A water-soluble myricetin derivative and its preparation method and application

Abstract

The present invention relates to the field of biomedicine technology, and discloses a preparation method and application of a water-soluble myricetin derivative. The present invention uses horseradish peroxidase oxidation combined with extraction and purification to prepare a water-soluble myricetin derivative for the first time. The derivative has good water solubility and anti-protein amyloid fibrosis activity. Compared with myricetin, it has a stronger inhibitory effect on lysozyme and bovine insulin amyloid fibrosis, and can more effectively prevent Aβ than myricetin. 1‑42 Aggregation and accumulation have an important impact on the neurotoxicity caused by GMC101. It can be used as a functional factor in anti-protein amyloid fibrillation functional foods, which has important practical significance and commercial value.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a water-soluble myricetin derivative and a preparation method and application thereof. Background Art

[0002] To date, nearly 40 types of amyloid fibril deposits associated with degenerative diseases have been discovered, which are mainly formed by protein or polypeptide aggregation. These proteins have different functions under normal physiological conditions, and the amyloid pathology of these proteins will cause abnormalities in the body, thereby inducing the occurrence and development of various neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). Therefore, inhibiting the production of related protein amyloid aggregation and eliminating amyloid aggregation are the main strategies for treating these diseases.

[0003] Many natural active small molecule compounds have good inhibitory and depolymerization effects on protein amyloid aggregation, making them a research hotspot for the prevention and treatment of protein conformational diseases. Such as natural polyphenols, flavonoids, and small molecule active peptides; all flavonoids can interact with Aβ and tend to destroy the structure of Aβ peptides. Among flavonoids, myricetin is the most widely and deeply studied one, but due to the particularity of its structure, its water solubility and stability are poor, which limits its wide application in food and medicine.

[0004] Flavonoid polymers have attracted wide attention in recent years due to their good stability. Natural flavonoid polymers, such as theaflavins, proanthocyanidins and tannins, have higher water solubility and biological activity than their flavonoid monomers. Studies have shown that the water solubility of polyrutin is 4200 times higher than that of rutin, and its biological activity is improved; catechin polymers have better inhibitory ability against tyrosinase and xanthine oxidase than the corresponding monomers. This shows that polymerization reactions can improve the water solubility of flavonoids and enhance their certain biological activities. Common polymerization methods for flavonoids are chemical polymerization and enzymatic polymerization. However, chemical polymerization has many problems, such as poor regioselectivity, large raw material loss, low product yield, toxic and side effects, and environmental unfriendliness. Enzymatic polymerization has become an attractive alternative method for preparing flavonoid polymers because of its strong specificity, greenness and safety.

[0005] Therefore, providing a method for biocatalytically improving the water solubility of myricetin and enhancing its pharmacology has important practical significance and commercial value. Summary of the invention

[0006] In view of the above problems, the present invention provides a water-soluble myricetin derivative and a preparation method and application thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The invention provides a water-soluble myricetin derivative, which is obtained by separating and purifying myricetin after catalytic oxidation with horseradish peroxide, and has a molecular weight of about 634.

[0009] The present invention provides a method for preparing a water-soluble myricetin derivative, comprising the following steps:

[0010] The myricetin oxidation product was dissolved in dimethyl sulfoxide, centrifuged, and the supernatant after centrifugation was taken, and water and ethyl acetate were added to it for repeated extraction, and the obtained aqueous extract was eluted on a macroporous resin HPD-100 column, and the eluate was collected and vacuum freeze-dried to obtain a water-soluble myricetin derivative dry powder. Using high performance liquid chromatography-mass spectrometry technology, based on the obtained quasi-molecular ion peak, it is inferred that the molecular weight of the water-soluble myricetin derivative is about 634.

[0011] Furthermore, the centrifugal speed is 12000r / min and the time is 5min.

[0012] Furthermore, the number of repeated extractions is 6-10 times.

[0013] Furthermore, the eluent of the macroporous resin HPD-100 column is ethanol with a volume fraction of 30-50%.

[0014] The water-soluble myricetin derivative provided by the present invention has good in vitro anti-lysozyme and bovine insulin amyloid fibrillation activity and better inhibitory effect than myricetin. The derivative almost completely inhibits the amyloid fibrillation of Lys and BI at final concentrations of 32 μg / mL and 16 μg / mL respectively.

[0015] The water-soluble myricetin derivative provided by the present invention can delay the paralysis rate of AD-type Caenorhabditis elegans GMC101 in a concentration-dependent manner, and the effect is more obvious than that of myricetin. After the dimer with a final concentration of 32 μg / mL was treated with nematodes for 8 days, the paralysis rate was reduced by 86%.

[0016] The water-soluble myricetin derivative obtained by the invention has better anti-protein amyloid fibrillation activity than myricetin through in vivo and in vitro detection.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] The present invention is the first to use horseradish peroxidase oxidation combined with extraction and purification to prepare a water-soluble myricetin derivative, which has good water solubility and significantly delays the paralysis rate of Caenorhabditis elegans, and can effectively prevent Aβ 1-42 Aggregation and accumulation have a great impact on the neurotoxicity caused by GMC101. The discovery of this derivative has great economic value for the rational and safe use of myricetin and the effective transformation of myricetin. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the HPLC graph of MYRox and MYRow;

[0020] Figure 2 This is the HPLC-MS graph of MYRow;

[0021] Figure 3 The 3D structures of MYR (left) and its derivatives (right) after DFT optimization;

[0022] Figure 4 It is a possible form of polymerization of water-soluble myricetin derivatives;

[0023] Figure 5 is the effect of MYR and MYRow on the growth kinetics of Lys amyloid fibrils;

[0024] Figure 6 is the effect of MYR and MYRow on the growth kinetics of BI amyloid fibrils;

[0025] Figure 7 The effect of MYR and MYRow on the rigidification of GMC101 at 25°C. Where, a: 8 μg / mL, b: 32 μg / mL. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0027] Unless otherwise specified, the reagents and methods in the present invention are conventional reagents and methods.

[0028] The water-soluble myricetin derivative is obtained by separating and purifying the myricetin oxidation product obtained by horseradish peroxidase-catalyzed oxidation of myricetin. The specific method can refer to the method described in Patent 202211425630.4.

[0029] Example 1

[0030] The preparation method of the water-soluble myricetin derivative comprises the following steps:

[0031] 0.3182 g of myricetin oxidation product (MYRox) was weighed and dissolved in 4 mL of DMSO, then centrifuged at 12000 r / min for 5 min. The supernatant after centrifugation was extracted repeatedly with water and ethyl acetate for 8 times. The obtained aqueous extract was eluted with 40% ethanol on a macroporous adsorption resin HPD-100 column to obtain a water-soluble myricetin derivative (MYRow), which was freeze-dried in a freeze dryer and placed at -20°C for use.

[0032] Example 2

[0033] Characterization of water-soluble myricetin derivatives

[0034] 2.1 High performance liquid chromatography

[0035] The obtained MYRow was detected by high performance liquid chromatography (HPLC) at a detection wavelength of 300 nm, chromatographic conditions: C18 column, mobile phase: A (methanol): B (0.1% acetic acid), flow rate 0.5 mL / min. Gradient conditions: 0-4 min: 30% A liquid, 70% B liquid; 4-15 min: 40% A liquid, 60% B liquid; 15-20 min: 60% A liquid, 40% B liquid; 20-30 min: 70% A liquid, 30% B liquid.

[0036] Figure 1 This is the HPLC chromatogram of MYRox before and after separation and purification. Figure 1 The results showed that the oxidation product was a mixture of multiple substance peaks before separation and purification, and a single substance peak was obtained after separation and purification, namely, a water-soluble myricetin derivative (RT = 6.9 min).

[0037] 2.2 High performance liquid chromatography-mass spectrometry

[0038] Weigh an appropriate amount of MYRow and add anhydrous ethanol to dissolve it. After passing through a 0.45μm filter, detect it on a liquid chromatography-mass spectrometer (HPLC-MS). Detection conditions: PDA detector 190-400nm full wavelength scan, UPLC C18 column (50×2.0mm, 3μm, phenomenex), mobile phase: A: 0.1% formic acid water; B: 0.1% formic acid acetonitrile, flow rate 0.2mL / min. Gradient conditions: 0-8min: 95% A solution, 5% B solution; 8-8.01min: 5% A solution, 95% B solution; 8.01-10min: 95% A solution, 5% B solution.

[0039] Figure 2 It is the HPLC-MS chart of water-soluble myricetin derivatives. Figure 2 The results show that based on the quasi-molecular ion peak of [M+2COOH-12H], the actual molecular weight of MYRow was analyzed to be 634, which is a water-soluble myricetin derivative.

[0040] 2.3 Density functional theory (DFT) calculations

[0041] Search for the 3D structure of MYR in PubChem, download the sdf file and open it with GaussianView 6.0, set the parameters and calculate with Gaussian 09W. Select DFT for the specific settings, and use the B3LYP hybrid functional, where B3 represents the Becke type 3-parameter DFT model and LYP is the Lee-Yang-Parr function. The ground state structure of the MYR molecule was optimized and analyzed by vibration using the 6-31G(d) basis set.

[0042] Figure 3 This is the 3D structure of MYR and its derivatives after DFT optimization. In the hybrid DFT calculation, B3LYP / 6-31G(d) was used to optimize the geometric structure of MYR and its derivatives. The optimization results showed no imaginary frequency, indicating that the structure was desirable. The spatial interaction in the water-soluble myricetin derivatives leads to the loss of planarity between catechol and the remaining part in the myricetin molecule, while MYR has only one rotatable carbon-carbon bond. When MYR is polymerized, the 2-1' bond rotates away from the plane to 49.3. The loss of the planar conformation of polymerized myricetin leads to a blue shift in the absorption wavelength. This loss of planarity will affect the UV-visible absorption, λ max It is about 300nm, and its polymerization method may be as follows Figure 4 .

[0043] Example 3

[0044] 3.1 Preparation of lysozyme (bovine insulin) amyloid fibril samples

[0045] Weigh an appropriate amount of lysozyme (bovine insulin) and dissolve it in HCl at pH = 2 to ensure a final concentration of 1 mg / mL. Add an appropriate amount of NaCl to ensure a final concentration of 150 mM (the final concentration of bovine insulin amyloid fiber sample is 100 mM). Add an appropriate amount of MYR and MYRow to make their final concentrations of 0, 8, 16, 32, and 64 μg / mL, respectively. DMSO (final concentration of 0.5%) is present in MYR and MYRow as a cosolvent to ensure complete dissolution. Place the sample in a 65°C constant temperature water bath and incubate for 8 days. The sample that is not placed in the water bath is taken out first as 0 day, and the remaining samples are sampled every 1 day, successively 1, 2, 3, 4, 5, 6, 7 and 8 days (the bovine insulin amyloid fiber sample is incubated for 7 hours and sampled every 1 hour). After the samples are taken out, they are immediately placed in a 4°C refrigerator. 0.5% DMSO was used to replace MYR and MYRow as a blank control to avoid the influence of DMSO, and no lysozyme (insulin) was used as a background control to avoid the interference of sample autofluorescence.

[0046] 3.2 ThT fluorescence spectrum analysis

[0047] ThT fluorescence analysis of the growth curve of lysozyme (Lys) fibers: 33 μL of sample and 950 μL Tris-HCl solution (pH=8.0, 10 mM) were added to a 1.5 mL centrifuge tube. 20 μL of ThT fluorescent dye was added before measurement. The solution was quickly shaken several times to mix evenly. The fluorescence intensity was measured with a multifunctional microplate reader at an excitation wavelength of 440 nm and an emission wavelength of 484 nm. The sample without sample and only with ThT fluorescent dye was subtracted as the background. Each sample was repeated three times and the average value was taken.

[0048] ThT fluorescence analysis of bovine insulin (BI) fiber growth curve: 5μL sample and 1480μL Tris-HCl solution (pH=8.0, 10mM) were added to a 2mL centrifuge tube. 15μL ThT fluorescent dye was added before measurement. The solution was mixed evenly by rapid shaking several times. The fluorescence intensity was measured with a multifunctional microplate reader at an excitation wavelength of 440nm and an emission wavelength of 484nm. The sample without sample and only with ThT fluorescent dye was subtracted as the background. Each sample was repeated three times and the average value was taken.

[0049] Figure 5 The effects of MYR and MYRow on the growth kinetics of Lys amyloid fibrils. Figure 5 The results showed that the addition of MYR and MYRow significantly reduced the fluorescence intensity of ThT, which indicates that both of them have a good inhibitory effect on the fibrillization of lysozyme. Moreover, with the increase of sample concentration, the fluorescence intensity of the MYR and MYRow treatment groups gradually decreased. At 32 μg / mL, MYRow almost completely inhibited the amyloid fibrillization of Lys, and at 64 μg / mL, MYR completely inhibited the fibrillization of Lys, and the effect of MYRow was stronger. Figure 6 is the effect of MYR and MYRow on the growth kinetics of BI amyloid fibrils. Figure 6 The results showed that after incubation of bovine insulin in a constant temperature water bath at pH = 2.0 and 65 °C for 6 hours, the fluorescence intensity of ThT increased significantly, indicating that the β-folding content of bovine insulin increased and amyloid fibrillation occurred in bovine insulin. The addition of MYR and MYRow significantly reduced the fluorescence intensity of ThT, indicating that both of them had a good inhibitory effect on the fibrillation of bovine insulin, and with the increase of sample concentration, the fluorescence intensity of MYR and MYRow gradually decreased. At 16 μg / mL, MYRow almost completely inhibited the amyloid fibrillation of BI, and at 32 μg / mL, MYR completely inhibited the amyloid fibrillation of BI, and MYRow had a stronger effect.

[0050] Example 4

[0051] Effects of MYR and MYRow on rigidity of GMC101 nematodes

[0052] GMC101 nematodes grown synchronously to the L4 stage (48h after egg hatching) at 20℃ were transferred to fresh NMG plates (containing FUdR) coated with E.coil OP50 containing 0, 8, and 32μg / mL MYR and MYRow. After 24h of incubation at 25℃, the rigid nematodes were counted. The rigidity criteria were: 1) complete loss of movement ability; 2) only the head can move, and due to the swinging of the head to feed, obvious bacterial circles (halos) will be formed around the head, or eggs will accumulate next to the body; 3) after a slight touch, it cannot make a complete sinusoidal movement.

[0053] Figure 7 Table 1 and Table 2 show the effects of MYR and MYRow on the rigidification of GMC101 at 25°C. The results showed that compared with the control group, the average survival time of nematodes in the 8μg / mL MYR and MYRow treatment groups was extended by 4.7% and 8.1% (P<0.05), respectively, and that in the 32μg / mL treatment group was extended by 6.3% (P<0.05) and 30% (P<0.01), respectively. On the 8th day, compared with the control group, the paralysis rate of nematodes in the 8μg / mL MYR and MYRow treatment groups was reduced by 23% and 37%, respectively, and that in the 32μg / mL treatment group was reduced by 48% and 86%, respectively. In summary, treatment with MYR and MYRow can effectively prevent Aβ 1-42 Aggregation and accumulation have a dose-dependent effect on the neurotoxicity caused by GMC101, and consistent with in vitro experiments, MYRow has a better effect.

[0054] Table 1 Effects of MYR and MYRow on paralysis of GMC101 nematodes at 25°C

[0055]

[0056] Example 5

[0057] The preparation method of the water-soluble myricetin derivative comprises the following steps:

[0058] 0.3182 g of myricetin oxidation product (MYRox) was weighed and dissolved in 4 mL of DMSO, then centrifuged at 12000 r / min for 5 min. The supernatant after centrifugation was extracted repeatedly with water and ethyl acetate for 6 times. The obtained aqueous extract was eluted with 30% ethanol on a macroporous adsorption resin HPD-100 column to obtain a water-soluble myricetin derivative (MYRow), which was freeze-dried in a freeze dryer and placed at -20°C for use.

[0059] The water-soluble myricetin derivative prepared in this example can also achieve a technical effect similar to that of Example 1.

[0060] Example 6

[0061] The preparation method of the water-soluble myricetin derivative comprises the following steps:

[0062] 0.3182 g of myricetin oxidation product (MYRox) was weighed and dissolved in 4 mL of DMSO, then centrifuged at 12000 r / min for 5 min. The supernatant after centrifugation was extracted repeatedly with water and ethyl acetate 10 times. The obtained aqueous extract was eluted with 50% ethanol on a macroporous adsorption resin HPD-100 column to obtain a water-soluble myricetin derivative (MYRow), which was freeze-dried in a freeze dryer and placed at -20°C for use.

[0063] The water-soluble myricetin derivative prepared in this example can also achieve a technical effect similar to that of Example 1.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a water-soluble myricetin derivative in the preparation of a drug for delaying paralysis of Caenorhabditis elegans, characterized in that: The preparation method of the water-soluble myricetin derivative comprises the following steps: The myricetin oxidation product is dissolved in dimethyl sulfoxide, centrifuged, and the supernatant is taken after centrifugation, and water and ethyl acetate are added to it for repeated extraction, and the obtained aqueous phase extract is eluted on a macroporous resin HPD-100 column, and the eluate is collected and vacuum freeze-dried to obtain a water-soluble myricetin derivative dry powder; The myricetin oxidation product is obtained by oxidizing myricetin catalyzed by horseradish peroxidase, and the specific method is as follows: accurately weigh 0.04774 g of a standard sample of myricetin, dissolve it in 6 mL of anhydrous ethanol, and dissolve HRP in 50 mM phosphate buffer, mix the two and adjust the pH to 6.5, then add H2O2 to start the reaction, and react at room temperature in the dark for 6 hours; wherein the molar ratio of myricetin to HRP and H2O2 is 1:2×10 -5 : 1.22, the volume ratio of ethanol to phosphate buffer is 2:8; after the reaction, the absorbance photometric curve of the reaction solution in the wavelength range of 200-600nm is monitored by UV-visible spectrophotometer and no longer changes, indicating that the reaction is complete; then, a high performance liquid chromatograph is used to monitor whether myricetin is completely oxidized; when no myricetin is detected, it means that myricetin has been completely oxidized, a 30kD ultrafiltration tube is used for centrifugation at 3000r / min at 4℃ for 30min to remove residual HRP, and the product is stored at -20℃ after vacuum freeze-drying for later use; the myricetin oxidation product obtained is called MYRox.

2. The use of a water-soluble myricetin derivative according to claim 1 in the preparation of a drug for delaying paralysis of Caenorhabditis elegans, characterized in that: In the method for preparing the water-soluble myricetin derivative, the centrifugal speed is 12000 r / min and the time is 5 min.

3. The use of a water-soluble myricetin derivative according to claim 1 in the preparation of a drug for delaying paralysis of Caenorhabditis elegans, characterized in that: The number of repeated extractions is 6-10 times.

4. The use of a water-soluble myricetin derivative according to claim 1 in the preparation of a drug for delaying paralysis of Caenorhabditis elegans, characterized in that: The eluent of the macroporous resin HPD-100 column is ethanol with a volume fraction of 30-50%.

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