Method for efficiently preparing hesperidin

Abstract

The invention discloses a method for efficiently preparing hesperidin, which comprises the following steps: mixing grapefruit outer skin powder with petroleum ether, and carrying out ultrasonic-assisted extraction, suction filtration, concentration and drying to obtain a crude extract; dissolving the crude extract in petroleum ether, loading a sample to an SNAP KP-Sil normal-phase silica gel column, carrying out forward rapid liquid chromatography separation by taking petroleum ether-ethyl acetate as a mobile phase, identifying separated hesperelactone components through thin-layer chromatography, and merging the same components; carrying out reversed-phase preparative high-pressure liquid chromatography purification on the purified hesperidin component by virtue of a reversed-phase preparative high-pressure liquid chromatography column, and identifying the purified hesperidin component by virtue of thin-layer chromatography; carrying out structure identification on the collected components by using nuclear magnetic resonance and mass spectrometry, and determining the purity of the components by using high performance liquid chromatography; therefore, high-purity hesperidin can be rapidly prepared in a low-energy-consumption and solvent-saving manner.

Description

Technical Field

[0001] The invention relates to the technical field of active substance extraction, and in particular to a method for efficiently preparing hesperidin. Background Art

[0002] Meranzin is a coumarin substance from citrus plants with a molecular formula of C 15 H 16 O4. Hesperidin has multiple biological functions, including regulating α2-adrenaline receptors, participating in AMPA-ERK1 / 2-BDNF signaling, inhibiting acetylcholinesterase, preventing atherosclerosis and depression complications, etc. Hesperidin has good application potential because of its multiple biological functions.

[0003] At present, the preparation of hesperidin is mainly prepared by extracting from citrus fruits using ethanol as solvent. For example, He et al. extracted Fructus Aurantii Immaturus powder with 60%-80% ethanol under light-proof room temperature conditions, stirred for 24 hours, collected the filtrate, extracted twice, concentrated by rotary evaporation to obtain a concentrate, passed through a chromatographic column containing a macroporous resin at a rate of 2-3 bv / h, washed the resin until it was colorless (about 5 bv), and then eluted with 10 bv 70% ethanol and 10 bv 95% ethanol respectively, and concentrated in vacuo to obtain a crude extract, and separated the two ethanol eluates with n-hexane-ethyl acetate-n-butanol-methanol-acetic acid and n-hexane-n-butanol-methanol-acetic acid two-phase solvent systems using HSCCC. This method takes too long to extract and requires more types of organic solvents for separation. Zhu Tailin et al. used 80% ethanol as solvent to extract hesperidin from citrus fruits. In this process, the solid-liquid ratio was 1:20, and the extraction was repeated 3 times under ultrasonic-assisted conditions. The amount of solvent used was large (a total of 60 volumes of ethanol), and the concentration consumed a lot of energy. After preliminary separation using a solid phase extraction column, segmented powders were obtained, and then further purified by Semi-prep HPLC. The mobile phases of segmented products I and II were water and methanol, and the gradient elution time was 95 minutes; the mobile phases of segmented product III were water and acetonitrile, and the gradient elution time was 53 minutes. This method used a large amount of solvent and the separation and preparation time was too long. Therefore, the extraction and preparation methods of hesperidin reported in relevant studies have the disadvantages of large solvent usage, complex solvent types, and high energy consumption for solvent recovery. Summary of the invention

[0004] To solve the above problems, the present invention proposes a method for efficiently preparing hesperidin, which solves the problems of large amount of solvent used, complex solvent types and high energy consumption for solvent recovery in the preparation process of hesperidin in the prior art.

[0005] A method for efficiently preparing hesperidin according to the present invention comprises the following steps:

[0006] (1) mixing the outer skin powder of grapefruit fruit with petroleum ether, performing ultrasonic-assisted extraction, filtering, concentrating and drying to obtain a crude extract;

[0007] (2) dissolving the crude extract in petroleum ether, loading the mixture onto a SNAP KP-Sil normal phase silica gel column at a flow rate of 15 mL / min, separating the mixture by normal phase rapid liquid chromatography using petroleum ether-ethyl acetate as the mobile phase, identifying the separated hesperidin components by thin layer chromatography, and combining the same components;

[0008] (3) loading the components collected in step (2) onto a reverse phase preparative high pressure liquid chromatography column, performing reverse phase preparative high pressure liquid chromatography purification, and identifying the purified hesperidin component by thin layer chromatography;

[0009] (4) The components collected in step (3) are structurally identified by nuclear magnetic resonance and mass spectrometry, and their purity is determined by high performance liquid chromatography.

[0010] According to the invention, a method for efficiently preparing hesperidin is provided. The method uses pomelo fruit outer peel powder as a raw material, adopts petroleum ether as an extraction solvent for extraction, separates through normal rapid chromatography and purifies through reverse phase preparative high pressure liquid chromatography, and undergoes nuclear magnetic resonance and mass spectrometry analysis and HPLC analysis to obtain hesperidin with a purity of ≥95%. The method can prepare high-purity hesperidin quickly, with low energy consumption and in a solvent-saving manner.

[0011] Optionally, in step (1), the pomelo fruit outer skin powder is mixed with the petroleum ether in a solid-liquid ratio of 1:3, and ultrasonic-assisted extraction is performed three times, each time for 30 minutes, and the ultrasonic temperature is 35°C.

[0012] Optionally, in step (2), gradient elution is performed with petroleum ether-ethyl acetate solutions in different proportions, and the concentration gradient of ethyl acetate during the gradient elution of the petroleum ether-ethyl acetate solution is 0%, 0-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-100%, each gradient elution is 2 column volumes, and all the eluate is collected.

[0013] Optionally, in step (2), components with an Rf value of 0.56 are combined.

[0014] Optionally, in step (3), the chromatographic column is an Ultimate XB-C18 chromatographic column; water (A)-methanol (B) is used as the mobile phase for gradient elution; the gradient elution program is: 0-5min 15%-45% B; 5-10min 45%-75% B; 10-20min 75%-90% B; 20-30min 90%-45% B; 25-35min 45%-15% B; the sample flow rate is 3mL / min, and the sample concentration is 4mg / mL; and the separated components within 22-23min are collected according to the spectrum collected by the chromatographic detector.

[0015] Optionally, in step (2) and step (3), identification by thin layer chromatography is: using a hesperidin standard as a control, performing a TLC qualitative analysis, and the component with the same ratio transfer value as the standard is the separated hesperidin component.

[0016] Optionally, in step (1), the pomelo is Guanxi honey pomelo, and the boiling point of petroleum ether is 60-90°C.

[0017] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a process flow for preparing hesperidin according to an embodiment of the present invention;

[0019] Figure 2 Ultrasonic assisted extraction optimization according to the embodiments and comparative examples of the present invention;

[0020] Figure 3 The TLC chromatography results according to the embodiment of the present invention;

[0021] Figure 4 : is a nuclear magnetic resonance spectrum of hesperidin monomer according to an embodiment of the present invention, wherein A is a hydrogen spectrum (500 MHz, methanol-d4), and B is a carbon spectrum (126 MHz, methanol-d4);

[0022] Figure 5 is a mass spectrum of a hesperidin monomer compound according to an embodiment of the present invention;

[0023] Figure 6 The liquid chromatogram of the hesperidone monomer compound obtained after purification by preparative high pressure liquid chromatography according to an embodiment of the present invention. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to limit the scope of the present invention. The change or adjustment of the relative relationship thereof shall also be regarded as the scope of the present invention without substantially changing the technical content.

[0025] In order to better understand the above technical scheme, the exemplary embodiments of the present invention are described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0026] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0027] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0028] Example 1

[0029] Preparation of pomelo fruit outer skin powder: Take a 1 mm thick outer skin layer of Guanxi pomelo, place it in a forced air drying oven, dry it at 37°C, and grind it through a 40-mesh sieve to obtain the pomelo fruit outer skin powder for later use.

[0030] Extraction: Extract the outer skin powder of grapefruit fruit with petroleum ether at a solid-liquid ratio of 1:2. Mix 140g of the outer skin powder of grapefruit fruit with 280mL of petroleum ether (boiling point 60-90°C), and perform ultrasonic-assisted extraction 3 times, 30 minutes each time, at an ultrasonic temperature of 35°C. Filter by suction, concentrate and dry the filtrate on a rotary evaporator, and weigh 1.8g of the crude extract. The purity was 5.17% (measured by high performance liquid chromatography). Figure 2 A). In addition, the extraction process used 6 mL of petroleum ether for three extractions of 1 g of grapefruit fruit outer skin powder, the extraction time was 90 min, and 0.1656 J of energy was consumed for the recovery of petroleum ether.

[0031] Rapid liquid chromatography separation: The crude extract was dissolved in 5 mL of petroleum ether and separated by rapid liquid chromatography, with a SNAP KP-Sil normal phase silica gel column as the stationary phase, the mass of the SNAP KP-Sil normal phase silica gel column being 30 times that of the sample to be separated, petroleum ether-ethyl acetate as the mobile phase, the sample loading flow rate being 15 mL / min, and gradient elution with 0%, 0-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-100% ethyl acetate solution, each elution time being 1:1. The sample was eluted with a gradient of 2 column volumes and all the eluents were collected. The hesperidin standard was used as a control for TLC qualitative analysis. The separated fractions were adsorbed on a F254 thin-layer silica gel plate coated with a fluorescent indicator, and petroleum ether / ethyl acetate (2:1, v / v) was used as the mobile phase. After development in the chromatography cylinder, the sample was fumigated with iodine for 10 minutes, and the ratio of the sample movement distance to the solvent front distance was used to calculate the sample's relative migration value (Rf). The fractions with the same Rf value were combined. Figure 3 As shown, the component with the same Rf value as the standard (Rf=0.56) is the separated hesperidin component.

[0032] Preparative high pressure liquid chromatography purification: The hesperidin component collected by rapid liquid chromatography was concentrated by rotary evaporation and dissolved in chromatographic grade methanol at a concentration of 4 mg / mL. After passing through a 0.22 μm filter membrane, it was loaded onto an Ultimate XB-C18 column (10 mm×250 mm, 5 μm); water (A)-methanol (B) was used as the mobile phase for gradient elution. The loading conditions were: flow rate 3 mL / min, loading concentration 4 mg / mL, loading volume 300 μL; the gradient elution program was: 0-5 min 15%-45% B; 5-10 min 45%-75% B; 10-20 min 75%-90% B; 20-30 min 90%-45% B; 25-35 min 45%-15% B. The separated components within 22-23 min were collected according to the spectra collected by the chromatographic detector. Using hesperidin standard as a control, TLC qualitative analysis was performed, and the separated fractions were adsorbed on a F254 thin layer silica gel plate coated with a fluorescent indicator, and petroleum ether / ethyl acetate (2:1, v / v) was used as the mobile phase. After development in the chromatography cylinder, the sample was fumigated with iodine for 10 minutes, and the ratio of the sample movement distance to the solvent front distance was used to calculate the sample's relative migration value (Rf). The fractions with the same Rf value were combined, such as Figure 3 As shown, the component with the same Rf value as the standard (Rf=0.56) is the purified hesperidin component.

[0033] Structural identification: The hesperidin components separated by preparative high pressure liquid chromatography were concentrated and dried on a rotary evaporator for nuclear magnetic resonance analysis, such as Figure 4 As shown, the NMR data are as follows: 1H NMR (500MHz, Methanol-d4) δ7.87 (d, J = 9.5Hz, 1H), 7.53 (d, J = 8.6Hz, 1H), 7.06 (d, J = 8.7Hz, 1H), 6.24 (d, J = 9.5Hz, 1H), 3.97 (s, 3H), 3.18 (dd, J = 13.3, 4.8Hz, 1H), 3.02 (dd, J = 6.9, 4.8Hz, 1H), 2.97 (dd, J = 13.3, 6.9Hz, 1H), 1.49 (s, 3H), 1.28 (s, 3H). 13C NMR (126MHz, Methanol-d4) δ162.98, 162.18, 154.41, 146.03, 129.08, 114.54, 114.37, 113.32, 109.01, 64.53, 61.06, 56.77, 24.87, 23.26, 19.32. Further mass spectrometry analysis, such as Figure 5 As shown, the results show that the m / z of the characteristic ion fragment is 261[M+H]+, and the relative molecular weight is determined to be 260. Through comparative analysis, it can be seen that its NMR data and molecular weight are consistent with those of the hesperidone standard. Therefore, the sample is determined to be hesperidone.

[0034] Purity determination: The target component peak was determined by high performance liquid chromatography, such as Figure 6 As shown, the peak purity was measured to be 96.47%.

[0035] As shown in Table 1, in this embodiment, the outer skin of Guanxi honey pomelo is subjected to ultrasonic-assisted extraction of petroleum ether, rapid liquid chromatography and preparative high pressure liquid chromatography separation and purification. It is calculated that 1559.45 g of pomelo fruit outer skin powder, 1824.56 mL of ethyl acetate, 13411.31 mL of petroleum ether, and 24155.95 mL of 79% methanol are required to prepare 1 g of hesperidin, a total of 39391.82 mL of organic reagents, and a total of 1897.70 J of energy is consumed for recovery, with a purity of 96.47%.

[0036] Example 2

[0037] Preparation of pomelo fruit outer skin powder: Take a 1 mm thick outer skin layer of Guanxi pomelo, place it in a forced air drying oven, dry it at 37°C, and grind it through a 40-mesh sieve to obtain the pomelo fruit outer skin powder for later use.

[0038] Extraction: Extract the outer skin powder of grapefruit fruit with petroleum ether at a solid-liquid ratio of 1:3. Mix 140g of the outer skin powder of grapefruit fruit with 420mL of petroleum ether (boiling point 60-90°C), and perform ultrasonic-assisted extraction 3 times, 30 minutes each time, at an ultrasonic temperature of 35°C. The filtrate obtained by suction filtration was concentrated and dried on a rotary evaporator and weighed to obtain 2.4g of crude extract, which was measured by high performance liquid chromatography to have a purity of 6.22% ( Figure 2 B). In addition, the extraction process used 9 mL of petroleum ether for three extractions of 1 g of grapefruit fruit outer skin powder, and the extraction time was 90 min. The energy required for the recovery of petroleum ether was 0.2484 J.

[0039] Rapid liquid chromatography separation: the obtained crude extract was dissolved in 5 mL of petroleum ether and separated by rapid liquid chromatography, with a SNAP KP-Sil normal phase silica gel column as the stationary phase, the mass of the SNAP KP-Sil normal phase silica gel column being 30 times that of the sample to be separated, petroleum ether-ethyl acetate as the mobile phase, the loading flow rate being 15 mL / min, and gradient elution with 0%, 0-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-100% ethyl acetate solution in sequence, each gradient elution being 2 column volumes, all eluents being collected, and the same fractions (components with an Rf value of 0.56) being combined and concentrated to dryness.

[0040] Preparative high pressure liquid chromatography purification: The hesperidin component collected by rapid liquid chromatography was concentrated by rotary evaporation and dissolved in chromatographic grade methanol at a concentration of 4 mg / mL. After passing through a 0.22 μm filter membrane, it was loaded onto an Ultimate XB-C18 column (10 mm×250 mm, 5 μm); water (A)-methanol (B) was used as the mobile phase for gradient elution. The loading conditions were: flow rate 3 mL / min, loading concentration 4 mg / mL, loading volume 300 μL; the gradient elution program was: 0-5 min 15%-45% B; 5-10 min 45%-75% B; 10-20 min 75%-90% B; 20-30 min 90%-45% B; 25-35 min 45%-15% B. The separated components within 22-23 min were collected according to the spectra collected by the chromatographic detector.

[0041] Structural identification: The hesperidin components separated by preparative high pressure liquid chromatography were concentrated and dried on a rotary evaporator for nuclear magnetic resonance analysis, such as Figure 4As shown, the NMR data are as follows: 1H NMR (500MHz, Methanol-d4) δ7.87 (d, J = 9.5Hz, 1H), 7.53 (d, J = 8.6Hz, 1H), 7.06 (d, J = 8.7Hz, 1H), 6.24 (d, J = 9.5Hz, 1H), 3.97 (s, 3H), 3.18 (dd, J = 13.3, 4.8Hz, 1H), 3.02 (dd, J = 6.9, 4.8Hz, 1H), 2.97 (dd, J = 13.3, 6.9Hz, 1H), 1.49 (s, 3H), 1.28 (s, 3H). 13C NMR (126MHz, Methanol-d4) δ162.98, 162.18, 154.41, 146.03, 129.08, 114.54, 114.37, 113.32, 109.01, 64.53, 61.06, 56.77, 24.87, 23.26, 19.32. Further mass spectrometry analysis, such as Figure 5 As shown, the results show that the m / z of the characteristic ion fragment is 261[M+H]+, and the relative molecular weight is determined to be 260. Through comparative analysis, it can be seen that its NMR data and molecular weight are consistent with those of the hesperidone standard. Therefore, the sample is determined to be hesperidone.

[0042] Purity determination: The target component peak was determined by high performance liquid chromatography, such as Figure 6 As shown, the peak purity was measured to be 96.47%.

[0043] As shown in Table 1, in this embodiment, the outer skin of Guanxi pomelo was subjected to ultrasonic-assisted extraction of petroleum ether, rapid liquid chromatography and preparative high pressure liquid chromatography separation and purification. It was calculated that 972.15 g of pomelo fruit outer skin powder, 1137.41 mL of ethyl acetate, 11276.92 mL of petroleum ether, and 15058.57 mL of 79% methanol were required to prepare 1 g of hesperidin, a total of 27472.90 mL of organic reagents, and a total of 1263.50 J of energy was consumed for recovery, with a purity of 96.47%.

[0044] Example 3

[0045] Preparation of pomelo fruit outer skin powder: Take a 1 mm thick outer skin layer of Guanxi pomelo, place it in a forced air drying oven, dry it at 37°C, and grind it through a 40-mesh sieve to obtain the pomelo fruit outer skin powder for later use.

[0046] Extraction: Extract the outer skin powder of grapefruit fruit with petroleum ether at a solid-liquid ratio of 1:4. Mix 140g of grapefruit fruit outer skin powder with 560mL of petroleum ether (boiling point 60-90°C), and perform ultrasonic-assisted extraction 3 times, 30 minutes each time, at an ultrasonic temperature of 35°C. The filtrate obtained by suction filtration was concentrated and dried on a rotary evaporator, and 2.4g of crude extract was weighed. The purity was 5.51% (measured by high performance liquid chromatography). Figure 2 C). In addition, the extraction process used 12 mL of petroleum ether for three extractions of 1 g of grapefruit fruit outer skin powder, and the extraction time was 90 min. The energy required for the recovery of petroleum ether was 0.3312 J.

[0047] Rapid liquid chromatography separation: the obtained crude extract was dissolved in 5 mL of petroleum ether, and separated by rapid preparative liquid chromatography, with a SNAP KP-Sil normal phase silica gel column as the stationary phase, the mass of the SNAP KP-Sil normal phase silica gel column being 30 times that of the sample to be separated, petroleum ether-ethyl acetate as the mobile phase, the loading flow rate being 15 mL / min, and gradient elution with 0%, 0-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-100% ethyl acetate solution in sequence, each gradient elution being 2 column volumes, and all eluents being collected, and the same fractions (components with an Rf value of 0.56) being combined and concentrated to dryness.

[0048] Preparative high pressure liquid chromatography purification: The hesperidin component collected by rapid liquid chromatography was concentrated by rotary evaporation and dissolved in chromatographic grade methanol at a concentration of 4 mg / mL. After passing through a 0.22 μm filter membrane, it was loaded onto an Ultimate XB-C18 column (10 mm×250 mm, 5 μm); water (A)-methanol (B) was used as the mobile phase for gradient elution. The loading conditions were: flow rate 3 mL / min, loading concentration 4 mg / mL, loading volume 300 μL; the gradient elution program was: 0-5 min 15%-45% B; 5-10 min 45%-75% B; 10-20 min 75%-90% B; 20-30 min 90%-45% B; 25-35 min 45%-15% B. The separated components within 22-23 min were collected according to the spectra collected by the chromatographic detector.

[0049] Structural identification: The hesperidin components separated by preparative high pressure liquid chromatography were concentrated and dried on a rotary evaporator for nuclear magnetic resonance analysis, such as Figure 4As shown, the NMR data are as follows: 1H NMR (500MHz, Methanol-d4) δ7.87 (d, J = 9.5Hz, 1H), 7.53 (d, J = 8.6Hz, 1H), 7.06 (d, J = 8.7Hz, 1H), 6.24 (d, J = 9.5Hz, 1H), 3.97 (s, 3H), 3.18 (dd, J = 13.3, 4.8Hz, 1H), 3.02 (dd, J = 6.9, 4.8Hz, 1H), 2.97 (dd, J = 13.3, 6.9Hz, 1H), 1.49 (s, 3H), 1.28 (s, 3H). 13C NMR (126MHz, Methanol-d4) δ162.98, 162.18, 154.41, 146.03, 129.08, 114.54, 114.37, 113.32, 109.01, 64.53, 61.06, 56.77, 24.87, 23.26, 19.32. Further mass spectrometry analysis, such as Figure 5 As shown, the results show that the m / z of the characteristic ion fragment is 261[M+H]+, and the relative molecular weight is determined to be 260. Through comparative analysis, it can be seen that its NMR data and molecular weight are consistent with those of the hesperidone standard. Therefore, the sample is determined to be hesperidone.

[0050] Purity determination: The target component peak was determined by high performance liquid chromatography, such as Figure 6 As shown, the peak purity was measured to be 96.47%.

[0051] As shown in Table 1, in this embodiment, the outer skin of Guanxi honey pomelo is subjected to ultrasonic-assisted extraction of petroleum ether, rapid liquid chromatography and preparative high pressure liquid chromatography separation and purification. It is calculated that 1097.42 g of pomelo fruit outer skin powder, 1283.98 mL of ethyl acetate, 16022.30 mL of petroleum ether, and 16999.00 mL of 79% methanol are required to prepare 1 g of hesperidin, a total of 34305.28 mL of organic reagents, and a total of 1517.18 J of energy is consumed for recovery, with a purity of 96.47%.

[0052] Comparative Example 1

[0053] As shown in Table 1, according to the traditional extraction method (Bioactivity-Guided Separation of PotentialD2 Dopamine Receptor Antagonists from Aurantii Fructus based on MolecularDocking Combined with High-Speed ​​Counter-Current Chromatography), the Fructus Aurantii powder was extracted with 70% ethanol, and then separated by macroporous resin and purified by two-step HSCCC separation. To prepare 1g of hesperidin, 1029.24g of Fructus Aurantii powder, 13308.08mL of 70% ethanol, 6051.94mL of 95% ethanol, 19740.84mL of n-hexane, 28417.34mL of ethyl acetate, 20131.95mL of n-butanol, 14604.93mL of methanol, and 62773.39mL of 0.05% acetic acid were required. Six organic reagents were required, totaling 165028.47mL, and a total of 13053.65J of energy was consumed for solvent recovery.

[0054] Comparison between Comparative Example 1 and Examples 1-3 shows that the preparation of high-purity hesperidin using grapefruit peel powder as raw material in the present application has the advantages of low solvent usage and low energy consumption; the solvent usage and recovery energy consumption for extracting 1g of raw material are much less than those of other citrus raw materials and corresponding extraction and purification methods. For example, the total amount of solvent in Comparative Example 1 (165028.47 mL) is 6 times that of Example 2, and the energy consumption for recovering solvent (13053.65 J) is 10.3 times that of Example 2.

[0055] Comparative Example 2

[0056] Preparation of grapefruit fruit inner peel powder: Take a 1 mm thick inner epidermis layer of Guanxi honey pomelo, place it in a forced air drying oven, dry it at 37° C., and grind it through a 40-mesh sieve to obtain the grapefruit fruit inner epidermis powder for later use.

[0057] Extraction: The grapefruit fruit inner peel powder is extracted with petroleum ether in a solid-liquid ratio of 1:3. Take 140g of grapefruit fruit inner peel powder and mix it with 420mL of petroleum ether (boiling point is 60-90℃), and perform ultrasonic-assisted extraction 3 times, 30 minutes each time, and the ultrasonic temperature is 35℃. The filtrate obtained by filtration is concentrated and dried on a rotary evaporator and weighed to obtain 0.5g of crude extract. In addition, this extraction process uses 9mL of petroleum ether for three extractions of 1g of grapefruit fruit inner peel powder. The extraction time is a total of 90min, and the recovery of petroleum ether consumes 0.2484J of energy. The crude extract was measured by high performance liquid chromatography, and the results are as follows Figure 2 As shown in D, there is no hesperidin, that is, the inner epidermis of Guanxi pomelo does not contain hesperidin compounds.

[0058] Comparative Example 2 is compared with Examples 1-3 to illustrate that the present application uses the outer peel of Guanxi pomelo as a raw material to extract hesperidin, and removes the inner peel that does not contain hesperidin, which is different from the relevant literature reports that use the whole citrus fruit or all the peel as raw materials to extract hesperidin.

[0059] Comparative Example 3

[0060] Preparation of pomelo pulp powder: weigh Guanxi pomelo pulp, place it in a forced air drying oven, dry it at 37°C, and grind it through a 40-mesh sieve to obtain pomelo pulp powder for later use.

[0061] Extraction: Extract the grapefruit pulp powder with petroleum ether at a solid-liquid ratio of 1:3. Mix 140 g of grapefruit fruit pulp powder with 420 mL of petroleum ether (boiling point of 60-90°C), perform ultrasonic-assisted extraction three times, 30 minutes each time, and the ultrasonic temperature is 35°C; filter, concentrate and dry the filtrate on a rotary evaporator and weigh to obtain 1.2 g of crude extract. In addition, the extraction process uses 9 mL of petroleum ether for three extractions of 1 g of grapefruit fruit pulp powder, and the extraction time is 90 minutes in total. The recovery of petroleum ether consumes 0.2484 J of energy. The crude extract was determined by high performance liquid chromatography, and its purity was determined by the peak area normalization method. The results are as follows Figure 2 As shown in E, the peak purity was measured to be 0.211%, that is, the purity of hesperidin in the crude extract extracted from Guanxi pomelo pulp was 0.211%.

[0062] Rapid liquid chromatography separation: the obtained crude extract was dissolved in 5 mL of petroleum ether, and separated by rapid preparative liquid chromatography, with a SNAP KP-Sil normal phase silica gel column as the stationary phase, the mass of the SNAP KP-Sil normal phase silica gel column being 30 times that of the sample to be separated, petroleum ether-ethyl acetate as the mobile phase, the loading flow rate being 15 mL / min, and gradient elution with 0%, 0-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-100% ethyl acetate solution in sequence, each gradient elution being 2 column volumes, and all eluents being collected, and the same fractions (components with an Rf value of 0.56) being combined and concentrated to dryness.

[0063] Preparative high pressure liquid chromatography purification: The hesperidin component collected by rapid liquid chromatography was concentrated by rotary evaporation and dissolved in chromatographic grade methanol at a concentration of 4 mg / mL. After passing through a 0.22 μm filter membrane, it was loaded onto an Ultimate XB-C18 column (10 mm×250 mm, 5 μm); water (A)-methanol (B) was used as the mobile phase for gradient elution. The loading conditions were: flow rate 3 mL / min, loading concentration 4 mg / mL, loading volume 300 μL; the gradient elution program was: 0-5 min 15%-45% B; 5-10 min 45%-75% B; 10-20 min 75%-90% B; 20-30 min 90%-45% B; 25-35 min 45%-15% B. The separated components within 22-23 min were collected according to the spectra collected by the chromatographic detector.

[0064] Structural identification: The hesperidin components separated by preparative high pressure liquid chromatography were concentrated and dried on a rotary evaporator for nuclear magnetic resonance analysis, such as Figure 4 As shown, the NMR data are as follows: 1H NMR (500MHz, Methanol-d4) δ7.87 (d, J = 9.5Hz, 1H), 7.53 (d, J = 8.6Hz, 1H), 7.06 (d, J = 8.7Hz, 1H), 6.24 (d, J = 9.5Hz, 1H), 3.97 (s, 3H), 3.18 (dd, J = 13.3, 4.8Hz, 1H), 3.02 (dd, J = 6.9, 4.8Hz, 1H), 2.97 (dd, J = 13.3, 6.9Hz, 1H), 1.49 (s, 3H), 1.28 (s, 3H). 13C NMR (126MHz, Methanol-d4) δ162.98, 162.18, 154.41, 146.03, 129.08, 114.54, 114.37, 113.32, 109.01, 64.53, 61.06, 56.77, 24.87, 23.26, 19.32. Further mass spectrometry analysis, such as Figure 5 As shown, the results show that the m / z of the characteristic ion fragment is 261[M+H]+, and the relative molecular weight is determined to be 260. Through comparative analysis, it can be seen that its NMR data and molecular weight are consistent with those of the hesperidone standard. Therefore, the sample is determined to be hesperidone.

[0065] Purity determination: The target component peak was determined by high performance liquid chromatography, such as Figure 6 As shown, the peak purity was measured to be 96.47%.

[0066] As shown in Table 1, in this comparative example, the Guanxi honey pomelo pulp is subjected to ultrasonic-assisted extraction of petroleum ether, rapid liquid chromatography and preparative high pressure liquid chromatography separation and purification. It is calculated that 57339.45 g of Guanxi honey pomelo pulp powder, 67087.16 mL of ethyl acetate, 665137.61 mL of petroleum ether, and 888188.07 mL of 79% methanol are required to prepare 1 g of hesperidin, a total of 1620412.84 mL of organic reagents, and a total of 74524.08 J of energy is consumed for recovery, with a purity of 96.47%.

[0067] Comparative Example 3 compared with Examples 1-3 shows that when using grapefruit pulp as a raw material to prepare 1 g of hesperidin, the amount of raw materials used (57339.45 g), the amount of solvent used (1620412.84 mL), and the energy consumption for solvent recovery (74524.08 J) are much greater than the corresponding values ​​of using grapefruit fruit outer peel to prepare hesperidin.

[0068] Comparative Example 4

[0069] Preparation of pomelo fruit outer skin powder: Take a 1 mm thick outer skin layer of Guanxi pomelo, place it in a forced air drying oven, dry it at 37°C, and grind it through a 40-mesh sieve to obtain the pomelo fruit outer skin powder for later use.

[0070] Extraction: The grapefruit fruit outer skin powder is extracted with ethanol at a solid-liquid ratio of 1:3. Take 140g of grapefruit fruit outer skin powder and mix it with 420mL of ethanol (boiling point 60-90°C), and perform ultrasonic-assisted extraction 3 times, 30 minutes each time, and the ultrasonic temperature is 35°C. The filtrate obtained by filtration is concentrated and dried on a rotary evaporator and weighed to obtain 1.8g of crude extract. In addition, this extraction process uses 9mL of ethanol for three extractions of 1g of grapefruit fruit outer skin powder, the extraction time is a total of 90min, and 0.3456J of energy is consumed for ethanol recovery. The crude extract was determined by high performance liquid chromatography, and its purity was determined by the peak area normalization method. The results are as follows Figure 2 As shown in Figure F, the peak purity was measured to be 3.28%, that is, the purity of hesperidin in the crude extract extracted from the outer skin of Guanxi pomelo was 3.28%.

[0071] Rapid liquid chromatography separation: the obtained crude extract was dissolved in 5 mL of petroleum ether, and separated by rapid preparative liquid chromatography, with a SNAP KP-Sil normal phase silica gel column as the stationary phase, the mass of the SNAP KP-Sil normal phase silica gel column being 30 times that of the sample to be separated, petroleum ether-ethyl acetate as the mobile phase, the loading flow rate being 15 mL / min, and gradient elution with 0%, 0-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-100% ethyl acetate solution in sequence, each gradient elution being 2 column volumes, and all eluents being collected, and the same fractions (components with an Rf value of 0.56) being combined and concentrated to dryness.

[0072] Preparative high pressure liquid chromatography purification: The hesperidin component collected by rapid liquid chromatography was concentrated by rotary evaporation and dissolved in chromatographic grade methanol at a concentration of 4 mg / mL. After passing through a 0.22 μm filter membrane, it was loaded onto an Ultimate XB-C18 column (10 mm×250 mm, 5 μm); water (A)-methanol (B) was used as the mobile phase for gradient elution. The loading conditions were: flow rate 3 mL / min, loading concentration 4 mg / mL, loading volume 300 μL; the gradient elution program was: 0-5 min 15%-45% B; 5-10 min 45%-75% B; 10-20 min 75%-90% B; 20-30 min 90%-45% B; 25-35 min 45%-15% B. The separated components within 22-23 min were collected according to the spectra collected by the chromatographic detector.

[0073] Structural identification: The hesperidin components separated by preparative high pressure liquid chromatography were concentrated and dried on a rotary evaporator for nuclear magnetic resonance analysis, such as Figure 4As shown, the NMR data are as follows: 1H NMR (500MHz, Methanol-d4) δ7.87 (d, J = 9.5Hz, 1H), 7.53 (d, J = 8.6Hz, 1H), 7.06 (d, J = 8.7Hz, 1H), 6.24 (d, J = 9.5Hz, 1H), 3.97 (s, 3H), 3.18 (dd, J = 13.3, 4.8Hz, 1H), 3.02 (dd, J = 6.9, 4.8Hz, 1H), 2.97 (dd, J = 13.3, 6.9Hz, 1H), 1.49 (s, 3H), 1.28 (s, 3H). 13C NMR (126MHz, Methanol-d4) δ162.98, 162.18, 154.41, 146.03, 129.08, 114.54, 114.37, 113.32, 109.01, 64.53, 61.06, 56.77, 24.87, 23.26, 19.32. Further mass spectrometry analysis, such as Figure 5 As shown, the results show that the m / z of the characteristic ion fragment is 261[M+H]+, and the relative molecular weight is determined to be 260. Through comparative analysis, it can be seen that its NMR data and molecular weight are consistent with those of the hesperidone standard. Therefore, the sample is determined to be hesperidone.

[0074] Purity determination: The target component peak was determined by high performance liquid chromatography, such as Figure 6 As shown, the peak purity was measured to be 96.47%.

[0075] As shown in Table 1, in this comparative example, the outer skin of Guanxi pomelo was subjected to ethanol ultrasonic-assisted extraction, rapid liquid chromatography and preparative high pressure liquid chromatography separation and purification. It was calculated that 2458.03 g of Guanxi pomelo fruit outer skin powder, 2875.89 mL of ethyl acetate, 6390.88 mL of petroleum ether, 38074.87 mL of 79% methanol, and 22122.26 mL of ethanol were required to prepare 1 g of hesperidin, a total of 69463.90 mL of organic reagents, and a total of 3433.62 J of energy was consumed for recovery, with a purity of 96.47%.

[0076] Comparison of Comparative Example 4 and Examples 1-3 shows that, compared with the traditional method of extracting with 70% ethanol, extracting the outer skin of grapefruit with petroleum ether as solvent can significantly reduce the amount of raw materials, the amount of preparation solvent and the energy consumption of solvent recovery. For example, in Comparative Example 4, ethanol is used as solvent for extraction, and 1g of hesperidin is prepared by rapid liquid chromatography separation and preparative liquid chromatography purification. The required Guanxi honey grapefruit fruit outer skin powder is 2458.03g, the organic solvent is 69463.90mL, and the energy consumption of solvent recovery is 3433.62J, which are 2.53 times, 2.53 times and 2.72 times of the raw material amount (972.15g), the organic solvent amount (27472.90mL), and the energy consumption of solvent recovery (1263.50J) of Example 2, respectively.

[0077] Table 1 Comparison of separation and purification steps, raw material mass required per gram of hesperidin, total solvent usage and total energy consumption of the embodiment and the comparative example

[0078]

[0079] In summary, according to the embodiments of the present invention, it is possible to directly separate and purify hesperidin from the outer skin of Guanxi pomelo, and the obtained compound has high purity and few impurities; the extraction solvent is easy to recover, consumes little energy, requires less solvent, takes a short time, and is simple to operate.

[0080] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily being directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0081] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for efficiently preparing hesperidin, characterized in that: The following steps are involved: (1) mixing the outer skin powder of grapefruit fruit with petroleum ether, performing ultrasonic-assisted extraction, filtering, concentrating and drying to obtain a crude extract; (2) dissolving the crude extract in petroleum ether, loading the mixture onto a SNAP KP-Sil normal phase silica gel column at a flow rate of 15 mL / min, separating the mixture by normal phase rapid liquid chromatography using petroleum ether-ethyl acetate as the mobile phase, identifying the separated hesperidin components by thin layer chromatography, and combining the same components; (3) loading the components collected in step (2) onto a reverse phase preparative high pressure liquid chromatography column, performing reverse phase preparative high pressure liquid chromatography purification, and identifying the purified hesperidin component by thin layer chromatography; (4) The components collected in step (3) are structurally identified by nuclear magnetic resonance and mass spectrometry, and their purity is determined by high performance liquid chromatography.

2. The method according to claim 1, characterized in that In step (1), the pomelo fruit outer skin powder and the petroleum ether are mixed in a solid-liquid ratio of 1:3, and ultrasonic-assisted extraction is performed three times, each time for 30 minutes, and the ultrasonic temperature is 35°C.

3. The method according to claim 1, characterized in that In step (2), gradient elution is performed with petroleum ether-ethyl acetate solution in different proportions, and the concentration gradient of ethyl acetate during the gradient elution of petroleum ether-ethyl acetate solution is 0%, 0-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-100%, each gradient elution is 2 column volumes, and all the eluents are collected.

4. The method according to claim 1, characterized in that In step (2), the fractions with an Rf value of 0.56 were combined.

5. The method according to claim 1, characterized in that In step (3), the chromatographic column is an Ultimate XB-C18 chromatographic column; water (A)-methanol (B) is used as the mobile phase for gradient elution; the gradient elution program is: 0-5min 15%-45% B; 5-10min 45%-75% B; 10-20min 75%-90% B; 20-30min 90%-45% B; 25-35min 45%-15% B; the sample flow rate is 3mL / min, and the sample concentration is 4mg / mL; and the separated components within 22-23min are collected according to the spectrum collected by the chromatographic detector.

6. The method according to claim 1, characterized in that In step (2) and step (3), identification by thin layer chromatography is: using the hesperidin standard as a control, performing TLC qualitative analysis, and the component with the same ratio transfer value as the standard is the separated hesperidin component.

7. The method according to claim 1, characterized in that In step (1), the pomelo is Guanxi honey pomelo, and the boiling point of petroleum ether is 60-90°C.

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