Application of immature bitter orange extract in preparation of antioxidant and anti-inflammatory drugs

Abstract

The invention provides application of an immature bitter orange extract in preparation of anti-oxidation and anti-inflammatory drugs. The fructus aurantii immaturus is extracted by using different solvents, and the extract is researched, so that the fructus aurantii immaturus extract has the capability of removing superoxide anion free radicals, ABTS free radicals and DPPH free radicals; in addition, the immature bitter orange extract can also reduce the content of nitric oxide, tumor necrosis factor-alpha, interleukin-1beta and interleukin-6 released by LPS-induced macrophages. The fructus aurantii immaturus extract has anti-oxidation and anti-inflammatory effects, and is used as an anti-oxidation and anti-inflammatory drug to develop a new application of an old drug of the fructus aurantii immaturus extract.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceuticals, and particularly to the application of extracts from Fructus Aurantii Immaturus in the preparation of antioxidant and anti-inflammatory drugs. Background Art

[0002] Fructus Aurantii Immaturus is a qi-regulating herb in traditional Chinese medicine. It is the dried immature fruit of Citrus aurantium L. and its cultivated varieties or sweet orange, Rutaceae, and has the effects of promoting qi circulation to relieve stagnation, resolving phlegm and dispelling mass. In the era of precision medicine and under the new guidance of "new uses for old drugs", it is very necessary to improve the extraction method of Fructus Aurantii Immaturus, further study the extracted active substances, and explore their new applications. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of extracts from Fructus Aurantii Immaturus in the preparation of antioxidant and anti-inflammatory drugs.

[0004] Specifically, the present invention adopts the following technical solutions:

[0005] The present invention provides the application of extracts from Fructus Aurantii Immaturus in the preparation of antioxidant and anti-inflammatory drugs.

[0006] Furthermore, the extracts from Fructus Aurantii Immaturus include active ingredients with different polarities of Fructus Aurantii Immaturus; the preparation method of the active ingredients with different polarities of Fructus Aurantii Immaturus includes: drying Fructus Aurantii Immaturus and grinding it into powder, passing through a 50-mesh sieve to obtain a sample; weighing 5 portions of the sample, respectively mixing 1 portion of the sample with 70% ethanol, pure water, ethyl acetate, n-butanol, and n-hexane as solvents according to a solid-liquid ratio of 1:10, using an ultrasonic cleaner to perform ultrasonic extraction at a frequency of 53 kHz, and filtering to obtain extraction solutions with different polar solvents; concentrating the extraction solutions with different polar solvents under reduced pressure and drying to constant weight to obtain extraction pastes with different solvents, namely the active ingredients with different polarities of Fructus Aurantii Immaturus.

[0007] Furthermore, the active ingredients with different polarities of Fructus Aurantii Immaturus are used to scavenge superoxide anion radicals; and within the concentration range of 0.0125 - 0.4 mg / mL, the ability of the active ingredients with different polarities of Fructus Aurantii Immaturus to scavenge superoxide anion radicals increases with the increase in concentration.

[0008] Furthermore, the active ingredients with different polarities of Fructus Aurantii Immaturus are used to scavenge ABTS radicals; and within the concentration range of 0.0125 - 0.1 mg / mL, the ability of the active ingredients with different polarities of Fructus Aurantii Immaturus to scavenge ABTS radicals increases with the increase in concentration.

[0009] Furthermore, the active ingredients with different polarities of Fructus Aurantii Immaturus are used to scavenge DPPH radicals; and within the concentration range of 0.05 - 0.5 mg / mL, the ability of the active ingredients with different polarities of Fructus Aurantii Immaturus to scavenge DPPH radicals increases with the increase in concentration.

[0010] Furthermore, the Fructus Aurantii Immaturus extract is obtained by extracting Fructus Aurantii Immaturus with 70% ethanol or pure water as the extraction solvent.

[0011] Furthermore, the Fructus Aurantii Immaturus extract is used to reduce the contents of nitric oxide, tumor necrosis factor-α, interleukin-1β and interleukin-6 released by macrophages induced by LPS.

[0012] Furthermore, the Fructus Aurantii Immaturus extract includes total flavonoids of Fructus Aurantii Immaturus; the extraction method of the total flavonoids of Fructus Aurantii Immaturus includes: using ultrasonic extraction method, using ethanol with a concentration of 63.01% as the extraction solvent, the material-liquid ratio is 1:31.91, the extraction temperature is 41.88 °C, and the extraction time is 29.71 min.

[0013] Furthermore, the average extraction rate of the total flavonoids in the extraction method of the total flavonoids of Fructus Aurantii Immaturus is 2.912%.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects:

[0015] The present invention provides the application of Fructus Aurantii Immaturus extract in the preparation of antioxidant and anti-inflammatory drugs. By extracting Fructus Aurantii Immaturus with different solvents and studying the extracts, it is found that the Fructus Aurantii Immaturus extract has the ability to scavenge superoxide anion radicals, ABTS radicals and DPPH radicals; and, the Fructus Aurantii Immaturus extract can also reduce the contents of nitric oxide, tumor necrosis factor-α, interleukin-1β and interleukin-6 released by macrophages induced by LPS. That is, the Fructus Aurantii Immaturus extract has antioxidant and anti-inflammatory effects, and its new use of old drug new use has been developed by using it as an antioxidant and anti-inflammatory drug. Description of the Drawings

[0016] Figure 1 For the in vitro antioxidant activity and total flavonoid content of different extracts;

[0017] Figure 2 For the effects of different solvent extracts on the viability of RAW cells;

[0018] Figure 3 For the effects of different extracts of Fructus Aurantii Immaturus on the release of NO by RAW264.7 cells; Note: #### P<0.0001 compared with Control; **P<0.01, ***P<0.001, ****P<0.0001 compared with Model.;

[0019] Figure 4 For the effects of different extracts of Fructus Aurantii Immaturus on reducing the release of TNF-α by RAW264.7 cells; Note: ####P < 0.0001 compared with Control; **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with Model.;

[0020] Figure 5 Effect of different extracts of Fructus Aurantii Immaturus on reducing the release of IL-6 by RAW264.7 cells; Note: #### P < 0.0001 compared with Control; **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with Model.;

[0021] Figure 6 Effect of different extracts of Fructus Aurantii Immaturus on reducing the release of IL-1β by RAW264.7 cells; Note: #### P < 0.0001 compared with Control; **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with Model.;

[0022] Figure 7 Effect of different extracts of Fructus Aurantii Immaturus on intracellular ROS in RAW264.7 cells; Note: #### P < 0.0001 compared with Control; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with Model.;

[0023] Figure 8 Single-factor experiment on total flavonoids of Fructus Aurantii Immaturus

[0024] Figure 9 Response surface 3D diagrams of various factors of total flavonoids of Fructus Aurantii Immaturus Detailed implementation manners

[0025] The present invention will be described below in conjunction with specific embodiments.

[0026] I. Preparation of different polar active components of Fructus Aurantii Immaturus and screening of in vitro antioxidant activities

[0027] 1 Experimental materials

[0028] Immature bitter orange powder, from Dazhou, Sichuan; absolute ethanol, ethyl acetate, n-butanol, n-hexane, sodium nitrite, aluminum nitrate, sodium hydroxide, from Shanghai Chemical Reagent Co., Ltd., Sinopharm Group; 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), nicotinamide adenine dinucleotide (NADH), nitroblue tetrazolium (NBT), phenazine methosulfate (PMS), rutin standard, ascorbic acid, from Shanghai Macklin Biochemical Co., Ltd.

[0029] 2 Experimental instruments

[0030] HH-W420 constant temperature water bath, from Baita Xinbao Instrument Factory, Jintan; BSA124S electronic analytical balance, from Beijing Sartorius Instrument System Co., Ltd.; ultra-micro ultraviolet spectrophotometer, from Thermo Fisher Scientific; electrothermal constant temperature forced air drying oven, from Shanghai Senxin Experimental Instrument Co., Ltd.; ultrasonic cleaner, from Shanghai Kedao Ultrasonic Instrument Co., Ltd.

[0031] 3 Experimental methods

[0032] 3.1 Preparation of active components with different polarities from immature bitter orange

[0033] The dried immature bitter orange was ground into powder, passed through a 50-mesh sieve. Five samples were accurately weighed and mixed with 70% ethanol, pure water, ethyl acetate, n-butanol, and n-hexane as solvents at a solid-liquid ratio of 1:10. Ultrasonic extraction was carried out using an ultrasonic cleaner at a frequency of 53 kHz. The extracts with different polar solvents were obtained by suction filtration, concentrated under reduced pressure, and dried to constant weight in an oven to obtain extracts with different solvents. The extracts were dissolved in 60% ethanol to prepare the sample solutions for subsequent antioxidant experiments and stored at 4°C.

[0034] 3.2 Study on in vitro antioxidant activities of active components with different polarities from immature bitter orange

[0035] 3.2.1 Determination of superoxide anion scavenging ability

[0036] Relevant reagents were accurately weighed on an electronic balance to prepare 2.52 mmol / L NBT, 624 μmol / L NADH, and 120 μmol / L PMS solutions. 1 mL of the sample solution, 1 mL of NBT, 1 mL of NADH, and 1 mL of PMS were mixed evenly and incubated in a water bath at 25°C for 5 min. The absorbance was measured at 560 nm using an ultraviolet spectrophotometer as Ac. The sample solution was replaced with pure water, and the absorbance was measured under the same conditions as As. The superoxide anion scavenging rate was determined according to formula (1).

[0037] Superoxide anion scavenging rate / % = (As - Ac) / As * 100% (1)

[0038] 3.2.2 ABTS radical scavenging ability

[0039] Accurately weigh ABTS and dissolve it in 2.45 mmol / L potassium persulfate solution to make the ABTS concentration reach 7 mmol / L. Let it stand in the dark for 16 h, and dilute it with PBS buffer solution to make the absorbance value around 0.70 when detected at 734 nm, with a positive and negative error of 0.02. This value is Ac. Mix 1 mL of ABTS dilution and 1 mL of the sample evenly, let it stand in the dark for 10 min, measure the absorbance As at 734 nm, and calculate the ABTS radical scavenging ability of the sample according to formula (2).

[0040] ABTS radical scavenging rate / % = (Ac - As) / Ac * 100% (2)

[0041] 3.2.3 DPPH radical scavenging ability

[0042] Take 1 mL of the sample solution, 250 μL of 0.02% DPPH and 1 mL of ethanol, mix them evenly, and after reacting in the dark for 30 min, measure the absorbance A at 517 nm i , measure the absorbance of 1 mL of water, 250 μL of 0.02% DPPH and 1 mL of ethanol as A0 in the same way, and measure the absorbance of 1 mL of the sample and 1.25 mL of ethanol as A j , calculate the scavenging rate of the sample solution to DPPH radicals according to formula (3). The experiment uses vitamin C as a control and repeats three times.

[0043] DPPH radical scavenging rate / % = (A0 + A j - A i ) / A0 * 100% (3)

[0044] 3.2.4 Determination of total flavonoid content in extracts with different solvents

[0045] Determination is carried out by the sodium nitrite - aluminum nitrate method. Prepare a rutin standard solution of 0.2 mg / mL. Respectively take 9, 8, 7, 6, 5, 4, 3, 2, 1 mL of the standard solution, make up the volume to 10 mL with 60% ethanol. Take 1 mL of the diluted standard solution, add 0.4 mL of 5% sodium nitrite solution, shake well and let it stand for 6 min, add 0.4 mL of 10% aluminum nitrate solution, shake well and let it stand for 6 min, add 2 mL of 4% sodium hydroxide, make up the volume to 10 mL with 60% ethanol, let it stand for 10 min and then measure the absorbance at 510 nm to establish a standard curve. Measure the OD value of the sample solution in the same way and then calculate the total flavonoid content.

[0046] 4 Experimental results

[0047] 4.1 Extraction rates of different active components in Fructus Aurantii Immaturus

[0048] Table 1 Extraction rates of different active components in Fructus Aurantii Immaturus

[0049]

[0050]

[0051] As can be seen from Table 1, the weight of the extracts with different polar solvents is the highest for the 70% ethanol extract, which is 7.61 g; followed by the pure water extract with a weight of 6.32 g; and the weight of n-hexane is the lowest, which is 0.37 g. This indicates that highly polar compounds are the main components of Fructus Aurantii Immaturus, and the content of non-polar compounds is relatively low.

[0052] 4.2 Study on the in vitro antioxidant activities of different polar components of Fructus Aurantii Immaturus

[0053] The results of detecting the superoxide anion scavenging activity through the NADH-PMS-NBT system are as Figure 1 shown. As can be seen from the figure, there is a dose-effect relationship between the scavenging ability of different solvent extracts of Fructus Aurantii Immaturus on superoxide anion radicals. In the concentration range of 0.0125 - 0.4 mg / mL, as the concentration of the extract increases, the scavenging ability on superoxide anion radicals increases accordingly. The order of the scavenging ability of different solvent extracts on superoxide anion radicals from large to small is: 70% ethanol > ethyl acetate > deionized water > n-butanol > n-hexane.

[0054] The ABTS radical scavenging method is a method for measuring the total antioxidant capacity of natural products. The ability of different solvent extracts of Fructus Aurantii Immaturus to scavenge ABTS radicals is as Figure 1 shown. As can be obtained from the figure, there is a dose-effect relationship between the scavenging ability of different solvent extracts of Fructus Aurantii Immaturus on ABTS radicals. In the concentration range of 0.0125 - 0.1 mg / mL, as the concentration of the extract increases, the scavenging ability on ABTS radicals increases accordingly. The scavenging rates of the extracts of 70% ethanol and ethyl acetate are similar. The order of the scavenging ability of different solvent extracts on ABTS radicals from large to small is: 70% ethanol > ethyl acetate > deionized water > n-butanol > n-hexane.

[0055] DPPH radicals are widely used to evaluate the antioxidant activities of antioxidants. The higher the DPPH radical scavenging rate, the stronger the ability of the test substance to interrupt the lipid peroxidation chain reaction. The ability of different solvent extracts of Fructus Aurantii Immaturus to scavenge DPPH radicals is as Figure 1 shown. There is a dose-effect relationship between the scavenging ability of different solvent extracts of Fructus Aurantii Immaturus on DPPH radicals. In the concentration range of 0.05 - 0.5 mg / mL, as the concentration of the extract increases, the DPPH radical scavenging ability increases accordingly, but the change in the DPPH radical scavenging ability of the n-hexane extract is not significant. As Figure 1 can be seen, the order of the scavenging ability of different solvent extracts on DPPH radicals from large to small is: 70% ethanol > ethyl acetate > deionized water > n-butanol > n-hexane.

[0056] The total flavonoid content in different polar solvent extracts of Fructus Aurantii Immaturus is asFigure 1 As shown. It can be seen from the figure that different solvents have varying degrees of influence on the extraction effect of total flavonoids. The order of total flavonoid content from high to low is: 70% ethanol > ethyl acetate > deionized water > n-butanol > n-hexane. The total flavonoid content in the 70% ethanol extract is the highest, and the total flavonoid content in the n-hexane extract is the lowest. There is no significant difference in the total flavonoid content between the n-butanol and n-hexane extracts. This difference may be due to the structural characteristics of flavonoids affecting the dissolution rate of flavonoids in different solvents. Table 2 shows the antioxidant activity Ec 50 value.

[0057] Table 2 Antioxidant activity EC of different extracts of Fructus Aurantii Immaturus 50 value

[0058]

[0059]

[0060] II. Determination of anti-inflammatory activity of different extracts of Fructus Aurantii Immaturus

[0061] 1 Experimental materials

[0062] DMEM high-glucose medium, FBS fetal bovine serum, penicillin / streptomycin, Wuhan Punosai Biotechnology Co., Ltd.; lipopolysaccharide (LPS), nitric oxide (NO) detection kits: Shanghai Beyotime Biotechnology Co., Ltd. Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) enzyme-linked immunosorbent assay kits: Hangzhou Leyi Biotechnology Co., Ltd.; ROS kit, Nanjing Jiancheng Bioengineering Institute.

[0063] 2 Experimental instruments

[0064] BSA124S electronic analytical balance, Beijing Sartorius Instrument System Co., Ltd.; TI-S inverted fluorescence biological microscope, Nikon Corporation; cell incubator, -80°C refrigerator, MultiskanGo microplate reader, Thermo Fisher Scientific; TGL-16G high-speed centrifuge, Shanghai Anting Scientific Instrument Co., Ltd.

[0065] 3 Experimental methods

[0066] 3.1 Cytotoxicity experiment

[0067] Resuscitate RAW264.7 cells in a culture flask and culture them in a 37°C, 5% CO2 incubator. When the cell growth density reaches about 80%, passage the cells. Gently pipette the adherent cells down. Collect cells in the logarithmic growth phase and adjust the cell number to 5×10 4Cells were inoculated at a density of 2.5×10 cells / mL into 96-well plates, 200 μL per well. After incubation for 24 hours, the supernatant was discarded. A control group and a drug administration group were set up. The control group was added with DMEM medium containing 0.5% FBS, and the drug administration group was added with DMEM medium containing extracts at different concentrations (25, 50, 100, 200, 400 μg / mL), 200 μL per well. After incubation for 24 h, the supernatant was discarded, and then 100 μL of DMEM medium containing 10% CCK-8 was added to each well. After incubation at 37 °C for 1 h, the absorbance was measured at a wavelength of 450 nm using a microplate reader.

[0068] Cell survival rate % = OD 给药组 / OD 对照组 ×100%

[0069] 3.2 Determination of the effect of Fructus Aurantii Immaturus extract on the release of NO from RAW cells

[0070] RAW264.7 cells were selected, and cells in the logarithmic growth phase with good growth status were collected. The cell number was adjusted to 2.5×10 5 cells / mL and inoculated into 96-well plates, 200 μL per well. After incubation for 24 hours, the supernatant was discarded. The LPS-induced group was added with 200 μL of DMEM medium containing LPS (1 μg / mL), the control group was added with ordinary DMEM medium, and the sample groups were added with DMEM medium containing LPS (1 μg / mL) and samples at different concentrations (25, 50, 100 μg / mL), 200 μL per well. After incubation for 24 h, 50 μL of the supernatant was taken, and the operation was carried out according to the NO kit. The OD value was measured at 540 nm using a microplate reader and calculated.

[0071] 3.3 Effect of Fructus Aurantii Immaturus extract on the release of inflammatory factors from RAW cells

[0072] RAW264.7 cells were selected, and cells in the logarithmic growth phase with good growth status were collected. The cell number was adjusted to 2.5×10 5 cells / mL and inoculated into 96-well plates, 200 μL per well. After incubation for 24 hours, the supernatant was discarded. The LPS-induced group was added with 200 μL of DMEM medium containing LPS (1 μg / mL), the control group was added with ordinary DMEM medium, and the sample groups were added with DMEM medium containing LPS (1 μg / mL) and samples at different concentrations (25, 50, 100 μg / mL), 200 μL per well. After incubation for 24 h, the supernatant was taken, and the contents of TNF-α, IL-1β, and IL-6 were measured according to the respective kits.

[0073] 3.4 Effect of Fructus Aurantii Immaturus extract on intracellular ROS in RAW cells

[0074] RAW264.7 cells were selected, and cells in the logarithmic growth phase with good growth status were collected. The cell number was adjusted to 2.5×10 5 cells / mL and inoculated into 24-well plates at 500 μL per well. After incubation for 24 hours, the supernatant was discarded. In the LPS-induced group, 500 μL of DMEM medium containing 0.5% serum and LPS (1 μg / mL) was added. In the control group, 0.5% serum DMEM medium was added. In the sample groups, 0.5% serum DMEM medium containing LPS (1 μg / mL) and samples at different concentrations (25, 50, 100 μg / mL) was added. After incubation at 37°C for 24 hours, the medium was discarded. Each well was washed twice with PBS. Under light-shielded conditions, a DCFH-DA staining solution with a concentration of 10 μmol / L was prepared with DMEM medium. 500 μL was added to each well and incubated for 60 min in the dark. Subsequently, it was rinsed three times with PBS to thoroughly remove the DCFH-DA staining solution that did not enter the cells. The fluorescence intensity was observed and photographed under a fluorescence microscope.

[0075] 4 Experimental results

[0076] 4.1 Cytotoxicity experiment

[0077] It can be seen from Figure 2 that as the concentration increased, the different polar extracts of Fructus Aurantii Immaturus gradually had a significant inhibitory effect on cell viability. Among them, the cells treated with ethyl acetate, n-butanol, and n-hexane extracts had significant differences in viability compared with the control group in the concentration range of 25-400 μg / mL. The 70% ethanol and pure water extracts had no significant effect on cell viability in the concentration range of 25-100 μg / mL. Therefore, 70% ethanol extract and pure water extract were selected for subsequent inflammatory activity determination experiments, and 25, 50, and 100 μg / mL were set as the experimental concentrations.

[0078] 4.2 Determination of the effect of Fructus Aurantii Immaturus extract on NO release from RAW cells

[0079] It can be seen from Figure 3It can be seen that NO is involved in the regulation of various physiological functions, is closely related to inflammation, and plays an important role in the process of the acute inflammation model of macrophages induced by LPS. NO is produced by catalyzing L-arginine by nitric oxide synthase (iNOS). Excessive NO will make the inflammatory response more persistent and intense. Therefore, the content of NO is one of the important indicators for measuring inflammatory lesions. As can be seen from the figure, after RAW264.7 cells are stimulated by LPS, the released NO content is significantly higher than that of the control group, which is a sign of inflammation in RAW264.7 cells. Within a certain concentration range, after the 70% ethanol and pure water extracts of Fructus Aurantii Immaturus act on the inflammatory RAW264.7 cells, the amount of NO generated decreases with the increase in the extract concentration, showing a certain dose-dependence. It can be preliminarily judged that the extracts have a certain anti-inflammatory effect. Each extract shows a certain anti-inflammatory effect at low concentrations, but the inhibitory effects are different. When the concentration is 25 μg / mL, the NO release amount in the 70% ethanol extract treatment group decreases by 16.7% compared with the LPS-induced group, while the pure water extract treatment group decreases by 13.3% compared with the LPS group, indicating that the anti-inflammatory activity of the 70% ethanol extract is better than that of the pure water extract at this concentration.

[0080] 4.3 Effects of Fructus Aurantii Immaturus Extracts on the Release Amount of Inflammatory Factors in RAW264.7 Cells

[0081] It can be Figure 4 seen that the TNF-α secretion in the cell supernatant of the control group is less. After being stimulated with 1 μg / mL LPS for 24 h, the secretion amount reaches 1549 pg / mL, indicating that the inflammation model is successfully established. Compared with the LPS treatment group, as the extract concentration increases, the TNF-α secreted by the treatment group decreases significantly, and there is a certain dose-dependence. When the concentration is 100 μg / mL, the TNF-α content in the 70% ethanol extract treatment group decreases by 753.97 pg / mL compared with the LPS-induced group, and the pure water extract treatment group decreases by 390.36 pg / mL. According to the experimental results, it can be speculated that Fructus Aurantii Immaturus extracts can inhibit the occurrence of the inflammatory response of LPS-induced RAW264.7 cells by reducing the release of TNF-α, and the anti-inflammatory effect of the 70% ethanol extract is better than that of the pure water extract.

[0082] It can be Figure 5It can be seen that the ethanol extract and pure water extract of Citrus aurantium can inhibit the IL-6 secretion of RAW264.7 cells stimulated by LPS in a concentration-dependent manner. Compared with the blank group, the IL-6 release of cells in the LPS-induced group increased significantly, reaching 118.3pg / mL. Under the action of the two extracts, the amount of IL-6 decreased significantly. When the intervention concentration reached 100μg / mL, the 70% ethanol extract reduced the secretion to 54.8pg / mL, while the pure water extract could reduce it to 73.5pg / mL, indicating that both extracts can reduce the damage to cells caused by inflammatory response by inhibiting the secretion of IL-6 by cells, and the ethanol extract is better than the pure water extract.

[0083] Depend on Figure 6 It can be seen that when the body produces an inflammatory response, it will promote cell inflammation by secreting TNF-α, IL-6, and IL-1β, thereby leading to cell proliferation or tissue degeneration. Compared with the blank control group, the amount of IL-1β secreted by the cells in the model group after LPS stimulation reached 149.6pg / mL, which was significantly different, proving that the model was successfully established. Compared with the model group, the intervention groups of Citrus aurantium extract showed an inhibitory effect on IL-1β secretion. The ethanol extract had a certain inhibitory effect at concentrations of 25, 50, and 100μg / mL, and the inhibition rate reached 47.5% at 100μg / mL; the pure water extract had an inhibitory effect at 50μg / mL and 100μg / mL, and 28.9% at 100μg / mL, and the inhibitory effect of both was concentration-dependent.

[0084] Depend on Figure 7 It can be seen that reactive oxygen species (ROS) are a class of highly reactive oxidative molecules that can cause oxidative stress and cell damage when accumulated excessively. The DCFH-DA staining method can observe and calculate the changes in ROS content in LPS-induced RAW264.7 cells induced by different concentrations of Citrus aurantium extract. Figure 6 It can be seen that compared with the control group, the cells in the model group showed high-intensity green fluorescence, and the average fluorescence intensity was significantly higher than that of the control group, indicating that there were more ROS in the model group and the cells were in a state of oxidative damage. After intervention with the Citrus aurantium extract, the average fluorescence intensity of the cells decreased significantly, and it had a better effect at a concentration of 100 μg / mL, indicating that the Citrus aurantium extract can reduce the production of cellular ROS, reduce the damage to cells caused by oxidative damage, and enhance the antioxidant capacity of cells.

[0085] 3. Optimization of Extraction Process of Total Flavonoids from Citrus Aurantium

[0086] 1 Experimental Materials

[0087] Citrus aurantium powder, Dazhou, Sichuan; anhydrous ethanol, sodium nitrite, aluminum nitrate, sodium hydroxide, Shanghai Sinopharm Chemical Reagent Co., Ltd.

[0088] 2 Experimental Instruments

[0089] HH-W420 constant temperature water bath, Baita Xinbao Instrument Factory, Jintan City; BSA124S electronic analytical balance, Sartorius Instrument Systems Co., Ltd., Beijing; ultra-micro ultraviolet spectrophotometer, Thermo Fisher Scientific; ultrasonic cleaner, Shanghai Kedao Ultrasonic Instrument Co., Ltd.

[0090] 3 Experimental Methods

[0091] 3.1 Single-factor Experiments

[0092] Fix the solid-liquid ratio at 1:10 g / mL, the extraction time at 30 min, and the extraction temperature at room temperature, and investigate the effects of different ethanol concentrations (50%, 60%, 70%, 80%, 90%) on the total flavonoid content.

[0093] Fix the ethanol concentration at 60%, the extraction time at 30 min, and the extraction temperature at room temperature, and investigate the effects of different solid-liquid ratios (1:10, 1:20, 1:30, 1:40, 1:50 g / mL) on the total flavonoid content.

[0094] Fix the solid-liquid ratio at 1:10 g / mL, the ethanol concentration at 60%, and the extraction time at 30 min, and investigate the effects of different temperatures (20, 30, 40, 50, 60 °C) on the total flavonoid content.

[0095] Fix the solid-liquid ratio at 1:10 g / mL, the ethanol concentration at 60%, and the extraction temperature at room temperature, and investigate the effects of different extraction times (10, 20, 30, 40, 50 min) on the total flavonoid content.

[0096] 3.2 Response Surface Experimental Design

[0097] On the basis of single-factor experiments, select the main factors affecting the total flavonoid content of Fructus Aurantii Immaturus, namely solid-liquid ratio, extraction temperature, extraction time, and ethanol concentration, as independent variables, and the total flavonoid content of Fructus Aurantii Immaturus as the response value. Using the Box-Behnken experimental principle in software Design-Expert.V8.0.6.1, design an optimization experiment with four factors and three levels. Table 3 shows the optimization experiment of the response value with the solid-liquid ratio, extraction temperature, extraction time, and ethanol concentration as independent variables.

[0098] Table 3 Optimization Experiment of Response Value with Solid-Liquid Ratio, Extraction Temperature, Extraction Time, and Ethanol Concentration as Independent Variables

[0099]

[0100] 4 Experimental Results

[0101] 4.1 Single-factor Experiments

[0102] From Figure 8 It can be seen that the total flavonoid content of Fructus Aurantii Immaturus shows a trend of first increasing and then decreasing with the increase of ethanol concentration. When the ethanol concentration is 60%, the total flavonoid content is the highest. After that, with the increase of ethanol concentration, the total flavonoid content gradually decreases. It may be that in the low concentration range of ethanol, according to the principle of "like dissolves like", the flavonoid compounds with larger polarity in Fructus Aurantii Immaturus are easily soluble in strong polar solvents. However, when the ethanol concentration continues to increase, due to the decrease of solvent polarity and the simultaneous extraction of some impurities, the total flavonoid content decreases.

[0103] The total flavonoid content of Fructus Aurantii Immaturus first increases and then slightly decreases with the increase of the solid-liquid ratio, reaching the maximum value at 1:30 g / mL. However, when the solid-liquid ratio continues to increase, the total flavonoid content decreases slightly instead. The reason may be that when the volume of the extraction solvent is small, the contact area between the solid and the liquid is small, and less total flavonoids are obtained. Therefore, the extraction rate increases with the increase of the solid-liquid ratio. Then, when the solid-liquid ratio is greater than 1:30 g / mL, too much extraction solvent will consume part of the ultrasonic energy, resulting in the weakening of the ultrasonic wave acting on the Fructus Aurantii Immaturus powder and reducing the ability of flavonoid components to dissolve, thus causing the total flavonoid content to decrease.

[0104] The extraction rate of total flavonoids from Fructus Aurantii Immaturus first increases and then decreases with the increase of temperature, reaching the highest at the extraction temperature of 40°C. The reason for the increase may be that increasing the temperature makes the molecular movement speed faster, resulting in a faster dissolution rate of flavonoids in the powder. The reason for the decrease may be that too high a temperature causes the denaturation of soluble proteins in the extract, increasing the viscosity of the system, affecting the dissolution of flavonoid substances, or some unstable flavonoids decompose due to high temperature.

[0105] With the increase of extraction time, the total flavonoid content of Fructus Aurantii Immaturus shows a linear upward trend from 10 min to 30 min, reaching the maximum at 30 min. With the extension of extraction time, the total flavonoid content shows a downward trend instead. The reason may be that when the extraction time is too short, the flavonoid components in the Fructus Aurantii Immaturus powder are not completely dissolved, and the total flavonoid content is low. After 30 min, with the increase of extraction time, other impurities are gradually dissolved, or some unstable flavonoid components are damaged due to long-term ultrasonic oscillation, resulting in a decrease in the total flavonoid content.

[0106] 4.2 Response Surface Experimental Design

[0107] Table 4 Response Surface Experiment on the Effects of Ethanol Concentration (A), Solid-Liquid Ratio (B), Temperature (C), and Extraction Time (D) on the Total Flavonoid Content

[0108]

[0109]

[0110] Table 5 Response value calculation of the effects of ethanol concentration (A), solid-liquid ratio (B), temperature (C), and extraction time (D) on the total flavonoid content

[0111]

[0112]

[0113] Combining Table 4 and Table 5, based on the experimental results obtained by the Box-Behnken response surface method, the experimental data were subjected to multiple quadratic regression using Design-Expert 8.0.6 software to obtain the total flavonoid content: Y = 232.63 + 20.84A + 13.2B + 10.25C + 4.68D + 2.03AB - 12.76AC - 14.17AD + 2.6BC + 0.04BD + 1.35BD - 28.11A 2 -27.44B 2 -20.38C 2 -13.92D 2 . Analysis of variance was performed on the regression model. The model F = 26.16, P < 0.0001, indicating that the regression model is extremely significant. The lack-of-fit term P = 0.1806 > 0.05, indicating that the model fits the data well. This regression equation can be used to replace the experimental true points to describe the relationship between each variable and the response value; R 2 = 0.9632, indicating a strong correlation; R 2 adj and R 2 pred are consistent within a reasonable range, and the difference is less than 0.4, indicating that the model can well explain the changes in the response value caused by most factor changes. This model can be used to predict the optimal extraction process conditions for total flavonoids in Fructus Aurantii Immaturus. Among the linear terms, the effects of ethanol concentration (A), solid-liquid ratio (B), and temperature (C) on the extraction amount of flavonoids in Fructus Aurantii Immaturus are extremely significant, and the effect of extraction time (D) is significant; among the quadratic terms, the effects of A 2 , B 2 , C 2 and D 2 are all extremely significant; among the interaction terms, the effects of AB, BC, BD, and CD on the extraction amount of flavonoids are not significant, and the effects of AC and AD are significant. Referring to the magnitude of the F value, the conclusion can be drawn that the influence degree of the four factors on the extraction of total flavonoids in Fructus Aurantii Immaturus is ethanol concentration (A) > solid-liquid ratio (B) > extraction temperature (C) > extraction time (D).

[0114] Figure 9To generate the response surface plot and contour plot of the interaction between different factors pairwise using software, which can more intuitively reflect the influence trend and variation range of the interaction of each factor on the total flavonoid content in Fructus Aurantii Immaturus, and at the same time keep other variables at the 0 level. The response surface and contour plot can intuitively reflect the influence degree of the interaction on the response value. The steeper the surface and the denser the contour lines, the more significant the influence. The closer the contour lines are to an ellipse, the stronger the interaction between the two factors. The results show that each surface plot opens downward, indicating that there is a maximum value for the total flavonoid content in Fructus Aurantii Immaturus. The 3D response surface plot shows that the slopes of the four factors are: A > B > C > D, indicating that the influence of the four factors on the total flavonoid content is in the order of: ethanol concentration > solid-liquid ratio > temperature > time. It can be seen from the figure that the contour lines are dense, indicating that the four factors have a significant influence on the total flavonoid content. The interaction plots of ethanol concentration (A) with extraction temperature (C) and ethanol concentration (A) and extraction time (D) show elliptical contour lines, indicating that the interactions of AC and AD are significant, while the interactions of other factors are not significant. These results are consistent with the conclusions of the variance analysis.

[0115] The theoretical optimal process parameters given by the model are ethanol concentration of 64%, solid-liquid ratio of 1:33, extraction temperature of 41°C, and extraction time of 30 min. The theoretical total flavonoid content is 238.75 mg / g. Considering the feasibility of actual operation, the theoretical parameters are adjusted to ethanol concentration of 65%, solid-liquid ratio of 1∶30, temperature of 40°C, and extraction time of 30 min. Three repeated experiments are carried out, and the average extraction rate of total flavonoids is measured to be 238.93 mg / g, which is close to the theoretical predicted value, indicating that the equation is in good agreement with the actual situation.

[0116] In summary, the polarity of different solvents has a great influence on the content of extracts and chemical components. The content of flavonoid components in Fructus Aurantii Immaturus is relatively high, accounting for about 22.45% - 33.76%. They are mainly dihydroflavonoids and flavonoids, and most of them are polymethoxyflavonoids. Flavonoids have been reported to have good antioxidant and anti-inflammatory activities. In this invention, five different polar solvents, namely n-butanol, ethyl acetate, deionized water, 70% ethanol, and n-hexane, were used to extract Fructus Aurantii Immaturus. Three in vitro antioxidant indexes, including the ability to scavenge DPPH free radicals, the ability to scavenge ABTS free radicals, and the ability to scavenge superoxide anion free radicals, were used to evaluate each extract, and the total flavonoid content was determined. The results showed that when 70% ethanol and pure water were used as solvents, the extraction rate was relatively high, indicating that the active ingredients in Fructus Aurantii Immaturus may mostly belong to high-polarity components. The level of total flavonoid content may be the main reason affecting the strength of antioxidant activity. There are research reports on the total flavonoid content and antioxidant activity of extracts from different solvents of blueberry leaves. The results showed that there were significant differences in the antioxidant activities of extracts obtained with different solvents, and there was a correlation between total flavonoids and antioxidant activity. Some scholars found that the 50% ethanol extract and pure water extract of Rubus idaeus had good in vitro antioxidant activities. The extracts of five different polar solvents in this invention showed different degrees of antioxidant activities in different antioxidant detection systems, and there was a certain dose-effect relationship. The order of antioxidant activity was 70% ethanol > ethyl acetate > water > n-butanol > n-hexane, which was basically consistent with the total flavonoid content.

[0117] Lipopolysaccharide (LPS) is a prominent component of the cell wall of Gram-negative bacteria. It mimics the early stage of the inflammatory response, and the body will secrete various inflammatory factors such as TNF-α, IL-1β, and IL-6. At the same time as the body produces an inflammatory response, it will also promote the occurrence of oxidative stress. At this time, the balance between oxidation and antioxidant effects is lost, and reactive oxygen species in the body will be produced and accumulated in large amounts in cells, and then oxidative stress will occur. Some studies have shown that the total flavonoid extract from Taraxacum mongolicum Hand.-Mazz. can significantly reduce the production of NO and the expression of iNOS mRNA in cells after LPS stimulation, and has potential anti-inflammatory activity. In this invention, LPS was used to establish an inflammation model to evaluate the antioxidant and anti-inflammatory effects of the extracts. The results showed that the 70% ethanol extract and pure water extract could significantly reduce the contents of NO, TNF-α, IL-1β, and IL-6 released by LPS-induced macrophages, and there was a certain dose-effect relationship. The fluorescence intensity of cellular ROS decreased significantly after the intervention of the extracts, indicating that the two types of extracts could relieve the inflammatory response, and the anti-inflammatory effect of the 70% ethanol extract was better than that of the pure water extract.

[0118] The ethanol solution was selected as the solvent to optimize the extraction process of total flavonoids from Fructus Aurantii Immaturus. The ultrasonic extraction method, which is widely used for flavonoid compounds, was adopted. Through single-factor and Box-Behnken response surface methods, combined with the actual situation, the optimal extraction process obtained was as follows: the ethanol concentration was 63.01%, the solid-liquid ratio was 1:31.91, the extraction temperature was 41.88 °C, the extraction time was 29.71 min, the theoretical total flavonoid content was 2.88%, and the average extraction rate of the actually measured total flavonoids was 2.912% (RSD = 0.169%). It was close to the theoretical predicted value, indicating that the equation was in good agreement with the actual situation.

[0119] Those of ordinary skill in the art can understand that the above embodiments are specific examples for implementing the present application. In actual applications, various changes can be made in form and details without departing from the spirit and scope of the present application. Any person skilled in the art can make their respective changes and modifications without departing from the spirit and scope of the present application. Therefore, the protection scope of the present application should be subject to the scope defined by the claims.

Claims

1. Use of Fructus Aurantii Immaturus extract in the preparation of antioxidant and anti-inflammatory drugs.

2. The application according to claim 1, characterized in that, The Fructus Aurantii Immaturus extract includes active ingredients with different polarities from Fructus Aurantii Immaturus; the preparation method of the active ingredients with different polarities from Fructus Aurantii Immaturus includes: drying Fructus Aurantii Immaturus and grinding it into powder, passing through a 50-mesh sieve to obtain a sample; weighing 5 portions of the sample, respectively mixing 1 portion of the sample with 70% ethanol, pure water, ethyl acetate, n-butanol, and n-hexane as solvents according to a solid-liquid ratio of 1:10, using an ultrasonic cleaner to perform ultrasonic extraction at a frequency of 53 kHz, and filtering to obtain extraction solutions with different polar solvents; concentrating the extraction solutions with different polar solvents under reduced pressure and drying to constant weight to obtain extraction pastes with different solvents, namely the active ingredients with different polarities from Fructus Aurantii Immaturus.

3. The application according to claim 2, wherein The active ingredients with different polarities from Fructus Aurantii Immaturus are used to scavenge superoxide anion radicals; and within the concentration range of 0.0125 - 0.4 mg / mL, the ability to scavenge superoxide anion radicals of the active ingredients with different polarities from Fructus Aurantii Immaturus increases with the increase in concentration.

4. The application according to claim 2, characterized in that The active ingredients with different polarities from Fructus Aurantii Immaturus are used to scavenge ABTS radicals; and within the concentration range of 0.0125 - 0.1 mg / mL, the ability to scavenge ABTS radicals of the active ingredients with different polarities from Fructus Aurantii Immaturus increases with the increase in concentration.

5. The application according to claim 2, wherein The active ingredients with different polarities from Fructus Aurantii Immaturus are used to scavenge DPPH radicals; and within the concentration range of 0.05 - 0.5 mg / mL, the ability to scavenge DPPH radicals of the active ingredients with different polarities from Fructus Aurantii Immaturus increases with the increase in concentration.

6. The application according to claim 1, wherein The Fructus Aurantii Immaturus extract is obtained by extracting Fructus Aurantii Immaturus with 70% ethanol or pure water as the extraction solvent.

7. The application according to claim 6, characterized in that, The Fructus Aurantii Immaturus extract is used to reduce the contents of nitric oxide, tumor necrosis factor-α, interleukin-1β, and interleukin-6 released by macrophages induced by LPS.

8. The application according to claim 1, characterized in that, The Fructus Aurantii Immaturus extract includes total flavonoids from Fructus Aurantii Immaturus; the extraction method of the total flavonoids from Fructus Aurantii Immaturus includes: using ultrasonic extraction method, with ethanol at a concentration of 63.01% as the extraction solvent, a solid-liquid ratio of 1:31.91, an extraction temperature of 41.88 °C, and an extraction time of 29.71 min.

9. The application according to claim 8, wherein The average extraction rate of total flavonoids in the extraction method of the total flavonoids from Fructus Aurantii Immaturus is 2.912%.

Images