter residue;
and step five, combining the first filtrate and the second filtrate, adding 30g of yeast powder, stirring at the temperature of 35 ℃ at 50r/min for 22min, centrifuging at 6000r/min for 8min, taking supernatant, and concentrating under reduced pressure to 1000ml to obtain the compound.
Example 3:
the method for extracting and purifying the senecio dianthus total flavonoids comprises the following steps:
step one, crushing the groundsel herb, and sieving the ground groundsel herb with a 60-mesh sieve to obtain groundsel herb powder;
step two, adding 3000ml of 70% ethanol solution with volume concentration into 100g of senecio scandens powder, carrying out reflux extraction at 70 ℃ for 1.5h, and filtering to obtain a first filtrate and a first filter residue;
adding 500ml of citric acid solution with the mass concentration of 13% into the first filter residue, stirring for 1h at normal temperature at 200r/min, then transferring the mixed solution to-18 ℃ for freezing for 8h, then transferring the mixed solution to 30 ℃ for unfreezing, transferring the unfrozen mixed solution to-18 ℃ for next freeze-thaw cycle treatment, and performing the freeze-thaw cycle treatment for 3 times;
step four, adding 2000ml of ethanol solution with the volume concentration of 70% into the unfrozen mixed solution, performing ultrasonic extraction at 60 ℃ for 20min, wherein the ultrasonic power is 600w, the frequency is 30KHz, and filtering to obtain a second filtrate and a second filter residue;
and step five, combining the first filtrate and the second filtrate, adding 22g of yeast powder, stirring at the temperature of 30 ℃ for 25min at the speed of 50r/min, centrifuging at the speed of 6000r/min for 8min, taking supernatant, and concentrating under reduced pressure to 1000ml to obtain the yeast extract.
Example 4:
the method for extracting and purifying the senecio dianthus total flavonoids comprises the following steps:
step one, crushing the groundsel herb, and sieving the ground groundsel herb with a 60-mesh sieve to obtain groundsel herb powder;
step two, adding 3000ml of 70% ethanol solution with volume concentration into 100g of senecio scandens powder, carrying out reflux extraction at 70 ℃ for 1.5h, and filtering to obtain a first filtrate and a first filter residue;
adding 500ml of citric acid solution with the mass concentration of 13% into the first filter residue, stirring for 1h at normal temperature at 200r/min, then transferring the mixed solution to a magnetizer with the magnetic field intensity of 700mT at the flow rate of 120ml/s for freezing for 8h at-18 ℃, then transferring the mixed solution to a magnetizer with the flow rate of 120ml/s for unfreezing at 30 ℃, transferring the magnetized mixed solution to-18 ℃, performing next freeze-thaw cycle treatment, and performing 3 times of freezing-thawing magnetization cycle treatment;
step four, adding 2000ml of ethanol solution with the volume concentration of 70% into the unfrozen mixed solution, performing ultrasonic extraction at 60 ℃ for 20min, wherein the ultrasonic power is 600w, the frequency is 30KHz, and filtering to obtain a second filtrate and a second filter residue;
and step five, combining the first filtrate and the second filtrate, adding 22g of yeast powder, stirring at the temperature of 30 ℃ for 25min at the speed of 50r/min, centrifuging at the speed of 6000r/min for 8min, taking supernatant, and concentrating under reduced pressure to 1000ml to obtain the yeast extract.
Example 5:
the method for extracting and purifying the senecio dianthus total flavonoids comprises the following steps:
step one, crushing the groundsel herb, and sieving the ground groundsel herb with a 60-mesh sieve to obtain groundsel herb powder;
step two, adding 3000ml of 70% ethanol solution with volume concentration into 100g of senecio scandens powder, carrying out reflux extraction at 70 ℃ for 1.5h, and filtering to obtain a first filtrate and a first filter residue;
adding 500ml of citric acid solution with the mass concentration of 13% into the first filter residue, stirring for 1h at normal temperature at 200r/min, then transferring the mixed solution to a magnetizer with the magnetic field intensity of 700mT at the flow rate of 120ml/s for freezing for 8h at-18 ℃, then transferring the mixed solution to a magnetizer with the flow rate of 120ml/s for unfreezing at 30 ℃, transferring the magnetized mixed solution to-18 ℃, performing next freeze-thaw cycle treatment, and performing 3 times of freezing-thawing magnetization cycle treatment;
step four, adding 2000ml of ethanol solution with the volume concentration of 70% into the unfrozen mixed solution, performing ultrasonic extraction at 60 ℃ for 20min, wherein the ultrasonic power is 600w, the frequency is 30KHz, and filtering to obtain a second filtrate and a second filter residue;
and step five, combining the first filtrate and the second filtrate, adding 22g of yeast powder into 500ml of sucrose solution with the mass concentration of 4%, oscillating in a water bath at 30 ℃ for 30min, then adding 300ml of the first filtrate, continuing to oscillate in the water bath for 15min, centrifuging at 6000r/min for 10min, taking the precipitate, washing, adding the obtained wet yeast into the combined solution of the first filtrate and the second filtrate, stirring at 30 ℃ for 25min at 50r/min, centrifuging at 6000r/min for 8min, taking the supernatant, and concentrating under reduced pressure to 1000ml to obtain the finished product.
Comparative example 1:
the method for extracting and purifying the senecio dianthus total flavonoids comprises the following steps:
step one, crushing the groundsel herb, and sieving the ground groundsel herb with a 60-mesh sieve to obtain groundsel herb powder;
step two, adding 3000ml of 70% ethanol solution with volume concentration into 100g of senecio scandens powder, carrying out reflux extraction at 80 ℃ for 2h, filtering, and concentrating the filtrate under reduced pressure to 1000ml to obtain the senecio scandens powder.
Comparative example 2:
the other steps are the same as example 3, except that after the first filter residue is obtained by filtration in the second step, the process directly proceeds to the fourth step, 2000ml of ethanol solution with volume concentration of 70% is added to the first filter residue, and ultrasonic extraction is performed.
Comparative example 3:
the other steps were the same as in example 3 except that in step five, the first filtrate and the second filtrate were combined and directly concentrated to 1000ml under reduced pressure.
Comparative example 4:
the other steps are the same as example 3, except that in the fifth step, the first filtrate and the second filtrate are combined, 22g of yeast powder is added, the mixture is stirred for 50min at the temperature of 30 ℃ at the speed of 50r/min, the mixture is centrifuged for 8min at the speed of 6000r/min, and supernate is taken and concentrated to 1000ml under reduced pressure.
Comparative example 5:
the other steps are the same as the example 3, except that in the fifth step, the first filtrate and the second filtrate are combined, the filtrate is added into an AB-8 macroporous adsorption resin column to adsorb the filtrate, then ethanol solution with the volume concentration of 65% is adopted for elution, the eluent is collected, and the pressure reduction concentration is carried out to 1000 ml.
Comparative example 6:
the other steps are the same as the example 3, except that 435ml of water is added into the first filter residue in the third step, after uniform mixing, the mixed solution is transferred to-18 ℃ for freezing for 8h, then transferred to 30 ℃ for unfreezing, the unfrozen mixed solution is transferred to-18 ℃ for next freeze-thaw cycle treatment, the freeze-thaw cycle treatment is carried out for 3 times, 65g of citric acid is added into the unfrozen mixed solution, and the mixture is stirred for 1h at the normal temperature at 200 r/min.
Comparative example 7:
the other steps are the same as the embodiment 3, except that in the third step, 435ml of water is added into the first filter residue, the mixture is uniformly mixed, the mixture is frozen for 8 hours at the temperature of minus 18 ℃, then the mixture is unfrozen at the temperature of 30 ℃, the unfrozen mixture is then frozen at the temperature of minus 18 ℃, and the next freezing-thawing cycle treatment is carried out for 3 times; in the fourth step, 65g of citric acid and 2000ml of 70% ethanol solution by volume are added into the thawed mixed solution, and ultrasonic extraction is carried out.
Example 1:
firstly, the content of total flavone in the concentrated solution obtained in the examples 1 to 5 and the comparative examples 1 to 7 is measured by adopting a spectrophotometry method, and the yield is calculated, wherein the specific test method comprises the following steps:
1. weighing 12.0mg of rutin standard substance, placing in a 50ml volumetric flask, adding 70% ethanol solution for dissolving and diluting to scale, and shaking up to obtain standard substance solution;
2. measuring 0, 1.0, 2.0, 3.0, 4.0 and 5.0ml of standard solution, respectively adding the standard solution into 25ml volumetric flasks, then adding 1ml of 5% sodium sulfite solution into each volumetric flask, shaking up the mixture by shaking, standing the mixture for 6min, then respectively adding 1ml of 10% aluminum nitrate solution, standing the mixture for 6min, then adding 10ml of 5% sodium hydroxide solution, finally fixing the volume by using 70% ethanol solution, shaking up the mixture, standing the mixture for 15min, measuring the absorbance at the wavelength of 510nm, and performing regression to obtain a standard curve equation: y 9.923x + 0.007; r2＝0.9995。
3. Respectively taking 1.0ml of the concentrated solution obtained in the examples 1-5 and the comparative examples 1-7, putting the concentrated solution into a 10ml volumetric flask, fixing the volume by using 70% ethanol solution, and shaking up to obtain a solution to be detected;
4. respectively putting 1.0ml of the solution to be detected into a 25ml volumetric flask, developing according to the method 2, measuring absorbance, substituting into a standard curve equation to obtain the concentration C of the total flavonoids, wherein the concentration C of the total flavonoids in the solution to be detected is 25C, and the yield of the total flavonoids in the senecio dianthus is as follows: d (%) (C × n × v/1000)/w × 100, where C is the total flavone concentration (mg/ml) in the solution to be tested, n is the dilution factor of the concentrate, v is the total volume (ml) of the concentrate, and w is the mass (g) of senecio dianthus powder, the results are shown in table 1.
TABLE 1
| Example 1 | Example 2 | Example 3 | Example 4 | Example 5 | Comparative example 1 | |
| Total Flavonoids yield (%) | 12.86 | 13.47 | 13.63 | 15.14 | 15.22 | 9.46 |
| Comparative example 2 | Comparative example 3 | Comparative example 4 | Comparative example 5 | Comparative example 6 | Comparative example 7 | |
| Total Flavonoids yield (%) | 9.94 | 13.55 | 12.27 | 13.28 | 11.12 | 10.49 |
As can be seen from Table 1, the yield of total flavonoids can reach 12.86-15.22% by adopting the method of the invention, and compared with the method of extracting by ethanol reflux in the comparative example 1, the yield of total flavonoids is improved by 35.9-60.9%, which shows that the method of the invention can obviously improve the yield of total flavonoids in senecio dianthus; compared with the embodiment 3, the yield of the total flavonoids in the embodiments 4 and 5 is respectively improved by 11.1% and 11.7%, which is probably because in the third step, in the process of freeze-thaw cycle treatment, the unfrozen mixed solution is magnetized by a magnetizer, so that the activity of water molecules in the mixed solution is improved, and the solvent can enter cell walls through capillary tubes or cell wall pores formed during the freeze-thaw treatment, so that on one hand, flavonoid components in the cells can be dissolved, on the other hand, the damage degree of the cell walls during the next freeze-thaw treatment can be increased, and the synergistic effect of magnetization and freeze-thaw treatment is realized, so that the dissolution rate of the total flavonoids is improved; compared with the example 3, the yield of the total flavone in the comparative examples 2, 6 and 7 is respectively reduced by 27.1%, 18.4% and 23.0%, which shows that the sequence of the citric acid solution soaking and the freeze-thaw treatment has obvious influence on the yield of the total flavone, when the first filter residue is treated, the citric acid solution is firstly added for soaking, then the freeze-thaw treatment is carried out, and then the ultrasonic extraction is carried out, so that the yield of the total flavone can be obviously improved, probably because the citric acid solution is firstly used for soaking the first filter residue, the acid solution can loosen the cell structure, and partially degrade cellulose, the damage degree of the cell structure is promoted during the subsequent freeze-thaw treatment, and the dissolution rate of the total flavone during the ultrasonic extraction is further improved; compared with example 3, the total flavone yield of comparative example 3 and comparative example 5 has no significant difference, and the total flavone yield of comparative example 4 is reduced by 10.0%, which is probably because the yeast fermentation time is too long, and the flavonoid component in the filtrate is consumed, so that the total flavone yield is reduced.
Secondly, the concentrates in example 3, example 4, example 5, comparative example 3 and comparative example 5 are freeze-dried to prepare total flavone freeze-dried powder, and the total flavone freeze-dried powder is weighed and the purity of the total flavone freeze-dried powder is calculated, and the result is shown in table 2.
TABLE 2
| Example 3 | Example 4 | Example 5 | Comparative example 3 | Comparative example 5 | |
| Lyophilized powder quality (g) | 21.76 | 24.51 | 22.20 | 25.80 | 16.12 |
| Purity (%) | 62.64 | 61.77 | 68.57 | 52.52 | 82.38 |
As can be seen from table 2, compared with comparative example 3, the total flavone purity of the lyophilized powder in examples 3, 4, 5 and 5 is significantly increased, but the increase range of examples 3, 4 and 5 is smaller than that of comparative example 5, which shows that proper yeast fermentation can reduce the content of impurities such as soluble protein and saccharide in the filtrate, improve the total flavone purity of the lyophilized powder, and the proper yeast fermentation can partially replace the existing resin column adsorption purification, and compared with the resin column adsorption purification, the yeast fermentation has simple operation and less pollution.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Claims (7)
1. The extraction and purification method of the senecio dianthus total flavonoids is characterized by comprising the following steps:
step one, crushing the groundsel herb, and sieving the ground groundsel herb with a 60-mesh sieve to obtain groundsel herb powder;
step two, adding 10-40 parts by weight of 60-80% ethanol solution in volume concentration into 1 part by weight of senecio scandens powder, performing reflux extraction at 60-80 ℃ for 1-2h, and filtering to obtain a first filtrate and a first filter residue;
adding 3-5 parts by weight of citric acid solution with the mass concentration of 10-15% into the first filter residue, stirring at normal temperature for 1-2h, and then performing freeze-thaw cycle treatment;
step four, adding 5-25 parts by weight of 60-80% ethanol solution in volume concentration into the unfrozen mixed solution, performing ultrasonic extraction at 50-70 ℃ for 15-25min, and filtering to obtain a second filtrate and a second filter residue;
and step five, combining the first filtrate and the second filtrate, adding 0.2-0.3 part by weight of yeast powder, stirring for 22-30min at 25-35 ℃, centrifuging, taking supernatant, and concentrating under reduced pressure to obtain the finished product.
2. The method for extracting and purifying senecio dianthus total flavonoids according to claim 1, wherein in the second step, the volume concentration of the ethanol solution is 70%, the addition amount is 30 parts by weight, the reflux extraction temperature is 70 ℃, and the extraction time is 1.5 h.
3. The method for extracting and purifying senecio dianthus total flavonoids according to claim 1, wherein in the third step, the citric acid solution has a mass concentration of 13%, an addition amount of 5 parts by weight, a stirring speed of 200r/min, and a stirring time of 1 h.
4. The method for extracting and purifying senecio dianthus franch total flavonoids according to claim 1, wherein in the third step, the freeze-thaw cycle treatment specifically comprises: the mixed solution is firstly transferred to be frozen for 8h at minus 18 ℃, then transferred to be unfrozen at 30 ℃, after unfreezing, the mixed solution passes through a magnetizer with the magnetic field intensity of 600 and 800mT at the flow rate of 120ml/s, and then the magnetized mixed solution is transferred to be minus 18 ℃ for next freeze-thaw cycle treatment, wherein the freeze-thaw cycle treatment is carried out for at least 3 times.
5. The method for extracting and purifying senecio dianthus chinensis total flavonoids according to claim 1, wherein in the fourth step, the volume concentration of the ethanol solution is 70%, the addition amount is 20 parts by weight, the ultrasonic extraction temperature is 60 ℃, the ultrasonic power is 600w, the frequency is 30KHz, and the extraction time is 20 min.
6. The method for extracting and purifying senecio dianthus chinensis total flavonoids according to claim 1, wherein in the fifth step, the yeast powder is activated before adding the filtrate, specifically, 0.2-0.3 part by weight of the yeast powder is added into 5 parts by weight of the sucrose solution with the mass concentration of 4%, the mixture is subjected to water bath oscillation at 30 ℃ for 30min, then 3 parts by weight of the first filtrate is added, the water bath oscillation is continued for 15min, and the precipitate is centrifuged and washed.
7. The method for extracting and purifying senecio dianthus total flavonoids according to claim 1, wherein in the fifth step, the addition amount of the yeast powder is 0.22 parts by weight, the stirring temperature is 30 ℃, the stirring speed is 50r/min, and the stirring time is 25 min.
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