Method for extracting polymethoxylated flavonoids from immature bitter oranges

Abstract

The invention discloses a method for extracting polymethoxylated flavonoids from immature bitter oranges, and belongs to the technical field of extraction and separation of traditional Chinese medicines. According to the method, the extraction efficiency of the flavonoid compounds is improved through cellulase and pectinase enzymolysis and cell wall destroying, and then the polymethoxylated flavone extract is obtained through extraction and multiple column chromatography. The fructus aurantii immaturus polymethoxylated flavone extract shows good anti-human lung cancer cell invasion and migration activity in vitro, and has a potential application prospect in the aspect of preparing medicines for treating lung cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction and separation of traditional Chinese medicines, and particularly relates to a method for extracting polymethoxyflavonoids from Citrus aurantium immaturus. Background Art

[0002] Fructus Aurantii Immaturus is a plant of the Rutaceae family. Citrus aurantium L. and its cultivars or sweet orange Citrus sinensis The dried young fruit of Osbeck tastes bitter, pungent, and sour, and is non-toxic. It has the effects of breaking up qi and eliminating stagnation, resolving phlegm, and dispersing lumps, and can directly resolve solid knots and toxic evils. This effect is particularly important for tumor treatment. Because Chinese medicine believes that the formation of tumors is often related to pathological factors such as qi stagnation and blood stasis, and phlegm turbidity and internal retention. In the occurrence and development of cancer, toxic knots are the most important pathological mechanism. Therefore, in the classical prescriptions, Zhishi and its similar prescriptions mostly have the effect of removing the toxicity of solid knots, and Zhishi is widely used in traditional Chinese medicine prescriptions for treating tumors.

[0003] Citrus aurantium mainly contains volatile oils, alkaloids, flavonoids and other ingredients. Its main flavonoids are hesperidin, neohesperidin, naringin, rutin, etc. Although these ingredients are present in high levels in Citrus aurantium, their activity is far less than that of polymethoxyflavonoids. Polymethoxyflavonoids are a class of flavonoid components that contain multiple methoxy groups, are low in polarity, and have strong biological activity; the content of such components in Citrus aurantium is very low, such as sweet orange flavonoids, nobiletin, tangeretin, 5-demethylnobiletin, etc., among which the content of a single polymethoxyflavonoid in Citrus aurantium is often about one thousandth. Based on this, it is of great significance to develop a method for rapid enrichment of polymethoxyflavonoids in Citrus aurantium. Summary of the invention

[0004] In view of the problems raised by the background technology, the purpose of the present invention is to provide a method for extracting polymethoxyflavonoids from Citrus aurantium. The present invention uses cellulase and pectinase enzymolysis to destroy the cell wall, improve the extraction efficiency of flavonoid compounds, and then obtains a polymethoxyflavonoid extract through extraction and multiple column chromatography. The polymethoxyflavonoid extract from Citrus aurantium exhibits good anti-invasion and anti-migration activity of human lung cancer cells in vitro, and has potential application prospects in the preparation of drugs for treating lung cancer.

[0005] The present invention specifically adopts the following technical solutions: The present invention provides a method for extracting polymethoxyflavonoids from Citrus aurantium, comprising the following steps: Step 1: crush the immature bitter orange, soak it in water, adjust the pH value and add cellulase and pectinase, keep warm and then add ethanol to reflux for extraction, filter to obtain a thick paste; Step 2: dissolving the obtained thick paste in water, and extracting with a water-saturated n-butanol solution to obtain an n-butanol extract; Step 3: dissolving the obtained n-butanol extract in water, passing it through polyamide column chromatography, eluting it to obtain an eluate, and drying it to obtain an extract; Step 4: Take the extract obtained in step 3 and mix it with silica gel, load it onto a column, elute and recover the solvent, and obtain the polymethoxyflavonoids extract of Citrus aurantium.

[0006] Furthermore, in step 1, the solid-liquid ratio of Citrus aurantium and water is 1:2, and the soaking time is 2 h.

[0007] Furthermore, in step 1, the pH value is adjusted to a range of 4-5.

[0008] Furthermore, in step 1, the added amounts of cellulase and pectinase are 0.5% and 1% of the weight of the bitter orange, respectively.

[0009] Furthermore, in step 3, the polyamide column chromatography is eluted with 10 column volumes of water, 5 column volumes of 20% ethanol, and 5 column volumes of 60% ethanol in sequence, and the eluate with 60% ethanol is collected.

[0010] Furthermore, in step 4, the column is filled with silica gel 10 times the amount of the sample loaded; eluted with 5 times the volume of dichloromethane:methanol=10:1, dichloromethane:methanol=5:1, and dichloromethane:methanol=1:1 in sequence, and the eluate with dichloromethane:methanol=5:1 is collected.

[0011] The beneficial effects of the present invention are: The invention can rapidly enrich the polymethoxyflavonoid components with extremely low content in the immature bitter orange, and the obtained extract has strong biological activity, exhibits good anti-invasion and anti-migration activity of human lung cancer cells, and has potential application prospects in the preparation of drugs for treating lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the chromatogram of the mixed reference substance under the chromatographic conditions described in Example 2.

[0013] Figure 2 This is a typical chromatogram of the n-butanol extract obtained in step 2 of Example 1.

[0014] Figure 3 This is the chromatogram of the mixed reference substance under the chromatographic conditions described in Example 3.

[0015] Figure 4 This is a typical chromatogram of the polymethoxyflavonoids extract of Citrus aurantium obtained in step 4 of Example 1.

[0016] Figure 5 This is a typical chromatogram of the extract test sample obtained in Comparative Example 7.

[0017] Figure 6 is the inhibition rate of Citrus aurantium polymethoxyflavonoids and Citrus aurantium total flavonoids on lung cancer cell proliferation.

[0018] Figure 7 The results of in vitro anti-tumor migration experiments in different groups.

[0019] Figure 8 Comparison of the non-migrated cell areas (n=3).

[0020] Fig. 9 The results of in vitro anti-tumor invasion experiments in different groups.

[0021] Fig.10 Comparison of non-invaded cell areas (n=3). DETAILED DESCRIPTION

[0022] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0024] The Citrus aurantium used in the experiment of the present invention was collected from Zhangshu, Jiangxi Province, with batch numbers 20220530, 20230604, and 20230610.

[0025] Example 1 A method for extracting polymethoxyflavonoids from Citrus aurantium immaturus, comprising the following steps: 1. Take 10 kg of Citrus aurantium and crush it into coarse powder; add water at a solid-liquid ratio of 1:2 and soak for 2 hours, adjust the pH value to 4.5, then add 50 g and 100 g of cellulase and pectinase respectively, and mix well; after keeping warm at 45℃ for 2 hours, add 5 times the volume of 75% ethanol solution, heat and reflux to extract twice, each time for 30 minutes, filter and combine the extracts, and reduce the pressure of the extracts to recover ethanol to make an alcohol-free thick paste.

[0026] 2. Dissolve the obtained alcohol-free thick paste in water, extract it three times by shaking with an equal amount of water-saturated n-butanol solution, combine the n-butanol extracts, recover the n-butanol solution under reduced pressure, and obtain the n-butanol extract.

[0027] 3. Dissolve the obtained n-butanol extract in water, and chromatograph on a polyamide column (80-120 mesh). Elute with 10 column volumes of water, 5 column volumes of 20% ethanol solution, and 5 column volumes of 60% ethanol solution in sequence. Collect the 60% ethanol eluate, and concentrate and dry it under reduced pressure.

[0028] 4. Purification by silica gel column chromatography; the extract obtained in step 3 was mixed with 1-2 times silica gel and loaded onto the column, wet-packed, the column was filled with silica gel 10 times the amount of the sample, and eluted with 5 times the column volume of dichloromethane:methanol=10:1, dichloromethane:methanol=5:1, and dichloromethane:methanol=1:1, respectively, and the eluate with dichloromethane:methanol=5:1 was collected, the solvent was recovered by reduced pressure, and dried to obtain the polymethoxyflavonoids extract of Citrus aurantium.

[0029] Three batches of Citrus aurantium were used to make a group of examples respectively; the comparative examples all used Citrus aurantium with batch number 20230610.

[0030] Comparative Example 1 Refer to the step parameters in Example 1, except that step 1 is adjusted to: Take 10 kg of Citrus aurantium and crush it into coarse powder; add 5 times the volume of 75% ethanol solution, heat and reflux to extract 3 times, each time for 30 minutes, filter and combine the extracts, and reduce the pressure of the extracts to recover ethanol to make an alcohol-free thick paste.

[0031] Comparative Example 2 The step parameters are as in Example 1, except that only 50 g of cellulase is added after adjusting the pH in step 1, and no pectinase is added.

[0032] Comparative Example 3 The step parameters are as in Example 1, except that only 100 g of pectinase is added after adjusting the pH in step 1, and no cellulase is added.

[0033] Comparative Example 4 The step parameters are as in Example 1, except that 100 g of cellulase and 50 g of pectinase are added after adjusting the pH in step 1.

[0034] Comparative Example 5 Refer to the step parameters in Example 1, except that the pH value in step 1 is adjusted to 3.5.

[0035] Comparative Example 6 Refer to the step parameters in Example 1, except that the pH value in step 1 is adjusted to 5.5.

[0036] Comparative Example 7 Refer to the step parameters in Example 1, except that step 4 is not performed.

[0037] Example 2 Liquid Phase Separation of n-Butanol Extract in Step 2 Instrument: Thermo SCIENTIFIC DIONEX UltiMate 3000 Liquid Chromatograph Reference substances: sweet orange flavonoids (batch number: AFBL0902, purity 98%) were purchased from Chengdu Aifa Biotechnology Co., Ltd.; 5-demethylnobiletin (batch number: wkq21052610, purity 98%) was purchased from Sichuan Weikeqi Biotechnology Co., Ltd.; nobiletin (batch number: 112055-202102, purity 99.7%) and tangeretin (batch number: 112054-202102, purity 99.7%) were purchased from China Food and Drug Administration The chromatographic column was Waters Symmetry C18 (4.6 mm×250 mm, 5 μm), the mobile phase was acetonitrile (A)-0.1% formic acid aqueous solution (B), the flow rate was 1.0 mL / min, the column temperature was 40°C, the injection volume was 5 μL, and the detection wavelength was 330 nm; gradient elution program: 0-40 min, 6%→50% A; 40-50 min, 50%→80% A; 50-60 min, 80% A.

[0038] Preparation of reference solution: Take appropriate amount of 5-demethylnobiletin reference, nobiletin reference, tangeretin reference, and sweet orange flavonoids reference, accurately weigh, and add methanol to prepare a mixed reference solution containing 55 μg of 5-demethylnobiletin, 70 μg of nobiletin, 60 μg of tangeretin, and 40 μg of sweet orange flavonoids per 1 mL.

[0039] Preparation of the test solution: Take about 100 mg of the n-butanol extract obtained in step 2 of Example 1, place it in a stoppered conical flask, accurately add 50 mL of methanol, weigh the weight, ultrasonically treat (power: 250 W; frequency: 25 kHz) for 30 minutes, cool, weigh again, make up the lost weight with methanol, shake well, and obtain.

[0040] The chromatograms of the mixed reference substance and the test substance with n-butanol extract are shown in Figure 2. Figure 1 and Figure 2 shown.

[0041] Example 3 Determination of the content of polymethoxyflavonoids extracts from Citrus aurantium in the examples and comparative examples Instruments: Thermo SCIENTIFIC DIONEX UltiMate 3000 liquid chromatograph; Sartorius Secura 125-1CN electronic balance (Sartorius Scientific Instruments Beijing Co., Ltd.); Milli-Q Direct 16 pure water system (Millipore, USA); KQ-500E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.). Sweet orange flavonoids (batch number: AFBL0902, purity 98%) were purchased from Chengdu Aifa Biotechnology Co., Ltd.; 5-demethylnobiletin (batch number: wkq21052610, purity 98%) was purchased from Sichuan Weikeqi Biotechnology Co., Ltd.; nobiletin (batch number: 112055-202102, purity 99.7%) and tangeretin (batch number: 112054-202102, purity 99.7%) were purchased from China Food and Drug Administration.

[0042] Chromatographic conditions: The chromatographic column was waters SunFire (C18 4.6×250 mm, 5 μm), acetonitrile-0.1% formic acid (50:50) was used as the mobile phase, the column temperature was 30°C, the detection wavelength was 330 nm, and the injection volume was 10 μL.

[0043] Preparation of reference solution: Take appropriate amount of 5-demethylnobiletin reference, nobiletin reference, tangeretin reference, and sweet orange flavonoids reference, accurately weigh them, and add methanol to make a mixed reference solution containing 15 μg of 5-demethylnobiletin, 80 μg of nobiletin, 60 μg of tangeretin, and 10 μg of sweet orange flavonoids per 1 mL.

[0044] Preparation of the test solution: Take about 10 mg of the polymethoxyflavonoids of Citrus aurantium obtained in step 4 of Example 1 and the extract obtained in Comparative Example 7, place them in a stoppered conical flask, accurately add 50 mL of methanol, weigh the weight, ultrasonically treat (power: 250 W; frequency: 25 kHz) for 30 minutes, cool, weigh again, make up the lost weight with methanol, shake well, and obtain.

[0045] The content of polymethoxylated flavonoids in the extract of Citrus aurantium was determined by using four polymethoxylated flavonoids reference substances, namely, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin. The content percentages of the four polymethoxylated flavonoids in the extract of Citrus aurantium were calculated in three batches of test samples. The results are shown in Figure 3 , 4 , 5 and Table 1.

[0046] Table 1 Results of determination of polymethoxylated flavonoids extracted from Citrus aurantium

[0047] After preparing three batches of polymethoxyflavonoids extract samples of Citrus aurantium in the examples, the total proportions of 5-demethylnobiletin, nobiletin, tangeretin, and sweet orange flavonoids in the polymethoxyflavonoids extract were 90.81%, 88.99%, and 87.37%, respectively. Taking into account the differences in approval documents, the proportion limit of 5-demethylnobiletin, nobiletin, tangeretin, and sweet orange flavonoids in the total polymethoxyflavonoids extract in the extract should not be less than 85%; the content of sweet orange flavonoids ranges from 5% to 15%, nobiletin 25% to 45%, tangeretin 35% to 45%, and 5-demethylnobiletin 5% to 10%.

[0048] The extract obtained in Comparative Example 1 has no significant difference in the proportion of the four polymethoxyflavonoid compounds in the total extract and that of Sample 3 (Batch No. 20230610), but the yield is quite different; the extraction of Citrus aurantium with 75% ethanol after enzymolysis with cellulase and pectinase is 6 g more than that with direct extraction with 75% ethanol; at the same time, the number of extractions in the embodiment is also one less than that in Comparative Example 1, which saves solvent and reduces energy consumption; the above advantages indicate that after the addition of cellulase and pectinase, the cell wall of Citrus aurantium can be destroyed, so that its polymethoxyflavonoids are released, and the extraction efficiency of polymethoxyflavonoids is increased. Comparative Examples 2 and 3 respectively add cellulase and pectinase enzymolysis separately, which is higher than the direct extraction efficiency with 75% ethanol, but far less than the efficiency of the embodiment adding cellulase and pectinase enzymolysis at the same time. Considering that Citrus aurantium contains both cellulose and a large amount of pectin, the use of a single enzyme is not as effective as adding two enzymes at the same time in destroying the cell wall of Citrus aurantium, and cellulase and pectinase play a synergistic role in the enzymolysis process. Comparative Example 4 adds cellulase and pectinase at a ratio of 1% and 0.5%, and compared with the example, the extraction rate is also affected by adding at a ratio of 0.5% and 1%. Considering that Fructus Aurantii Immaturus is a fruit-based medicinal material, pectin is relatively large in it, and increasing the amount of pectinase is beneficial to the extraction of flavonoid compounds. Therefore, the amount of pectinase added is preferably larger than the amount of cellulase added. Comparative Examples 5 and 6 adjust the pH value of the enzymolysis solution to 3.5 and 5.5 respectively. When the pH of the enzymolysis solution is 3.5, there is no significant difference in the extraction yield with directly adding 75% ethanol. It is speculated that when the pH value is 3.5, it exceeds the optimal pH value of cellulase and pectinase, resulting in enzymolysis failure; when the pH is 5.5, although the extraction yield is improved compared with the example, it is still not as good as the extraction efficiency when the pH is 4.5. Therefore, the optimal enzymolysis pH value is 4.5. Comparative Example 7 omits the silica gel column chromatography process. Although the extraction efficiency is high and the total amount of polymethoxyflavonoids extract is more than doubled, the purity is reduced by at least half. The reduced purity will directly affect the efficacy of the polymethoxyflavonoids extract.

[0049] Example 4 Activity test of polymethoxyflavonoids extract from Citrus aurantium 1 Materials and methods 1.1 Cell lines A549 (human lung cancer cells) were purchased from Wuhan Prosai Life Science Technology Co., Ltd.

[0050] 1.2 Experimental samples and reagents 1640 complete culture medium, CCK-8 cell assay kit, recombinant human epidermal growth factor (EGF), Oris™ Universal Cell Migration Assembly Kit, Oris™ Collagen I CellInvasion Assay, Batimastat (BB-94); Citrus aurantium total flavonoids extract was extracted from the laboratory, and its main components include non-polymethoxylated flavonoids such as naringin, hesperidin, neohesperidin, and naringin, and the content of polymethoxylated flavonoids is less than 20%.

[0051] 1.3 Experimental instruments CO2 constant temperature incubator, microplate reader, cell counter, fluorescence inverted microscope 2 Inhibitory effect of polymethoxylated flavonoids extract from Citrus aurantium on lung cancer cells 2.1 Experimental Methods The polymethoxyflavonoids extract of Citrus aurantium was placed in a sterile plastic tube, and a certain amount of DMSO was added to dissolve it into a mother solution. In a clean bench, the polymethoxyflavonoids extract of Citrus aurantium and the total flavonoids extract of Citrus aurantium were diluted to 1500 μg / mL, 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.2 μg / mL and 15.6 μg / mL (the final concentration of DMSO did not exceed 5‰) with 1640 complete medium.

[0052] Take A549 cells in the logarithmic growth phase, digest them, and fully blow them into a single cell suspension. After counting, dilute them into 1×10 4 / mL, inoculated in a 96-well culture plate, 100 μL of cell suspension was added to each well, and the cells were cultured in a 37°C, 5% CO2 incubator for 24 hours, and the original culture medium was discarded. 100 μL of culture medium containing the above-mentioned concentration gradient of polymethoxyflavonoids extract of Citrus aurantium and total flavonoids extract of Citrus aurantium was added to the test wells, and 6 wells were parallel for each concentration; an equal volume of solvent was added to the control group. After the 96-well culture plate was cultured in a 37°C, 5% CO2 incubator for 72 hours, the drug in each well was aspirated, and 100 μL of 1640 complete culture medium and 10 μL of CCK-8 solution were added to each well. After continuing to culture for 2 hours, the absorbance was measured at a wavelength of 450 nm according to the instructions of the CCK-8 kit. Calculation of tumor cell inhibition rate (%) = (1-determination value of experimental well / determination value of control well) × 100%. The IC50 value of polymethoxyflavonoids extract of Citrus aurantium was calculated by SPSS software.

[0053] 2.2 Inhibitory effect of polymethoxylated flavonoids extract from Citrus aurantium on lung cancer cell proliferation The results showed that the polymethoxylated flavonoids extract of Citrus aurantium had an inhibitory effect on the proliferation of lung cancer cells A549, with an IC50 of 98.07 μg / mL. The total flavonoids extract of Citrus aurantium also had an inhibitory effect on the proliferation of lung cancer cells A549, with an IC50 of 132.77 μg / mL. Figure 6 At concentrations of 15.6 μg / mL and 31.2 μg / mL, the two extracts had no significant effect on the cell proliferation of lung cancer A549 cells (the inhibition rates were both below 30%), so 15.6 μg / mL and 31.2 μg / mL were selected as low and high doses for subsequent experiments.

[0054] 3 In vitro anti-tumor migration experiment of polymethoxylated flavonoids extract from Citrus aurantium 3.1 Experimental Grouping The experimental settings included EGF group (25 ng / mL), EGF + low-dose Citrus aurantium polymethoxyflavonoids extract (15.6 μg / mL), EGF + high-dose Citrus aurantium polymethoxyflavonoids extract (31.2 μg / mL), EGF + low-dose Citrus aurantium total flavonoids extract (15.6μg / mL), EGF + high-dose Citrus aurantium total flavonoids extract (31.2 μg / mL), blank group (without EGF), and BB-94 positive drug group, with 3 replicate wells in each group.

[0055] 3.2 Experimental methods Take out the oris-compatible 96-well plate, insert the matching stoppers vertically, check the stoppers to ensure that they are completely vertically inserted into the corresponding wells and tightly sealed at the bottom of the wells, and then put them into the 37°C incubator to preheat for 1 hour.

[0056] Collect cells in the logarithmic growth phase to prepare cell suspension and adjust the cell density to 3×10 5 Take 100 μL of the cells adjusted above and add them to the preheated oris-compatible 96-well plate containing stoppers. The final cell concentration is 3×10 4 / well, after adding, gently shake the 96-well plate horizontally to evenly distribute the cell suspension, and place it in a 5% CO2, 37°C cell culture incubator for culture. After the cells adhere to the wall, remove the stoppers vertically, carefully aspirate the culture medium, and wash twice with PBS. Add different concentrations of drug-containing culture medium and culture for 72 h. After 72 h, remove the culture plate, wash once with PBS, add 100 μL of methanol to each well for 10 min, stain with 0.5% crystal violet for 30 min, take pictures under the same magnification of an inverted microscope, use Image J software to calculate the area of ​​cells that have not migrated, and use SPSS software to statistically compare the area.

[0057] 3.3 Experimental Results The results of the cell migration experiment show that the number of A549 cells that migrated to the central blank circle after 72 hours of EGF treatment was significantly greater than that of the blank group without EGF. After incubation with the polymethoxylated flavonoids extract of Citrus aurantium and the total flavonoids extract for 72 hours, the migration of A549 cells to the central blank circle was significantly prevented, and both the low-dose and high-dose groups showed a dose-dependent relationship, and the polymethoxylated flavonoids extract of Citrus aurantium was more effective than the total flavonoids extract (P < 0.05) (see Figure 7 ; Figure 8 : **, extremely significant difference compared with EGF group, P<0.01; #, significant difference compared with high / low dose groups of total flavonoids from Citrus aurantium, P<0.05).

[0058] 4 In vitro anti-tumor invasion experiment of polymethoxylated flavonoids extract from Citrus aurantium 4.1 Experimental Grouping The experimental settings included EGF group (25 ng / mL), EGF + low-dose Citrus aurantium polymethoxyflavonoids extract (15.6 μg / mL), EGF + high-dose Citrus aurantium polymethoxyflavonoids extract (31.2 μg / mL), EGF + low-dose Citrus aurantium total flavonoids extract (15.6μg / mL), EGF + high-dose Citrus aurantium total flavonoids extract (31.2 μg / mL), blank group (without EGF), and BB-94 positive drug group, with 3 replicate wells in each group.

[0059] 4.2 Experimental methods Collect cells in logarithmic phase to prepare cell suspension and adjust the cell density to 3×10 5Take 100 μL of the cells adjusted above and add them to a 4°C pre-cooled oris-compatible 96-well plate containing stoppers and coated with collagen I. The final cell concentration is 3×10 4 / well, after adding, gently shake the 96-well plate horizontally to evenly distribute the cell suspension, and place it in a 5% CO2, 37℃ cell culture incubator for culture. After the cells adhere to the wall, remove the stoppers vertically, carefully aspirate the culture medium, and wash twice with PBS. Take the diluted Oris™ Pro Collagen I Overlay, add 40 μL to each well, put the 96-well plate into a 37℃ cell culture incubator for 1 h, take out the 96-well plate after the Collagen I gel solidifies, add different concentrations of drug-containing culture medium, and culture for 72 h. After 72 h, take out the culture plate, wash once with PBS, add 100 μL of methanol to each well for 10 minutes, stain with 0.5% crystal violet for 30 minutes, take pictures at the same magnification of an inverted microscope, use Image J software to calculate the non-invasion area of ​​the cells, and use SPSS software to statistically compare the area.

[0060] 4.3 Experimental Results The results of the cell invasion experiment show that the number of A549 cells that invaded the central blank circle after 72 hours of EGF treatment was significantly greater than that of the blank group without EGF. After incubation with high-dose total flavonoids extract and Fructus Aurantii Immaturus for 72 hours, the invasion of A549 cells to the central blank circle was significantly prevented, and the effect of Fructus Aurantii Immaturus polymethoxyflavonoids extract was stronger than that of total flavonoids extract (P < 0.05) (see Fig. 9 ; Fig.10 : **, extremely significant difference compared with EGF group, P<0.01; #, significant difference compared with high / low dose group of Citrus aurantium total flavonoids, P<0.05; ##, significant difference compared with high dose group of Citrus aurantium total flavonoids, P<0.01).

[0061] The embodiments described above only express several preferred embodiments of the present invention, and the descriptions thereof are relatively specific and detailed, but are not intended to limit the present invention. It should be noted that for those skilled in the art, the present invention may also have various changes and modifications, and any modifications, equivalent substitutions, improvements, etc. made within the concept and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for extracting polymethoxyflavonoids from Citrus aurantium, characterized in that: The steps include: Step 1: crush the immature bitter orange, soak it in water, adjust the pH value and add cellulase and pectinase, keep warm and then add ethanol to reflux for extraction, filter to obtain a thick paste; Step 2: dissolving the obtained thick paste in water, and extracting with a water-saturated n-butanol solution to obtain an n-butanol extract; Step 3: dissolving the obtained n-butanol extract in water, passing it through polyamide column chromatography, eluting it to obtain an eluate, and drying it to obtain an extract; Step 4: Take the extract obtained in step 3 and mix it with silica gel, load it onto a column, elute and recover the solvent, and obtain the polymethoxyflavonoids extract of Citrus aurantium.

2. The method for extracting polymethoxyflavonoids from Citrus aurantium according to claim 1, characterized in that: Step 1: The solid-liquid ratio of Citrus aurantium and water is 1:2, and the soaking time is 2 h.

3. The method for extracting polymethoxyflavonoids from Citrus aurantium according to claim 1, characterized in that: In step 1, the pH value is adjusted to a range of 4-5.

4. The method for extracting polymethoxyflavonoids from Citrus aurantium according to claim 1, characterized in that: In step 1, the added amounts of cellulase and pectinase are 0.5% and 1% of the weight of the bitter orange, respectively.

5. The method for extracting polymethoxyflavonoids from Citrus aurantium according to claim 1, characterized in that: In step 3, the polyamide column chromatography is eluted with 10 column volumes of water, 5 column volumes of 20% ethanol, and 5 column volumes of 60% ethanol in sequence, and the eluate with 60% ethanol is collected.

6. The method for extracting polymethoxyflavonoids from Citrus aurantium according to claim 1, characterized in that: Step 4: Fill the column with silica gel 10 times the amount of the sample loaded; elute with 5 volumes of dichloromethane:methanol = 10:1, dichloromethane:methanol = 5:1, and dichloromethane:methanol = 1:1 in sequence, and collect the eluate with dichloromethane:methanol = 5:1.

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