Method for extracting sonchus oleraceus glycoside A in cichorium intybus as well as product and application of sonchus oleraceus glycoside A

Abstract

The invention discloses a method for extracting sonchus oleraceus glycoside A in cichorium intybus as well as a product and application of the sonchus oleraceus glycoside A. The method comprises the following steps: (1) crushing the overground part of the cichorium intybus, adding an ethanol solution for soaking and extracting, separating out alcohol extracts after extraction, combining the alcohol extracts, and performing reduced pressure distillation to recover ethanol, so as to obtain a cichorium intybus extracting solution; (2) extracting the cichorium intybus extracting solution with an ethyl acetate solvent to obtain an ethyl acetate extracting part, and carrying out reduced pressure distillation on the ethyl acetate extracting part to recover ethyl acetate to obtain an extract; (3) dissolving the extract, mixing with silica gel, drying in the air, filling into a glass chromatographic column, separating by using a glass chromatography method, detecting the flowing-out sample by thin-layer chromatography, and combining to obtain an extract; and (4) taking the extract, and performing medium-pressure C18 column chromatography separation to obtain the sonchus oleraceus glycoside A. The invention also discloses a preparation method of the sonchus oleraceus glycoside A. According to the invention, sonchus oleraceus glycoside A is extracted and prepared from chicory, and the sonchus oleraceus glycoside A has potential inhibitory activity on xanthine oxidase.

Description

Technical Field

[0001] The present invention relates to the technical field of plant extraction, and in particular to a method for extracting sonchuside A from chicory, and its products and applications. Background Art

[0002] Hyperuricemia has become a global health problem. Among basic metabolic diseases, its incidence rate ranks fourth, second only to hypertension, hyperlipidemia, and hyperglycemia, and is closely related to the occurrence and progression of gout, kidney diseases, and cardiovascular diseases, which has been confirmed in relevant studies. The root cause of hyperuricemia is mainly the disorder of purine metabolism, which leads to excessive uric acid production and insufficient uric acid secretion. The production process of uric acid is closely related to xanthine oxidase mainly present in the liver. This enzyme plays a key role in the purine metabolism process. Inhibiting the activity of xanthine oxidase is crucial for improving the condition of hyperuricemia because it can promote the oxidation reaction of xanthine or hypoxanthine to generate uric acid. With the improvement of people's living standards and the change of diet structure, the prevalence of hyperuricemia shows an increasing trend year by year. Therefore, there is an urgent need to explore safe and effective anti-hyperuricemic drugs, which is urgent in current clinical treatment.

[0003] Chicory (Cichorii Herba / Cichorii Radix) belongs to the dried aerial parts or roots of the Compositae plants Cichorium glandulosum Boiss.et Huet. or Cichorium intybus L., and is a traditional medicinal and edible plant. According to relevant records, chicory has the effects of clearing the liver and gallbladder, strengthening the stomach and promoting digestion, and diuretic and detumescence, and has a long application history in the field of folk medicine. Modern scientific research shows that chicory has potential therapeutic effects on diseases such as hyperuricemia, hyperglycemia, and hyperlipidemia. Existing research results show that chicory is rich in various bioactive components such as flavonoids and phenolic acids, and these components may exert anti-hyperuricemic activity by interfering with the production and excretion processes of uric acid. For example, some flavonoid compounds can inhibit the activity of xanthine oxidase, thereby reducing the amount of uric acid produced. However, the material basis for chicory to play an anti-hyperuricemic role and the anti-hyperuricemic mechanism of specific compounds therein have not been fully elucidated and still need to be further explored.

[0004] In-depth research on the anti-hyperuricemic active components in chicory and clarification of its anti-hyperuricemic mechanism can provide a more solid theoretical basis for the medicinal development of chicory, and it is very necessary for expanding the development ideas of anti-hyperuricemic drugs. Summary of the Invention

[0005] The present invention discloses a method for extracting sonchuside A from chicory, its product and application. Sonchuside A is extracted and prepared from chicory, and the sonchuside A has potential inhibitory activity against xanthine oxidase.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A method for extracting sonchuside A from chicory, comprising the following steps:

[0008] (1) Take the dried above-ground part of chicory, crush it, add 3 - 10 times the volume of an ethanol solution with a volume fraction of 60 - 65% for soaking extraction. After extraction, separate the ethanol extract. Extract 2 - 5 times, combine the ethanol extracts, and recover ethanol by reduced pressure distillation to obtain a chicory extraction solution;

[0009] (2) Take the chicory extraction solution obtained in step (1), extract it with an ethyl acetate solvent to obtain an ethyl acetate extraction part. Take the ethyl acetate extraction part and recover ethyl acetate by reduced pressure distillation to obtain an extract;

[0010] (3) Take the extract obtained in step (2), dissolve it and mix it with silica gel with a weight 2 - 5 times that of the extract, then dry it in the air, load it into a glass chromatography column, use a mixed solvent of dichloromethane and methanol as an eluent, and perform separation by glass chromatography. The samples flowing out are combined after being detected by thin-layer chromatography to obtain an extract;

[0011] (4) Take the extract obtained in step (3), and perform separation by medium-pressure C18 column chromatography to obtain sonchuside A.

[0012] Preferably, in step (1), the soaking extraction time with the ethanol solution is 48 - 96 h.

[0013] Preferably, in step (3), the volume ratio of dichloromethane to methanol is 9:1.

[0014] Preferably, in step (3), the extract is dissolved in methanol and then mixed with silica gel.

[0015] Preferably, in step (4), the eluent for medium-pressure C18 column chromatography separation is a methanol - aqueous solution, and gradient elution is adopted, with a gradient of 0 - 100%.

[0016] The present invention also provides sonchuside A prepared by the method for extracting sonchuside A from chicory described above.

[0017] The present invention also provides the application of the above-mentioned sonchuside A in the preparation of drugs for reducing uric acid.

[0018] The present invention also provides the application of the above-mentioned sonchuside A in the preparation of drugs for inhibiting the activity of xanthine oxidase.

[0019] The present invention provides a method for extracting sonchuside A from chicory, which can effectively extract the active ingredient sonchuside A.

[0020] It has been experimentally confirmed that sonchuside A extracted from chicory has potential inhibitory activity against xanthine oxidase and has a good affinity with xanthine oxidase, indicating that sonchuside A has potential uric acid-lowering effects.

[0021] The present invention clarifies that sonchuside A, one of the compounds in chicory with uric acid-lowering activity, is beneficial to improving the safety and effectiveness of the medicinal use of chicory and provides a solid theoretical basis for the development of new uric acid-lowering drugs or functional food ingredients. Description of the Drawings

[0022] Figure 1 is the hydrogen spectrum of Compound 1.

[0023] Figure 2 is the carbon spectrum of Compound 1.

[0024] Figure 3A is a schematic diagram of the inhibitory effect of allopurinol at different mass concentrations on xanthine oxidase.

[0025] Figure 3B is a schematic diagram of the inhibitory effect of kaempferol at different mass concentrations on xanthine oxidase.

[0026] Figure 4 is a schematic diagram of the molecular docking result of kaempferol and xanthine oxidase. Detailed Embodiments

[0027] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0028] Instrument materials: 1D and 2D NMR were measured on a Bruker DRX-400MHz nuclear magnetic resonance spectrometer; Agilent preparative HPLC system (Agilent LC1260 infinity, Agilent Technologies, USA); microplate reader (Thermo Scientific, USA); D101 macroporous resin (Shanghai Yuanye Bio-Technology Co., Ltd.); thin-layer chromatography silica gel, column chromatography silica gel (purchased from Qingdao Marine Chemical Factory); reverse-phase silica gel RP-18 (purchased from Merk, USA); chromatographically pure methanol (purchased from Fisher, USA); analytically pure methanol, dichloromethane, ethyl acetate, absolute ethanol (purchased from Xilong Chemical Co., Ltd.); gel chromatography column SephadexLH-20 (40–70μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden); PBS pH 7.5 (JR25996A, Shanghai Yuanye Bio-Technology Co., Ltd.); xanthine oxidase (JS277056, Shanghai Yuanye Bio-Technology Co., Ltd.); allopurinol (X27J10Y91606, Shanghai Yuanye Bio-Technology Co., Ltd.); xanthine (M06GB140821, Shanghai Yuanye Bio-Technology Co., Ltd.).

[0029] Preparation of Compound 1 in Example 1

[0030] The chicory used in this example was collected from Xinjiang Uygur Autonomous Region in August 2023.

[0031] A method for extracting sonchuside A from chicory, comprising the following steps:

[0032] (1) Take 5 kg of the above-ground part of chicory, crush it, add 5 times the volume of 65% ethanol solution, soak and extract at room temperature for 72 h. After extraction, separate the alcohol extract. Extract 3 times, combine the alcohol extracts, and recover ethanol by vacuum distillation until there is no ethanol smell to obtain a chicory extraction solution;

[0033] (2) Take the chicory extraction solution obtained in step (1), extract it with ethyl acetate solvent to obtain the ethyl acetate extraction part. Take the ethyl acetate extraction part and recover ethyl acetate by vacuum distillation to obtain 37.0 g of extract;

[0034] (3) Take the extract obtained in step (2), dissolve it in methanol and mix it with 2 - 5 times the weight of the extract of silica gel, then dry it. First load 500 g of silica gel (100 mesh) into a glass chromatography column, then put in the silica gel mixed with the extract. Use a mixed solvent of dichloromethane and methanol with a volume ratio of 9:1 as the eluent, and separate it with a 10 cm × 80 cm glass chromatography. The outflowing samples are detected by thin-layer chromatography and then combined to obtain 12.36 g of extract;

[0035] (4) Take the extract obtained in step (3), separate it by medium-pressure C18 column chromatography (5 cm × 30 cm), with the eluent being a methanol-aqueous solution, using gradient elution with a gradient of 0 to 100%, a flow rate of 30 mL / min, and the eluted samples were determined by thin-layer chromatography to obtain 42.9 mg of compound 1.

[0036] Example 2 Identification of Compound 1

[0037] Compound 1: 1 HNMR (400 MHz, DMSO-d 6 ) δ H 4.81 (3H, m, H-1,3,5), 4.32 (1H, t, J = 8.1 Hz, H-6), 4.00 (1H, d, J = 7.8 Hz, H-1'), 1.67 (3H, s, H-15), 1.42 (3H, s, H-14), 1.11 (3H, d, J = 7.0 Hz, H-13) 13 CNMR (125 MHz, DMSO-d 6 ) δ c 124.8 (C-1), 33.1 (C-2), 82.8 (C-3), 140.1 (C-4), 126.8 (C-5), 80.5 (C-6), 53.6 (C-7), 27.9 (C-8), 40.9 (C-9), 138.0 (C-10), 41.7 (C-11), 178.7 (C-12), 13.4 (C-13), 16.6 (C-14), 12.1 (C-15), 101.7 (C-1′), 73.9 (C-2′), 77.2 (C-3′), 70.5 (C-4′), 77.4 (C-5′), 61.5 (C-6′).

[0038] Combined Figure 1 with Figure 2 as shown, the NMR data of compound 1 was compared with the data in Document 1 (Peng Deqian, Gao Juan, Guo Xiumei, Wang Jinlan, Zhang Shujun. Study on the Chemical Constituents of the Root of Taraxacum mongolicum Hand.-Mazz. Chinese Traditional Patent Medicine, 2014, 36(07): 1462-1466), and they were basically the same. Therefore, compound 1 was identified as sonchuside A.

[0039]

[0040] Example 3 Testing the Hypouricemic Activity of Sonchuside A Prepared in this Example

[0041] 3.1 Determination of Hypouricemic Activity

[0042] The xanthine oxidase inhibitory activity of sonchuside A was tested according to the method of Reference 2 (Zhao Shouhuan, Yang Hui, Shi Guanying, Wang Xiaomin, Zhao Hongyuan, Wang Zhaogai. Optimization of the inhibitory effects of three natural products on xanthine oxidase by response surface methodology. Science and Technology of Food Industry, 2018, 39(5): 230-234.). Allopurinol was used as the positive control. First, 50 μL of the sample to be tested and 25 μL of xanthine oxidase solution with a concentration of 0.02 U / mL were successively added to a 96-well plate. After shaking and mixing for 30 s, it was incubated at 25 °C for 5 min. Then, 150 μL of xanthine solution with a concentration of 0.48 mmol / L was added, and it was shaken again for 30 s. Subsequently, it was continued to be incubated at 25 °C for 25 min. Finally, the absorbance value at 290 nm was measured. Formula (1) was used to calculate the inhibition rate of XOD activity. With the help of IBM SPSS Statistics 23 software, the half-maximal inhibitory concentration (IC 50 value) of compound onchuside A against XOD was calculated based on the inhibitory concentration and inhibition rate of different samples.

[0043] Inhibition rate of enzyme activity / % = [1 - (F1–F2) / (F3 - F4)] × 100% (1)

[0044] The meanings of the parameters in the formula are as follows: F1 represents the absorbance of the sample group; F2 represents the absorbance of the control group (the absorbance measured by replacing the XOD solution with an equal volume of buffer); F3 is the absorbance of the standard group (the absorbance obtained by replacing the sample solution with an equal volume of PBS buffer); F4 is the absorbance of the standard control group (the absorbance measured by replacing the sample XOD solution with an equal volume of PBS buffer).

[0045] All experiments were repeated 3 times, and IBM SPSS Statistics 23 was used for statistical analysis.

[0046] The inhibitory ability of allopurinol at different mass concentrations on xanthine oxidase is as Figure 3A shown. The inhibitory ability of sonchuside A at different mass concentrations on xanthine oxidase is as Figure 3B shown. It can be observed from Figure 3B that as the mass concentration of sonchuside A gradually increases, its inhibitory effect on xanthine oxidase shows an increasingly significant enhancement trend. Combining with Table 1, the IC 50 value of sonchuside A is 49.0 μg / mL. Comparing it with the positive control drug allopurinol (IC 50When compared with allopurinol (at a concentration of 30.0 μg / mL), it can be clearly seen that the two are relatively close in inhibitory activity. Such results strongly confirm the potential uric acid-lowering activity of sonchuside A. This finding has certain similarities with the inhibitory effect of flavonoids on xanthine oxidase in previous studies, further providing strong evidence to support the view that certain compounds in chicory have potential uric acid-lowering effects.

[0047] Table 1 IC of sonchuside A and allopurinol on xanthine oxidase 50

[0048]

[0049] 3.2 Molecular docking

[0050] The crystal structure of xanthine oxidase (PBD ID: 1FIQ) was obtained from the Protein Data Bank PDB (http: / / www.rcsb.org / ). The PyMOL software was used to preprocess the protein, separating and extracting the original ligand from the crystal structure and removing the water molecules therein. Subsequently, the AutoDock software was used to add hydrogen and Gasteriger charges to the protein and save it in the PDBQT format. For the ligand structure, conformational optimization was performed using the MM2 force field in the ChemBio3D Ultra software and saved after output in the MOL2 format. Similarly, it was processed using AutoDock and saved in the PDBQT format. The docking experiment was carried out using the AutoDock Vina algorithm, and then PyMOL was used to draw the conformation of the ligand-enzyme combination. When the binding energy < -5 kJ·mol -1 -1, it means that the ligand molecule can spontaneously bind to the receptor protein.

[0051] As Figure 4 shown, sonchuside A can accurately enter the active core region of xanthine oxidase and mainly achieve the effect of inhibiting the activity of xanthine oxidase through the form of hydrogen bond interaction with the amino acid residues Gln-349 and Ser-347 of xanthine oxidase. The data shows that the binding energy between sonchuside A and xanthine oxidase reaches -8.0 kJ / mol. Given that this binding energy is lower than the index of 0 kJ / mol, this indicates that this component has the ability to spontaneously bind to the target protein in its natural state, strongly proving that sonchuside A has good affinity with xanthine oxidase, which also means that it has strong potential efficacy in inhibiting the activity of xanthine oxidase.

[0052] Those skilled in the art should note that the embodiments described in this invention are merely exemplary, and various other substitutions, changes, and improvements can be made within the scope of this invention. Therefore, this invention is not limited to the above embodiments, but is only defined by the claims.

Claims

1. A method for extracting chicory glycoside A from chicory, characterized in that The following steps are involved: (1) Crush the above-ground part of dried chicory, add 3 to 10 times the volume fraction of 60 to 65% ethanol solution for soaking and extraction, separate the ethanol extract after extraction, extract 2 to 5 times, combine the ethanol extracts, and recover ethanol by vacuum distillation to obtain a chicory extract solution; (2) extracting the chicory extract solution obtained in step (1) with ethyl acetate to obtain an ethyl acetate extract portion, and subjecting the ethyl acetate extract portion to vacuum distillation to recover ethyl acetate to obtain an extract; (3) taking the extract obtained in step (2), dissolving it and mixing it with 2 to 5 times the weight of silica gel, then drying it, loading it into a glass chromatography column, using a mixed solvent of dichloromethane and methanol as an eluent, separating it by glass chromatography, and combining the effluent samples after thin layer chromatography detection to obtain an extract; (4) The extract obtained in step (3) is separated by medium-pressure C18 column chromatography to obtain choleroside A.

2. The method for extracting chicoryside A from chicory according to claim 1, characterized in that: In step (1), the ethanol solution soaking extraction time is 48 to 96 hours.

3. The method for extracting chicoryside A from chicory according to claim 1, characterized in that: In step (3), the volume ratio of dichloromethane to methanol is 9:

1.

4. The method for extracting chicoryside A from chicory according to claim 1, characterized in that: In step (3), the extract is dissolved in methanol and then mixed with silica gel.

5. The method for extracting chicoryside A from chicory according to claim 1, characterized in that: In step (4), the eluent for medium-pressure C18 column chromatography separation is a methanol-water solution, and gradient elution is adopted with a gradient of 0-100%.

6. Chicoryside A prepared by the method for extracting chicoryside A from chicory according to any one of claims 1 to 5.

7. Use of choleroside A according to claim 6 in the preparation of uric acid-lowering drugs.

8. Use of the cholerae glycoside A according to claim 6 in the preparation of a drug for inhibiting xanthine oxidase activity.

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