Preparation method of active ingredients in fructus psoraleae and application of active ingredients in anti-inflammatory products

Abstract

The invention relates to the technical field of natural medicines, in particular to a preparation method of active ingredients in fructus psoraleae and application of the active ingredients in anti-inflammatory products, firstly, a fructus psoraleae reflux extract is properly enriched by using an extraction method, then selective separation is performed by using different separation modes of HSCCC, and the active ingredients in the fructus psoraleae are obtained. And 10 monomeric compounds are separated by combining a prepared liquid phase. Through cell experiments, four compounds of psoralen, neopsoralen isoflavone, psoralen flavanone and psoralen flavanone methyl ether, which can well inhibit NO release, are screened out; the four monomeric compounds can reduce secretion of proinflammatory factors by weakening mRNA transcriptional ability and weaken over-phosphorylation expression of pathway protein, so that an anti-inflammatory effect is achieved; through verification, the fructus psoraleae flavanone and the fructus psoraleae flavanone methyl ether can play an anti-inflammatory role through MAPK and NF-kappa B signal channels.

Description

Technical Field

[0001] The present invention relates to the technical field of natural medicines, in particular to a method for preparing an active ingredient in psoralea corylifolia and an application thereof in anti-inflammatory products. Background Art

[0002] Psoralea corylifolia L. is the dried mature fruit of the leguminous plant Psoralea corylifolia L. It has a pungent and slightly bitter taste and enters the kidney and spleen meridians. It is mainly used to treat kidney yang deficiency, cold pain in the waist and knees, and diarrhea. It can also be used externally to treat skin diseases such as vitiligo and bald spots [1]. The growing conditions of Psoralea corylifolia are relatively common, and the planting area covers many provinces in China. The low planting cost and easy planting difficulty have created conditions for the widespread and economical use of Psoralea corylifolia. Psoralea corylifolia can nourish the kidney and strengthen bones, warm the spleen and moisten dryness, promote blood circulation and replenish qi, promote qi and relieve asthma, and can also be used to treat diseases caused by wind evil. The innovation and continued use of new and old prescriptions with Psoralea corylifolia as an important ingredient, such as Qing'e Pills, Taohong Xiaobai Decoction, Bushen Huoxue Decoction, Bushen Huatan Recipe, Bufei Huoxue Capsules, and Xianling Gubao Capsules, have to some extent demonstrated the pharmacological activity and potential medicinal value of Psoralea corylifolia. The specific chemical components of Psoralea corylifolia deserve further study.

[0003] Related research has shown that psoralea corylifolia contains chemical components such as coumarins, flavonoids, terpenes, glycosides, lipids, and volatile oils, and possesses pharmacological activities such as treating osteoporosis, providing anti-inflammatory, antioxidant, and anti-tumor effects, and acting as an estrogen analog. Due to structural differences, different types of compounds, or even compounds of the same type, exhibit significant differences in physical and chemical properties. Using one or a combination of extraction and separation methods to extract and separate multiple compounds, based on their specific characteristics, ultimately yielding a variety of monomers is of great significance for subsequent research.

[0004] Liquid-liquid partition chromatography (LPC) is a basic chromatographic separation method commonly used for separating natural products. Its key difference from solid-phase and liquid chromatography is the lack of a solid stationary phase, effectively ensuring the utilization of the separated substances during the separation process. Extraction is the most basic separation method based on LLP. A stable two-phase solution, composed of at least two different solvents, is essential for successful extraction. The properties of the two-phase solution directly influence the separation efficiency. High-speed countercurrent chromatography (HSC) is a highly efficient LPC technique that can effectively separate monomeric components of traditional Chinese medicines (TCMs). Further research on the separation of TCM monomers is warranted. Inflammation is a symptom and potential cause of numerous diseases. Different types of inflammation may develop synergistically, and abnormal inflammation can affect normal life activities. The side effects and emergence of drug resistance to traditional anti-inflammatory drugs highlight the importance of identifying novel anti-inflammatory agents.

[0005] In this study, Psoralea corylifolia was isolated and purified to obtain monomeric compounds, and the anti-inflammatory effects of the related compounds were verified in vitro, providing a reference for the further development and utilization of the chemical components in Psoralea corylifolia. Summary of the Invention

[0006] In response to the above problems, the present invention provides a method for preparing the active ingredients in Psoralea corylifolia and its application in anti-inflammatory products. Psoralea corylifolia is extracted and its chemical components are separated and purified, and the anti-inflammatory effects of each component are evaluated, providing a reference for the separation and purification of monomeric compounds in Psoralea corylifolia and further development of pharmacological research.

[0007] The technical solution adopted by the present invention is: a method for preparing the active ingredient in Psoralea corylifolia, characterized in that the isolated compound comprises compound 1-10 represented by the following formula: The method comprises the following steps: S1: After the Psoralea corylifolia was crushed, it was reflux extracted with 95% ethanol at a solid-liquid ratio of 1:10 to obtain a crude product; S2: Extract the crude product according to target compounds 1-10, and select a two-phase solvent system, wherein the first two-phase solvent system has a volume ratio of n-hexane / ethyl acetate / methanol / water of 5-6:5-6:4-5:5-6, and the second two-phase solvent system has a volume ratio of n-hexane / ethyl acetate / methanol / water of 5-6:5-6:7-8:3-6; S3: Conventional HSCCC separation, using the first two-phase solvent system to separate the extracted sample. After the peak of the mixture of compounds 2 and 3 ends, the mobile phase of the separation system is immediately replaced with the lower phase of the first two-phase solvent system, and the separation is continued until completion; S4: using a second two-phase solvent system to separate the mixture of compounds 7, 9 and 8; S5: In preparative liquid chromatography, compounds 5 and 6 were separated using 52% acetonitrile-48% 0.1% formic acid in water as the mobile phase elution conditions.

[0008] As an optimization, in steps S3 and S4, compounds 2 and 3, and compounds 7 and 9 were separated using IRCCC mode: Using the first two-phase solvent system, the flow rate was set at 5-7 mL / min to circulate and separate compounds 2 and 3; Using the second two-phase solvent system, the flow rate was set to 2 mL / min. Compounds 7, 9, and 8 were first subjected to conventional high-speed countercurrent separation. When the mixed components of compounds 7 and 9 were about to be detected by the UV detector for the first time, the six-way valve was adjusted to switch to the circulation mode, allowing the eluent to flow back to the separation pipeline for subsequent circulation separation. When the mixed components of compounds 7 and 9 were detected by the UV detector for the first time, the six-way valve was adjusted to switch to conventional HSCCC separation. After compound 8 was collected, the valve was switched back to the circulation mode until the separation was completed.

[0009] As an optimization, in step S1, reflux extraction was performed three times, with extraction times of 2 h, 2 h, and 1.5 h, respectively. After filtration, the extracts were combined and concentrated.

[0010] As an optimization, in step S2, 10 g of the crude product after extraction was weighed, and 500-550 mL of the upper phase and lower phase of the first two-phase solvent system were used for the first set of extractions. The lower phase of each extraction was retained in the separatory funnel, and a new upper phase was added for the next extraction. The upper phase in each extraction was transferred and collected, and extracted 3-4 times in total. The upper phase collected in the first set of extractions was evaporated to dryness, and the second set of extractions was continued using 500-550 mL of the upper phase and lower phase of the second two-phase solvent system. The upper phase of each extraction was retained in the separatory funnel, and a new lower phase was added for the next extraction. The lower phase in each extraction was transferred and collected, and extracted 9-10 times in total.

[0011] As an optimization, in step S3, the prepared sample solution is injected into the pipeline through the six-way injection valve, and the flow rate is opened for separation after the injection. After the injection, the sample solution passes through the internal pipeline of the countercurrent chromatograph and the ultraviolet detector in sequence and is collected in the centrifuge tube of the fraction collector.

[0012] As an optimization, in the IRCCC mode, before the component that needs to be circulated is about to be eluted, the eluate is pumped back into the countercurrent chromatograph pipeline through an external six-way valve via a constant flow pump for the next separation. After the successful separation of each component is observed through the UV detection spectrum, the six-way valve mode is switched to end the circulation separation mode, allowing the eluate to flow out, and the eluate is collected and subsequent operations are performed according to the conventional HSCCC method.

[0013] As an optimization, in step S3, the liquid chromatography detection conditions are as follows: the chromatographic column is Agilent Extend-C18, the mobile phase is acetonitrile and 0.1% formic acid in water, the flow rate is 1.0-1.5 mL / min, the injection volume is 10-12 μL, the column temperature is 20-25 °C, and the detector wavelength is 240-246 nm.

[0014] The anti-inflammatory components were extracted using a method for preparing active components from Psoralea corylifolia. (1) The effects of the extracted and separated components on the number of RAW264.7 cells were investigated. Psoralea corylifolia isoflavones and isopsoralea corylifolia chalcone could reduce NO release. (2) Effects on cell growth morphology: cells treated with psoralen, neopsoralen isoflavones, psoralen dihydroflavone, and psoralen dihydroflavone methyl ether showed relatively uniform cell growth and an increased proportion of round cells, indicating that the intervention of each compound had a certain alleviating effect on cell inflammation; (3) Effect on RNA expression content, reducing the expression levels of IL-6, IL-1β, TNF-α and TNF-α mRNA to varying degrees, playing a role in alleviating inflammation; (4) The effect on the expression of related proteins, exerting potential anti-inflammatory ability in the NF-κB or p38 signaling pathway.

[0015] In addition, the present invention also proposes a method for preparing active ingredients in Psoralea corylifolia and the use of the extracted compounds in the preparation of anti-inflammatory products.

[0016] The beneficial effects of the present invention are as follows: the method for preparing the active ingredients in Psoralea corylifolia provided by the present invention first uses an extraction method to appropriately enrich the Psoralea corylifolia reflux extract, then uses different separation modes of HSCCC for selective separation, and combines with the preparation of liquid phase to complete the separation of 10 monomer compounds. Through cell experiments, four compounds that can effectively inhibit NO release were screened out: psoralen, neopsoralen isoflavones, psoralen dihydroflavone, and psoralen dihydroflavone methyl ether; the four monomer compounds can reduce the secretion of inflammatory factors and reduce the excessive phosphorylation expression of pathway proteins by weakening mRNA transcription ability, thereby exerting anti-inflammatory effects; it has been verified that psoralen dihydroflavone and psoralen dihydroflavone methyl ether may exert anti-inflammatory effects through the MAPK and NF-κB signaling pathways. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 HPLC detection chart of the first set of extractions: A: HPLC detection chart of the lower phase of the first extraction with a solvent system of n-hexane: ethyl acetate: methanol: water = 5:5:4:6 (v / v); B: HPLC detection chart of the lower phase of the third extraction with a solvent system of n-hexane: ethyl acetate: methanol: water = 5:5:4:6 (v / v); Arabic numerals represent the corresponding compounds, such as 1 represents compound 1, and so on; Figure 2 HPLC detection chart of the second set of extractions: A: HPLC detection chart of the upper phase of the first extraction with a solvent system of n-hexane: ethyl acetate: methanol: water = 5:5:7:3 (v / v); B: HPLC detection chart of the upper phase of the ninth extraction with a solvent system of n-hexane: ethyl acetate: methanol: water = 5:5:7:3 (v / v); Figure 3 HPLC test chart of Psoralea corylifolia before and after extraction; Figure 4 Effect of rotation speed on retention rate (solvent system n-hexane: ethyl acetate: methanol: water = 5:5:4.5:5.5 (v / v)); Figure 5 Effect of flow rate on retention rate: A: solvent system n-hexane: ethyl acetate: methanol: water = 5:5:4.5:5.5 (v / v); B: solvent system n-hexane: ethyl acetate: methanol: water = 5:5:7:3 (v / v); C: solvent system n-hexane: ethyl acetate: methanol: water = 5:5:5:5 (v / v); Figure 6 HSCCC separation diagram of the sample after Psoralea corylifolia extraction; Figure 7 HPLC detection chart of each component in HSCCC separation of sample after Psoralea corylifolia extraction; Figure 8 Component E HSCCC separation diagram; Figure 9 IRCCC separation diagram of component B; Figure 10 IRCCC separation diagram of component E; Figure 11 Preparative liquid chromatography detection chart of component D; Figure 12 HPLC detection chart of samples after extraction of Psoralea corylifolia and each compound; Figure 13 Schematic diagram of the structures of each compound; Figure 14 Effects of different drug concentrations on RAW264.7 cell viability: C represents the control group, the drug concentration is 0, compared with the control group, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001; Figure 15 Effects of various drugs on NO content in RAW264.7 cells induced by LPS: C represents the control group, with a drug concentration of 0; M represents the inflammatory model group induced by 1.5 μg / mL LPS; D10 and D20 represent the positive drug groups with a concentration of 10 μM and 20 μM dexamethasone sodium phosphate, respectively; compared with the control group, the model group **** p <0.0001; compared with the model group, the other experimental groups #p <0.05, ## p <0.01, ### p <0.001, #### p <0.0001; Figure 16 Schematic diagram of the growth status of RAW264.7 cells in each group: A: Normal growth of RAW264.7 cells without intervention; B: 1.5 μg / mL LPS-induced inflammatory RAW264.7 cells; C: LPS-induced cells treated with 20 μM dexamethasone sodium phosphate; D: LPS-induced cells treated with 100 μM psoralen; E: LPS-induced cells treated with 12.5 μM psoralen isoflavone; F: LPS-induced cells treated with 12.5 μM psoralen dihydroflavonoids; G: LPS-induced cells treated with 12.5 μM psoralen dihydroflavonoids methyl ether; Scale bar, 50 μm; Figure 17 mRNA transcriptional expression of inflammatory factors in RAW264.7 cells: C: Normally grown RAW264.7 cells without intervention; M: 1.5 μg / mL LPS-induced inflammatory RAW264.7 cells; D: LPS-induced cells treated with 20 μM dexamethasone sodium phosphate; 1: LPS-induced cells treated with 100 μM psoralen; 3: LPS-induced cells treated with 12.5 μM psoralen isoflavones; 4: LPS-induced cells treated with 12.5 μM psoralen dihydroflavonoids; 8: LPS-induced cells treated with 12.5 μM psoralen dihydroflavonoids methyl ether; Compared with the control group, the model group **** p <0.0001; compared with the model group, the other experimental groups # p <0.05, ## p <0.01, ### p <0.001, #### p <0.0001; Figure 18Expression of related proteins in RAW264.7 cells Ⅰ: related protein band diagram. Ⅱ: Quantification of related protein phosphorylation expression; C: normal growth RAW264.7 cells without intervention; M: 1.5 μg / mL LPS-induced inflammatory RAW264.7 cells; D: LPS-induced cells treated with 20 μM dexamethasone sodium phosphate; 1: LPS-induced cells treated with 100 μM psoralen; 3: LPS-induced cells treated with 12.5 μM new psoralea isoflavone; 4: LPS-induced cells treated with 12.5 μM psoralea corylifolia; 8: LPS-induced cells treated with 12.5 μM psoralea corylifolia methyl ether. Compared with the control group, the model group **** p <0.0001; compared with the model group, the other experimental groups ## p <0.01, ### p <0.001, #### p <0.0001. DETAILED DESCRIPTION

[0018] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0019] Psoralea corylifolia contains numerous compounds. While compounds of the same type may have similar solubility properties, compounds of different types can differ significantly, making it difficult to obtain pure monomers through a single separation. This chapter first extracts Psoralea corylifolia extracts. A method combining high-speed countercurrent chromatography (HSCCC) and preparative liquid chromatography was established to separate the Psoralea corylifolia. The separated components were analyzed by high-performance liquid chromatography (HPLC), and their chemical structures were identified by nuclear magnetic resonance and mass spectrometry. This resulted in the successful targeted separation of 10 compounds from Psoralea corylifolia.

[0020] After reflux extraction of Psoralea corylifolia, some impurities were removed using extraction methods. In a high-speed countercurrent chromatography separation, two solvent systems were first used in a single countercurrent separation to obtain three monomers (compounds 1, 4, and 10) and multiple combinations of compounds with similar polarity. Each combination of compounds was then subjected to a corresponding countercurrent separation mode, combined with preparative liquid chromatography, to ultimately obtain 10 monomers with a purity exceeding 95%.

[0021] The purpose of using extraction is to remove the most polar and least polar parts of the reflux extraction sample, reduce the content of non-target components, and appropriately increase the effective amount of injection in the subsequent HSCCC separation, thereby improving the separation efficiency. The first group of extractions (using the solvent system of n-hexane: ethyl acetate: methanol: water = 5:5:4:6 ( v / v )) ended, HPLC test results showed that the polar components (possibly coumarin glycosides) were effectively removed; in the second set of extractions (using the solvent system of n-hexane: ethyl acetate: methanol: water = 5:5:7:3 ( v / v During the process, oil droplets or small flocculent floating objects (possibly oil or insoluble impurities) can be seen floating on the upper layer of the solution. When the extracted sample is loaded for HSCCC separation, Figure 6 As shown in the figure, before the target component (component A) eluted, there was a component with a shorter retention time, which is theoretically a component with greater polarity. Based on the countercurrent chromatography results, it is speculated that when HPLC detects the components at each stage of extraction, it may not be able to effectively detect all the high-polarity and low-polarity components because the concentration is too low or the detection and analysis method is not optimal. Although the extraction did not achieve the best target effect, the target component (component A) was separated in HSCCC ( Figure 6 ) can be well separated from highly polar non-target components. Less polar components, such as lipids, present in the Psoralea corylifolia sample before and after extraction are the substances that remain longest in the stationary phase during HSCCC separations. This can affect the true solution state of the stationary phase, and emulsification can alter the solubility properties of the original solvent system, affecting the actual separation. These results demonstrate the positive effects of extraction, as demonstrated by HPLC detection and countercurrent separation during the extraction step.

[0022] When selecting the separation parameters of HSCCC, it is necessary to consider both the separation retention rate and the separation efficiency. The retention rate of the solvent system used in this method is directly proportional to the rotation speed. Under the premise of ensuring the normal operation of the countercurrent chromatograph, the centrifugal force brought by the rotation speed has a retention effect on the stationary phase in the spiral coil. Increasing the rotation speed helps to vigorously mix the two-phase solvent and thus promote distribution, which can increase the retention rate of the stationary phase. The components to be separated are fully exchanged and distributed between the two-phase solvent in the pipeline, thereby improving the separation effect; the flow rate of the mobile phase will affect the elution time. Too slow a flow rate will lead to a longer elution time and reduced separation efficiency. Too fast a flow rate will cause the stationary phase to be lost, and the two-phase solvent in the separation pipeline cannot fully perform liquid-liquid distribution, affecting the separation effect of the components in the pipeline. Temperature will affect the viscosity and interfacial tension of the solvent system, thereby affecting the retention rate of the stationary phase. Temperature will also affect the solubility of the sample. Under normal circumstances, the separation temperature is close to room temperature or slightly above room temperature.

[0023] Conventional HSCCC separations struggle to fully separate multiple components with similar or significantly different properties in a single pass. This often necessitates selecting a different separation mode or combining other separation methods tailored to the specific separation situation. For components with relatively similar properties, the IRCCC separation mode, where the mixed components of multiple target compounds re-enter the separation pipeline via a six-port valve, increases the number of separations and enables the gradual separation of different compounds. The multiple cycles of the IRCCC separation mode can theoretically be roughly understood as increasing the separation column volume, effectively separating multiple compounds within a longer column volume. Compared to simply increasing column volume, the IRCCC separation mode avoids excessive peak elongation and solvent consumption. The IRCCC separation mode is a commonly used separation mode in HSCCC separations and is undergoing continuous optimization and innovation. This method can achieve a single separation of multiple similar compounds, improving separation efficiency. The IRCCC used in this method is based on conventional HSCCC, with modifications to accommodate the separation of diverse compounds. Diversified separation modes offer more practical possibilities for complex component combinations and separations, providing more distinctive and feasible separation methods while maintaining optimal separation performance.

[0024] Example 1 1 Extraction of Psoralea corylifolia samples Weigh 500 g of salted Psoralea corylifolia, crush it, add 95% ethanol at a solid-liquid ratio of 1:10, and reflux extract three times with extraction times of 2 h, 2 h, and 1.5 h, respectively. After filtration, combine the extracts and concentrate to obtain the crude product (148 g) for later use.

[0025] 2 Extraction of crude product after extraction of Psoralea corylifolia Weigh 10 g of the crude product after extraction and use a solvent system of n-hexane: ethyl acetate: methanol: water = 5:5:4:6 ( v / v ) were used for the first set of extractions, with 500 mL of each of the upper and lower phases. The lower phase of each extraction was retained in the separatory funnel, and a new upper phase was added for the next extraction. The upper phase of each extraction was transferred and collected, and extracted three times in total to remove some non-target components with high polarity in the sample. The upper phase collected in the first set of extractions was evaporated to dryness and then the solvent system of n-hexane: ethyl acetate: methanol: water = 5:5:7:3 ( v / v) and 500 mL of the upper and lower phases were used to continue the second set of extractions. The upper phase of each extraction was retained in the separatory funnel, and a new lower phase was added for the next extraction. The lower phase of each extraction was transferred and collected. A total of 9 extractions were performed to remove some non-target components with less polarity in the sample. The lower phase collected in the second set of extractions was evaporated to dryness to obtain the crude products after the two sets of extractions for later use. The lower phase in the first set of extractions was freeze-dried, and the upper phase in the second set of extractions was evaporated to dryness for calculating the extraction efficiency. The content of the solution during the extraction process was detected by HPLC. 20 µL of the upper phase or lower phase was quantitatively transferred to a centrifuge tube. After the upper phase was evaporated to dryness, 1000 µL of methanol was added, and 980 µL of methanol was added to the lower phase sample. The filter membrane was fully dissolved before detection.

[0026] 3 Isolation of HSCCC from Psoralea corylifolia 3.1 Selection of HSCCC instrument separation parameters When selecting separation parameters, without adding sample, fix one solvent system and gradually accelerate from a low speed of 600 rpm to a high speed of 800 rpm. Compare the retention rates of the selected systems at different speeds (the proportion of the upper phase of the solvent system in the separation solenoid), and select the speed corresponding to the high retention rate as the separation speed. Fix the rotor speed at 800 rpm, change the mobile phase flow rate, compare the retention rates of different solvent systems, and select the appropriate flow rate for countercurrent separation.

[0027] 3.2 Selection of HSCCC solvent system Take 2 mg of the extracted sample in a 10 ml centrifuge tube, add 4 ml of the upper phase and lower phase of the solvent system to be tested, shake thoroughly, let it stand for stratification, take 100 μL of the upper phase and lower phase solution into different 1.5 ml centrifuge tubes, add 1 ml of methanol to dissolve the upper phase after evaporation, and add 900 μL of methanol to the lower phase to ensure full dissolution. Use HPLC to detect, the peak area occupied by the target peak in the upper phase is A 上 , the peak area occupied by the target peak in the lower phase is A 下 , the distribution coefficient K = A 上 / A 下 .

[0028] 3.3 Preparation of solvent system and sample solution Prepare the target solvent system by adding n-hexane, ethyl acetate, methanol, and water to a 2 L separatory funnel in the corresponding volume ratios. Shake thoroughly and allow to stand until a distinct and stable phase interface forms. Transfer the upper and lower phases to separate beakers and ultrasonically degas for 15 minutes before subsequent high-speed countercurrent chromatography separation. Dissolve the sample to be separated in equal amounts of the upper and lower phases of the selected solvent system for subsequent separation.

[0029] 3.4 High-Speed ​​Countercurrent Chromatography In this study, the separation modes can be divided into the following two modes according to different operation methods: (1) Conventional HSCCC separation mode Start the cold trap in advance and control the temperature at 25°C. Preheat the UV detector. Select the upper phase of the corresponding solvent system as the stationary phase and pump it into the tubing at 30 ml / min. When the upper phase flows out of the tubing at the corresponding rate, the chromatographic separation column is filled with the stationary phase. Select forward-flow-in (FWD-IN) mode and start the rotational speed. Pump the lower phase of the corresponding solvent system at the set flow rate to equilibrate before injection. A steady flow of the lower phase at the set flow rate indicates that the separation system has reached equilibrium. Turn off the flow rate to stop pumping the lower phase. Inject the prepared sample solution into the tubing through the six-port injection valve. Once the injection is complete, start the flow rate and perform separation. After injection, the sample solution passes through the countercurrent chromatograph tubing and the UV detector, and is collected in a centrifuge tube in the fraction collector. After each separation, blow out the countercurrent liquid. The volume fraction occupied by the stationary phase is the retention rate for that separation. Rinse the tubing with ethanol after each separation.

[0030] (2) Circular (IRCCC) separation mode In this mode, equilibration and injection procedures are identical to those for conventional countercurrent chromatography. Based on the real-time image captured by the UV detector, just before the components to be recycled are eluted, the eluate is pumped back into the countercurrent chromatograph via a constant-flow pump via an external six-way valve for the next separation. Once successful separation of the components is confirmed by UV-detection, the six-way valve is switched to terminate the cyclic separation mode, allowing the eluate to flow out. The eluate is then collected and subsequently processed according to conventional HSCCC methods.

[0031] The eluate collected in the centrifuge tube of the fraction collector was filtered through a 0.22 µm nylon filter membrane and analyzed by HPLC. The same fractions were combined and evaporated to dryness or freeze-dried.

[0032] 4 Preparative liquid phase separation and purification Compounds 5 and 6 were not successfully separated by high-speed countercurrent chromatography. The mixed components were dissolved in methanol and separated by preparative liquid chromatography. The conditions for preparative liquid chromatography separation of the components were as follows: the chromatographic column was YMC-PackODS-A (250 mm × 10 mm, 5.0 µm); the mobile phase was acetonitrile: 0.1% formic acid in water = 52:48 ( v / v The flow rate was 3.0 mL / min, the injection volume was 50 μL, the column temperature was 25 °C, and the detector wavelength was 246 nm.

[0033] 5 High-performance liquid chromatography and mass spectrometry analysis Liquid chromatography detection conditions were as follows: an Agilent Extend-C18 column (250 mm × 4.6 mm, 5.0 µm); the mobile phase consisted of acetonitrile and 0.1% formic acid in water; the flow rate was 1.0 mL / min, the injection volume was 10 µL, the column temperature was 25°C, and the detector wavelength was 246 nm. The mobile phase conditions are shown in Table 3. Mass spectrometric analysis was performed using an LC-MS-9030 Q-TOF quadrupole-time-of-flight mass spectrometer (Shimadzu).

[0034] Table 3 HPLC mobile phase detection conditions 6 Experimental Results 6.1 Extraction and extraction of Psoralea corylifolia 500 g of salt psoralea corylifolia was extracted with 95% ethanol to obtain about 148 g of extract. The extract was extracted. In the first set of extractions, except for compounds 1 and 2, the other 8 compounds were extracted in the solvent system of n-hexane: ethyl acetate: methanol: water = 5:5:4:6 ( v / v ) tends to dissolve in the upper phase. After extraction 3 times, Figure 1 As shown, more than 90% of compounds 1 and 2 can be separated from the large polar components.

[0035] In the second set of extractions, except for compound 10, the other 9 compounds were extracted in the solvent system of n-hexane: ethyl acetate: methanol: water = 5:5:7:3 ( v / v ) tends to dissolve in the lower phase. After 9 extractions, Figure 2 As shown, the peak area ratio of compound 10 in HPLC detection is about 10:1, which can ensure that 90% of compound 10 is separated from some small polar components.

[0036] The extracted samples were compared with the samples after two sets of extraction, e.g. Figure 3The results show that extraction can effectively remove most of the high-polarity components and some of the low-polarity components. Approximately 10 g of the extract was extracted each time. The first set of extractions removed approximately 3.2 g of high-polarity components, while the second set removed approximately 1.8 g of low-polarity components, for an extraction rate of approximately 50%. After two sets of extractions, approximately 5.0 g of sample was obtained for subsequent countercurrent separation.

[0037] 6.2 Separation of Psoralea corylifolia by High-Speed ​​Countercurrent Chromatography 6.2.1 Selection of Separation Parameters for High-Speed ​​Countercurrent Chromatography The maximum speed of the high-speed countercurrent chromatography instrument used in this study is 850 rpm. The relationship between speed and retention rate is as follows: Figure 4 As shown in the figure, to ensure the separation effect and the safe and stable operation of the instrument, 800 rpm is selected as the subsequent speed. Figure 5 As shown in the figure, at 800 rpm, the retention rate of each solvent system gradually decreased with the gradual increase of flow rate. The solvent system was n-hexane: ethyl acetate: methanol: water = 5:5:5:5 ( v / v ) The retention rate at 5 mL / min is already close to 50%. A retention rate too low will reduce the separation effect. At flow rates below 5 mL / min, the retention rates of all three solvent systems can exceed 50%. Therefore, 5 mL / min was selected as the maximum mobile phase flow rate for countercurrent chromatography in this study. The cold trap was operated to maintain the line temperature at 25°C.

[0038] 6.2.2 Selection of solvent system K The value represents the solubility distribution of the compound in different phases in a two-phase solvent system. K >2 and the larger the value, the slower the compound elutes in the countercurrent chromatography separation with the above phase as the stationary phase, the longer the retention time, and the lower the separation efficiency; K <0.5 indicates that the compound has a short retention time and may be difficult to separate effectively; K When the value is between 0.5 and 2, the separation is relatively reasonable. To determine whether two compounds can be separated, it is necessary to judge based on the separation coefficient α. 1,2 = K 1 / K 2 ( K 1> K 2) When α>1.5, it indicates that the compounds can be well separated. When α<1.5, countercurrent chromatography circulation mode is required for separation in this study.

[0039] The high-speed countercurrent chromatography separation of compounds in Psoralea corylifolia requires the separation of components between different solvent systems. K The values ​​were used as the basis to detect each compound according to HPLC.K The results are shown in Table 4. K The values ​​show that the polarities of the compounds in Psoralea corylifolia vary greatly, making it difficult to separate all target components at one time. Compounds with similar polarities can be classified and collected in segments before continuing to separate single compounds.

[0040] In the solvent system of n-hexane: ethyl acetate: methanol: water = 5:5:6:4 ( v / v ), 7 compounds K Although the value is between 0.5-1.5, the number of target compounds is large, and the peaks are early and concentrated, which may cause signal overlap when multiple components elute, affecting the judgment of the actual separation and the reception of the eluent, thereby affecting the effective separation. Compounds 1 and 4 in the solvent system of n-hexane: ethyl acetate: methanol: water = 5:5:5:5 ( v / v ) K The value is relatively appropriate in theory. However, in the subsequent actual countercurrent separation process, even after some impurities are removed by extraction, there are still non-target components with short retention times, which are easily mixed with compounds 1 and 4, which may affect the separation effect; compounds 5, 6, 7, 8, 9 and 10, 6 compounds in the solvent system of n-hexane: ethyl acetate: methanol: water = 5:5:4.5:5.5 ( v / v )middle K The value is too large, the elution time is long, and the separation efficiency is low. In summary, in order to ensure the separation effect, for the single separation of the extracted sample, this study also selected K The values ​​are relatively large for n-hexane:ethyl acetate:methanol:water = 5:5:4.5:5.5 and 5:5:5:5 ( v / v ) 2 solvent systems for preliminary separation.

[0041] As shown in Table 5, the α 2,3 The value cannot meet the condition of being greater than 1.5 in the designed solvent systems, and the IRCCC mode is planned to be used for separation. K The values ​​show that the two compounds are in the solvent system of n-hexane: ethyl acetate: methanol: water = 5:5:4.5:5.5 ( v / v ) system relative to the solvent system n-hexane: ethyl acetate: methanol: water = 5:5:5:5 ( v / v ) is slightly larger, which is convenient for collecting eluent, and is relatively less likely to cause mixing of components between different cycles during multiple cycle separations with large injection volumes. After comparison, the solvent system was selected: n-hexane: ethyl acetate: methanol: water = 5:5:4.5:5.5 (v / v ) was used for the cyclic separation of compounds 2 and 3.

[0042] α of compounds 7 and 9 7,9 The value cannot meet the condition of being greater than 1.5 in the several designed solvent systems. It is planned to use IRCCC separation mode for separation. There are few other non-target components in the mixed components of compounds 7 and 9. In order to improve the separation efficiency, a solvent system with relatively short retention time is selected: n-hexane: ethyl acetate: methanol: water = 5:5:7:3 ( v / v ) for cyclic separation.

[0043] Compound 10 has a large K The value indicates that its polarity is relatively small and is quite different from other compounds. It is the last to elute in the HSCCC separation.

[0044] Table 4 Partition coefficients of samples extracted from Psoralea corylifolia in different solvent systems Table 5 Separation factors of some compounds in different solvent systems 6.2.3 HSCCC Isolation Procedure (1) Conventional HSCCC separation (a) First, use the solvent system of n-hexane: ethyl acetate: methanol: water = 5:5:4.5:5.5 ( v / v ) Separate the extracted sample with a flow rate of 5 mL / min. After the peak of component B is eluted, replace the mobile phase of the separation system with a solvent system of n-hexane: ethyl acetate: methanol: water = 5:5:5:5 ( v / v ) and continue separation until the end. Figure 6 As shown, the single separation injection amount was 100.0 mg, the retention rate was 50.2%, and 4.8 mg of compound 1 monomer (component A), 11.2 mg of a mixture of compounds 2 and 3 (component B), 4.5 mg of compound 4 monomer (component C), 7.4 mg of a mixture of compounds 5 and 6 (component D), 8.5 mg of a mixture of compounds 7, 8 and 9 (component E), and 11.7 mg of compound 10 monomer (component F) were obtained.

[0045] (b) Using a solvent system of n-hexane: ethyl acetate: methanol: water = 5:5:7:3 ( v / v), set the flow rate to 2 mL / min, and separate the mixture of component E (compounds 7, 9 and 8). The separation results are shown in Figure 8 As shown, a single injection of 10.0 mg was performed, and after cyclic separation, the retention rate was 64.3%, and 4.9 mg of a mixture of compounds 7 and 9 and 2.3 mg of compound 8 monomer were obtained.

[0046] (2) IRCCC separation model Compounds 2 and 3 in component B and compounds 7 and 9 in component E have similar polarities and cannot be effectively separated by a single separation. They can be separated using the IRCCC mode.

[0047] (a) Use the solvent system of n-hexane: ethyl acetate: methanol: water = 5:5:4.5:5.5 ( v / v ), set the flow rate to 5mL / min, and perform cyclic separation on component B (compounds 2 and 3). Figure 9 As shown, 71.0 mg was injected, and after cyclic separation, the retention rate was 50.7%, and 30.9 mg of compound 2 monomer and 34.4 mg of compound 3 monomer were obtained.

[0048] (b) Using a solvent system of n-hexane: ethyl acetate: methanol: water = 5:5:7:3 ( v / v ), set the flow rate to 2 mL / min, first perform conventional high-speed countercurrent separation on component E (compounds 7, 9, and 8). When the mixed components of compounds 7 and 9 are about to be detected by the UV detector for the first time, adjust the six-way valve to switch to the circulation mode, so that the eluent flows back to the separation pipeline for subsequent circulation separation; when the mixed components of compounds 7 and 9 are detected by the UV detector for the first time (before compound 8 is eluted from the pipeline), adjust the six-way valve to switch to conventional HSCCC separation. After compound 8 is collected, return to the circulation mode until the separation is completed. The separation results are shown as follows: Figure 10 As shown, 20.5 mg was injected, and after cyclic separation, the retention rate was 61.2%, and 4.3 mg of compound 7 monomer, 5.4 mg of compound 9 monomer, and 5.0 mg of compound 8 monomer were obtained.

[0049] 6.3 Preparative Liquid Chromatographic Separation of Psoralea corylifolia In preparative liquid chromatography, component D (compounds 5 and 6) was separated using (52% acetonitrile-48% 0.1% formic acid water) as the mobile phase elution condition. The separation results are shown in FIG. Figure 11 As shown, after separation of 30.2 mg of sample, 8.2 mg of compound 5 monomer and 11.0 mg of compound 6 monomer were obtained.

[0050] 6.4 HPLC Test Results of Psoralea corylifolia Psoralea corylifolia was extracted and 10 compounds were separated using different HSCCC modes combined with preparative liquid phase. The HPLC test results of each compound are as follows: Figure 12 shown.

[0051] 6.5 Structural Identification of Isolated Compounds Comparison of the compounds with relevant literature revealed that the 10 compounds isolated from Psoralea corylifolia were: psoralen, isopsoralen, neopsoralen isoflavone, psoralen dihydroflavonoid, D(3)-2-Hydroxylbakuchiol, psoralen isoflavone, isopsoralen chalcone, psoralen dihydroflavonoid methyl ether, chalcone glycosides, and bakuchiol. The analytical data are described below.

[0052] Compound 1, ESI-MS m / z 185.0815 [MH] - , 1 H-NMR (DMSO- d 6, 400 MHz) δ: 6.45(1H, d, J = 9.6 Hz, H-3), 7.11 (1H, dd, J = 2.2, 1.3 Hz, H-3'), 7.74 (1H, s, H-8), 8.02 (1H, s, H-5), 8.13 (1H, d, J = 2.2 Hz, H-2'), 8.19 (1H, d, J = 9.6 Hz, H-4); 13 C-NMR (DMSO- d 6, 101 MHz) δ: 99.8 (C-8), 107.2 (C-3'), 114.6 (C-3), 115.8(C-10), 121.1 (C-5), 125.0 (C-6), 145.5(C-4), 148.4 (C-2'), 152.0 (C-9),156.2 (C-7), 160.6 (C-2). The above data were compared with the literature and identified as psoralen, with the molecular formula C 11 H6O3.

[0053] Compound 2, ESI-MS m / z 185.0813 [MH] - , 1 H-NMR (DMSO- d6, 400 MHz) δ: 6.47(1H, d, J = 9.6 Hz, H-3), 7.29 (1H, m,H-3'), 7.64 (1H, d, J = 8.6 Hz, H-5), 7.67(1H, d, J = 8.6 Hz, H-6), 8.18 (1H, d, J = 2.0 Hz, H-2'), 8.19 (1H, d, J = 9.6 Hz,H-4); 13 C-NMR (DMSO- d 6, 101 MHz) δ: 104.1 (C-3'), 109.2 (C-6), 113.9 (C-10),114.2 (C-3), 116.4 (C-8), 125.1 (C-5),145.8 (C-4), 147.8 (C-2'), 148.3 (C-9),151.7 (C-7), 160.3 (C-2). The above data were compared with the literature and identified as isopsoralen, with the molecular formula C 11 H6O3.

[0054] Compound 3, ESI-MS m / z 321.1736 [MH] - , 1 H-NMR (DMSO- d 6, 400 MHz) δ: 8.25(1H, s, H-2), 7.96 (1H, d, J = 8.8 Hz, H-5), 7.24 (1H, d, J = 2.1 Hz, H-2'), 7.19 (1H, dd, J = 8.2, 2.1 Hz, H-6'), 6.93 (1H, dd, J = 8.8, 2.1 Hz, H-6), 6.86 (1H, d, J = 2.1 Hz, H-8), 6.82 (1H, d, J = 8.2Hz, H-5'), 5.31 (1H, t, J= 7.2 Hz, H-2''), 3.25 (2H, d, J = 7.2 Hz, H-1''), 1.69 (6H, s, H-4'', 5'');13C-NMR (DMSO- d6, 101 MHz) δ: 153.2 (C-2), 124.2 (C-3), 175.2 (C-4), 127.7 (C-5), 115.6 (C-6), 163.1 (C-7), 102.5 (C-8), 155.3 (C-9), 117.0 (C-10), 127.8(C-1'), 130.5 (C-2'), 131.6 (C-3'), 157.9 (C-4'), 115.0 (C-5'), 127.6 (C-6'), 26.0 (C-1”), 123.0 (C-2”), 123.4 (C-3”), 18.1 (C-4”), 28.7 (C-5”). The above data were compared with the literature and identified as neobavaisoflavone, with the molecular formula C 20 H 18 O4.

[0055] Compound 4, ESI-MS m / z 323.1530 [MH] - , 1 H-NMR (DMSO- d 6, 400 MHz) δ: 5.39(1H, dd, J = 12.8, 2.6 Hz, H-2), 3.07 (1H, dd, J = 16.7, 12.9 Hz, Ha-3), 2.59(1H, dd, J = 16.7, 2.8 Hz, Hb-3), 7.44 (1H,s, H-5), 6.37 (1H, s, H-8), 7.31(2H, d, J = 8.5 Hz, H-2', 6'), 6.79 (2H, d, J = 8.5 Hz, H-3', 5'), 3.16 (2H,d, J = 7.3 Hz, H-1”), 5.26 (1H, t, J = 7.4 Hz, H-2”), 1.66 (3H, s, H-4”), 1.72 (3H, s, H-5”); 13C-NMR (DMSO- d6, 101 MHz) δ: 79.4 (C-2), 43.75 (C-3),190.6 (C-4),122.7 (C-5), 123.1 (C-6), 158.1 (C-7), 102.6 (C-8), 161.9 (C-9),113.5 (C-10), 132.3 (C-1'), 128.7 (C-2', 6'), 115.6 (C-3', 5'), 163.0 (C-4'),27.6 (C-1"), 127.2 (C-2"), 129.9 (C-3"), 26.0 (C-4"), 18.1 (C-5"). The above data were compared with the literature and identified as bavachin, with the molecular formula C 20 H 20 O4.

[0056] Compound 5, ESI-MS m / z 271.1696 [MH] - , 1 H-NMR (DMSO- d 6, 400 MHz) δ: 7.20(2H, d, J = 7.6 Hz, H-2,6), 6.70 (2H, d, J = 7.6 Hz, H-3,5), 6.18 (1H, d, J = 16.2Hz, H-7), 6.05 (1H, d, J = 16.2 Hz, H-8), 5.89 (1H, dd, J = 17.2, 10.8 Hz, H-17), 5.50 (2H, m, H-11, 12), 4.99 (2H, m, H-18), 2.14 (2H, d, J = 6.2 Hz,, H-10),1.13 (6H, s, H-14,15), 1.12 (3H, s, H-16); 13 C-NMR (DMSO- d6, 101 MHz) δ: 128.8(C-1), 127.6 (C-2,6), 115.8 (C-3,5), 157.2 (C-4), 134.4 (C-7), 127.0(C-8),44.0 (C-9), 42.7 (C-10), 121.6 (C-11), 142.7 (C-12), 69.4 (C-13), 30.7 (C-14,15), 23.6 (C-16), 146.3 (C-17), 112.3 (C-18). The above data were compared with the literature and identified as delta3,2-hydroxybakuchiol with the molecular formula C 18 H 24 O2.

[0057] Compound 6, ESI-MS m / z 319.0945 [MH] - , 1 H-NMR (DMSO- d 6, 400 MHz) δ: 8.34(1H, s, H-2), 7.95 (1H, d, J = 7.4 Hz, H-5), 7.24-7.35(2H, m, H-2',6'), 6.96(1H, dd, J = 7.4, 2.1 Hz, H-6), 6.90 (1H, d, J = 2.1 Hz, H-8), 6.79 (1H, d, J= 8.2 Hz, H-5'), 6.44 (1H, d, J = 8.9 Hz, H-1''), 5.78 (1H, d, J = 8.9 Hz,H-2''), 1.40 (6H, s, CH3×2); 13C-NMR (DMSO- d6, 101 MHz) δ: 152.6 (C-2), 122.2(C-3), 175.0 (C-4),127.4 (C-5), 116.0 (C-6), 163.4 (C-7), 102.6 (C-8), 157.9(C-9),116.0 (C-10), 123.6 (C-1'), 131.7 (C-2'), 125.0 (C-3'), 153.6 (C-4'), 121.0 (C-5'), 127.6 (C-6'), 116.0 (C-1”), 130.1 (C-2”), 76.7 (C-3”), 28.2(CH3×2). The above data were compared with the literature and identified as corylin, with the molecular formula C 20 H 16 O4.

[0058] Compound 7, ESI-MS m / z 325.1452 [M+H] + , 1 H-NMR (DMSO- d 6, 400 MHz) δ: 8.04(1H,d,J=9.0 Hz,H-6'), 7.76 (4H, m, H-α, β, 2,6), 6.85 (2H,d,J=8.5 Hz,H-3, 5), 6.48 (1H,d,J=9.0 Hz,H-5'), 5.18(1H,t, J=6.9 Hz,H-2''), 3.24 (2H, d, J= 7.0Hz, H-1''), 1.73 (3H,s,H-5''), 1.62 (3H, s,H-4''); 13 C-NMR (DMSO- d6, 101 MHz)δ: 18.2(C-4''), 21.8 (C-1''), 26.0 (C-5''), 107.8(C-5'), 113.2 (C-1'), 114.9(C-3'), 116.3 (C-3, 5), 117.9 (C-α), 122.9 (C-2''), 126.3 (C-1), 130.3 (C-6'),130.9(C-3''), 131.6 (C-2, 6), 144.6 (C-β), 160.7(C-4), 162.9 (C-2'),164.1 (C-4'), 192.2 (C=O). The above data were compared with the literature and identified as isobavachalcone, with the molecular formula C 20 H 20 O4.

[0059] Compound 8, ESI-MS m / z 339.1606 [M+H] + , 1 H-NMR (DMSO- d 6, 400 MHz) δ: 5.46(1H, dd, J = 13.0, 2.6 Hz, H-2), 3.12 (1H, dd, J = 16.8, 12.8 Hz, Ha-3), 2.64(1H, dd, J = 16.8, 2.8 Hz, Hb-3), 7.48 (1H,s, H-5), 6.61 (1H, s, H-8), 7.34(2H, d, J = 8.5 Hz, H-2', 6'), 6.79 (2H, d, J = 8.5 Hz, H-3', 5'), 3.18(2H,d, J = 7.7 Hz, H-1”), 5.23 (1H, t, J = 7.3 Hz, H-2”), 1.70 (3H, s, H-5”), 1.65 (3H, s, H-4”); 13 C-NMR (DMSO- d6, 101 MHz) δ: 79.7 (C-2), 43.6 (C-3), 190.8 (C-4), 126.5 (C-5), 123.9 (C-6), 163.9 (C-7), 99.8 (C-8), 158.1 (C-9), 114.0 (C-10), 129.7 (C-1'), 128.7 (C-2', 6'), 115.6 (C-3', 5'), 162.4 (C-4'), 27.7(C-1”), 122.4 (C-2”), 132.7 (C-3”), 26.0 (C-4”), 18.0 (C-5”). The above data were compared with the literature and identified as bavachinin, with the molecular formula C 21 H 22 O4.

[0060] Compound 9, ESI-MS m / z 389.1753 [MH] - , 1 H NMR (DMSO- d 6, 400 MHz) δ 1.53 (3H,s, H-8ʺ), 1.56 (3H, s, H-9ʺ), 1.66 (3H, s, H-10ʺ), 2.00 (2H, m, H-4ʺ), 2.04(2H,m, H-5ʺ), 3.25 (2H, d, J =7.2 Hz, H-1ʺ), 5.07 (1H, t, J =6.3 Hz, H-6ʺ), 5.31(1H, t, J =7.2 Hz,, H-2ʺ), 6.82 (1H, d, J =8.3 Hz, H-5ʹ), 6.85 (1H, d, J =2.0 Hz,H-8) 6.93 (1H, dd, J =8.8, 2.0 Hz, H-6), 7.18 (1H, dd, J =8.4, 2.0 Hz, H-6ʹ),7.23 (1H, d, J =2.0 Hz, H-2ʹ), 7.95 (1H, d, J =8.8 Hz, H-5), 8.24 (1H, s, H-2); 13 CNMR (DMSO- d6, 101 MHz) δ 16.4 (C-10ʺ), 18.0 (C-8ʺ), 25.9 (C-9ʺ), 26.6 (C-5ʺ), 28.4 (C-1ʺ), 39.8 (C-4ʺ), 102.6 (C-8), 115.0 (C-5ʹ), 115.6(C-6), 117.0 (C-10), 123.0 (C-2ʺ), 123.1 (C-1ʹ), 124.3 (C-6ʺ), 124.6 (C-3), 127.5 (C-6ʹ), 127.6 (C-5), 127.8 (C-3ʹ), 130.4 (C-2ʹ), 131.2 (C-7ʺ), 135.4(C-3ʺ), 153.1 (C-2), 155.3 (C-4ʹ), 157.9 (C-9), 163.2 (C-7), 175.2 (C-4). The above data were compared with the literature and identified as corylifol A, with the molecular formula C 25 H 26 O4.

[0061] Compound 10, ESI-MS m / z 255.2322 [MH] - , 1 H-NMR (DMSO- d 6, 400 MHz) δ: 7.21(2H, d, J = 8.4 Hz, H-2,6), 6.70 (2H, d, J = 8.4 Hz, H-3,5), 6.19 (1H, d, J = 16.3Hz, H-7), 6.04 (1H, d, J = 16.3 Hz, H-8), 5.88 (1H, dd, J = 17.5, 10.7 Hz, H-17),5.10 (1H, t, J = 6.7 Hz, H-12), 5.00 (2H, m, H-18), 1.89 (2H, m, H-10), 1.63(3H, s, H-14), 1.53 (3H, s, H-14), 1.44 (2H, m, H-11), 1.15 (3H, s, H-16); 13 C-NMR (DMSO- d6, 101 MHz) δ: 128.8 (C-1), 127.6 (C-2,6), 115.8 (C-3,5), 157.1(C-4), 134.5 (C-7), 127.0 (C-8), 42.5 (C-9), 41.4 (C-10), 23.3(C-11), 125.2(C-12), 130.9 (C-13), 17.9 (C-14), 25.9 (C-15), 23.5 (C-16), 146.4 (C-17),112.3 (C-18). The above data were compared with the literature and identified as bakuchiol, with the molecular formula C 18 H 24 O.

[0062] The specific process of evaluating the cell anti-inflammatory activity of the present invention is as follows: 1. Cell culture DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin was used as the complete culture medium.

[0063] 2 Cytotoxicity experiments RAW264.7 cells (1×10 5 Cells were seeded in 96-well plates (100 μL / well, 100 μL / well) for control and experimental groups, and cultured in complete medium. After 24 hours, the original medium was replaced with fresh medium. Experimental groups were supplemented with different concentrations of sample, while the control group received an equal volume of the corresponding solvent. After 24 hours of incubation at 37°C and 5% CO2, the original medium was discarded and washed twice with PBS. Fresh medium containing 10% CCK8 reagent (100 μL / well) was added to each well. After 2 hours of incubation at 37°C, absorbance at 450 nm was measured using a microplate reader to calculate cell viability. The concentration that achieved a cell viability of 90% or higher was selected for subsequent NO content analysis.

[0064] 3 NO content detection RAW264.7 cells (4×10 5Cells were inoculated with 100 μL of LPS per well (100 μg / mL, 100 μL / well) in a 96-well culture plate. A control, model, positive drug, and experimental groups were assigned and cultured in complete medium. After 24 hours, the original medium was replaced with fresh medium. LPS solution with a final concentration of 1.5 μg / mL was added to the model, positive drug, and experimental groups, while an equal volume of medium was added to the control group. Different concentrations of drugs or samples were added to the positive drug and experimental groups, while an equal volume of the corresponding solvent was added to the control and model groups. After 24 hours of incubation at 37°C and 5% CO2, an equal volume of Griess reagent (Griess A:Griess B = 1:1) was added and allowed to react at room temperature for 30 minutes. The absorbance of the supernatant was measured at 548 nm using a microplate reader. A standard curve was generated according to the kit instructions, and NO content was calculated based on the absorbance.

[0065] 4 RT-qPCR detection of RNA expression RAW264.7 cells (4×10 5 Cells were seeded with 1 mL / well (1 μg / mL) in a 12-well culture plate. A control group, model group, positive drug group, and experimental group (psoralen, neopsoralen isoflavones, psoralen flavonoids, and psoralen flavonoid methyl ether) were set up and cultured in complete medium. After 24 hours, the original medium was replaced with fresh medium. For each experimental group, the sample concentration corresponding to the minimum NO release in Section 2.3 was used as the sample loading concentration. The final LPS loading concentration was 1.5 μg / mL. Cells were exposed to each sample and LPS for 24 hours at 37°C and 5% CO2. After observing cell morphology under a microscope, the culture medium was aspirated, rinsed once with PBS, and 1 mL of Trizol was added. Cells were lysed by pipetting the bottom of the plate to allow the cells to float. The lysate was collected in a 1.5 mL centrifuge tube and stored at -80°C until ready for use.

[0066] 5. Western blot detection of protein expression The cell plating and drug intervention conditions are the same as those in 2.4.1 of this chapter. On the third day of culture, aspirate the culture medium, rinse once with PBS, add 250 μL of protein extract, mix well to ensure that the extract is in full contact with the cells at the bottom of the well, collect in a centrifuge tube after the reaction, heat at 95°C for 5 min, and store at -20°C until use.

[0067] Experimental results 1 Effects of samples on RAW264.7 cell number The CCK-8 kit was used to detect the effects of dexamethasone sodium phosphate, the extracted samples isolated in Chapter 1, and 10 compound monomers at different concentrations on the number of RAW264.7 cells. The results are as follows: Figure 14The viability of RAW264.7 cells was above 90% at the following concentrations: psoralen (0-100 μM), isopsoralen (0-50 μM), neopsoralen (0-25 μM), psoralen (0-12.5 μM), delta 3,2-hydroxybakuchiol (0-25 μM), psoralen (0-50 μM), isopsoralen chalcone (0-3.125 μM), psoralen (0-12.5 μM), hypoglycine chalcone (0-100 μM), bakuchiol (0-12.5 μM), extracted sample (0-6.25 μg / mL), and dexamethasone sodium phosphate (0-30 μM).

[0068] 2 Effect of samples on NO release Under the induction of LPS, macrophages will significantly increase the expression of inducible nitric oxide synthase (iNOS) gene and increase the release of NO. Using the LPS-induced RAW264.7 cell inflammation model, the concentration with a cell survival rate of more than 90% in the toxicity experiment was selected as the sample experimental concentration, and the NO release was used as the standard to evaluate the anti-inflammatory activity. The results are as follows Figure 15 As shown. Psoralen, neopsoralen isoflavones, psoralen flavonoids and psoralen flavonoid methyl ether as well as the extracted samples can significantly reduce NO release at the selected experimental concentrations ( p <0.0001), psoralen isoflavones and isopsoralen chalcone showed some effect in reducing NO release, while the remaining compounds had little effect on NO release at safe concentrations. The half-maximal inhibitory concentrations (IC50) for NO release were as follows: psoralen (178.53 μM), neopsoralen isoflavones (15.26 μM), psoralen dihydroflavonoids (72.81 μM), psoralen dihydroflavonoid methyl ether (271.92 μM), the extracted sample (32.09 μg / mL), and dexamethasone sodium phosphate (22.49 μM). The remaining compounds had IC50 values ​​greater than 1000 μM, exceeding the actual safe concentration, indicating a weak effect in reducing NO release.

[0069] 3. Effects of samples on cell growth morphology The morphological changes of cells in each group were observed under a microscope. Figure 16As shown, the cells in the control group without LPS induction grew evenly and were round or oval in shape, which can be considered as normal growth; the cells in the model group after LPS induction showed a trend of aggregated growth, produced cell clumps that grew adherently, and had fibrous pseudopodia. The proportion of cells deformed into spindle shape increased significantly, indicating that the cells had an inflammatory response; after 20 μM dexamethasone sodium phosphate intervention in LPS-induced cells, the trend of cell aggregation growth was weakened, the number of cell clumps decreased, and the number of spindle cells was significantly reduced, indicating that the intervention of dexamethasone sodium phosphate had a positive effect on the cell morphology of inflammatory cells; the cells treated with psoralen, neopsoralen isoflavones, psoralen dihydroflavone and psoralen dihydroflavone methyl ether showed relatively uniform cell growth and an increased proportion of round cells, indicating that the intervention of each compound had a certain alleviating effect on cell inflammation.

[0070] 4. Effect of samples on RNA expression The results are as follows Figure 17 The results show that the four compounds affect cellular inflammation at the transcriptional level. The results showed that psoralen, new psoralea isoflavones, and psoralea flavonoids reduced the expression of TNF-α and COX-2 in cells after treatment, but the inhibitory effect was not significant. They also significantly inhibited the expression of IL-6 and IL-1β to varying degrees. Psoralea flavonoid methyl ether had a relatively stronger inhibitory effect on the expression of the four inflammatory mediators. Although the effects of the four compounds were not as strong as those of dexamethasone sodium phosphate, they reduced the expression of IL-6, IL-1β, TNF-α, and COX-2 mRNA to varying degrees, suggesting a role in alleviating inflammation.

[0071] 5. Effect of samples on the expression of related proteins After the intervention of four compounds in Psoralea corylifolia on LPS-induced RAW264.7 cells, they had different effects on the phosphorylation of proteins in the NF-κB, JNK, Erk and p38 signaling pathways. Figure 18 As shown in (II), the protein phosphorylation levels of the four pathways in the model group were significantly different from those in the control group ( p< 0.0001). There was no significant difference in the phosphorylation expression of Erk in each experimental group compared with the model group. Psoralen, flavone and methyl flavone all reduced the phosphorylation expression in the four pathways and were superior to dexamethasone sodium phosphate in inhibiting the phosphorylation expression of NF-κB. The effects of flavone and methyl flavone on p38 were similar to those of positive drugs and were significantly different from those in the model group ( p< 0.0001), indicating that these two compounds may play a potential anti-inflammatory role in the NF-κB or p38 signaling pathway.

[0072] The above-mentioned specific embodiments are only specific cases of the present invention. The patent protection scope of the present invention includes but is not limited to the product form and style of the above-mentioned specific embodiments. Any appropriate changes or modifications made to them by ordinary technicians in the relevant technical field in accordance with the claims of the present invention shall fall within the patent protection scope of the present invention.

Claims

1. A method for preparing the active ingredient in Psoralea corylifolia, characterized in that: The isolated compounds include compounds 1-10 represented by the following formulas: The method comprises the following steps: S1: After the Psoralea corylifolia was crushed, it was reflux extracted with 95% ethanol at a solid-liquid ratio of 1:10 to obtain a crude product; S2: Extract the crude product according to target compounds 1-10, and select a two-phase solvent system, wherein the first two-phase solvent system has a volume ratio of n-hexane / ethyl acetate / methanol / water of 5-6:5-6:4-5:5-6, and the second two-phase solvent system has a volume ratio of n-hexane / ethyl acetate / methanol / water of 5-6:5-6:7-8:3-6; S3: Conventional HSCCC separation, using the first two-phase solvent system to separate the extracted sample. After the peak of the mixture of compounds 2 and 3 ends, the mobile phase of the separation system is immediately replaced with the lower phase of the first two-phase solvent system, and the separation is continued until completion; S4: using a second two-phase solvent system to separate the mixture of compounds 7, 9 and 8; S5: In preparative liquid chromatography, compounds 5 and 6 were separated using 52% acetonitrile-48% 0.1% formic acid in water as the mobile phase elution conditions.

2. The method for preparing the active ingredient in Psoralea corylifolia according to claim 1, wherein: In steps S3 and S4, compounds 2 and 3, and compounds 7 and 9 were separated using IRCCC mode: Using the first two-phase solvent system, the flow rate was set at 5-7 mL / min to circulate and separate compounds 2 and 3; Using a second two-phase solvent system with a flow rate set at 1-2 mL / min, compounds 7, 9, and 8 were first subjected to conventional high-speed countercurrent separation. When the mixed components of compounds 7 and 9 were about to be detected by the UV detector for the first time, the six-way valve was adjusted to switch to the circulation mode, allowing the eluent to flow back to the separation pipeline for subsequent circulation separation. When the mixed components of compounds 7 and 9 were detected by the UV detector for the first time, the six-way valve was adjusted to switch to conventional HSCCC separation. After compound 8 was collected, the valve was switched back to the circulation mode until the separation was completed.

3. The method for preparing the active ingredient in Psoralea corylifolia according to claim 1, wherein: In step S1, reflux extraction is performed three times, with extraction times of 2 h, 2 h, and 1.5 h, respectively. After filtration, the extracts are combined and concentrated.

4. The method for preparing the active ingredient in Psoralea corylifolia according to claim 1, wherein In step S2, 10 g of the crude product after extraction was weighed, and 500-550 mL of the upper phase and lower phase of the first two-phase solvent system were used for the first set of extractions. The lower phase of each extraction was retained in the separatory funnel, and a new upper phase was added for the next extraction. The upper phase in each extraction was transferred and collected, and extracted 3-4 times in total. The upper phase collected in the first set of extractions was evaporated to dryness, and the second set of extractions was continued using 500-550 mL of the upper phase and lower phase of the second two-phase solvent system. The upper phase of each extraction was retained in the separatory funnel, and a new lower phase was added for the next extraction. The lower phase in each extraction was transferred and collected, and extracted 9-10 times in total.

5. The method for preparing the active ingredient in Psoralea corylifolia according to claim 1, wherein: In step S3, the prepared sample solution is injected into the pipeline through the six-way injection valve, and the flow rate is opened for separation after the injection. After the injection, the sample solution passes through the internal pipeline of the countercurrent chromatograph and the ultraviolet detector in sequence and is collected in the centrifuge tube of the fraction collector.

6. The method for preparing the active ingredient in Psoralea corylifolia according to claim 2, wherein: In the IRCCC mode, before the component that needs to be circulated is about to be eluted, the eluate is pumped back into the countercurrent chromatograph pipeline through an external six-way valve via a constant flow pump for the next separation. After the successful separation of each component is observed through the UV detection spectrum, the six-way valve mode is switched to end the circulation separation mode, allowing the eluate to flow out, and the eluate is collected and subsequent operations are performed according to the conventional HSCCC method.

7. The method for preparing the active ingredient in Psoralea corylifolia according to claim 1, wherein: In step S3, the liquid chromatography detection conditions are as follows: the chromatographic column is Agilent Extend-C18, the mobile phase is acetonitrile and 0.1% formic acid in water, the flow rate is 1.0-1.5 mL / min, the injection volume is 10-12 μL, the column temperature is 20-25 °C, and the detector wavelength is 240-246 nm.

8. The anti-inflammatory component obtained by extracting the active component from Psoralea corylifolia according to claim 1, characterized in that: (1) Effects of the extracted and separated components on the number of RAW264.7 cells. Psoralea corylifolia isoflavones and isopsoralea corylifolia chalcone can reduce NO release; (2) Effects on cell growth morphology: cells treated with psoralen, neopsoralen isoflavones, psoralen dihydroflavone, and psoralen dihydroflavone methyl ether showed relatively uniform cell growth and an increased proportion of round cells, indicating that the intervention of each compound had a certain alleviating effect on cell inflammation; (3) Effect on RNA expression content, reducing the expression levels of IL-6, IL-1β, TNF-α and TNF-α mRNA to varying degrees, playing a role in alleviating inflammation; (4) The effect on the expression of related proteins, exerting potential anti-inflammatory ability in the NF-κB or p38 signaling pathway.

9. Use of the compound extracted by the preparation method according to any one of claims 1 to 7 in the preparation of anti-inflammatory products.

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