CN111875483B - Method for extracting lignanoid compounds from schisandra chinensis - Google Patents
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Abstract
The invention provides a method for extracting lignanoid compounds from schisandra chinensis, and belongs to the technical field of traditional Chinese medicine extracts. The method comprises soaking fructus Schisandrae sample powder with appropriate amount of anhydrous ethanol, and magnetically stirring for supercritical CO extraction 2 Extracting lignanoid compounds in schisandra chinensis by an extraction method to obtain a solid-liquid mixture after extraction is finished, transferring the obtained solid-liquid mixture into a centrifugal tube for centrifugal separation, collecting all supernate, concentrating the supernate under reduced pressure to obtain an extract, and drying the extract in vacuum and crushing the extract to form powder to obtain the lignanoid extract of schisandra chinensis. The invention firstly mixes magnetic stirring and supercritical CO 2 Extraction combines together, and magnetic stirring is favorable to with extraction solvent and sample intensive mixing, increases the contact frequency between extraction solvent and the sample to improve extraction efficiency. In addition, in supercritical CO 2 Before extraction, a small amount of absolute ethyl alcohol is adopted as an auxiliary extraction solvent to dip the sample in advance, so that the supercritical CO in the extraction process is enhanced 2 The dissolving power and the selectivity of the method reduce the extraction pressure.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine extracts, and particularly relates to a method for extracting lignanoid compounds from schisandra chinensis.
Background
Schisandra chinensis, a plant of Schisandra of Magnoliaceae, can be classified into Schisandra chinensis Baill and Schisandra sphenanthera Baill due to their different growth regions. The schisandra chinensis is in an irregular spherical or oblate spherical shape, and the diameter is 5-8 mm. The surfaces of the five flavors are red, mauve or dark red, the peels are shriveled and oily, the pulps are soft, and some surfaces are black red or white frost. The particles of the schisandra sphenanthera are small, the surface is brownish red or dark brown, the peel is dry and shrivelled, and the pulp is always clung to the seeds. The Chinese medicine considers that the schisandra chinensis is acid-warm and nontoxic, and is generally applied to treating cough and asthma caused by lung and kidney deficiency of the elderly, diabetes and polyuria, qi deficiency and spontaneous perspiration, yin deficiency and night sweat and the like; has good effects of improving senile insomnia and palpitation caused by heart blood deficiency, heart qi deficiency and heart-kidney imbalance. The Western medicine usually uses fructus Schisandrae or fructus Schisandrae compound medicine for treating chronic hepatitis, relieving cough, eliminating phlegm, asthenia and insomnia, and delaying aging of the elderly. The lignan compounds have wide biological activity, such as central nervous system protection, liver and kidney protection, cardiovascular protection, blood sugar and blood fat reduction, in vivo free radical elimination, inflammation diminishing and calming, tumor resistance, HIV resistance and the like, and can be used as plant germination inhibitors, growth inhibitors, bactericides and the like.
At present, there are many methods for extracting lignans from schisandra chinensis, such as a pharmacopeia method (i.e., an ultrasonic extraction method), a thermal reflux extraction method, and a microwave extraction method, wherein the thermal reflux extraction method is a traditional extraction method and is often applied to industrial production, but the method uses a large amount of organic solvents in the extraction process, and has a long extraction time and a relatively low extraction rate. The ultrasonic extraction method and the microwave extraction method, as new extraction methods appearing in recent years, have the advantages of simple operation, short time, high extraction rate and the like, but the two methods cannot process a large amount of samples, are not suitable for industrial production, and need a large amount of organic solvents.
Disclosure of Invention
The invention aims to solve the problems that the existing extraction method of lignan compounds in schisandra chinensis needs a large amount of solvents, has long extraction time and low extraction rate, and provides the extraction method of the lignan compounds in schisandra chinensis.
The invention provides a method for extracting lignans compounds from schisandra chinensis, which is to stir by magnetic forceBlending assisted supercritical CO 2 Realized in the extraction device specifically comprises:
the method comprises the following steps: opening the low-temperature constant-temperature tank to set the temperature at 3-8 deg.C, and opening the high-purity air valve and high-purity CO when the set temperature is reached 2 Valve for introducing high-purity air and CO 2 Filling the system with gas, weighing fructus Schisandrae sample powder in extraction kettle, adding anhydrous ethanol, screwing, sealing, preheating the extraction kettle in heat-collecting constant-temperature magnetic stirrer, opening inlet valve and outlet valve, and introducing CO 2 Discharging air in the extraction kettle, closing an outlet valve, checking the air tightness of the device, simultaneously opening a booster pump and a heat-collecting type constant-temperature magnetic stirrer, setting the rotating speed to be 25-100r/min, stopping pressurizing when the pressure in the extraction kettle reaches 3-12MPa, extracting for 15-180min at 35-80 ℃, after the extraction is finished, sequentially closing the heat-collecting type constant-temperature magnetic stirrer, a high-purity air valve, a high-purity carbon dioxide valve and an extraction kettle inlet valve, manually rotating the booster pump to start pressure release, after the extraction kettle is cooled to the room temperature, opening an outlet valve of the extraction kettle, opening the extraction kettle, and flushing the extraction kettle with ethanol to obtain a solid-liquid mixture;
step two: transferring the solid-liquid mixture obtained in the step one into a centrifugal tube, centrifuging to enable schisandra chinensis sample powder to be deposited at the bottom of the centrifugal tube, discarding, and taking out all supernatant for later use;
step three: and (4) concentrating the supernatant obtained in the second step into an extract under reduced pressure, and drying and crushing the extract in vacuum to form powder to obtain the schisandra lignan compound extract.
Preferably, the temperature set in the cryostat is 5 ℃.
Preferably, the mass (g) of the schisandra chinensis sample powder is: volume of absolute ethanol (mL) was 1.
Preferably, the preheating time of the extraction kettle is 10min.
Preferably, the rotating speed of the heat collection type constant temperature magnetic stirrer is 50r/min.
Preferably, the pressure in the extraction kettle is 9MPa.
Preferably, the extraction temperature is 35 ℃, and the extraction time is 30min.
Preferably, the centrifugal speed in the second step is 5000r/min, and the centrifugal time is 5min.
Preferably, the vacuum drying temperature in the third step is 60 ℃, and the drying time is 48h.
Preferably, the lignanoid compounds in schisandra chinensis are schizandrol A, gomisin D, schizandrol B, schisanhenol, schizandrin A, ampelopsin, schisandrin B and schisandrin C.
The invention has the advantages of
The invention provides a method for extracting lignans compounds from schisandra, which is based on self-assembled magnetic stirring assisted supercritical CO 2 An extraction device for magnetically stirring and supercritical CO 2 In combination with extraction, magnetic stirring is beneficial to fully mixing the extraction solvent with the sample, and the contact frequency between the extraction solvent and the sample is increased, so that the extraction efficiency is improved; meanwhile, the method adopts a small amount of absolute ethyl alcohol as an auxiliary extraction solvent to dip the sample in advance, and adopts supercritical CO 2 In the extraction process, the fructus Schisandrae sample, ethanol and supercritical CO are mixed by magnetic stirring 2 The uniform mixing and the addition of a small amount of ethanol enhance the supercritical fluid CO in the extraction process 2 The dissolving power and the selectivity of the method reduce the extraction pressure, form a mixture in a solid-liquid-flow state, and improve the extraction efficiency of the lignanoid compounds in the schisandra chinensis;
the extraction process is completely closed, the environment is not polluted, the lignan compounds in the schisandra chinensis are extracted by adopting a static extraction method, and the CO is greatly saved on the premise of ensuring the extraction rate 2 Compared with the method reported in the literature, the method saves CO 2 The dosage can reach about 40 percent, and the extraction cost is reduced. Therefore, the method has the advantages of low cost, simple operation, high extraction rate and the like, and can be widely applied to extraction and separation of lignanoid compounds in other traditional Chinese medicines.
Drawings
FIG. 1 shows magnetically assisted supercritical CO of the present invention 2 The structure of the extraction device is shown schematically.
Wherein, 1, high-purity air valve; 2. a high purity carbon dioxide valve; 3. a low-temperature constant-temperature tank; 4. a booster pump; 5. a heat collection type constant temperature magnetic stirrer; 6. an extraction kettle; 7. an extraction kettle outlet valve; 8. an extraction kettle inlet valve; 9. and a pressure gauge.
FIG. 2 is a standard graph of lignan compounds;
FIG. 3 is a liquid chromatogram of a lignan standard solution and a sample extract solution prepared in example 1;
FIG. 4 is a graph showing the effect of different extraction pressures on the extraction rate of lignans in examples 1 and 2 of the present invention;
FIG. 5 is a graph showing the effect of different ethanol concentrations on the extraction rate of lignans in examples 1 and 3 of the present invention;
FIG. 6 is a graph showing the effect on the extraction rate of lignan compounds in different volumes of ethanol in examples 1 and 4 according to the present invention;
FIG. 7 is a graph showing the effect of different magnetic stirring speeds on the extraction rate of lignans in examples 1 and 5 of the present invention;
FIG. 8 is a graph showing the effect of different extraction temperatures on the extraction rate of lignans in examples 1 and 6 of the present invention;
FIG. 9 is a graph showing the effect of extraction time on the lignan compound extraction rate in examples 1 and 7 of the present invention.
Detailed Description
The invention provides a method for extracting lignanoid compounds from schisandra, which is characterized in that supercritical CO assisted by magnetic stirring 2 Supercritical CO assisted by magnetic stirring in an extraction apparatus 2 As shown in figure 1, the extraction device comprises a low-temperature constant-temperature tank 3, a booster pump 4, a heat-collecting constant-temperature magnetic stirrer 5 and an extraction kettle 6, wherein the low-temperature constant-temperature tank 3 is connected with the booster pump 4 through a pipeline, the heat-collecting constant-temperature magnetic stirrer 5 is arranged on the booster pump 4, the extraction kettle 6 is arranged on the heat-collecting constant-temperature magnetic stirrer 5, a pressure gauge 9 is also arranged above the extraction kettle 6,the high-purity air device is respectively connected with the low-temperature thermostatic bath 3 and the booster pump 4 through a first pipeline, a high-purity air valve 1 is arranged on the first pipeline, the high-purity carbon dioxide device is connected with the low-temperature thermostatic bath 3 through a second pipeline, and a high-purity carbon dioxide valve 2 is arranged on the second pipeline; the booster pump 4 is also connected with a pressure gauge 9 through a third pipeline, and the third pipeline is also provided with an extraction kettle outlet valve 7 and an extraction kettle inlet valve 8.
The method specifically comprises the following steps:
the method comprises the following steps: opening the low-temperature constant-temperature tank 3 with the set temperature of 3-8 deg.C, preferably 5 deg.C, and opening the high-purity air valve 1 and high-purity CO when the set temperature is reached 2 A valve 2 for introducing high-purity air and CO 2 Filling the system with gas, weighing fructus Schisandrae sample powder in extraction kettle 6, adding anhydrous ethanol with concentration of 40-100%, preferably 100%, screwing, sealing, preheating the extraction kettle in heat-collecting type constant temperature magnetic stirrer 5, opening inlet valve 8 and outlet valve 7, and introducing CO 2 Discharging air in the extraction kettle, closing an outlet valve 7, checking the air tightness of the device, simultaneously opening a booster pump 4 and a heat-collecting type constant-temperature magnetic stirrer 5, setting the rotation speed to be 25-100r/min, preferably 50r/min, stopping pressurizing when the pressure in the extraction kettle reaches 3-12MPa, preferably 9MPa, extracting at 35-80 ℃, preferably 35 ℃ for 15-180min, preferably 30min, after the extraction is finished, sequentially closing the heat-collecting type constant-temperature magnetic stirrer 5, a high-purity air valve 1, a high-purity carbon dioxide valve 2 and an extraction kettle inlet valve 8, manually rotating the booster pump to start pressure release, after the extraction kettle is cooled to room temperature, opening an outlet valve of the extraction kettle, opening the extraction kettle, and flushing the extraction kettle with ethanol to obtain a solid-liquid mixture; the mass (g) of the schisandra chinensis sample powder is as follows: the volume (mL) of the anhydrous ethanol is preferably 1;
step two: transferring the solid-liquid mixture obtained in the step one into a centrifugal tube, centrifuging to enable schisandra chinensis sample powder to be deposited at the bottom of the centrifugal tube, discarding, and taking out all supernatant for later use; the centrifugal rotating speed is preferably 5000r/min, and the centrifugal time is preferably 5min;
step three: concentrating the supernatant obtained in the second step into an extract under reduced pressure, and performing vacuum drying and crushing on the extract to form powder to obtain a schisandra lignan compound extract; the vacuum drying temperature is preferably 60 ℃, and the drying time is preferably 48 hours.
According to the invention, the lignanoid compound extract in schisandra chinensis is schisandrin A, gomisin D, schisandrin B, schisanhenol, deoxyschizandrin, anwuzhin, schisandrin B and schisandrin C.
Dissolving the obtained fructus Schisandrae lignanoid extract with methanol, filtering with 0.22 μm filter membrane, and measuring the extraction rate of lignanoid compounds in fructus Schisandrae by high performance liquid chromatography under the following conditions: the mobile phase consists of chromatographic grade methanol (A) and water (B), and a gradient elution mode is adopted: 0 to 10min,68 percent (A); 10-15min, 68-80% (A); 15-40min, 80% (A); 40-42min, 80-68% (A); 42-45min, 68% (A). Flow rate of mobile phase: 0.5mL/min; sample injection volume: 10 mu L of the solution; column temperature: 35 ℃; detection wavelength: 254nm.
The invention is described in further detail below with reference to specific examples, wherein the specific sources of the raw materials used in the experiments are:
methanol (chromatographically pure), purchased from Fisher, usa; ethanol (analytical grade), purchased from beijing chemical plant, china; schisandrin A, schisandrin B, deoxyschizandrin and schisandrin B with purity of more than 98% are purchased from Shanghai Aladdin reagent company in China, and schisandrin, schisanhenol, gomisin D and schisandrin C with purity of more than 98% are purchased from Chengdu Manster reagent company Limited; high purity air, high purity carbon dioxide (purity > 0.99999), purchased from vinca megasonia nitrogen limited.
Model 1100 liquid chromatograph equipped with a photodiode detector, agilent, usa; acclaim TM120-C18 column (250 mm. Times.4.6mmL.D., 5 μm), agilent, USA; 0.22 μm nylon filter, tianjin Jinteng instruments ltd; milli-Q water purification systems, millipore corporation (Millipore Co., USA); KQ3200E ultrasonic generator (40KHz, 150W), ultrasonic instruments, inc. of Kunshan; RE-52AA vacuum rotary evaporator, shanghai Yangrong, inc.; allegra 64R high speed centrifuge, beckmann coulter, usa; DF-101S heat collection type magnetic stirrer, steve City Zeihua Instrument Limited liability company; supercritical extraction apparatus, applied Separations, usa; cryostat, bio-technology ltd, beijing new.
Example 1
Respectively taking 0.5mg of schisandrin A, gomisin D, schisandrin B, schisanhenol, deoxyschizandrin, anwuzhin, schisandrin B and schisandrin C in a 10mL brown volumetric flask, adding chromatographic grade methanol to constant volume to scale, performing ultrasonic treatment to better dissolve the eight lignan compounds in the methanol, and storing the dissolved solution in a refrigerator at 4 ℃ for later use. The concentration is plotted on the abscissa and the peak area is plotted on the ordinate, as shown in FIG. 2.
The low-temperature constant-temperature tank 3 is opened, the temperature is set to be 5 ℃, and when the set temperature is reached, the high-purity air valve 1 and the high-purity CO are opened 2 Valve 2, high purity air and CO are introduced 2 Filling the system with gas, weighing 5g of fructus Schisandrae sample powder in extraction kettle 6, adding 10mL of anhydrous ethanol with concentration of 100%, screwing and sealing, preheating the extraction kettle in heat-collecting type constant temperature magnetic stirrer 5, opening inlet valve 8 and outlet valve 7, and introducing CO 2 Discharging air in the extraction kettle, closing an outlet valve 7, checking the air tightness of the device, simultaneously opening a booster pump 4 and a heat-collecting type constant-temperature magnetic stirrer 5, setting the rotating speed to be 50r/min, stopping pressurizing when the pressure in the extraction kettle reaches 9MPa, extracting for 30min at 35 ℃, sequentially closing the heat-collecting type constant-temperature magnetic stirrer 5, a high-purity air valve 1, a high-purity carbon dioxide valve 2 and an extraction kettle inlet valve 8 after extraction is finished, manually rotating the booster pump to start pressure release, opening the outlet valve of the extraction kettle after the extraction kettle is cooled to room temperature, opening the extraction kettle, flushing the extraction kettle with ethanol to obtain a solid-liquid mixture;
transferring the obtained solid-liquid mixture into a centrifuge tube, centrifuging at 5000r/min for 5min to deposit fructus Schisandrae chinensis powder at the bottom of the centrifuge tube, collecting all supernatant, concentrating under reduced pressure to obtain extract, vacuum drying the extract at 60 deg.C for 48 hr, and pulverizing to obtain fructus Schisandrae chinensis lignanoid extract.
Accurately weighing 0.1g of extract powder, dissolving with methanol, diluting to a constant volume to 10mL volumetric flask, filtering with 0.22 μm filter membrane to obtain solution to be detected, and performing high performance liquid chromatography to obtain extraction rates of schisandrin A, gomisin D, schisandrin B, schisanhenol, deoxyschizandrin, anwuzhin, schisandrin B and schisandrin C; the high performance liquid chromatography conditions are as follows: the mobile phase consists of chromatographic grade methanol (A) and water (B), and a gradient elution mode is adopted: 0 to 10min,68 percent (A); 10-15min, 68-80% (A); 15-40min, 80% (A); 40-42min, 80-68 percent (A); 42-45min, 68% (A). Flow rate of mobile phase: 0.5mL/min; sample introduction volume: 10 mu L of the solution; column temperature: 35 ℃; detection wavelength: 254nm. The obtained chromatogram is shown in FIG. 3, wherein a is the chromatogram of the lignan standard solution, b is the chromatogram of the sample extract solution obtained in example 1, wherein, 1, schizandrol A; 2. gomisin D;3. schizandrol B; 4. schisanhenol; 5. schizandrin A; 6. adding pentalipin; 7. schizandrin B; 8. schisandrin C.
Example 2
The specific steps and conditions are the same as those in example 1, except that the extraction pressure in the extraction kettle is 0, 3, 7.5 and 12MPa respectively; the effect of different extraction pressures on the extraction rate of lignans in examples 1 and 2 is shown in fig. 4.
FIG. 4 illustrates: in the absence of CO 2 In the case of (2), the extraction rate of the target substance is low, but as the extraction pressure increases, supercritical CO is used 2 The sample is continuously fed into the sample, so that the mass transfer efficiency is greatly improved, and when the extraction pressure is 9MPa, the extraction rate of the target analyte reaches the maximum value. Illustrating supercritical CO 2 As an extraction solvent, the dosage of the extraction solvent has a crucial influence on the extraction rate of the target product.
Example 3
The specific steps and conditions were the same as those in example 1 except that the concentrations of ethanol were 40, 60 and 80%, respectively, and the effect on the extraction rate of lignan compounds at different ethanol concentrations in examples 1 and 3 is shown in fig. 5.
Fig. 5 illustrates that the higher the concentration of ethanol, the higher the extraction yield of the target compound. On one hand, the ethanol molecules have larger polarity, so that the supercritical CO of the lignanoid compounds is greatly increased 2 Solubility in (c); on the other hand, ethanol is used as an organic solvent, is easy to form hydrogen bonds with lignans compounds, and can remarkably improve the extraction efficiency. Therefore, the use of absolute ethanol as the impregnation solvent has a good effect.
Example 4
The specific procedures and conditions were the same as in example 1, except that the volumes of ethanol were 0, 5, 15 and 20mL, respectively, and the effects on the extraction rate of lignans compounds in the different volumes of ethanol in examples 1 and 4 are shown in fig. 6.
FIG. 6 illustrates the use of supercritical CO directly when the sample is not impregnated with ethanol 2 When a sample is extracted, the extraction rate of the lignan compound is only 0.05-1.43 mg/g, and the extraction rate is increased along with the increasing of the volume of ethanol, probably because a target analyte has a biphenyl cyclooctadiene mother nucleus structure, ethanol is used as the target object and supercritical CO is adopted 2 The miscible reagent has great strengthening effect on the supercritical extraction effect. When the amount of ethanol is 10mL, the extraction rate reaches the maximum value, and is about 0.13-4.45 mg/g. When the amount of ethanol is further increased, the extraction rate of the target compound is slightly decreased. This is probably because some of the schisandra samples were adhered to the extraction still wall and top cover due to the increase of ethanol dosage during the extraction process, and could not contact with the extraction solvent, thus slightly decreasing the extraction yield. Therefore, a suitable ratio of sample mass to immersion solvent facilitates extraction of lignan compounds.
Example 5
The specific steps and conditions are the same as those of example 1, except that the set rotating speeds in the heat collection type constant temperature magnetic stirrer 5 are 0, 25 and 100r/min, respectively, and the influence on the extraction rate of lignans compounds in examples 1 and 5 at different rotating speeds is shown in fig. 7.
FIG. 7 shows that when magnetic stirring is not used, the extraction rate of the target analyte is low, the total lignan extraction rate is less than 5mg/g, the extraction rate increases with the increasing of the rotating speed, the extraction rate reaches the highest when the rotating speed reaches 50r/min, at the moment, the extraction rate of the total lignan compound is about 11.8mg/g, and the extraction rate is almost unchanged when the rotating speed is increased continuously. The invention is helpful to fully mix the sample with the extraction solvent and the absolute ethyl alcohol through magnetic stirring, and increases the contact frequency between the extraction solvent and the sample, thereby improving the mass transfer rate and the extraction efficiency.
Example 6
The specific steps and conditions were the same as those in example 1 except that the extraction temperatures were set at 50, 65 and 80 ℃ respectively, and the effects on the extraction rate of lignan compounds at different extraction temperatures in examples 1 and 6 are shown in fig. 8.
Fig. 8 illustrates that the extraction rate of the target analyte is almost constant with increasing temperature. This is probably because the supercritical extraction has reached equilibrium over a relatively long time frame, so that the temperature does not have a significant effect on the extraction yield.
Example 7
The specific steps and conditions were the same as in example 1, except that the extraction times were set at 15, 60, 120 and 180min, respectively, and the effects on the extraction rate of lignan compounds in examples 1 and 7 at different extraction times are shown in fig. 9.
FIG. 9 illustrates that the extraction rate becomes higher with increasing extraction time, and when the extraction time reaches 30min, the extraction rate of the target analyte no longer increases significantly, which indicates that supercritical CO 2 The extracted lignan compound has reached an equilibrium state.
The extract extracted in example 1 was subjected to performance analysis as follows:
the formula for calculating the extraction rate is shown in formula (1), and the formula for calculating the recovery rate is shown in formula (2). Data for optimization of extraction conditions and methodology were statistically analyzed by Excel tables, with the method precision expressed as Relative Standard Deviation (RSD).
Wherein C is the concentration of the extract and V is the total volume of the extract.
1. Performance analysis
Preparing a series of mixed standard solutions containing 8 lignans compounds with different concentrations, carrying out chromatographic analysis, drawing a standard curve by taking the concentration (c) of a target analyte as a horizontal coordinate and a peak area (A) as a vertical coordinate, and obtaining a linear regression equation through Origin 8.0.6 software. The experimental results are shown in table 1, where each target analyte has a good linear relationship in the linear range, with a correlation coefficient r >0.9998. The detection Limit (LOD) and quantitation Limit (LOQ) refer to the lowest concentration of the target analyte that can be detected or accurately quantified by the instrument at signal-to-noise ratios of 3 and 10, respectively. The results of the experiments show that the LOD of eight target analytes are 41.0, 52.9, 63.4, 27.3, 21.3, 990.2, 45.40 and 49.9ng/mL in sequence, and the LOQ is 122.3, 160.2, 186.2, 80.8, 60.8, 2992.8, 135.4 and 145.8ng/mL in sequence. The Relative Standard Deviation (RSD) of the extraction rates obtained by continuously analyzing 6 samples within 1 day using the analytical detection method established in this study was intra-day precision, the Relative Standard Deviation (RSD) of the extraction rates obtained by continuously analyzing 2 samples per day for 3 days was inter-day precision, and the intra-day and inter-day precision for each target analyte were lower than 5.85% and 7.63%, respectively, indicating that the reproducibility of this study method is acceptable.
TABLE 1 analytical Properties
2. Determination of recovery
To study the accuracy of the method, the recovery of the spiked samples was analyzed in this experiment. The experimental results are shown in table 2, the recovery rate of each target compound is 75.00-97.78%, and the Relative Standard Deviation (RSD) is less than 5.36%, which shows that the method has satisfactory recovery rate and precision, and can be used for extracting and detecting lignans in traditional Chinese medicines.
TABLE 2 recovery of the Compounds
Comparative example 1 pharmacopoeia method (i.e., ultrasonic extraction method)
According to Chinese pharmacopoeia (2015 edition), taking about 0.25g of dried schisandra sample powder, precisely weighing, putting into a 25mL volumetric flask, adding about 18mL of methanol, ultrasonically extracting for 20min after a bottle mouth is sealed, taking out, cooling to room temperature, adding methanol to the scale, mixing, shaking uniformly, filtering, taking 2mL of filtrate, filtering through a 0.22 mu m filter membrane, and performing high performance liquid chromatography analysis.
Comparative example 2 hot reflux extraction method
Adding 0.5g of schisandra chinensis sample powder and 25mL of 50% ethanol solution into a round-bottom flask, heating and refluxing for extraction for 2h, taking out all solid-liquid mixtures to a 50mL centrifuge tube, centrifuging for 5min at 5000r/min, discarding residues, taking all supernatants in a 50mL volumetric flask, fixing the volume to a scale, accurately measuring 2mL of the solution, drying under reduced pressure, dissolving the residues back with 2mL of methanol, filtering the obtained solution through a 0.22-micrometer filter membrane, and performing high performance liquid chromatography.
Comparative example 3 microwave extraction method
Adding 0.5g of schisandra chinensis sample powder and 25mL of 50% ethanol solution into a 50mL centrifuge tube, performing microwave extraction for 1min under the condition of 420W, taking out the centrifuge tube, cooling to room temperature, centrifuging for 5min at 5000r/min, taking supernatant, fixing the volume to 50mL, taking 2mL of solution, drying under reduced pressure, dissolving residues back by using 2mL of methanol, filtering the obtained solution through a 0.22-micron filter membrane, and performing high performance liquid chromatography.
Comparing several methods of example 1 of the present invention and comparative examples 1 to 3, the results are shown in table 3,
TABLE 3
a Standard deviation, n =3.
Table 3 shows that the hot reflux extraction method is a conventional extraction method, and is often applied to industrial production, however, the method uses a large amount of organic solvent in the extraction process, and has long extraction time and relatively low extraction rate. The ultrasonic extraction method and the microwave extraction method, which are new extraction methods appearing in recent years, have the advantages of simple operation, short time, high extraction rate and the like, but the two methods cannot process a large amount of samples, are not suitable for industrial production, and need to use a large amount of organic solvents. Compared with other methods, the extraction rate obtained by the method is not much different from that obtained by a pharmacopoeia method, and is slightly higher than that obtained by a reflux extraction method and a microwave extraction method. The method uses supercritical CO 2 The traditional organic solvent is replaced as an extracting agent, the using amount of the organic solvent is greatly reduced, the sample processing capacity is large, the operation is simple, and the extraction time is short.
Claims (10)
1. A method for extracting lignanoid compounds from schisandra chinensis is characterized in that the method is supercritical CO assisted by magnetic stirring 2 Realized in the extraction device specifically includes:
the method comprises the following steps: opening the low-temperature constant-temperature tank to set the temperature at 3-8 deg.C, and opening the high-purity air valve and high-purity CO when the set temperature is reached 2 Valve for introducing high-purity air and CO 2 Filling the system with gas, weighing fructus Schisandrae sample powder in extraction kettle, adding anhydrous ethanol, screwing, sealing, preheating the extraction kettle in heat-collecting constant-temperature magnetic stirrer, opening inlet valve and outlet valve, and introducing CO 2 Discharging air in the extraction kettle, closing an outlet valve, checking the air tightness of the device, simultaneously starting a booster pump and a heat-collecting type constant-temperature magnetic stirrer, setting the rotating speed to be 25-100r/min, stopping pressurizing when the pressure in the extraction kettle reaches 3-12MPa, extracting for 15-180min at 35-80 ℃, sequentially closing the heat-collecting type constant-temperature magnetic stirrer, a high-purity air valve, a high-purity carbon dioxide valve and an extraction kettle inlet valve after extraction is finished, manually rotating the booster pump, and starting to extractReleasing the pressure, opening an outlet valve of the extraction kettle after the extraction kettle is cooled to room temperature, opening the extraction kettle, and washing the extraction kettle with ethanol to obtain a solid-liquid mixture;
step two: transferring the solid-liquid mixture obtained in the step one into a centrifugal tube, centrifuging to enable schisandra chinensis sample powder to be deposited at the bottom of the centrifugal tube, discarding, and taking out all supernatant for later use;
step three: and D, concentrating the supernatant obtained in the step two under reduced pressure to obtain an extract, and drying and crushing the extract in vacuum to obtain powder to obtain the schisandra lignanoid compound extract.
2. The method according to claim 1, wherein the temperature of the cryostat is set at 5 ℃.
3. The method for extracting lignanoid compounds in schisandra chinensis according to claim 1, wherein the mass g of the schisandra chinensis sample powder is as follows: volume mL of absolute ethanol is 1.
4. The method for extracting lignans compounds from schisandra chinensis as claimed in claim 1, wherein the preheating time of the extraction kettle is 10min.
5. The method for extracting lignanoid compounds from schisandra chinensis as claimed in claim 1, wherein the rotation speed of the heat-collecting constant temperature magnetic stirrer is 50r/min.
6. The method for extracting lignans from schisandra chinensis according to claim 1, wherein the pressure in the extraction tank is 9MPa.
7. The method for extracting lignans compounds from schisandra chinensis as claimed in claim 1, wherein the extraction temperature is 35 ℃ and the extraction time is 30min.
8. The method for extracting lignanoids from schisandra chinensis as claimed in claim 1, wherein the centrifugation speed in step two is 5000r/min and the centrifugation time is 5min.
9. The method for extracting lignans compounds from schisandra chinensis according to claim 1, wherein the vacuum drying temperature in the third step is 60 ℃ and the drying time is 48h.
10. The method for extracting lignans in schisandra chinensis as claimed in claim 1, wherein the lignans in schisandra chinensis are schizandrin A, gomisin D, schizandrol B, schisanhenol, schizandrin A, ampelopsin, schizandrin B and schizandrin C.
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| CN101214278A (en) * | 2007-12-26 | 2008-07-09 | 沈阳药科大学 | New raw material for extracting fructus schizandrae total lignans and preparation technique and use |
| CN101744886A (en) * | 2010-02-24 | 2010-06-23 | 哈尔滨仁皇药业股份有限公司 | Method for extracting high-purity schisandra total lignan |
| CN103467438A (en) * | 2013-09-18 | 2013-12-25 | 浙江工业大学 | Method for extracting, separating and preparing lignin monomers from schisandra chinensis |
| CN107412336A (en) * | 2017-08-23 | 2017-12-01 | 佛山市三水区嘉华化学研究院（普通合伙） | Fruit of Chinese magnoliavine lignanoid and preparation method and application with antioxidant and anti-aging activity |
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| CN101214278A (en) * | 2007-12-26 | 2008-07-09 | 沈阳药科大学 | New raw material for extracting fructus schizandrae total lignans and preparation technique and use |
| CN101744886A (en) * | 2010-02-24 | 2010-06-23 | 哈尔滨仁皇药业股份有限公司 | Method for extracting high-purity schisandra total lignan |
| CN103467438A (en) * | 2013-09-18 | 2013-12-25 | 浙江工业大学 | Method for extracting, separating and preparing lignin monomers from schisandra chinensis |
| CN107412336A (en) * | 2017-08-23 | 2017-12-01 | 佛山市三水区嘉华化学研究院（普通合伙） | Fruit of Chinese magnoliavine lignanoid and preparation method and application with antioxidant and anti-aging activity |
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