Method for extracting caulis spatholobi flavone through ultrasonic and microwave synergism

Abstract

The invention belongs to the technical field of natural medicine extraction, and particularly discloses a method for extracting caulis spatholobi flavone through ultrasonic and microwave synergy, which comprises the following steps: sieving a caulis spatholobi medicinal material with a 60-mesh sieve to obtain medicinal material powder, mixing the medicinal material powder with a 50% ethanol solution, performing ultrasonic-microwave extraction, filtering, merging and collecting filtrate, and performing vacuum concentration to obtain the caulis spatholobi flavone. The invention also discloses a preparation method of the caulis spatholobi flavone extracted by the method, and provides an extraction method which is time-consuming, less in generated waste liquid and higher in flavone extraction rate.

Description

Technical Field

[0001] The present invention relates to the technical field of natural medicine extraction, in particular to a method for extracting flavonoids from Millettia repens with the help of ultrasound and microwave. Background Art

[0002] Millettia spatholobi (Spatholobi vine) is the dried stem of the leguminous plant, Miscanthus sinensis. It is bitter and sweet in nature, warming in nature, and primarily enters the liver and kidney meridians. It has the effects of promoting blood circulation, tonifying the tendons and tendons, and regulating menstruation and relieving pain. It is commonly used to treat symptoms such as irregular menstruation, dysmenorrhea, amenorrhea, rheumatic joint pain, numbness and paralysis, and chlorosis caused by blood deficiency. Millettia spatholobi is rich in various chemical components, including flavonoids, terpenes, sterols, and lignin. Flavonoids are the main active ingredients in Millettia spatholobi, exhibiting significant pharmacological activity.

[0003] At present, the main extraction methods of total flavonoids from Millettia reticulata include ultrasonic extraction, reflux extraction and immersion extraction. However, these methods still have room for improvement in extraction efficiency, environmental friendliness and sustainability. For example, the flavonoid extraction yield of the immersion method is 7.75%; while the extraction rates of the ultrasonic method and reflux method are slightly higher, at 29.54% and 34.90% respectively, both have the problem of long extraction time and the generation of a large amount of toxic, flammable and explosive organic waste liquid during the extraction process. In addition, the flavonoid extraction rate is significantly affected by a variety of factors in the process, including extraction time, temperature and material-liquid ratio. Therefore, optimizing the process condition combination of material-liquid ratio, extraction time and number of extractions can help improve the extraction rate and separation effect. Summary of the Invention

[0004] The present invention aims to provide a method for extracting flavonoids from Millettia spatholobi by synergistic ultrasound and microwave, so as to provide an extraction method which is time-consuming, generates less waste liquid and has a higher flavonoid extraction rate.

[0005] To achieve the above-mentioned purpose, the present invention provides the following technical solution: a method for ultrasonic-microwave synergistic extraction of flavonoids from Millettia spatholobi, comprising the following steps: crushing and sieving Millettia spatholobi medicinal materials through 60 mesh to obtain medicinal material powder, mixing it with 50% ethanol solution, extracting it using ultrasonic-microwave, filtering, and combining and collecting the filtrate.

[0006] Preferably, the material-liquid ratio of the medicinal material powder to the ethanol solution is 1:(15-25), preferably 1:20.

[0007] Preferably, the ultrasonic extraction time is 20 to 60 minutes, preferably 30 minutes.

[0008] Preferably, the microwave power of the microwave extraction is 100-700 W, preferably 462 W, ​​and the microwave time is 10-50 seconds, preferably 30 seconds.

[0009] A preparation method of flavonoids from Spatholobus suberectus after extraction comprises the following steps: preparing macroporous resin, removing white floating matter on the macroporous resin with deionized water, soaking the resin in anhydrous ethanol for 24 hours, washing with water until the resin has no alcohol smell, soaking the resin in a 40g / L HCl solution for 5 hours, washing with water until the resin is neutral, soaking the resin in a 40g / L NaOH solution for 5 hours, rinsing with water until the resin is neutral, and removing excess water from the surface of the resin with filter paper; filling the pretreated resin into a chromatography column, loading the sample, standing the column for 4 hours, eluting with distilled water until the resin has no alcohol smell, eluting with 8BV of 60% ethanol solution at an elution flow rate of 2BV / h, collecting the obtained liquid, concentrating the liquid by rotary evaporation to remove the solvent, and obtaining a powdered product of flavonoids from Spatholobus suberectus by freeze drying.

[0010] Preferably, the macroporous resin is AB-8 macroporous resin used to prepare the total flavonoids from Millettia reticulata.

[0011] Preferably, the loading concentration is about 5 mg / mL, the flow rate is 2 BV / h, and the loading volume is 7 BV.

[0012] The principle and beneficial effects of this technical solution:

[0013] The invention uses an ultrasonic and microwave simultaneous assisted extraction method to achieve an extraction rate of (34.71±0.02)%, with high extraction efficiency. The time of the entire extraction process can be controlled within 60 minutes. The materials used are simple, the fertilizer produced is less, and the impact on the human body and the environment is small. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the relationship diagram between the solid-liquid ratio and the total flavonoids extraction rate;

[0015] Figure 2 is the relationship between ultrasonic time and total flavonoid extraction rate;

[0016] Figure 3 is the relationship between microwave time and total flavonoid extraction rate;

[0017] Figure 4 is the relationship between microwave power and total flavonoid extraction rate;

[0018] Figure 5 is the contour and response surface stereogram of the interaction;

[0019] Figure 6 To verify the optimal conditions, the extraction rate of total flavonoids from Millettia repens was repeated;

[0020] Figure 7 is the total current diagram of positive ions;

[0021] Figure 8 This is the total flow diagram of negative ions. DETAILED DESCRIPTION

[0022] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments:

[0023] Example 1:

[0024] To investigate the effect of the solid-liquid ratio on the total flavonoids extraction from Millettia repens, a single-factor experiment was designed, repeated three times, and the average value was taken. Weigh 0.20 g of the medicinal material powder and set the solid-liquid ratio (1:10 g / mL, 1:15 g / mL, 1:20 g / mL, 1:25 g / mL, 1:30 g / mL) as a single-factor condition.

[0025] Depend on Figure 1 It can be seen that the extraction rate of total flavonoids from Millettia spatholobi is significantly correlated with the solid-liquid ratio. The extraction rate reaches its peak at a solid-liquid ratio of 1:20, indicating that the flavonoids are essentially dissolved and the solution is saturated. As the solid-liquid ratio increases, the dissolution of other alcohol-soluble substances affects the dissolution of flavonoids. The increase in other impurities may also interfere with the measurement of flavonoid absorbance. The final solid-liquid ratio for extracting total flavonoids from Millettia spatholobi is determined to be 1:20 mg / mL.

[0026] Example 2:

[0027] To investigate the effect of ultrasound time on the extraction of total flavonoids from Millettia repens, a single-factor experiment was designed, repeated three times, and the average value was taken. Weigh 0.20g of medicinal material powder and set the ultrasound time (10min, 20min, 30min, 40min, 50min).

[0028] Depend on Figure 2 It can be seen that the extraction rate of total flavonoids from Millettia spatholobi first increases and then decreases with the increase of ultrasonic time. When the ultrasonic time is 30 minutes, the extraction rate reaches its maximum. At this time, the solution is close to saturation, that is, the total flavonoids in Millettia spatholobi have been basically fully extracted. If the ultrasonic time is too short, the total flavonoids in Millettia spatholobi may not be fully dissolved, resulting in a low extraction rate. If the ultrasonic time is too long, the flavonoid structure changes and the extraction rate decreases. The final ultrasonic time for extracting total flavonoids from Millettia spatholobi was determined to be 30 minutes.

[0029] Example 3:

[0030] To investigate the effect of microwave time on the extraction of total flavonoids from Millettia repens, a single-factor experiment was designed, repeated three times, and the average value was taken. Weigh 0.20g of medicinal material powder and set the microwave time (10s, 20s, 30s, 40s, 50s).

[0031] Depend on Figure 3The results show a significant correlation between the extraction rate of total flavonoids from Spatholobus suberectus and microwave time. The highest extraction rate was achieved when the microwave time reached 30 seconds. After 30 seconds, the extraction rate of total flavonoids from Spatholobus suberectus began to decline. This is because if the microwave time is too short, the total flavonoids in Spatholobus suberectus cannot be fully dissolved. If the microwave time is too long, the flavonoids may decompose due to the heat generated by the microwave, thus reducing the extraction rate. Ultimately, the optimal microwave time for total flavonoid extraction from Spatholobus suberectus was determined to be 30 seconds.

[0032] Example 4:

[0033] To investigate the effect of microwave power on the extraction of total flavonoids from Millettia repens, a single-factor experiment was designed and repeated three times, with the average value taken. Weigh 0.20 g of the medicinal material powder and set the microwave power to 98 W, 259 W, 462 W, ​​595 W, or 700 W.

[0034] according to Figure 4 , indicating a significant correlation between the extraction rate of total flavonoids from Millettia spatholobi and microwave power. The extraction rate reached its peak when the microwave power reached 462W. As the microwave power continued to increase, the extraction rate began to decline. This is because when the microwave power is too low, the total flavonoids in Millettia spatholobi are not fully dissolved. When the power is too high, the temperature in the system rises, and the flavonoids decompose due to heat, resulting in a decrease in extraction efficiency. Ultimately, the optimal microwave power for extracting total flavonoids from Millettia spatholobi was determined to be 462W.

[0035] Box-Behnken Experimental Design:

[0036] 1. Response surface experimental design and results

[0037] Based on single-factor experiments, the effects of factors such as material-liquid ratio, ultrasonic time, microwave power, and microwave time on the extraction rate of total flavonoids from Millettia spatholobi were investigated. To determine the optimal combination of these factors, total flavonoid extraction rate was selected as the metric. A Box-Behnken design was performed using Design-Expert 13 software, as shown in Table 1. Each factor was set at three different levels, and the results are shown in Table 2.

[0038] Table 1 Box-Behnken design factor coding levels

[0039]

[0040] Table 2 Box-Behnken experimental design and results

[0041]

[0042]

[0043] 2. Establishment of regression equation model and variance analysis

[0044] As shown in Table 3, the F value is 9.96 and the P value is much less than 0.001, indicating that the overall effect of the model is significant, that is, the model can effectively explain the relationship between the dependent variable (total flavonoids extraction rate of Millettia reticulata) and the independent variable (extraction conditions), and this relationship is statistically valid. The F value of the model misfit term is 3.13 and the P value is greater than 0.05, which generally indicates that the residuals of the model are random and there is no specific pattern or trend, thus indicating that the model fits well. A coefficient of variation of less than 5% indicates that the variability of the experimental results is relatively low, that is, the experiment has good reproducibility. The signal-to-noise ratio is an important indicator of the predictive ability of the model, and its ideal value should be greater than 4. In this model, the signal-to-noise ratio is 11.6849, which is much higher than 4, indicating that the model has strong predictive ability and the experimental results are more accurate. The quadratic polynomial equation model between each extraction condition and the total flavonoids extraction rate of Millettia reticulata is:

[0045] Y=33.69+0.475A+0.1421B+0.3296C+11D-0.7879AB-0.4409AC-0.2614AD-1.25BC-0.9431BD-1.94CD-2.91A^24.16B^2-2.2C^2-3.53D^2

[0046] Table 3 Variance analysis table

[0047]

[0048] Based on the above model, the influence of various factors on the extraction rate of total flavonoids from Millettia spatholobi is as follows: microwave power > material-liquid ratio > ultrasonic time > microwave time.

[0049] 3. Response Surface Analysis

[0050] In the experiment to explore the influence of four factors A, B, C, and D on the flavonoid content, the response surface plot shows the combined effect of the other two factors on the flavonoid extraction rate when any two factors are zero. Figure 5 As shown, the response surface plot exhibits a typical saddle-shaped pattern, indicating that the total flavonoid content in Millettia repens does not increase monotonically with increasing levels of the relevant factors, but rather undergoes an initial rise followed by a decline. Contour plots, as an auxiliary representation of the response surface plot, clearly demonstrate this interaction trend by depicting the projections of points with the same response value onto the bottom surface. The contour lines for ultrasound time and microwave time, and for ultrasound time and microwave power, approximate ellipses, indicating a significant interaction between these two factors.

[0051] Optimal extraction conditions of total flavonoids from Millettia repens and verification of the results

[0052] In summary, the optimal process parameters for ultrasonic-microwave synergistic extraction were obtained: solid-liquid ratio of 20.37 mg / mL, microwave time of 29.92 s, ultrasonic time of 30.03 min, microwave power of 465.7 W, and the predicted extraction rate of 34.73%. Figure 6 As shown in the figure, the actual extraction rate was (34.71±0.02)% according to experimental verification, and the relative error between the measured value and the predicted value was 0.02%, indicating that the model has high accuracy and practicality.

[0053] Analysis of flavonoids in Millettia repens

[0054] The structure of flavonoids was preliminarily analyzed by high performance liquid chromatography-mass spectrometry (LC-MS), and the type and molecular weight information of the flavonoids contained were analyzed.

[0055] (1) Sample preparation: Accurately weigh 10 mg of powder and dissolve it in methanol solution to prepare a 1 mg / mL sample. After filtering through a 0.22 μm microporous filter membrane, load the sample and perform LC-MS analysis.

[0056] (2) Chromatographic conditions: Chromatography: Dinonex Ultimate 3000 UHPLC; Chromatographic column: Atlantis T3 100 mm × 3.0 mm, 3.0 μm; Column temperature: 30°C; Injection volume: 5.0; Mobile phase: A: 0.1% formic acid in water; B: acetonitrile.

[0057] (3) Ladder elution conditions:

[0058] Table 4 Ladder elution table

[0059]

[0060] (4) Mass spectrometry conditions: Samples were analyzed using a Thermo Scientific Q Exactive mass spectrometer. The ion source was set to HESI mode, the sheath gas rate was set to 40 arb, and the auxiliary gas rate was set to 10 arb to ensure stable and efficient ion transmission. The positive ion mode was 3.8 kV, and the negative ion mode was 3.0 kV. The capillary temperature was 320°C and the auxiliary gas temperature was 300°C. The scan mode was Full MS / DD-MS2top10. The primary scan was set to a high resolution of 70,000 and covered a wide mass range from 100 to 1500 m / z. In the secondary scan, the resolution was reduced to 17,500, the starting ion mass was set to 50 m / z, and NCE 15, 30, and 45 were selected for the collision voltage.

[0061] (5) Component analysis: According to the LC-MS results (Table 5, Figure 7 and Figure 7) It can be seen that the total flavonoids in Millettia reticulata mainly contain key flavonoid compounds such as Petunidin, Luteolin, LicochalconeaA, Vestitol, and Calycosin.

[0062] Table 5 Key flavonoid compounds

[0063]

[0064] The above is only an embodiment of the present invention, and common knowledge such as the specific technical solutions or characteristics in the solution is not described in detail here. For those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. A method for extracting flavonoids from Millettia spatholobi by ultrasonic and microwave synergistic extraction, characterized in that: The following steps are involved: The Millettia spatholobi medicinal material is crushed and sieved through 60 meshes to obtain medicinal material powder, which is mixed with 50% ethanol solution, extracted by ultrasonic-microwave, filtered, and the filtrate is combined and collected.

2. The method for extracting flavonoids from Millettia spatholobi by ultrasonic and microwave synergistic extraction according to claim 1, characterized in that: The solid-liquid ratio of the medicinal material powder to the ethanol solution is 1:(15-25), preferably 1:

20.

3. The method for extracting flavonoids from Millettia spatholobi by ultrasonic and microwave synergistic extraction according to claim 1, characterized in that: The ultrasonic extraction time is 20 to 60 minutes, preferably 30 minutes.

4. The method for extracting flavonoids from Millettia spatholobi by ultrasonic and microwave synergistic extraction according to claim 1, characterized in that: The microwave power of the microwave extraction is 100-700 W, preferably 462 W, ​​and the microwave time is 10-50 seconds, preferably 30 seconds.

5. A method for preparing the flavonoids of Millettia spatholobi after extraction according to claims 1-4, characterized in that: The following steps are involved: Prepare macroporous resin, remove white floating matter on the macroporous resin with deionized water, soak in anhydrous ethanol for 24 hours, wash with water until there is no alcohol smell, soak in 40g / L HCl solution for 5 hours, wash with water until neutral, soak in 40g / LNaOH solution for 5 hours, rinse with water until neutral, use filter paper to absorb excess water on the surface of the resin; fill the pretreated resin into a chromatography column and load the sample, let it stand for 4 hours, rinse with distilled water until there is no alcohol smell, elute with 8BV of 60% ethanol solution, the elution flow rate is 2BV / h, collect the obtained liquid, remove the solvent after rotary evaporation concentration, and obtain a powdered product of total flavonoids of Millettia spatholobi by freeze drying.

6. The preparation method after extraction of flavonoids from Millettia spatholobi according to claim 5, characterized in that: The macroporous resin is AB-8 type macroporous resin used to prepare the total flavonoids of Millettia spatholobi.

7. The preparation method of Caulis Spatholobi flavonoids after extraction according to claim 5, characterized in that: The loading concentration was about 5 mg / mL, the flow rate was 2 BV / h, and the loading volume was 7 BV.

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