Method for extracting flavonoid compounds in immature bitter oranges

Abstract

The invention discloses a method for extracting flavonoid compounds in fructus aurantii immaturus, which comprises the following steps: 1, preparing DESs by using choline chloride ethylene glycol, choline chloride cane sugar and choline chloride lactic acid as raw materials; 2, weighing 1 g of the immature bitter orange dry powder, putting the immature bitter orange dry powder into a beaker, adding a DESs solution, carrying out ultrasonic extraction under a 40 DEG C water bath condition for 60 min, cooling to room temperature, and supplementing weightlessness; filtering, discarding primary filtrate, transferring 1mL of filtrate into a 10mL volumetric flask, adding water to dilute and fix the volume to the scale, shaking uniformly, and preparing a test solution taking water and 60% ethanol as an extraction solvent through a filter membrane by the same method; 3, measuring the content of flavonoid compounds, and performing chromatographic analysis; and 4, respectively taking a proper amount of naringin and neohesperidin reference substances, and qualitatively analyzing the flavone compound of the immature bitter orange. The invention provides a sustainable and safe extraction process for analysis and preparation of flavonoid in a complex sample, and has potential to be used for large-scale industrial application.

Description

Technical Field

[0001] The invention relates to the technical field of extracting Citrus aurantium immaturus, and in particular to a method for extracting flavonoid compounds in Citrus aurantium immaturus. Background Art

[0002] Citrus aurantium is the young fruit of the Rutaceae plant Citrus aurantium L., which has the effect of lowering qi and eliminating stagnation, and has high medicinal value. Citrus aurantium contains hesperidin, neohesperidin, naringin, synephrine and N-methyltyramine.

[0003] As a new type of green and environmentally friendly solvent, DESs has the characteristics of low melting point, non-volatility, safety, non-toxicity, biodegradability, wide source of raw materials, low price, convenient preparation process, good biocompatibility, etc. In addition, DES as a new type of environmentally friendly solvent for the extraction of natural bioactive substances has excellent extraction efficiency for natural active compounds such as flavonoids, polysaccharides, phenolic acids, alkaloids, saponins and anthraquinones.

[0004] In the prior art, Chinese patent number CN 111057117 A discloses a method for extracting hesperidin from Citrus aurantium: using two enzymatic pretreatments and then extracting the enzymatic Citrus aurantium raw material. In an alkaline solution, hesperidin is in a soluble state and can be fully extracted by a solvent, and finally hesperidin is extracted. Although the process is simple, it still uses enzyme preparations for impurity removal and organic solvents for extraction, causing certain pollution and waste, and only hesperidin is extracted, and other flavonoid components in Citrus aurantium are not effectively processed. Summary of the invention

[0005] The purpose of the present invention is to provide a method for extracting flavonoid compounds in Citrus aurantium, which solves the problem of overcoming the inherent defects of the prior art such as high energy consumption, environmental pollution and solvent residue, and provides a method with simple operation, low equipment requirements, lower extraction cost and higher extraction rate.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for extracting flavonoids from Citrus aurantium immaturus, comprising the following steps: Step 1: using choline chloride ethylene glycol, choline chloride sucrose and choline chloride lactic acid as raw materials, preparing a low eutectic solvent DESs; Step 2: Weigh 1g of dried powder of Citrus aurantium in a beaker, add DESs solution, extract by ultrasonic in a 40℃ water bath for 60min, cool to room temperature, and make up for weight loss. Filter, discard the initial filtrate, transfer 1mL of filtrate into a 10mL volumetric flask, dilute to the mark with water, shake well, pass through a 0.22μm filter membrane, and prepare the test solution with water and 60% ethanol as extraction solvents in the same way; Step 3: Determine the content of flavonoids by UV spectrophotometry and perform chromatographic analysis on UHPLC Ultimate 3000; Step 4: Take appropriate amounts of naringin and neohesperidin reference substances, respectively, and use HPLC-MS to qualitatively analyze the flavonoid compounds in Citrus aurantium.

[0007] In some embodiments, preparing a deep eutectic solvent DESs comprises the following steps: S11. Raw material preparation: ensure that the purity of choline chloride glycol, choline chloride sucrose, and choline chloride lactic acid meets the experimental requirements, and accurately weigh the required raw materials; S12, molar ratio mixing: mixing choline chloride ethylene glycol and choline chloride sucrose, and an appropriate amount of choline chloride lactic acid in a molar ratio of 1:2; S13, heating and dissolving: Place the mixed raw materials in a suitable container and heat to 80°C; this temperature is usually lower than the melting point of the components, but is sufficient to promote their mixing and dissolution; S14. Stirring: During the heating process, the mixture is continuously stirred using a magnetic stirrer or other stirring equipment to ensure that the components are evenly mixed; S15, cooling and solidification: when the mixture reaches a uniform state, the temperature can be gradually lowered to cool and solidify to form a low eutectic solvent; S16. Post-processing: The cooled eutectic solvent may require further purification and characterization, such as removing insoluble matter by filtration, or characterizing the chemical structure using methods such as NMR and FTIR; S17. Performance testing: The prepared low eutectic solvent is subjected to performance testing, including its solubility, viscosity, thermal stability, etc., to evaluate its suitability as a solvent.

[0008] In some embodiments, the solid-liquid ratio of the added DESs solution is 1:20.

[0009] In some embodiments, the chromatographic analysis comprises the following steps: The sample was eluted with a mobile phase of 0.1% formic acid-water (A) and 0.1% formic acid-acetonitrile (B), gradient elution: 0-2 minutes: 5% B; 2-22 minutes: 5%-95% B; 22-30 minutes: 95% B; 30.01~35 minutes: 5% B; The equilibration time before each run was 10 min; the flow rate was 0.3 mL / min, and the column temperature was maintained at 40°C; the injection volume was 10 μL, and the detection wavelength was 360 nm.

[0010] Compared with the prior art, the present invention has the following beneficial effects: The method of extracting flavonoids from Citrus aurantium by DES can be successfully applied to the extraction of flavonoids in plant matrices, provides a sustainable and safe extraction process for the analysis and preparation of flavonoids in complex samples, and has the potential to be used in large-scale industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a schematic diagram of the extraction process of the present invention; DETAILED DESCRIPTION

[0012] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0013] See also Figure 1 , a method for extracting flavonoids from Citrus aurantium, comprising the following steps: Step 1: Using choline chloride ethylene glycol, choline chloride sucrose, and choline chloride lactic acid as raw materials, preparing a low eutectic solvent DESs; comprising the following steps: S11. Raw material preparation: ensure that the purity of choline chloride glycol, choline chloride sucrose, and choline chloride lactic acid meets the experimental requirements, and accurately weigh the required raw materials; S12, molar ratio mixing: mixing choline chloride ethylene glycol and choline chloride sucrose, and an appropriate amount of choline chloride lactic acid in a molar ratio of 1:2; S13, heating and dissolving: Place the mixed raw materials in a suitable container and heat to 80°C; this temperature is usually lower than the melting point of the components, but is sufficient to promote their mixing and dissolution; S14. Stirring: During the heating process, the mixture is continuously stirred using a magnetic stirrer or other stirring equipment to ensure that the components are evenly mixed; S15, cooling and solidification: when the mixture reaches a uniform state, the temperature can be gradually lowered to cool and solidify to form a low eutectic solvent; S16. Post-processing: The cooled eutectic solvent may require further purification and characterization, such as removing insoluble matter by filtration, or characterizing the chemical structure using methods such as NMR and FTIR; S17. Performance testing: The prepared low eutectic solvent is subjected to performance testing, including its solubility, viscosity, thermal stability, etc., to evaluate its suitability as a solvent.

[0014] Step 2: Weigh 1g of dried powder of Citrus aurantium in a beaker and add DESs solution, wherein the solid-liquid ratio of the added DESs solution is 1:20. Ultrasonic extraction was performed in a 40°C water bath for 60 min, cooled to room temperature, and weight loss was supplemented. Filter, discard the initial filtrate, transfer 1mL of the filtrate into a 10mL volumetric flask, dilute with water to the mark, shake well, pass through a 0.22μm filter membrane, and prepare the test solution with water and 60% ethanol as the extraction solvent in the same way; Step 3: Determine the content of flavonoids by UV spectrophotometry and perform chromatographic analysis on UHPLC Ultimate 3000; The sample was eluted with a mobile phase of 0.1% formic acid-water (A) and 0.1% formic acid-acetonitrile (B), gradient elution: 0-2 minutes: 5% B; 2-22 minutes: 5%-95% B; 22-30 minutes: 95% B; 30.01~35 minutes: 5% B; The equilibration time before each run was 10 min; the flow rate was 0.3 mL / min, and the column temperature was maintained at 40°C; the injection volume was 10 μL, and the detection wavelength was 360 nm.

[0015] Step 4: Take appropriate amounts of naringin and neohesperidin reference substances, and use HPLC-MS to qualitatively analyze the flavonoid compounds in Citrus aurantium. The steps include: 1. Sample preparation: Take an appropriate amount of Citrus aurantium sample and carry out appropriate pretreatment, such as drying, crushing, etc.

[0016] Flavonoids were extracted from Fructus Aurantii Immaturus using appropriate extraction methods (e.g. solvent extraction, ultrasound-assisted extraction, etc.).

[0017] 2. Preparation of reference substances: Take appropriate amounts of naringin and neohesperidin reference substances, respectively, and dissolve them in appropriate solvents (usually a mixture of methanol, acetonitrile or water) to prepare standard solutions.

[0018] 3. Chromatographic conditions setting: Select a suitable chromatographic column, such as a C18 column.

[0019] The composition and flow rate of the mobile phase are set, for example using a mixture of methanol and water, and possibly including a certain proportion of an acid (such as phosphoric acid) to improve the separation.

[0020] Set the column temperature, usually between room temperature and 40°C.

[0021] 4. Mass spectrometry condition settings: Select an appropriate ionization mode, such as ESI (electrospray ionization).

[0022] The scan range of the mass spectrometer was set to cover the possible mass range of flavonoids.

[0023] Optimize mass spectrometry parameters such as collision energy, spray voltage, etc. to obtain the best signal response.

[0024] 5. Sample analysis: The extracted Fructus Aurantii Immaturus sample solution was chromatographically separated by HPLC system.

[0025] The chromatographically separated compounds enter the mass spectrometer for ionization and detection.

[0026] 6. Data processing: The qualitative analysis of flavonoids was performed using mass spectrometry data, and the compounds were identified by comparing retention time and mass-to-charge ratio (m / z) with HPLC-MS data of reference substances.

[0027] Peak area integration was performed using software tools to assess the relative content of each compound.

[0028] 7. Interpretation of results: Based on the results of HPLC-MS, the flavonoids in the Fructus Aurantii Immaturus samples were identified and confirmed.

[0029] Combined with the analysis results of the reference substances, a qualitative analysis of the flavonoids in Fructus Aurantii Immaturus was carried out.

Claims

1. A method for extracting flavonoids from Citrus aurantium, characterized in that: The following steps are involved: Step 1: using choline chloride ethylene glycol, choline chloride sucrose and choline chloride lactic acid as raw materials, preparing a low eutectic solvent DESs; Step 2: Weigh 1g of dried powder of Citrus aurantium in a beaker, add DESs solution, extract by ultrasonic in a water bath at 40℃ for 60min, cool to room temperature, and make up for weight loss; filter, discard the initial filtrate, transfer 1mL of filtrate into a 10mL volumetric flask, dilute to the mark with water, shake well, pass through a 0.22μm filter membrane, and prepare the test solution with water and 60% ethanol as extraction solvents in the same way; Step 3: Determine the content of flavonoids by UV spectrophotometry and perform chromatographic analysis on UHPLC Ultimate 3000; Step 4: Take appropriate amounts of naringin and neohesperidin reference substances, respectively, and use HPLC-MS to qualitatively analyze the flavonoid compounds in Citrus aurantium.

2. The method for extracting flavonoids from Citrus aurantium according to claim 1, characterized in that: The preparation of the deep eutectic solvent DESs in step 1 comprises the following steps: S11. Raw material preparation: ensure that the purity of choline chloride glycol, choline chloride sucrose, and choline chloride lactic acid meets the experimental requirements, and accurately weigh the required raw materials; S12, molar ratio mixing: mixing choline chloride ethylene glycol and choline chloride sucrose, and an appropriate amount of choline chloride lactic acid in a molar ratio of 1:2; S13, heating and dissolving: putting the mixed raw materials into a suitable container and heating to 80°C; S14. Stirring: During the heating process, the mixture is continuously stirred using a magnetic stirrer or other stirring equipment to ensure that the components are evenly mixed; S15, cooling and solidification: when the mixture reaches a uniform state, the temperature can be gradually lowered to cool and solidify to form a low eutectic solvent; S16, post-processing: the cooled deep eutectic solvent needs further purification and characterization, and the insoluble matter is removed by filtration; S17. Performance test: Perform performance test on the prepared low eutectic solvent.

3. The method for extracting flavonoids from Citrus aurantium according to claim 1, characterized in that: The solid-liquid ratio of the added DESs solution is 1:

20.

4. The method for extracting flavonoids from Fructus Aurantii Immaturus according to claim 1, characterized in that: The chromatographic analysis comprises the following steps: The sample was eluted with a mobile phase of 0.1% formic acid-water (A) and 0.1% formic acid-acetonitrile (B), gradient elution: 0-2 minutes: 5% (B); 2-22 minutes: 5%-95% (B); 22-30 minutes: 95% (B); 30.01~35 minutes: 5% (B); The equilibration time before each run was 10 min; the flow rate was 0.3 mL / min, and the column temperature was maintained at 40°C; the injection volume was 10 μL, and the detection wavelength was 360 nm.

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