A highly water-soluble hyperoside product and its preparation method

Abstract

The present invention discloses a highly water-soluble hyperoside product, which is an inclusion compound formed by an open-ring cucurbituril and hyperoside. The open-ring cucurbituril is composed of a glycoluril tetramer and a terminal aromatic group, and the terminal aromatic group is modified by a sodium propanesulfonate side chain, and its water solubility is significantly improved relative to that of the cucurbituril. The present invention utilizes the adaptive inclusion complex of the C-shaped adjustable cavity of the open-ring cucurbituril and hyperoside, coordinated with hydrogen bonds and π-π interactions, etc., to obtain a stable inclusion compound. The product significantly improves the water solubility and in vitro antioxidant activity of hyperoside, is expected to expand its scope of use and promote its in-depth development in medicine and food applications. At the same time, the product preparation method is simple, the conditions are mild, and it is suitable for industrial production and market promotion applications.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine and food, and particularly relates to a highly water-soluble hyperoside product and a preparation method thereof. Background Art

[0002] Hyperoside (chemical structure as follows) is a natural flavonoid glycoside extracted from plants. It is widely found in the fruits or whole herbs of plants such as Hypericaceae, Campanulaceae, Rosaceae, Leguminosae and Ericaceae. It has a variety of pharmacological activities, including anti-inflammatory, antiviral, organ protection, antioxidant, and anti-tumor. However, due to the low water solubility of hyperoside and its almost insoluble in water, it has brought a series of adverse effects, such as extremely low oral bioavailability, making it difficult to develop it in depth. According to literature searches, nanoparticle delivery systems (Food Chem., 2021, 364: 130335.) and nanocrystals (LWT-Food Sci. Technol., 2020, 133: 109869.) are used to encapsulate hyperoside to improve its bioavailability, but the preparation method is relatively complicated and its application is more difficult. Recently, cyclodextrin (Molecules, 2022, 27:2761) has also been used to increase the water solubility of hyperoside. However, its binding to hyperoside is weak, and it can only increase the water solubility of hyperoside by 7 times at most. Therefore, the pursuit of highly water-soluble hyperoside products remains an urgent need for further development of hyperoside for medical and food applications.

[0003] Summary of the Invention

[0004] The invention provides a stable hyperoside product with high water solubility and high antioxidant activity. The inclusion compound is an inclusion compound of hyperoside and an open-ring cucurbituril. The inclusion compound contains hyperoside and the open-ring cucurbituril and is prepared by an aqueous solution method. The inclusion compound comprises the following steps: adding hyperoside to an aqueous solution of an open-ring cucurbituril at a concentration of 0.01 to 0.1 mol / L at a temperature of 15 to 50° C., stirring while adding, and continuing stirring in the dark for 36 to 72 hours after the addition. Insoluble matter is then filtered off, and the product is evaporated to dryness under reduced pressure or freeze-dried to obtain a powdery solid inclusion compound of hyperoside and the open-ring cucurbituril. The molar ratio of the hyperoside to the open-ring cucurbituril is 1 to 3:1.

[0005] The open-ring cucurbituril is divided into two types: M1 and M2, and its structural formula is as follows:

[0006]

[0007] The acyclic cucurbiturils (ACBs) described in this invention are composed of a glycoluril tetramer unit and two aryl termini bearing anionic sulfonate groups. This structure endows them with a tunable C-shaped cavity and excellent water solubility. The cavity size can be fine-tuned according to the size of the guest molecule. The aryl termini can form π-π interactions with the guest molecule, and the polycarbonyl structure can form multiple hydrogen bonds with the guest. Therefore, the acyclic cucurbiturils have a strong inclusion capacity for neutral molecules or cations.

[0008] When poorly soluble drug molecules form inclusion complexes with open-ring cucurbiturils, their water solubility and stability can be significantly improved. They can also mask their unpleasant odor, reduce gastrointestinal irritation and adverse reactions, achieve controlled release, and enhance their bioavailability and antioxidant activity. Furthermore, studies have shown that open-ring cucurbiturils exhibit low toxicity in both in vitro and in vivo tests, thus holding considerable promise for their application in drug development.

[0009] The open-ring cucurbituril is prepared by referring to the method of existing literature (Nat. Chem., 2012, 4: 503.).

[0010] The highly water-soluble hyperoside product disclosed in the present invention can improve the stability of hyperoside through host-guest inclusion complexation, greatly increasing the water solubility of hyperoside and effectively enhancing its in vitro antioxidant activity. Relatively speaking, cyclodextrins, including β-cyclodextrin, methyl-β-cyclodextrin and hydroxypropyl-β-cyclodextrin, have relatively weak inclusion capacities for hyperoside and their solubilization effects are also poor. The preparation method of the hyperoside product disclosed in the present invention uses water as a solvent, avoiding the use of organic solvents. At the same time, the preparation method is simple, the conditions are mild, and it is suitable for industrial applications. The hyperoside product prepared by the present invention can be used as a new formulation of hyperoside in the fields of medicine and food. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the H NMR spectrum of hyperoside / open-ring cucurbituril M1 inclusion complex (600 MHz, D2O);

[0012] Figure 2 This is the H NMR spectrum of hyperoside / open-ring cucurbituril M2 inclusion complex (600 MHz, D2O);

[0013] Figure 3 The X-ray diffraction patterns of the inclusion complex prepared in Example 1, open-ring cucurbituril M1, hyperoside, and the physical mixture of open-ring cucurbituril M1 and hyperoside are compared, wherein (a) open-ring cucurbituril M1; (b) hyperoside; (c) the physical mixture of open-ring cucurbituril M1 and hyperoside; (d) the inclusion complex of open-ring cucurbituril M1 and hyperoside;

[0014] Figure 4 Comparison of infrared spectra of the inclusion complex, open-ring cucurbituril M1, hyperoside, and the physical mixture of open-ring cucurbituril M1 and hyperoside prepared in Example 1, wherein (a) open-ring cucurbituril M1; (b) hyperoside; (c) the physical mixture of open-ring cucurbituril M1 and hyperoside; (d) the inclusion complex of open-ring cucurbituril M1 and hyperoside;

[0015] Figure 5 The X-ray diffraction patterns of the inclusion complex prepared in Example 2, the open-ring cucurbituril M2, hyperoside, and the physical mixture of the open-ring cucurbituril M2 and hyperoside are compared, wherein (a) is the open-ring cucurbituril M2; (b) is hyperoside; (c) is the physical mixture of the open-ring cucurbituril M2 and hyperoside; and (d) is the inclusion complex of the open-ring cucurbituril M2 and hyperoside.

[0016] Figure 6 Comparison of infrared spectra of the inclusion complex prepared in Example 2, open-ring cucurbituril M2, hyperoside, and the physical mixture of open-ring cucurbituril M2 and hyperoside, wherein (a) open-ring cucurbituril M2; (b) hyperoside; (c) the physical mixture of open-ring cucurbituril M2 and hyperoside; (d) the inclusion complex of open-ring cucurbituril M2 and hyperoside. DETAILED DESCRIPTION

[0017] The method described in the present invention is further described below by way of examples, but the scope of protection of the present invention is not limited by the examples. Unless otherwise specified, the reagents used in the examples are conventional commercial reagents or reagents prepared according to conventional methods, and the methods used are conventional methods unless otherwise specified.

[0018] Example 1: Preparation of hyperoside and open-ring cucurbituril M1 inclusion complex

[0019] 1. Preparation of methylated glycoluril

[0020] 10.4 g of urea was dissolved in hydrochloric acid (0.3 M, 30 mL), and then 5 g of 1,3-butanedione was added. The reaction solution was stirred at room temperature for 12 h and then filtered to obtain a white crude product. The crude product was washed twice with water (500 mL × 2), filtered, and the filter cake was vacuum-dried to obtain 9.5 g of white solid methylated glycoluril (yield 92%). 1 H NMR (600MHz, DMSO-d6, ppm): δ7.18 (s, 4H), 1.36 (s, 6H).

[0021] 2. Preparation of etherified methylated glycoluril

[0022] 5 g of methylated glycoluril was dissolved in hydrochloric acid (9 M, 25 mL), and 4.5 g of paraformaldehyde was slowly added. The reaction solution was stirred at room temperature for 24 h and then filtered to obtain a white crude product. The crude product was washed with water three times (500 mL × 3), filtered, and the filter cake was vacuum dried to obtain 5.5 g of a white solid (yield 67%); 1 H NMR (600MHz, DMSO-d6, ppm): δ5.25 (d, J=10.8Hz, 4H), 5.03 (d, J=10.8Hz, 4H), 1.83 (s, 6H).

[0023] 3. Preparation of dimers

[0024] 10 g of glycoluril was dissolved in hydrochloric acid (8 M, 30 mL), and 2.1 g of paraformaldehyde was slowly added. The reaction solution was reacted at 50°C for 48 h and then filtered to obtain a white crude product. The crude product was washed with water several times until neutral, filtered, and the filter cake was vacuum dried to obtain 4.7 g of a white solid (yield 43%). 1 H NMR (600MHz, DMSO-d6, ppm): δ7.66 (d, J=2.2Hz, 4H), 5.59 (d, J=14.5Hz, 2H), 5.36 (d, J=8.5Hz, 2H), 5.24 (dt, J=8.4, 2.1Hz, 2H), 4.04 (d, J=14.5Hz, 2H).

[0025] 4. Preparation of Tetramers

[0026] 2.2 g of dimer and 7.6 g of etherified methylated glycoluril were added to 15 mL of anhydrous methanesulfonic acid, and the reaction solution was reacted at 50° C. for 3 h. The reaction solution was then cooled to room temperature and added dropwise to 150 mL of acetone and stirred for 2 h. The mixture was filtered, and the filter cake was washed twice with acetone (100 mL × 2) and twice with water (100 mL × 2). The filter cake was filtered and dried in vacuo to obtain 4 g of a white solid (yield 80%). 1 HNMR (600MHz, DMSO-d6, ppm): δ5.72-5.40 (m, 10H), 5.15 (d, J=10.6Hz, 4H), 4 .82 (d, J=10.8Hz, 4H), 4.24 (dd, J=14.7, 9.5Hz, 6H), 1.8 (s, 6H), 1.62 (s, 6H).

[0027] 5. Preparation of benzene ring side wall

[0028] 5 g of hydroquinone was dissolved in a 1.5 M sodium hydroxide solution (60 mL), and then 12 g of 1,3-propane sultone was added. The reaction solution was reacted at 50°C for 12 h. After cooling to room temperature, the solution was poured into 150 mL of acetone to precipitate a solid. The solid was filtered and the filter cake was dried under vacuum to obtain 16.5 g of a light yellow solid (yield 91%). 1 H NMR (600MHz, D2O, ppm): δ6.96 (s, 4H), 4.10 (t, J=6.3Hz, 4H), 3.10-2.99 (m, 4H), 2.20-2.06 (m, 4H).

[0029] 6. Preparation of open-ring cucurbituril M1

[0030] 3 g of the tetramer was dissolved in a mixed solution of trifluoroacetic acid and acetic anhydride (28 mL, 1:1), and then 6 g of the benzene ring side wall was added to the reaction solution. The reaction solution was reacted at 70° C. for 3 h, cooled to room temperature, and then the reaction solution was dropped into 300 mL of methanol and filtered. The filter cake was washed three times with a mixed solution of water and acetone (v / v=1:3, 200 mL×3), filtered, and the filter cake was dispersed in 100 mL of acetone and the pH was adjusted to neutral with saturated sodium bicarbonate solution. The filter cake was filtered and vacuum dried to obtain 2.5 g of a white solid (yield 42%). 1 H NMR (600MHz, D2O, ppm): δ6.91 (d, J=10.6Hz, 4H), 5.68-5.29 (m, 14H), 4.27-3.92 (m, 18H), 3.12 (d ddd, J=23.6, 13.9, 9.7, 6.0Hz,, 8H), 2.20 (tdd, J= 15.8, 8.3, 3.8Hz, 8H), l.76 (d, J= 23.4Hz, 12H).

[0031] 7. Preparation of inclusion complex of hyperoside and open-ring cucurbituril M1

[0032] 0.8 g (0.51 mmol) of open-ring cucurbituril M1 was dissolved in 20 mL of ultrapure water, and 717 mg (1.53 mmol) of hyperoside was added thereto while stirring at 25 ° C. After stirring for 72 h at 25 ° C in the dark, the reactants were filtered with filter paper and microporous filter membrane in turn. The filtrate was freeze-dried to obtain a yellow powder inclusion compound. Its nuclear magnetic resonance hydrogen spectrum showed Figure 1 .

[0033] The powder X-ray diffraction comparison diagrams of the inclusion compound, open-ring cucurbituril M1, hyperoside, and the physical mixture of open-ring cucurbituril M1 and hyperoside prepared in this example are shown in FIG. Figure 3 , infrared spectrum comparison chart see Figure 4As can be seen from the figure, the X-ray diffraction pattern and infrared spectrum of the prepared inclusion complex are significantly different from those of hyperoside itself, and are different from the physical mixture of hyperoside and open-ring cucurbituril M1, so it can be confirmed that the inclusion complex has been successfully prepared.

[0034] Example 2: Preparation of hyperoside and open-ring cucurbituril M2 inclusion complex

[0035] 1. The preparation of methylated glycoluril, etherified methylated glycoluril, dimer and tetramer is the same as steps 1-4 of Example 1;

[0036] 2. Preparation of naphthalene ring sidewalls

[0037] 10 g of 1,4-naphthoquinone was dissolved in 400 mL of ether, and then 35 g of sodium thiosulfate was added to the reaction solution. The reaction solution was reacted under nitrogen for 1 h, extracted three times with ethyl acetate (200 mL×3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent to obtain 9.2 g of naphthol precursor (yield 90%); the naphthol precursor was dissolved in sodium hydroxide solution (10 wt %, 75 mL), and a 1,4-dioxane solution of 1,3-propane sultone (18 g of 1,3-propane sultone dissolved in 120 mL of 1,4-dioxane) was added to the reaction solution. The reaction solution was reacted at room temperature for 12 h and then filtered. The solid was dissolved in an appropriate amount of water and then added dropwise to 300 mL of acetonitrile to precipitate the solid. The solid was filtered, and the filter cake was vacuum dried to obtain 7.6 g of a light yellow solid (yield 29%). 1 H NMR (600MHz, D2O, ppm): δ8.25-8.02(m,2H), 7.64-7.43(m,2H), 6.88(d,J=1. 3Hz, 2H), 4.22 (dt, J = 4.9, 3.6Hz, 4H), 3.25-3.02 (m, 4H), 2.37-2.17 (m, 4H).

[0038] 3. Preparation of open-ring cucurbituril M2

[0039] 3 g of the tetramer was dissolved in a mixed solution of trifluoroacetic acid and acetic anhydride (28 mL, 1:1), and then 6.9 g of the naphthalene ring sidewall was added to the reaction solution. The reaction solution was reacted at 70 ° C for 3 h, cooled to room temperature, and then the reaction solution was dropped into 300 mL of methanol and filtered. The filter cake was washed 3 times with a mixed solution of water and acetone (v / v=1:3, 200 mL×3), filtered, and the filter cake was dispersed in 100 mL of acetone and adjusted to neutral pH with saturated sodium bicarbonate solution. The filter cake was then filtered and dried in vacuo to obtain 2.1 g of a white solid (33% yield). 1H NMR (600MHz, D2O, ppm): δ7.80 (dd, J=6.7, 3.3Hz, 4H), 7.33 (d, J=7.0Hz, 4H), 5.58-5.25 (m, 14H), 4.47-3.81 (m, 18H), 3.15 (dddd, J=42.7, 13.8, 8.9, 6.7Hz,, 8H), 2.23 (ddt, J=12.9, 9.9, 4.9Hz, 8H), 1.76 (d, J= 19.1Hz, 12H).

[0040] 4. Preparation of Hyperoside and Open-ring Cucurbituril M2 Inclusion Complex

[0041] 1.5 g (0.9 mmol) of open-ring cucurbituril M2 was dissolved in 20 mL of ultrapure water, and 835 mg (1.2 mmol) of hyperoside was added thereto while stirring at 50 ° C. After stirring at 50 ° C in the dark for 36 h, the reactants were filtered with filter paper and microporous filter membrane in turn. The filtrate was concentrated under reduced pressure to obtain a yellow powder inclusion compound. Its nuclear magnetic resonance hydrogen spectrum showed Figure 2 .

[0042] The powder X-ray diffraction comparison diagrams of the inclusion compound, open-ring cucurbituril M2, hyperoside, and the physical mixture of open-ring cucurbituril M2 and hyperoside prepared in this example are shown in FIG. Figure 5 , infrared spectrum comparison chart see Figure 6 As can be seen from the figure, the X-ray diffraction pattern and infrared spectrum of the prepared inclusion complex are significantly different from those of hyperoside itself, and are different from the physical mixture of hyperoside and open-ring cucurbituril M2, so it can be confirmed that the inclusion complex has been successfully prepared.

[0043] Example 3: Determination of water solubility of hyperoside after forming inclusion complex with open-ring cucurbituril

[0044] 1. Determination of hyperoside solubility standard curve

[0045] Hyperoside was dissolved in ethanol to prepare a concentration of 1×10 -4 mol / L hyperoside ethanol solution, and then diluted with ethanol to prepare a series of concentrations in the range of 1×10 -5 ~5×10 -5 mol / L solution, use UV spectrophotometer to measure the maximum absorbance (Abs) at 360nm at 25℃, and then use the concentration C (mol·L -1 ) as the horizontal axis, and the absorbance Abs as the vertical axis to fit the standard curve: Abs = 0.2192C-0.0115 (R 2 =0.9991).

[0046] 2. Determination of water solubility of hyperoside in inclusion complex

[0047] The excess inclusion compound prepared in Examples 1 and 2 was added to 2 mL of ultrapure water, and after ultrasonic vibration for 4 h, the inclusion compound was filtered through a 0.45 μm microporous membrane to obtain a saturated solution. The saturated solution was diluted by 10 4 times, measure its absorbance at 25℃ and 360nm, and finally substitute it into the standard curve to calculate its solubility;

[0048] The solubility of hyperoside in water (at 25°C) is 0.153 mg / mL (Molecules, 2022, 27(9):2761). The water solubility of the inclusion complex of hyperoside, cyclodextrin and hyperoside, and the inclusion complex of open-ring cucurbituril and hyperoside were compared. The results are shown in the following table:

[0049] Water solubility of hyperoside after inclusion complex formation (H2O, 25℃)

[0050]

[0051] Example 4: Determination of the in vitro antioxidant activity of hyperoside inclusion complex

[0052] The DPPH scavenging test was used to evaluate the in vitro antioxidant capacity of the inclusion complex of hyperoside and open-ring cucurbituril. The determination method was as follows: hyperoside was dissolved in ethanol to prepare a hyperoside ethanol solution with a concentration of 0.05 mg / mL, the inclusion complex prepared in Example 1-2 was dissolved in ultrapure water to prepare an inclusion complex aqueous solution with a concentration of 0.05 mg / mL, and then they (0.05 mL) were added to a 96-well plate, and then DPPH (0.1 mg / mL ethanol solution, 0.1 mL) was added. The plate was placed in the dark at 25°C for half an hour, and the ultraviolet absorbance at 518 nm was measured. The in vitro antioxidant activity was measured by the percentage of remaining DPPH (% DPPH rem ) was used to evaluate the antioxidant activity of the samples. The lower the percentage of residual DPPH, the higher the in vitro antioxidant activity, which was calculated by the following formula:

[0053]

[0054] Among them A C(0) and A A(t) The in vitro antioxidant activity of hyperoside was compared with that of the inclusion complex, and the results are shown in the following table:

[0055]

[0056] The results showed that the in vitro antioxidant activity of hyperoside was significantly enhanced after forming an inclusion complex with open-ring cucurbituril.

Claims

1. A highly water-soluble hyperoside product, characterized in that: Contains seco-cucurbituril and hyperoside; The open-ring cucurbituril is selected from the following structural formula: ； The preparation method of the highly water-soluble hyperoside product comprises the following steps: adding hyperoside to a 0.01-0.1 mol / L open-ring cucurbituril aqueous solution at 15-50° C. while stirring, continuing stirring in the dark for 36-72 hours after the addition, filtering out insoluble matter, and evaporating to dryness under reduced pressure or freeze-drying to obtain a powdery inclusion complex of hyperoside and open-ring cucurbituril.

2. The highly water-soluble hyperoside product according to claim 1, characterized in that: The molar ratio of hyperoside to open-ring cucurbituril is 1~3:1.

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