Method for extracting esculetin from cichorium intybus as well as product and application of esculetin

Abstract

The invention discloses a method for extracting esculetin 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, separating out water extract after extraction, extracting for 2-5 times, and merging the water extract to obtain a cichorium intybus water extract; (2) taking the chicory water extraction solution obtained in the step (1), and carrying out adsorption enrichment by using D101 macroporous resin chromatography; then eluting with a methanol solution with the volume fraction of 30%, carrying out reduced pressure distillation on the obtained eluent, and recovering the solvent to obtain two parts of extractum, namely a part I extractum and a part II extractum; and (3) taking the extract of the part I, taking 100% methanol as an eluent, and separating by gel chromatography to obtain esculetin. According to the invention, esculetin is extracted and prepared from cichorium intybus, and the esculetin has potential inhibitory activity on alpha-glucosidase.

Description

Technical Field

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

[0002] Chicory (Cichorium intybus L.), belonging to the genus Cichorium of the Compositae family, is a perennial herbaceous plant, which has a unique position in the fields of medicine, food, etc. Common aliases include coffee grass, coffee radish, blue chrysanthemum, etc. In the field of traditional Chinese medicine, chicory is used as medicine with its dried aerial parts or roots. It tastes slightly bitter, salty and cool, and has significant effects such as clearing the liver and gallbladder, strengthening the stomach and promoting digestion, and diuretic and detumescence. It is commonly used in the treatment of diseases such as damp-heat jaundice, stomachache with less food intake, and edema with less urine output.

[0003] With the in-depth study of modern scientific research, various pharmacological effects of chicory have been gradually revealed. Numerous studies have shown that chicory has important physiological activities such as hypoglycemic, liver-protecting and antioxidant effects. For example, in the study of "Effect of chicory extract on blood glucose and oxidative stress in diabetic mice", it was confirmed through animal experiments and cell experiments that chicory extract can effectively regulate blood glucose levels, improve insulin resistance, and has potential prevention and treatment effects on diabetes and its complications. Chicory is also widely used as high-quality forage in the fields of agriculture and animal husbandry, and is often used as a vegetable in the food industry. Moreover, health care drinks and functional foods developed from it have attracted much attention in the international health food industry, and the market prospect is broad.

[0004] The chemical composition of chicory is extremely rich, including polysaccharides, terpenoids (especially sesquiterpenoids), flavonoids, phenolic acid compounds, as well as various vitamins and metal elements, etc. To further deeply explore the medicinal value of chicory and expand its application in the field of medicine and health care, it is very necessary to conduct in-depth research on the hypoglycemic active ingredients in chicory and clarify the application potential of hypoglycemic active ingredients in reducing blood glucose, which will provide more ideas for the development of new hypoglycemic drugs or functional food ingredients. Summary of the Invention

[0005] The present invention discloses a method for extracting esculetin from chicory, its products and applications. Esculetin is extracted and prepared from chicory, and this esculetin has potential inhibitory activity against α-glucosidase.

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

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

[0008] (1) Take the dried aerial parts of chicory, crush them, add 3 to 10 times the amount of water for soaking extraction, separate the water extract after extraction, extract 2 to 5 times, and combine the water extracts to obtain a chicory water extract solution;

[0009] (2) Take the chicory water extract solution obtained in step (1), and adsorb and enrich it by D101 macroporous resin chromatography; then elute it with a methanol solution with a volume fraction of 30%, to obtain two components, Fr.1 and Fr.2. After recovering the solvent by reduced pressure distillation respectively, two parts of extract are obtained, corresponding to part I extract and part II extract;

[0010] (3) Take the part I extract obtained in step (2), use 100% methanol as the eluent, and separate it by gel chromatography to obtain esculetin.

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

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

[0013] The present invention also provides the application of the above-mentioned esculetin in the preparation of hypoglycemic drugs.

[0014] The present invention also provides the application of the above-mentioned esculetin in the preparation of drugs for inhibiting α-glucosidase activity.

[0015] The present invention provides a method for extracting esculetin from chicory. This method mainly uses water immersion extraction, which is simple in operation, low in cost and efficient in extraction.

[0016] It has been experimentally confirmed that the esculetin extracted from chicory has potential inhibitory activity against α-glucosidase, the inhibition type is non-competitive inhibition, and it has good binding property with α-glucosidase protein, indicating that this esculetin has potential hypoglycemic effect.

[0017] The present invention clarifies one of the compounds with hypoglycemic activity in chicory, which is more conducive to controlling the stability, safety and effectiveness of the compound, and provides a solid foundation for the development of new hypoglycemic drugs or functional food ingredients. Description of the Drawings

[0018] Figure 1A It is a graph showing the relationship between the concentration of esculetin and α-glucosidase.

[0019] Figure 1B It is a Lineweaver-Burk plot of the α-glucosidase inhibition of esculetin.

[0020] Figure 2A It is a schematic diagram of the molecular docking result of esculetin and α-glycosidase enzyme protein.

[0021] Figure 2B It is a schematic diagram of the molecular docking result of acarbose and α-glycosidase enzyme protein. Detailed implementation manners

[0022] 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, rather than 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.

[0023] Instrument materials: 1D and 2D NMR were measured on a Bruker DRX-400MHz nuclear magnetic resonance spectrometer; an Agilent preparative HPLC system (Agilent LC1260 infinity, Agilent Technologies, USA); an enzyme-linked immunosorbent assay instrument (Thermo Scientific, USA); a gel chromatography column Sephadex LH-20 (40–70μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden); methanol (AR, Guangdong Xilong Technology Co., Ltd.); PBS powder (Beijing Regene Biotechnology Co., Ltd.); α-glucosidase (Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.); acarbose (109A032, Beijing Solarbio Science & Technology Co., Ltd.); 4-pNPG (4-nitrophenyl-α-D-glucopyranoside) (N0493, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan).

[0024] Example 1 Preparation of Compound 1

[0025] The chicory used in this example was collected from Xinjiang in August 2023.

[0026] A method for extracting esculetin from chicory, comprising the following steps:

[0027] (1) Take the dried above-ground part of 5 kg of 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;

[0028] (2) Take the chicory aqueous extract solution prepared in step (1), adsorb and enrich it by chromatography on a 50 cm × 200 cm D101 macroporous resin; then elute it successively with methanol solutions with volume fractions of 30%, 60% and 100%. Among them, the methanol solution with a volume fraction of 30% is eluted, analyzed by thin-layer chromatography and combined to obtain 2 components, Fr.1 and Fr.2. After the solvents are recovered by reduced pressure distillation respectively, two parts of extracts are obtained, corresponding to part I extract and part II extract, where: part I extract is 118 g, and part II extract is 108 g;

[0029] (3) Take the I part of 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 2.0 g of compound 1.

[0030] In addition, in step (2), elute with a 100% methanol solution, and for the eluted part, after recovering the solvent by reduced pressure distillation, obtain the III part of the extract. Use 100% methanol as the eluent for the III part of the extract and separate it with a gel chromatograph to obtain 0.56 g of kaempferol.

[0031] Example 2 Identification of Compound 1

[0032] Compound 1: Pale yellow crystals, with the molecular formula C 9 H 6 O 4 , and the degree of unsaturation is 7. From the 1 H-NMR data, it can be known that the compound has two benzene ring singlet proton signals [δ H 6.98 (1H, s, H-5), 6.84 (1H, s, H-8)], and two cis-olefinic hydrogen proton signals [δ H 7.86 (1H, d, J = 9.3 Hz, H-4), 6.15 (1H, d, J = 9.1 Hz, H-3)]. From the 13 C-NMR data, it can be seen that there are 9 carbon signals, among which 1 is an α,β-unsaturated ketone carbon signal (δ C 161.4), and 8 aromatic carbon signals (δ C 151.5, 149.1, 144.9, 143.6, 112.5, 111.5, 110.9, 103.0). The specific NMR data of this compound are as follows: 1 H NMR (400 MHz, Pyridine-d 5 ) δ 6.98 (1H, s, H-5), 6.84 (1H, s, H-8), 7.86 (1H, d, J = 9.3 Hz, H-4), 6.15 (1H, d, J = 9.1 Hz, H-3). 13 C NMR (Pyridine-d 5 , 100 MHz) δ 161.4 (C-2), 151.5 (C-7), 149.1 (C-9), 144.9 (C-4), 143.6 (C-6), 112.5 (C-5), 111.5 (C-3), 110.9 (C-10), 103.0 (C-8).

[0033] By consulting the literature, it is known that the NMR data of this compound are basically consistent with the compound data reported in Literature 1 (Lu Xiaoli, Qiao Ying, Zhang Xianmin, et al. Chemical Constituents of Ceratophyllum demersum L. (English). Acta Botanica Yunnanica, 2007, 02: 263-264). Therefore, this compound was identified as esculetin.

[0034]

[0035] Example 3 Hypoglycemic Activity Test of Esculetin Prepared in this Example

[0036] 3.1 Hypoglycemic Activity Determination

[0037] The α-glucosidase inhibitory activity test of esculetin was carried out with reference to the method of Literature 2 (Zhao M, Xian X Y, Yan M Q, et al. A new oleanane-type triterpenoid saponin with α-glucosidase inhibitory activity from Camellia nitidissima. J Asian Nat Prod Res, 2023, 25(9): 890-898.). Acarbose was used as the positive control. Esculetin was made into a 1% DMSO stock solution, and then esculetin and acarbose were diluted into solutions with different concentrations with phosphate buffer (pH = 6.8) for the experiment. 50 μL of esculetin or acarbose with different concentrations (1, 10, 50, 100, 200, and 300 μM) was added to a 96-well plate, 50 μL of buffer was added, and then 100 μL of α-glucosidase solution with a concentration of 0.2 U / mL was added. The mixture was incubated at 37 °C for 15 minutes, then 50 μL of PNPG solution with a concentration of 2 mM was added, and the interaction was carried out again at 37 °C. The absorbance value was detected at 405 nm.

[0038] α-glucosidase Inhibition Rate (%) = [Ac - (As - Ab) / Ac] × 100%

[0039] Ac: represents the absorbance of the control group without the sample, Ab: represents the absorbance of the control group without PNPG, As: represents the absorbance of the sample to be tested.

[0040] Enzymatic inhibition kinetics assay of the compound: When the enzyme concentration was 0.2, 0.4, 0.6, 0.8 U / mL, the inhibitory activity of the enzyme against different concentrations of the compound (0 and 300 μM) was tested, and the reversibility of the inhibition was studied; The reaction rates were tested using four different concentrations of PNPG (0.5, 1.0, 1.5, 2.0 mM) and 4 different concentrations of the compound (0, 89.0, 198.0, 396.0 μM), the double-reciprocal curve of the compound was obtained, and the type of its inhibitory effect was evaluated.

[0041] All experiments were repeated 3 times and statistical analysis was performed using IBM SPSS Statistics 23.

[0042] The results showed that the IC 50 value of esculetin was 198.01 ± 8.40 μmol, and the IC 50 value of acarbose was 150.12 ± 3.63 μmol. Esculetin had potential inhibitory activity against α-glucosidase.

[0043] The inhibition kinetics of esculetin was analyzed. It could be seen from Figure 1A that all the straight lines passed through the origin, and the slope of the straight line decreased with the increase of the sample concentration. This indicated that the inhibitory effect of esculetin on α-glucosidase was reversible. In addition, Figure 1B esculetin intersected with the y-axis in

[0044] 3.2 Molecular docking

[0045] The crystal structure of α-glucosidase protein (PDB ID 3A4A) was downloaded from the Protein Data Bank, and the protein was preprocessed using PyMOL software. The original ligand was extracted from the crystal structure and water molecules were removed. Hydrogen and Gasteriger charges were added to the protein using AutoDock software and saved in PDBQT format. The ligand structure was conformationally optimized using the MM2 force field in ChemBio3D Ultra software, saved in MOL2 format and then output. Similarly, it was processed using AutoDock and saved in PDBQT format. To evaluate the accuracy of the docking method, the extracted native ligand molecule α-D-glucose was docked into the binding pocket of the receptor, and the RMSD value was calculated using Viaual Molecular Dynamics software. Generally, it was considered that It can be considered that the docking method has a relatively high accuracy. The ligand docking space is set with the active binding site of the co-crystal of α-glucosidase, and the docking experiment is carried out using the AutoDock Vina algorithm. Then, PyMOL is used to draw the conformational mode of the ligand binding to the enzyme. The binding energy < 0 indicates that the ligand molecule can spontaneously bind to the receptor protein, and the binding energy < -5.0 kJ·mol -1 , indicating good binding.

[0046] Such as Figure 2A , esculetin forms hydrogen bond interactions with Asp-242, Asp-307, His-280, LEU-313, THR-310 and TYR-158. The binding energy of esculetin is -7.2 kJ / mol, and the binding energy < -5.0 kJ / mol, indicating that compound 1 has good binding with α-glucosidase protein. The docking results of the binding energy of compound 1 are shown in Table 1. The molecular docking results of acarbose are as Figure 2B shown.

[0047] Table 1 Docking results of compounds

[0048]

[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 aescin 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 performing adsorption enrichment on D101 macroporous resin chromatography; then eluting with a 30% by volume methanol solution to obtain two components Fr. 1 and Fr. 2, respectively performing reduced pressure distillation to recover the solvent to obtain two parts of extracts, which correspond to part I extract and part II extract; (3) Taking the extract of part I obtained in step (2), separating it by gel chromatography using 100% methanol as eluent to obtain esculetin.

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

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

4. Use of the aescin according to claim 3 in the preparation of hypoglycemic drugs.

5. Use of the aescin according to claim 3 in the preparation of a drug for inhibiting α-glucosidase activity.

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