Method for extracting kaempferol from chicory as well as product and application thereof

Abstract

The invention discloses a method for extracting kaempferol from cichorium intybus and a product and application thereof, and the method comprises the following steps: (1) crushing the overground part of the cichorium intybus, adding water to soak and extract, and merging water extracts after extraction to obtain a cichorium intybus water extract; (2) taking the chicory water extraction solution prepared in the step (1), and carrying out adsorption enrichment by using D101 macroporous resin chromatography; sequentially eluting with methanol solutions with the volume fractions of 30%, 60% and 100%, and carrying out reduced pressure distillation on the elution part of the methanol solution with the volume fraction of 100% to recover the solvent, so as to obtain an extract; and (3) taking the extract prepared in the step (2), taking 100% methanol as an eluent, and separating by using a gel chromatograph to obtain kaempferol. The invention provides a method for extracting kaempferol from chicory, and verifies that kaempferol has a potential inhibition effect 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 kaempferol from chicory, as well as its products and applications. Background Art

[0002] Hyperuricemia has become a global health problem and is the fourth most common underlying metabolic disease after hypertension, hyperlipidemia, and hyperglycemia. It is closely related to the occurrence and development of gout, kidney diseases, and cardiovascular diseases. Hyperuricemia is mainly caused by disorders of purine metabolism, leading to excessive production of uric acid and insufficient secretion of uric acid. The production of uric acid is related to xanthine oxidase mainly present in the liver, which is a key enzyme related to purine metabolism. Inhibiting the activity of xanthine oxidase is the key to improving hyperuricemia because it can catalyze the oxidation of xanthine or hypoxanthine to produce uric acid. With the improvement of living standards and the change of dietary structure, the prevalence of hyperuricemia has been increasing year by year. Therefore, there is an urgent clinical need to find safe and effective drugs for reducing uric acid.

[0003] Chicory Cichorii Herba / Cichorii Radix is a traditional medicinal and edible plant, which is the dried aerial part or root of Cichorium glandulosum Boiss.et Huet. or Cichorium intybus L. of the Compositae family. It is recorded that chicory has the effects of clearing the liver and gallbladder, strengthening the stomach and promoting digestion, and diuretic and detumescence, and has a long history of folk medicine. Modern research shows that it has potential therapeutic effects on hyperuricemia, hyperglycemia, hyperlipidemia, etc. Existing research has shown that chicory is rich in various bioactive components, such as flavonoids, phenolic acids and other compounds, and these components may exert anti-hyperuricemic activity by intervening in the process of uric acid production and excretion. For example, certain flavonoid compounds can inhibit the activity of xanthine oxidase and reduce the production of uric acid. However, the material basis of the anti-hyperuricemic effect of chicory and the anti-hyperuricemic mechanism of its specific compounds have not been fully elucidated and still need in-depth research.

[0004] Therefore, it is very necessary to conduct in-depth research on the anti-hyperuricemic active components in chicory and clarify its anti-hyperuricemic mechanism for the safe utilization of chicory to develop new anti-hyperuricemic drugs or functional foods. Summary of the Invention

[0005] The present invention discloses a method for extracting kaempferol from chicory, as well as its products and applications. Kaempferol is extracted and prepared from chicory, and this kaempferol has potential anti-hyperuricemic activity.

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

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

[0008] (1) Grind the aerial part of dried chicory, add 3 - 10 times of water for soaking extraction. After extraction, separate the aqueous extract, extract 2 - 5 times, and combine the aqueous extracts to obtain the chicory aqueous extract solution.

[0009] (2) Take the chicory aqueous extract solution prepared in step (1), perform adsorption enrichment by D101 macroporous resin chromatography; then elute successively with methanol solutions with volume fractions of 30%, 60% and 100%. The elution part with 100% methanol solution is concentrated under reduced pressure to recover the solvent to obtain an extract.

[0010] (3) Take the extract prepared in step (2), use 100% methanol as the eluent, and separate by gel chromatography to obtain kaempferol.

[0011] Preferably, in step (1), the time for water soaking extraction is 48 - 96 h.

[0012] The present invention also provides kaempferol prepared by the method for extracting kaempferol from chicory as described above.

[0013] The present invention also provides the application of the above-mentioned kaempferol in the preparation of drugs for reducing uric acid.

[0014] The present invention also provides the application of the above-mentioned kaempferol in the preparation of drugs for inhibiting the activity of xanthine oxidase

[0015] The present invention provides a method for extracting kaempferol from chicory, which mainly uses water immersion extraction, has simple operation, low cost and low energy consumption.

[0016] It has been experimentally confirmed that kaempferol extracted from chicory has potential inhibitory activity against xanthine oxidase and has good affinity for p - coumaric acid and xanthine oxidase, indicating that kaempferol has potential uric acid - lowering effect.

[0017] The present invention identifies one of the compounds in chicory that has uric acid - lowering effect and clarifies its action mechanism, providing a theoretical basis for the development of new uric acid - lowering drugs or functional food ingredients. Description of the Drawings

[0018] Figure 1 is the hydrogen spectrum of compound 1.

[0019] Figure 2 is the carbon spectrum of compound 1.

[0020] Figure 3A is the schematic diagram of the inhibitory effect of allopurinol with different mass concentrations on xanthine oxidase.

[0021] Figure 3BIt is a schematic diagram of the inhibitory effect of kaempferol at different mass concentrations on xanthine oxidase.

[0022] Figure 4 It is a schematic diagram of the molecular docking result of kaempferol and xanthine oxidase. Detailed implementation manners

[0023] 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 part of the embodiments of the present invention, rather than all of the embodiments. 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 protection scope of the present invention.

[0024] Instruments and 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, petroleum ether, 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.).

[0025] Example 1 Preparation of Compound 1

[0026] The chicory used in this example was collected from Xinjiang in August 2023.

[0027] A method for extracting kaempferol from chicory, comprising the following steps:

[0028] (1) Take 5 kg of the above-ground part of dry chicory, crush it, add 5 times the amount of pure water, soak and extract at room temperature for 72 h, separate the aqueous extract after extraction, extract 3 times, and combine the aqueous extracts to obtain a chicory aqueous extract solution;

[0029] (2) Take the chicory aqueous extract solution obtained in step (1), and perform adsorption enrichment by chromatography on 50 cm × 200 cm D101 macroporous resin; then elute successively with methanol solutions with volume fractions of 30%, 60% and 100%. For the elution part with 100% methanol solution, after recovering the solvent by vacuum distillation, 162 g of extract is obtained;

[0030] (3) Take the extract obtained in step (2), use 100% methanol as the eluent, and separate it with a 5 cm × 200 cm Sephadex LH-20 gel chromatograph to obtain 0.56 g of compound 1.

[0031] In addition, for the elution part obtained by eluting with 30% methanol solution in step (2), after thin-layer chromatography analysis and merging, two parts are obtained. After recovering the solvent by vacuum distillation for the two parts respectively, two parts of extract are obtained, namely 118 g of part I extract and 108 g of part II extract. Take part I extract, use 100% methanol as the eluent, and separate it with a 5 cm × 200 cm Sephadex LH-20 gel chromatograph to obtain 2.0 g of esculetin.

[0032] Example 2 Identification of Compound 1

[0033] Compound 1: 1 HNMR (400 MHz, DMSO-d 6 ) δ H 8.06 (2H, d, J = 8.9 Hz, H-2',6'), 6.94 (2H, d, J = 8.9 Hz, H-3',5'), 6.45 (1H, d, J = 2.1 Hz, H-8), 6.20 (1H, d, J = 2.1 Hz, H-6); 13 CNMR (125 MHz, DMSO-d 6 ) δ c 176.4 (C-4), 164.4 (C-7), 161.2 (C-9), 159.6 (C-4'), 156.6 (C-5), 147.2 (C-2), 136.2 (C-3), 129.9 (C-2',6'), 122.2 (C-1'), 115.9 (C-3',5'), 103.5 (C-10), 98.7 (C-6), 93.9 (C-8).

[0034] Combined with Figure 1 and Figure 2As shown, the NMR data of Compound 1 were compared with those in Literature 1 (Zhang, H., Fang, W. T., Li, Y., Kong, Q. H., Fang, C. W., Luo, H., Liu, S. J.. Chemical constituents from the aerial parts of Rubia cordifolia L. with their NO inhibitory activity. Nat Prod Res, 2023, 38(5), 711–718), and they were basically the same. Therefore, the compound was identified as kaempferol.

[0035]

[0036] Example 3 Testing the uric acid-lowering activity of kaempferol prepared in this example

[0037] 3.1 Testing the inhibitory activity against xanthine oxidase

[0038] Refer to the method in Literature 2 (Zhao, S. H., Yang, H., Shi, G. Y., Wang, X. M., Zhao, H. Y., Wang, Z. G. 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.), with slight modifications. Allopurinol was used as the positive control. Add 50 μL of the sample to be tested and 25 μL of xanthine oxidase solution with a concentration of 0.02 U / mL into a 96-well plate, shake for 30 s, incubate at 25 °C for 5 min, then add 150 μL of xanthine solution with a concentration of 0.48 mmol / L, shake for 30 s, and incubate at 25 °C for 25 min. Measure the absorbance at 290 nm. Calculate the inhibition rate of XOD activity according to formula (1), and use IBM SPSS Statistics 23 software to calculate the half-maximal inhibitory concentration (IC 50 value) of kaempferol against XOD based on the inhibitory concentration and inhibition rate of different samples.

[0039] Inhibition rate of enzyme activity / % = [1 - (F 1 – F 2 ) / (F 3 - F 4 )] × 100% (1)

[0040] Where: F 1 is the absorbance of the sample group; F 2 is the absorbance of the control group (equal volume of buffer instead of XOD solution); F 3 is the absorbance of the standard group (equal volume of PBS buffer instead of the sample solution); F 4 is the absorbance of the standard control group (equal volume of PBS buffer instead of the sample and XOD solution).

[0041] All experiments were repeated three times, and the corresponding half-maximal inhibitory concentration (IC50) values were obtained using IBM SPSS Statistics 23 for statistical analysis.

[0042] The inhibitory ability of allopurinol at different mass concentrations against xanthine oxidase is as Figure 3A shown. The inhibitory ability of kaempferol at different mass concentrations against xanthine oxidase is as Figure 3B shown, and the inhibitory effect of kaempferol on xanthine oxidase gradually increases with the increase in mass concentration. As shown in Table 1, the IC 50 value of kaempferol against xanthine oxidase is 43.0 μg / mL, which is similar to the inhibitory activity of the positive control drug allopurinol (IC 50 value of 29.5 μg / mL). This result demonstrates the potential uric acid-lowering activity of kaempferol.

[0043] Table 1 IC 50

[0044]

[0045] 3.2 Molecular docking

[0046] Download the crystal structure of xanthine oxidase (PBDID: 1FIQ) from the Protein Data Bank PDB (http: / / www.rcsb.org / ), and preprocess the protein using PyMOL software. Extract the original ligand from the crystal structure and remove water molecules. Add hydrogen and Gasteriger charges to the protein using AutoDock software and save it in PDBQT format. Optimize the ligand structure using the MM2 force field in ChemBio3D Ultra software, save it in MOL2 format, and then output it. Similarly, process and save it in PDBQT format using AutoDock. Then set the ligand docking space based on the active binding site co-crystallized with xanthine oxidase, perform docking experiments using the AutoDock Vina algorithm, and then use PyMOL to draw the conformational mode of ligand and enzyme binding. The binding energy < -5.0 kJ·mol -1 , indicating that the ligand molecule can spontaneously bind to the receptor protein.

[0047] The binding energy of kaempferol to xanthine oxidase is -9.2 kJ / mol, and the binding energy is lower than -5.0 kJ / mol, suggesting that the component can spontaneously bind to the target protein, indicating that kaempferol and p-coumaric acid have good affinity for xanthine oxidase and strong ability to inhibit xanthine oxidase.

[0048] The interaction mode of kaempferol with xanthine oxidase is as follows Figure 4 As shown, kaempferol can enter the active center of xanthine oxidase and mainly form hydrogen bond interactions with the amino acid residues of xanthine oxidase, thereby inhibiting the activity of xanthine oxidase. It can form hydrogen bond forces with amino acid residues Ile-264, Glu-402, Leu-404, and Glu-263. The molecular docking results show that kaempferol has good binding affinity with xanthine oxidase, can block the interaction between the enzyme and the substrate, and thus achieve the effect of reducing enzyme activity.

[0049] Those skilled in the art should note that the embodiments described in the present invention are merely exemplary, and various other substitutions, changes, and improvements can be made within the scope of the present invention. Therefore, the present invention is not limited to the above embodiments, but is only defined by the claims.

Claims

1. A method for extracting kaempferol from chicory, characterized in that The following steps are involved: (1) taking the above-ground part of dried chicory, crushing it, adding 3 to 10 times of water to soak and extract, separating the water extract after extraction, extracting 2 to 5 times, combining the water extracts, and obtaining a chicory water extract solution; (2) taking the chicory water extract obtained in step (1), and enriching it by adsorption using D101 macroporous resin chromatography; then eluting it with methanol solutions with volume fractions of 30%, 60% and 100% in sequence, wherein the eluted portion of the 100% methanol solution is subjected to reduced pressure distillation to recover the solvent to obtain an extract; (3) Taking the extract obtained in step (2), separating it by gel chromatography using 100% methanol as eluent to obtain kaempferol.

2. The method for extracting kaempferol from chicory according to claim 1, characterized in that: In step (1), the water immersion extraction time is 48 to 96 hours.

3. Kaempferol prepared by the method for extracting kaempferol from chicory according to claim 1 or 2.

4. Use of the kaempferol according to claim 3 in the preparation of uric acid-lowering drugs.

5. Use of the kaempferol according to claim 3 in the preparation of a drug for inhibiting xanthine oxidase activity.

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