Composite extraction and rapid detection method for quercetin in pseudo-ginseng

Abstract

The invention belongs to the technical field of sample pretreatment, and particularly discloses a composite extraction and rapid detection method of quercetin in pseudo-ginseng, which comprises the following steps: precisely weighing a pseudo-ginseng powder sample, fully and uniformly mixing the pseudo-ginseng powder sample with a preferable good solvent, then carrying out enzymolysis reaction, then putting into a reduced pressure ultrasonic device, and carrying out ultrasonic treatment for 2-3 hours; by means of the synergistic mass transfer effect of freeze-drying, enzymolysis, pressure reduction and ultrasound, effective components in the pseudo-ginseng powder matrix can be rapidly dissolved out. And carrying out accurate analysis on effective components in high performance liquid chromatography on the pretreated sample. In addition, by adjusting the type and proportion of the mobile phase, the chromatographic analysis time of a single sample is remarkably shortened, and the detection flux is improved. According to the method, the experimental process and the process duration of the pretreatment and detection parts are optimized, the time for completely detecting a single sample can be shortened to about 15 minutes, the enrichment and analysis efficiency of quercetin in the pseudo-ginseng powder is remarkably improved, and the method is suitable for ultrafast analysis of a large batch of pseudo-ginseng samples.

Description

Technical Field

[0001] This invention belongs to the field of sample pretreatment technology, specifically a method for the compound extraction and rapid detection of quercetin in Panax notoginseng. Background Technology

[0002] Quercetin, a natural polyhydroxy flavonoid compound, is widely found in various traditional Chinese medicinal herbs, including Panax notoginseng. It possesses pharmacological properties such as expectorant, antitussive, antihypertensive, lipid-lowering, and anticancer effects, and is a major component of some traditional Chinese medicine preparations and health products. Therefore, its extraction from natural sources has attracted considerable attention. However, the content of quercetin in natural products is low; for example, the mass percentage of quercetin in natural Panax notoginseng is typically less than 0.1%. Furthermore, the extraction of active ingredients is highly susceptible to the influence of complex matrices. Polysaccharides and saponins in Panax notoginseng can form complexes with quercetin, making separation difficult and limiting further development and application. Reported total flavonoid extraction processes include solvent extraction, ultrasonic extraction, and microwave extraction.

[0003] Traditional solvent extraction methods require prolonged high-temperature reflux, which not only damages the hydroxyl structure of quercetin but also results in high solvent residue, affecting product quality and increasing process costs and complexity. While atmospheric pressure ultrasonic extraction shortens the extraction time, the temperature of the extract solution easily rises above 60°C under atmospheric pressure, potentially degrading the heat-sensitive quercetin during extraction. The working principle of vacuum ultrasonic extraction is to use vacuum reduction to lower the boiling point of the solution in the extraction tank, thereby achieving rapid and effective extraction of heat-sensitive components from the material at a lower temperature. Compared with traditional water extraction, alcohol extraction, or ultrasonic extraction, vacuum ultrasonic extraction offers significantly better extraction results, featuring low temperature and low pressure, and a high yield of active ingredients.

[0004] In view of the above-mentioned technical problems, this invention is proposed. Summary of the Invention

[0005] The present invention aims to provide a compound extraction and rapid detection method for quercetin in Panax notoginseng, which is used to determine the content of quercetin in Panax notoginseng and solves the problems of slow extraction speed, low extraction yield, unstable yield and large matrix interference in the existing extraction process of quercetin in Panax notoginseng.

[0006] The purpose of this invention is to provide a method for the compound extraction and rapid detection of quercetin in Panax notoginseng, comprising the following steps: S1: Take the pretreated Panax notoginseng powder and mix it with an ethanol aqueous solution at a solid-liquid ratio of 1:20-60 to ensure that the powder and solvent are in full contact. The pretreatment specifically involves: vacuum freeze-drying the lumpy Panax notoginseng and then pulverizing the lumpy Panax notoginseng to obtain Panax notoginseng powder. S2: The Panax notoginseng powder is enzymatically hydrolyzed using a compound enzyme, wherein the compound enzyme is 0.5%-1.5% of the mass of the Panax notoginseng powder, and the compound enzyme is composed of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase. The enzymatic hydrolysis conditions are: pH=5-6, enzymatic hydrolysis time is 1-1.5h, and enzymatic hydrolysis temperature is 45-55℃. S3: Heat the solution obtained in step S2 to 85°C for enzyme inactivation treatment; S4: Connect the three ports of the three-way valve to the vacuum pump, the sealed sample container, and the atmosphere respectively. First, open the container to the atmosphere. After starting the vacuum pump, switch the valve position to allow the vacuum pump to evacuate the container. When the required vacuum level is reached, turn the valve to the closed position to cut off the passage. Turn off the vacuum pump, disconnect the container connection, and quickly place it into an ultrasonic extractor containing an appropriate amount of water. Set the ultrasonic frequency to 40kHz and the ultrasonic temperature to 25℃ to extract the solution obtained in step S3 and obtain the extract. S5: Centrifuge the extract obtained in step S4, take the supernatant, filter it through a 0.45 μm filter membrane to remove residual fine particles and colloidal impurities in the extract for chromatographic analysis; S6: Perform high performance liquid chromatography analysis on the sample obtained in step S5, record the chromatogram, plot the standard curve with concentration as the abscissa and peak area as the ordinate, calculate the regression equation, calculate the quercetin content, and analyze the yield.

[0007] Preferably, the particle size of the Panax notoginseng powder in step S1 is 10-20 μm.

[0008] Using the above technical solution, quercetin in Panax notoginseng is preserved to the maximum extent during vacuum freeze-drying. After freeze-drying, the Panax notoginseng has a low moisture content, its properties are similar to fresh Panax notoginseng, it is in a puffed state, has a bright and glossy color, and a high rehydration rate, making it usable as fresh Panax notoginseng. In addition to retaining the original color and shape of fresh Panax notoginseng, freeze-dried Panax notoginseng also has a full appearance, bright color, low moisture content, is easy to preserve, has a loose texture, is easy to crush, and has good rehydration properties; it can absorb water and restore its original shape in a short time after soaking, which is beneficial for the dissolution of active ingredients.

[0009] Preferably, the amount of Panax notoginseng powder used in step S1 is 0.5 g.

[0010] Preferably, the alcohol-water solution in step S1 has a strength of 40%-80%.

[0011] Compared with the traditional method of using a solid-liquid ratio of 1:40 to 1:50, this method preferably uses a solid-liquid ratio of 1:30 and a 60% ethanol aqueous solution, which reduces the amount of solvent used by about 25%, thereby reducing experimental costs and the pressure of subsequent waste liquid treatment.

[0012] Preferably, the mass ratio of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase in step S2 is (2-3):(2-3):(1-2):(0.5-1):(0.5-1).

[0013] Preferably, the enzyme activity of any one of the enzymes in the complex enzyme is ≥5×10⁻⁶. 4 U / g.

[0014] Using the above technical solutions, cellulase mainly breaks down cellulose (the main structural component of plant cell walls) to produce small molecules such as glucose; pectinase breaks down pectin (the binding substance in the intercellular matrix of plants), helping to separate cells or soften tissues. Hemicellulase breaks down hemicellulose in plant cell walls into oligosaccharides or monosaccharides; amylase can break down large starch molecules into smaller molecules.

[0015] Enzymatic hydrolysis with compound enzymes can break down large molecules into smaller molecules. At the same time, the loose cell wall structure makes it easier for depressurized ultrasound energy to penetrate, increasing solvent penetration efficiency by 3-5 times, achieving an extraction synergy of "1+1>2".

[0016] Preferably, the ultrasonic extractor is set to 40 kHz and 25 °C in step S4.

[0017] Preferably, the ultrasonic extraction time in step S4 is 5-25 min.

[0018] Preferably, in step S5, the centrifugation speed is 3000-3500 r / min and the centrifugation time is 5-8 min.

[0019] Preferably, the chromatographic conditions in step S6 are as follows: a 0.2% aqueous solution of phosphoric acid and acetonitrile in a volume ratio of 50:50 are selected as the mobile phase, a C18 column is selected, the detection wavelength is 372 nm, the flow rate is 1 mL / min, the injection volume is 20 μL, and the analysis time is 7 min.

[0020] Using the above technical solution, this invention achieves rapid dissolution of the effective components in the Panax notoginseng powder matrix through the synergistic mass transfer effect of decompression and ultrasound. The pretreated sample is centrifuged to obtain the extract, which is then directly subjected to high-performance liquid chromatography (HPLC) analysis. This reduces the detection time of the target analyte to less than 5 minutes, while simultaneously increasing the analytical throughput, making it suitable for batch sample detection.

[0021] The beneficial effects of this invention are: (1) Faster analysis speed. Conventional high-performance liquid chromatography (HPLC) analysis of each quercetin sample requires approximately 20 minutes. This invention utilizes a depressurized ultrasonic extraction coupled with HPLC for ultra-fast and efficient analysis of quercetin in Panax notoginseng powder. By leveraging the synergistic mass transfer effect of depressurization and ultrasound, the effective components in the Panax notoginseng powder matrix are rapidly dissolved. The pretreated sample is centrifuged to obtain the extract, which is then directly analyzed by HPLC, reducing the detection time of the target analyte to less than 5 minutes and simultaneously increasing the analytical throughput, making it suitable for batch sample detection. This invention combines depressurized ultrasonic extraction with HPLC and simultaneously optimizes the experimental procedures for pretreatment and detection, shortening the complete detection time of a single sample to approximately 15 minutes. This method provides an ultra-fast analytical approach for the detection of quercetin in Panax notoginseng powder, significantly reducing the analysis time.

[0022] (2) Higher yield of target components. Compared with traditional solvent extraction methods such as maceration extraction, reflux extraction, and atmospheric pressure ultrasonic extraction, the reduced pressure environment can lower the boiling point of the extraction solvent and reduce the thermal degradation of quercetin. At the same time, the synergistic effect of freeze drying, enzymatic cell disruption, and reduced pressure ultrasonication can enhance the penetration of the solvent into Panax notoginseng powder, resulting in more complete dissolution of quercetin.

[0023] (3) Better extraction efficiency. Traditional solvent extraction methods generally take 30-60 min. This method combines the synergistic mass transfer effect of freeze drying-enzymatic hydrolysis-reduced ultrasound, and optimizes the extraction time to 15 min, which reduces the time by more than 50% and does not require long-term heating.

[0024] (4) The solvent is more economical and environmentally friendly. Compared with the 1:40~1:50 solid-liquid ratio commonly used in traditional methods, this method prefers a solid-liquid ratio of 1:30 and a 60% ethanol aqueous solution, which reduces the amount of solvent used by about 25%, thereby reducing experimental costs and the pressure of subsequent waste liquid treatment.

[0025] (5) This invention utilizes a combination of freeze-drying, enzymatic hydrolysis, vacuum extraction and ultrasonic technology. This method is universal and applicable to the rapid dissolution of active ingredients in traditional Chinese medicine, providing a reference for the efficient extraction of active ingredients from plant samples. Attached Figure Description

[0026] Figure 1 Standard curves were determined by high-performance liquid chromatography (HPLC) using quercetin standard solutions at concentrations of 0.05 mg / L, 0.1 mg / L, 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5 mg / L, and 10.0 mg / L in the test examples. X represents the concentration of the quercetin standard solution in mg / L, and Y represents the peak area of ​​quercetin in mA.

[0027] Figure 2 This is a UV wavelength scan of quercetin.

[0028] Figure 3 The chromatogram is the result of high performance liquid chromatography in Example 1, with the experimental conditions being: 60% ethanol aqueous solution, solid-liquid ratio of 1:30, and extraction time of 15 min.

[0029] Figure 4 The chromatogram is the result of high performance liquid chromatography in Example 2, with the experimental conditions being: 40% ethanol aqueous solution, solid-liquid ratio of 1:30, and extraction time of 15 min.

[0030] Figure 5 The chromatogram is the injection chromatogram of the high performance liquid chromatography pair in Example 3 under the experimental conditions of 80% ethanol aqueous solution, solid-liquid ratio of 1:30, and extraction time of 15 min.

[0031] Figure 6 The chromatogram is the result of high performance liquid chromatography in Example 4, with the experimental conditions being: 60% ethanol aqueous solution, solid-liquid ratio of 1:30, and extraction time of 5 min.

[0032] Figure 7 The chromatogram is the injection chromatogram of the high performance liquid chromatography pair in Example 5 under the experimental conditions of 60% ethanol aqueous solution, solid-liquid ratio of 1:30, and extraction time of 25 min.

[0033] Figure 8 The chromatogram is the injection chromatogram of the high performance liquid chromatography pair in Example 6 under the experimental conditions of 60% ethanol aqueous solution, solid-liquid ratio of 1:20, and extraction time of 15 min.

[0034] Figure 9 The chromatogram is the injection chromatogram of the high performance liquid chromatography pair in Example 7, with experimental conditions of 60% ethanol aqueous solution, solid-liquid ratio of 1:60, and extraction time of 15 min.

[0035] Figure 10 The images show the chromatograms of high-performance liquid chromatography (HPLC) in Example 1 under the following experimental conditions: 60% ethanol-water solution, solid-liquid ratio 1:30, and extraction time of 15 min, with extraction methods of reduced pressure ultrasound and normal pressure ultrasound, respectively. In the image, a is the chromatogram of the sample extracted by reduced pressure ultrasound, and b is the chromatogram of the sample extracted by normal pressure ultrasound. Detailed Implementation

[0036] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0037] The technical solution of the present invention is as follows: A method for the combined extraction and rapid detection of quercetin from Panax notoginseng includes the following steps: S1: Take the pretreated Panax notoginseng powder and mix it with an ethanol aqueous solution at a solid-liquid ratio of 1:20-60 (non-limiting examples: 1:20, 1:30, 1:40, 1:50, 1:60, etc.) to ensure that the powder and solvent are in full contact. The pretreatment is as follows: vacuum freeze-dry the lumpy Panax notoginseng and then pulverize the lumpy Panax notoginseng to obtain Panax notoginseng powder. S2: The Panax notoginseng powder is enzymatically hydrolyzed using a compound enzyme, wherein the compound enzyme is 0.5%-1.5% of the mass of the Panax notoginseng powder, and the compound enzyme is composed of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase. The enzymatic hydrolysis conditions are: pH=5-6, enzymatic hydrolysis time is 1-1.5h, and enzymatic hydrolysis temperature is 45-55℃. S3: Heat the solution obtained in step S2 to 85°C for enzyme inactivation treatment; S4: Connect the three ports of the three-way valve to the vacuum pump, the sealed sample container, and the atmosphere respectively. First, open the container to the atmosphere. After starting the vacuum pump, switch the valve position to allow the vacuum pump to evacuate the container. When the required vacuum level is reached, turn the valve to the closed position to cut off the passage. Turn off the vacuum pump, disconnect the container connection, and quickly place it into an ultrasonic extractor containing an appropriate amount of water. Set the ultrasonic frequency to 40kHz and the ultrasonic temperature to 25℃ to extract the solution obtained in step S3 and obtain the extract. S5: Centrifuge the extract obtained in step S4, take the supernatant, filter it through a 0.45 μm filter membrane to remove residual fine particles and colloidal impurities in the extract for chromatographic analysis; S6: Perform high performance liquid chromatography analysis on the sample obtained in step S5, record the chromatogram, plot the standard curve with concentration as the abscissa and peak area as the ordinate, calculate the regression equation, calculate the quercetin content, and analyze the yield.

[0038] In some embodiments, the Panax notoginseng powder in step S1 is pretreated by: vacuum freeze-drying the lumpy Panax notoginseng, and then pulverizing the lumpy Panax notoginseng to obtain Panax notoginseng powder.

[0039] In some embodiments, the particle size of Panax notoginseng powder in step S1 is 10-20 μm.

[0040] In some embodiments, the solid-liquid ratio in step S1 is preferably 1:30.

[0041] In some embodiments, the amount of Panax notoginseng powder used in step S1 is 0.5 g.

[0042] In some embodiments, the concentration of the ethanol-water solution in step S1 is 40%-80% (non-limiting examples: 40%, 50%, 60%, 70%, 80%), preferably 60%.

[0043] In some embodiments, the mass ratio of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase in step S2 is (2-3):(2-3):(1-2):(0.5-1):(0.5-1) (non-limiting examples: 2:2:1:0.5:1, 2.5:3:2:1:1, 2:2:2:1:0.5, 3:3:1:0.5:0.5, 2:2.5:1.5:1:1, etc.).

[0044] In some embodiments, the enzyme activity of any one of the enzymes in the complex enzyme is ≥5×10⁻⁶. 4 U / g.

[0045] In some embodiments, the ultrasonic extraction instrument is set to 40 kHz and 25 °C in step S2.

[0046] In some embodiments, the ultrasonic extraction time in step S2 is 5-25 min (non-limiting examples: 5 min, 10 min, 15 min, 20 min, 25 min), preferably 15 min.

[0047] In some embodiments, the centrifugation speed in step S3 is 3000-3500 r / min, and the centrifugation time is 5-8 min.

[0048] In some embodiments, the chromatographic conditions in step S4 are as follows: a 0.2% aqueous solution of phosphoric acid and acetonitrile in a volume ratio of 50:50 are selected as the mobile phase; a C18 column is selected; the detection wavelength is 372 nm; the flow rate is 1 mL / min; the injection volume is 20 μL; and the analysis time is 7 min.

[0049] Example 1 This embodiment provides a method for detecting the quercetin content in actual samples using a combined extraction and rapid detection method of quercetin from Panax notoginseng, including the following steps: S1: Accurately weigh 0.5g of pretreated Panax notoginseng powder, place the powder in a 100ml round-bottom flask, and add 15mL of 60% ethanol aqueous solution (solid-liquid ratio of 1:30). S2: The Panax notoginseng powder is enzymatically hydrolyzed using a compound enzyme, which accounts for 0.5% of the mass of the Panax notoginseng powder. The compound enzyme consists of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase. The enzymatic hydrolysis conditions are: pH=5, hydrolysis time is 1.5h, and hydrolysis temperature is 45℃. S3: Heat the solution obtained in step S2 to 85°C for enzyme inactivation treatment; S4: Use a three-way valve to connect a vacuum pump to reduce pressure and extract ultrasonically for 15 minutes. After extraction, transfer the extract to a centrifuge tube. S5: Centrifuge at 3000 r / min for 5 min, transfer the extract to a liquid chromatography vial for chromatographic analysis, and filter the sample through a 0.45 μm filter membrane before chromatographic analysis using a high performance liquid chromatograph. S6: Select 0.2% phosphoric acid aqueous solution and acetonitrile in a volume ratio of 50:50 as the mobile phase. Select a C18 column, a detection wavelength of 372nm, a flow rate of 1mL / min, an injection volume of 20μL, and an analysis time of 7min.

[0050] In this embodiment, the particle size of the pretreated Panax notoginseng powder in step S1 is 10 μm.

[0051] In this embodiment, the mass ratio of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase in step S2 is 2:2:2:1:1.

[0052] Quercetin standard solutions of different concentrations (0.05 mg / L, 0.1 mg / L, 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5 mg / L, and 10.0 mg / L) were prepared using quercetin standard. The peak areas were measured using high-performance liquid chromatography (HPLC). A standard curve was plotted with peak area (mAU) on the ordinate and quercetin concentration (mg / L) on the abscissa (e.g., ...). Figure 1 The linear equation results are shown in Table 1. The ultraviolet wavelength scan of quercetin is shown below. Figure 2 As shown, high-performance liquid chromatography (HPLC) analysis was performed according to the chromatographic conditions, and the chromatograms were recorded. The chromatographic results are as follows. Figure 3 As shown in the figure. A standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The regression equation was calculated, and the quercetin content was determined. The peak area was 0.1643, and the content was 0.072489 mg / g.

[0053] Table 1 Standard working curve of quercetin Example 2 This embodiment provides a method for detecting the quercetin content in actual samples using a combined extraction and rapid detection method of quercetin from Panax notoginseng, including the following steps: S1: Accurately weigh 0.5 g of pretreated Panax notoginseng powder, place the powder in a 100 mL round-bottom flask, and add 15 mL of 40% ethanol aqueous solution (solid-liquid ratio of 1:30). S2: The Panax notoginseng powder is enzymatically hydrolyzed using a compound enzyme, which accounts for 1.5% of the mass of the Panax notoginseng powder. The compound enzyme consists of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase. The enzymatic hydrolysis conditions are: pH=6, hydrolysis time is 1h, and hydrolysis temperature is 55℃. S3: Heat the solution obtained in step S2 to 85°C for enzyme inactivation treatment; S4: Use a three-way valve to connect a vacuum pump to reduce pressure and extract ultrasonically for 15 minutes. After extraction, transfer the extract to a centrifuge tube. S5: Centrifuge at 3000 r / min for 5 min, transfer the extract to a liquid chromatography vial for chromatographic analysis, and filter the sample through a 0.45 μm filter membrane before chromatographic analysis using a high performance liquid chromatograph. S6: Select 0.2% phosphoric acid aqueous solution and acetonitrile in a volume ratio of 50:50 as the mobile phase. Select a C18 column, a detection wavelength of 372 nm, a flow rate of 1 mL / min, an injection volume of 20 μL, and an analysis time of 7 min.

[0054] In this embodiment, the particle size of the pretreated Panax notoginseng powder in step S1 is 20 μm.

[0055] In this embodiment, the mass ratio of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase in step S2 is 3:2:2:0.5:1.

[0056] Perform high-performance liquid chromatography (HPLC) analysis according to the chromatographic conditions, record the chromatogram, and the chromatographic results are as follows: Figure 4 As shown in the figure. A standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The regression equation was calculated, and the quercetin content was determined. The peak area was 0.0787 mAU, and the content was 0.004803 mg / g.

[0057] Example 3 This embodiment provides a method for detecting the quercetin content in actual samples using a combined extraction and rapid detection method of quercetin from Panax notoginseng, including the following steps: S1: Accurately weigh 0.5 g of pretreated Panax notoginseng powder, place the powder in a 100 mL round-bottom flask, and add 15 mL of 80% ethanol aqueous solution (solid-liquid ratio of 1:30). S2: The Panax notoginseng powder is enzymatically hydrolyzed using a compound enzyme, which accounts for 1% of the mass of the Panax notoginseng powder. The compound enzyme consists of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase. The enzymatic hydrolysis conditions are: pH=6, hydrolysis time is 1.5h, and hydrolysis temperature is 50℃. S3: Heat the solution obtained in step S2 to 85°C for enzyme inactivation treatment; S4: Use a three-way valve to connect a vacuum pump to reduce pressure and extract ultrasonically for 15 minutes. After extraction, transfer the extract to a centrifuge tube. S5: Centrifuge at 3000 r / min for 5 min, transfer the extract to a liquid chromatography vial for chromatographic analysis, and filter the sample through a 0.45 μm filter membrane before chromatographic analysis using a high performance liquid chromatograph. S6: Select 0.2% phosphoric acid aqueous solution and acetonitrile in a volume ratio of 50:50 as the mobile phase. Select a C18 column, a detection wavelength of 372 nm, a flow rate of 1 mL / min, an injection volume of 20 μL, and an analysis time of 7 min.

[0058] In this embodiment, the particle size of the pretreated Panax notoginseng powder in step S1 is 15 μm.

[0059] In this embodiment, the mass ratio of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase in step S2 is 3:3:1:0.5:1.

[0060] Perform high-performance liquid chromatography (HPLC) analysis according to the chromatographic conditions, record the chromatogram, and the chromatographic results are as follows: Figure 5 As shown in the figure. A standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The regression equation was calculated, and the quercetin content was determined. The peak area was 0.0649 mAU, and the content was 0.0044085 mg / g.

[0061] Example 4 S1: Accurately weigh 0.5 g of pretreated Panax notoginseng powder, place the powder in a 100 mL round-bottom flask, and add 15 mL of 60% ethanol aqueous solution (solid-liquid ratio of 1:30). S2: The Panax notoginseng powder is enzymatically hydrolyzed using a compound enzyme, which accounts for 0.5% of the mass of the Panax notoginseng powder. The compound enzyme consists of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase. The enzymatic hydrolysis conditions are: pH=5.5, hydrolysis time is 1h, and hydrolysis temperature is 55℃. S3: Heat the solution obtained in step S2 to 85°C for enzyme inactivation treatment; S4: Use a three-way valve to connect a vacuum pump to reduce pressure and extract ultrasonically for 5 minutes. After extraction, transfer the extract to a centrifuge tube. S5: Centrifuge at 3000 r / min for 5 min, transfer the extract to a liquid chromatography vial for chromatographic analysis, and filter the sample through a 0.45 μm filter membrane before chromatographic analysis using a high performance liquid chromatograph. S6: Select 0.2% phosphoric acid aqueous solution and acetonitrile in a volume ratio of 50:50 as the mobile phase. Select a C18 column, a detection wavelength of 372 nm, a flow rate of 1 mL / min, an injection volume of 20 μL, and an analysis time of 7 min.

[0062] In this embodiment, the particle size of the pretreated Panax notoginseng powder in step S1 is 15 μm.

[0063] In this embodiment, the mass ratio of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase in step S2 is 3:3:1:0.5:0.5.

[0064] Perform high-performance liquid chromatography (HPLC) analysis according to the chromatographic conditions, record the chromatogram, and the chromatographic results are as follows: Figure 6 As shown in the figure. A standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The regression equation was calculated, and the quercetin content was determined. The peak area was 0.1167 mAU, and the content was 0.0058887 mg / g.

[0065] Example 5 S1: Accurately weigh 0.5 g of pretreated Panax notoginseng powder, place the powder in a 100 mL round-bottom flask, and add 15 mL of 60% ethanol aqueous solution (solid-liquid ratio of 1:30). S2: The Panax notoginseng powder was enzymatically hydrolyzed using a compound enzyme, which was 1.2% of the mass of the Panax notoginseng powder. The compound enzyme consisted of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase. The enzymatic hydrolysis conditions were: pH=5, hydrolysis time was 1h, and hydrolysis temperature was 45℃. S3: Heat the solution obtained in step S2 to 85°C for enzyme inactivation treatment; S4: Use a three-way valve to connect a vacuum pump to reduce pressure and extract ultrasonically for 25 minutes. After extraction, transfer the extract to a centrifuge tube. S5: Centrifuge at 3000 r / min for 5 min, transfer the extract to a liquid chromatography vial for chromatographic analysis, and filter the sample through a 0.45 μm filter membrane before chromatographic analysis using a high performance liquid chromatograph. S6: Select 0.2% phosphoric acid aqueous solution and acetonitrile in a volume ratio of 50:50 as the mobile phase. Select a C18 column, a detection wavelength of 372 nm, a flow rate of 1 mL / min, an injection volume of 20 μL, and an analysis time of 7 min.

[0066] In this embodiment, the particle size of the pretreated Panax notoginseng powder in step S1 is 10 μm.

[0067] In this embodiment, the mass ratio of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase in step S2 is 2.5:3:1.5:0.5:1.

[0068] Perform high-performance liquid chromatography (HPLC) analysis according to the chromatographic conditions, record the chromatogram, and the chromatographic results are as follows: Figure 7 As shown in the figure. A standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The regression equation was calculated, and the quercetin content was determined. The peak area was 0.1563 mAU, and the content was 0.0070203 mg / g.

[0069] Example 6 S1: Accurately weigh 0.5 g of pretreated Panax notoginseng powder, place the powder in a 100 ml round-bottom flask, and add 10 mL of 60% ethanol aqueous solution (solid-liquid ratio of 1:20). S2: The Panax notoginseng powder is enzymatically hydrolyzed using a compound enzyme, which accounts for 1.5% of the mass of the Panax notoginseng powder. The compound enzyme consists of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase. The enzymatic hydrolysis conditions are: pH=5.5, hydrolysis time is 1.5h, and hydrolysis temperature is 55℃. S3: Heat the solution obtained in step S2 to 85°C for enzyme inactivation treatment; S4: Use a three-way valve to connect a vacuum pump to reduce pressure and extract ultrasonically for 15 minutes. After extraction, transfer the extract to a centrifuge tube. S5: Centrifuge at 3000 r / min for 5 min, transfer the extract to a liquid chromatography vial for chromatographic analysis, and filter the sample through a 0.45 μm filter membrane before chromatographic analysis using a high performance liquid chromatograph. S6: Select 0.2% phosphoric acid aqueous solution and acetonitrile in a volume ratio of 50:50 as the mobile phase. Select a C18 column, a detection wavelength of 372 nm, a flow rate of 1 mL / min, an injection volume of 20 μL, and an analysis time of 7 min.

[0070] In this embodiment, the particle size of the pretreated Panax notoginseng powder in step S1 is 10 μm.

[0071] In this embodiment, the mass ratio of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase in step S2 is 2.5:2:1.5:0.5:0.5.

[0072] Perform high-performance liquid chromatography (HPLC) analysis according to the chromatographic conditions, record the chromatogram, and the chromatographic results are as follows: Figure 8 As shown in the figure. A standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The regression equation was calculated, and the quercetin content was determined. The peak area was 0.1269 mAU, and the content was 0.00412 mg / g.

[0073] Example 7 S1: Accurately weigh 0.5 g of pretreated Panax notoginseng powder, place the powder in a 100 mL round-bottom flask, and add 30 mL of 60% volume ethanol aqueous solution (solid-liquid ratio of 1:60). S2: The Panax notoginseng powder is hydrolyzed using a compound enzyme, which is 0.5% of the mass of the Panax notoginseng powder. The compound enzyme is composed of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase. The hydrolysis conditions are: pH=5, hydrolysis time is 1h, and hydrolysis temperature is 45℃. S3: Heat the solution obtained in step S2 to 85°C for enzyme inactivation treatment; S4: Use a three-way valve to connect a vacuum pump to reduce pressure and extract ultrasonically for 15 minutes. After extraction, transfer the extract to a centrifuge tube. S5: Centrifuge at 3000 r / min for 5 min, transfer the extract to a liquid chromatography vial for chromatographic analysis, and filter the sample through a 0.45 μm filter membrane before chromatographic analysis using a high performance liquid chromatograph. S6: Select 0.2% phosphoric acid aqueous solution and acetonitrile in a volume ratio of 50:50 as the mobile phase. Select a C18 column, a detection wavelength of 372 nm, a flow rate of 1 mL / min, an injection volume of 20 μL, and an analysis time of 7 min.

[0074] In this embodiment, the particle size of the pretreated Panax notoginseng powder in step S1 is 20 μm.

[0075] In this embodiment, the mass ratio of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase in step S2 is 2:2.5:2:1:1.

[0076] Perform high-performance liquid chromatography (HPLC) analysis according to the chromatographic conditions, record the chromatogram, and the chromatographic results are as follows: Figure 9 As shown in the figure. A standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The regression equation was calculated, and the quercetin content was determined. The peak area was 0.0529 mAU, and the content was 0.0081312 mg / g.

[0077] Comparative Example 1 The difference from Example 1 is that the Panax notoginseng powder is not subjected to vacuum freeze-drying pretreatment, but otherwise it is the same as Example 1.

[0078] Comparative Example 2 The difference from Example 1 is that steps S2 and S3 are not included; otherwise, they are the same as in Example 1.

[0079] Comparative Example 3 The difference from Example 1 is that the complex enzyme in step S2 does not include polysaccharide enzymes, but otherwise it is the same as in Example 1.

[0080] Comparative Example 4 The difference from Example 1 is that the complex enzyme in step S2 does not include hemicellulase, but otherwise it is the same as in Example 1.

[0081] Comparative Example 5 The difference from Example 1 is that the complex enzyme in step S2 does not include amylase, but otherwise it is the same as in Example 1.

[0082] Comparative Example 6 The difference from Example 1 is that the complex enzyme in step S2 does not include hemicellulase and polysaccharide enzyme, but otherwise it is the same as in Example 1.

[0083] Comparative Example 7 The difference from Example 1 is that the complex enzyme in step S2 does not include hemicellulase and amylase, but otherwise it is the same as in Example 1.

[0084] Comparative Example 8 The difference from Example 1 is that the complex enzyme in step S2 does not include polysaccharide enzyme and amylase, but otherwise it is the same as in Example 1.

[0085] Comparative Example 9 The difference from Example 1 is that the complex enzyme in step S2 does not include hemicellulase, polysaccharide enzyme and amylase, but otherwise it is the same as in Example 1.

[0086] The components of the complex enzymes in Comparative Examples 3-9 are detailed in Table 2.

[0087] Table 2 Comparative Example 8 The difference from Example 1 is that step S4 is normal pressure ultrasound, while the rest is the same as in Example 1.

[0088] Experimental Example 1 Experimental groups: Example 1 and Comparative Example 8; Perform high-performance liquid chromatography (HPLC) analysis according to the chromatographic conditions, record the chromatogram, and the chromatographic results are as follows: Figure 10 As shown in Table 3, a standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The regression equation was calculated to determine the quercetin content. Both RSD values ​​were within a reasonable range. The average quercetin content extracted by the depressurized ultrasound group was significantly higher than that extracted by the normal pressure group, approximately 1.5 times higher. Furthermore, the detection time for the depressurized ultrasound group was 15 minutes, while that for the normal pressure ultrasound group was 30 minutes, indicating that depressurized ultrasound can further shorten the detection time.

[0089] Table 3. Extraction of quercetin from Panax notoginseng powder by different extraction methods Experimental Example 2 Experimental groups: Example 1 and Comparative Example 1.

[0090] Experimental method: High performance liquid chromatography analysis was performed according to chromatographic conditions to calculate the quercetin content.

[0091] Experimental results: See Table 4 for details.

[0092] Table 4 Referring to Table 4, the quercetin content in the Panax notoginseng powder without vacuum freeze-drying was significantly lower than that in Example 1, indicating that quercetin in Panax notoginseng was preserved to the maximum extent during vacuum freeze-drying, which is beneficial for the dissolution of active ingredients during extraction. Meanwhile, the detection time in Example 1 was 15 min, and in Comparative Example 1 it was 25 min; the pretreated Panax notoginseng powder could further shorten the detection time. The synergistic effect of pretreatment and reduced-pressure ultrasound not only enhances the solvent's penetration into the Panax notoginseng powder, resulting in more complete dissolution of quercetin, but also further shortens the detection time.

[0093] Experimental Example 3 Experimental groups: Example 1 and Comparative Examples 3-9.

[0094] Experimental method: High performance liquid chromatography analysis was performed according to chromatographic conditions to calculate the quercetin content.

[0095] Experimental results: See Table 5 for details.

[0096] Table 5 As can be seen from Table 5, the synergistic effect of the components in the compound enzyme more effectively increases the content of quercetin. The synergistic effect of freeze drying, enzymatic hydrolysis, and depressurized ultrasound can enhance the penetration of the solvent into Panax notoginseng powder, making the dissolution of quercetin more complete.

[0097] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0098] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for the compound extraction and rapid detection of quercetin in Panax notoginseng, characterized in that, Includes the following steps: S1: Take the pretreated Panax notoginseng powder and mix it with an ethanol aqueous solution at a solid-liquid ratio of 1:20-60. The pretreatment specifically involves: vacuum freeze-drying the block Panax notoginseng, and then pulverizing the block Panax notoginseng to obtain Panax notoginseng powder. S2: The Panax notoginseng powder is enzymatically hydrolyzed using a compound enzyme, wherein the compound enzyme is 0.5%-1.5% of the mass of the Panax notoginseng powder, and the compound enzyme is composed of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase. The enzymatic hydrolysis conditions are: pH=5-6, enzymatic hydrolysis time is 1-1.5h, and enzymatic hydrolysis temperature is 45-55℃. S3: Heat the solution obtained in step S2 to 85°C for enzyme inactivation treatment; S4: Connect the three ports of the three-way valve to the vacuum pump, the sealed sample container, and the atmosphere respectively. First, open the container to the atmosphere. After starting the vacuum pump, switch the valve position to allow the vacuum pump to evacuate the container. When the required vacuum level is reached, turn the valve to the closed position to cut off the passage. Turn off the vacuum pump, disconnect the container connection, and quickly place it into an ultrasonic extractor containing an appropriate amount of water. Set the ultrasonic frequency to 40kHz and the ultrasonic temperature to 25℃ to extract the solution obtained in step S3 and obtain the extract. S5: Centrifuge the extract obtained in step S4, take the supernatant, filter it through a 0.45 μm filter membrane, and use it for chromatographic analysis; S6: Perform high-performance liquid chromatography analysis on the sample obtained in step S5, record the chromatogram, plot the standard curve with concentration as the abscissa and peak area as the ordinate, calculate the regression equation, calculate the quercetin content, and analyze the yield.

2. The method for the compound extraction and rapid detection of quercetin in Panax notoginseng as described in claim 1, characterized in that, The particle size of Panax notoginseng powder in step S1 is 10-20 μm.

3. The method for compound extraction and rapid detection of quercetin in Panax notoginseng according to claim 2, characterized in that, The amount of Panax notoginseng powder used in step S1 is 0.5 g.

4. The method for compound extraction and rapid detection of quercetin in Panax notoginseng according to claim 3, characterized in that, The alcohol-water solution in step S1 has a strength of 40%-80%.

5. The method for compound extraction and rapid detection of quercetin in Panax notoginseng according to claim 1, characterized in that, The mass ratio of cellulase, pectinase, polysaccharide enzyme, hemicellulase and amylase in step S2 is (2-3):(2-3):(1-2):(0.5-1):(0.5-1).

6. The method for compound extraction and rapid detection of quercetin in Panax notoginseng according to claim 4, characterized in that, The enzyme activity of any one of the enzymes in the complex enzyme is ≥5×10 4 U / g.

7. The method for compound extraction and rapid detection of quercetin in Panax notoginseng according to claim 1, characterized in that, In step S4, the ultrasonic extractor is set to 40 kHz and 25 ℃.

8. The method for compound extraction and rapid detection of quercetin in Panax notoginseng according to claim 7, characterized in that, The ultrasonic extraction time in step S4 is 5-25 min.

9. The method for compound extraction and rapid detection of quercetin in Panax notoginseng according to claim 1, characterized in that, In step S5, the centrifugation speed is 3000-3500 r / min and the centrifugation time is 5-8 min.

10. The method for compound extraction and rapid detection of quercetin in Panax notoginseng according to claim 1, characterized in that, The chromatographic conditions in step S6 are as follows: 0.2% phosphoric acid aqueous solution and acetonitrile in a volume ratio of 50:50 are selected as the mobile phase, a C18 column is selected, the detection wavelength is 372 nm, the flow rate is 1 mL / min, the injection volume is 20 μL, and the analysis time is 7 min.

Images