Preparation method and application of isoquercetin for inhibiting xanthine oxidase activity

Abstract

The present invention relates to a method for preparing isoquercetin that inhibits xanthine oxidase activity, characterized by comprising the following steps: (1) using dried tangerine peel as a raw material, extracting it with an organic liquid, combining the extracts, and draining the solvent using a rotary evaporator to obtain an organic layer extract AE; (2) separating and purifying the organic layer extract AE through a column to obtain component AE-3; (3) separating and purifying component AE-3 again to obtain component AE-3-7; and identifying component AE-3-7 as containing isoquercetin by nuclear magnetic resonance; and (4) conducting an enzyme activity inhibition experiment. The preparation process of the present invention is simple and easy to operate; the obtained isoquercetin is extracted, separated and purified from dried tangerine peel, and is found to have the ability to inhibit xanthine oxidase activity, providing a new source for the prevention and treatment of hyperuricemia, and having great significance for the research on the effective ingredients of natural products of Chinese medicinal materials with medicinal and edible properties.

Description

Technical Field

[0001] The present invention relates to the field of active drugs, and in particular to a preparation method of isoquercetin for inhibiting xanthine oxidase activity and an application thereof. Background Art

[0002] Tangerine peel ( Pericarpium Citri Reticulatae is a plant of the Rutaceae family Citrus reticulata The dried mature peel of the tangerine peel (Citrus blanco) and its cultivated varieties. First recorded in the Shennong Bencao Jing (Shen Nong's Classic of Materia Medica), it is listed under the "top-grade tangerine and pomelo" category. Its pungent and bitter flavor and warm nature enter the lung and spleen meridians, making it a common medicinal and edible herb used clinically to regulate central qi and treat cough and phlegm. Modern research indicates that tangerine peel possesses anti-inflammatory, anti-cancer, antioxidant, and lipid-lowering properties, and has a preventive and therapeutic effect on hypertension. It also has antispasmodic, antiasthmatic, and phlegm-thinning properties.

[0003] Isoquercetin, also known as quercetin-3-O-β-D-glucoside, is a glucose-bound derivative of quercetin, commonly found in herbs, fruits, vegetables, and plant-based foods and beverages. Recent studies have shown that isoquercetin exhibits anti-inflammatory, antioxidant, anti-allergic, antihypertensive, anti-hyperglycemic, and diuretic effects.

[0004] Xanthine oxidase, primarily found in the liver, is a key enzyme in purine catabolism. It catalyzes the oxidation of hypoxanthine to xanthine, which in turn oxidizes xanthine to uric acid (UA), while simultaneously generating reactive oxygen species (ROS). When uric acid levels rise in the blood, it crystallizes and deposits in joints and tissues, forming monosodium urate (MSU), which is closely associated with the development of gout and inflammation. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for preparing isoquercetin that inhibits xanthine oxidase activity and its application.

[0006] The purpose of the present invention is achieved by adopting the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing isoquercetin that inhibits xanthine oxidase activity, comprising the following steps:

[0008] (1) Using dried tangerine peel as raw material, after extraction with organic liquid, the extracts were combined and the solvent was extracted by rotary evaporation to obtain the organic layer extract AE;

[0009] (2) dissolving the organic layer extract AE, and then separating and purifying it by column, isocratically eluting it, and collecting the fractions, which are fraction AE-3;

[0010] (3) Dissolving the component AE-3 again, separating and purifying it, eluting it, and collecting the components to obtain component AE-3-7; nuclear magnetic resonance identification of the component AE-3-7 revealed that it contained isoquercetin;

[0011] (4) An enzyme activity inhibition experiment was conducted to detect the inhibition rate of component AE-3-7 on xanthine oxidase activity.

[0012] Preferably, in step (1), the leaching is performed by soaking in ethyl acetate and accelerating the leaching with the assistance of ultrasound, the ultrasonic frequency is 20-50 kHz, and the leaching is repeated 3 times.

[0013] Preferably, in step (2), a 50% by mass methanol aqueous solution is used for dissolution, a medium-low pressure reverse phase C18 column is used for separation and purification, the column flow rate is set to 30 mL / min, a 30% by mass methanol aqueous solution is used for elution, and the components collected are the components from 38 to 63 minutes.

[0014] Preferably, in step (3), pure methanol is used for dissolution, Sephadex LH-20 is used for separation and purification, the flow rate is set to 0.25 mL / min, methanol is used as the eluent, and the collected components are the components from 1635 to 1770 min.

[0015] Preferably, in step (4), the process of the enzyme activity inhibition experiment includes:

[0016] S1. Add 100 μL PBS buffer, 25 μL sample solution, and 25 μL 0.1 U / mL xanthine oxidase to a 96-well plate, shake well, and incubate at 37°C for 20 min.

[0017] S2. After incubation, add 50 μL of 1 mM xanthine reaction solution, shake to mix, and incubate at 37°C for 5 min.

[0018] S3. Then measure the absorbance of the sample well at 290 nm;

[0019] S4. The blank control group consisted of PBS buffer instead of the sample and xanthine reaction solution. In the complete reaction group, PBS buffer was used instead of the sample. In the sample control group, PBS buffer was used instead of xanthine oxidase. Allopurinol served as a positive control.

[0020] S5. The inhibition rate of xanthine oxidase in the sample group was calculated as follows:

[0021]

[0022] Wherein, C1 is the blank control group; C2 is the complete reaction group; C3 is the sample reaction group; and C4 is the sample control group.

[0023] Preferably, in step (4), isoquercetin inhibits xanthine oxidase by IC 50 The value was 101.12 ± 1.66 μg / mL.

[0024] In a second aspect, the present invention provides an isoquercetin for inhibiting xanthine oxidase activity, wherein the isoquercetin contains the flavonoid compound isoquercetin, which has C 21 H 20 O 12 The molecular formula and chemical structure are as follows:

[0025] .

[0026] In a third aspect, the present invention provides isoquercetin that inhibits xanthine oxidase activity and can be used to prevent and treat hyperuricemia.

[0027] The beneficial effects of the present invention are:

[0028] 1. The present invention provides a method for preparing isoquercetin that inhibits xanthine oxidase activity and its application. The method uses tangerine peel as a raw material for separation. The raw material is easily available and the tangerine peel can be fully recycled to avoid waste. The preparation process is simple and easy to operate. The obtained isoquercetin is extracted, separated and purified from tangerine peel, and is found to have the effect of inhibiting xanthine oxidase activity, providing a new source for the prevention and treatment of hyperuricemia. It is of great significance for the research on the effective ingredients of natural products of Chinese medicinal materials that are both medicinal and edible.

[0029] 2. The present invention optimizes the step of extracting isoquercetin using dried tangerine peel as a raw material. Compared with the traditional method of first ethanol precipitation and water extraction or water precipitation and alcohol extraction, and then organic extraction, the present invention directly uses an organic liquid for primary extraction, and ethyl acetate is used as the organic liquid. At the same time, ultrasound is used to assist. Compared with the traditional method of ethanol precipitation and water extraction or water precipitation and alcohol extraction, not only is the extraction method simpler but also the desired product is more easily obtained. In the process of extraction, elution is an important part of the present invention, comprising two elutions performed sequentially. The first elution adopts 30% methanol aqueous solution as eluent, controls the elution rate (30 mL / min) and the time (38-63 min) for collecting the eluted product, and the second elution adopts pure methanol as eluent, controls the elution rate (0.25 mL / min) and the time (1635-1770 min) for collecting the eluted product again, and finally completes the elution and obtains the desired product.

[0030] 3. The isoquercetin (component AE-3-7) finally obtained by the present invention has an inhibition rate of more than 98.5% on xanthine oxidase at a concentration of 0.623 mg / mL, which not only exceeds the inhibition rate of allopurinol, but also has a higher inhibition rate than the isoquercetin obtained by traditional methods. This shows that the isoquercetin (component AE-3-7) prepared by the method of the present invention is more active and has a higher inhibitory effect on xanthine oxidase. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention is further described with reference to the accompanying drawings. However, the embodiments in the accompanying drawings do not constitute any limitation to the present invention. A person skilled in the art can obtain other drawings based on the following drawings without creative effort.

[0032] Figure 1 HPLC analysis chart of compound AE-3-7 isoquercetin according to an embodiment of the present invention;

[0033] Figure 2 According to the embodiment of the present invention, the compound AE-3-7 isoquercetin 1 H-NMR spectrum;

[0034] Figure 3 According to the embodiment of the present invention, the compound AE-3-7 isoquercetin 13 C-NMR spectrum;

[0035] Figure 4 This is the inhibitory effect of the compound AE-3-7 isoquercetin on xanthine oxidase according to an example of the present invention. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the presence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to define the scope of the present invention. Changes or adjustments in their relative relationships, without substantially changing the technical content, should also be regarded as the scope of the present invention.

[0037] In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0038] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0039] The present invention is described below with reference to specific examples. It should be noted that these examples are merely illustrative and do not limit the present invention in any way. Example

[0040] A method for preparing isoquercetin for inhibiting xanthine oxidase activity comprises the following steps:

[0041] Step 1: Take 1000 g of dried tangerine peel, grind it in a grinder, place it in a 2 L conical flask, soak it with 10 times the volume of ethyl acetate and extract it with ultrasonic acceleration for 1 h, repeat the extraction 3 times, combine the extracts, and drain the solvent with a rotary evaporator to obtain 34.65 g of ethyl acetate layer extract.

[0042] Step 2: Take 34.65 g of the ethyl acetate extract (AE) obtained in step 1, dissolve it in 50 mL of 50% methanol water, and use a medium-low pressure reversed-phase C18 column (filler: YMC-GEL ODS-AQ-HG, s-50 μm; column length: 4.5 × 35 cm) with a flow rate of 30 mL / min. Elute with 30% methanol water and collect the 38-63 min component (AE-3).

[0043] Step 3: Dissolve 1.12 g of (AE-3) obtained in Step 2 in 20 mL of 100% methanol. Use Sephadex LH-20 (2.5 × 170 cm) and 100% methanol as the eluent at a flow rate of 0.25 mL / min. Collect the fractions (AE-3-7) from 1635 to 1770 min and concentrate to obtain approximately 56.8 mg of the compound.

[0044] The compound AE-3-7 prepared in this example was a yellow powder and was subjected to HPLC analysis. The HPLC parameters were as follows: HPLC model: Agilent 1260 Infinity II; detector: 1260 DAD WR; chromatographic column: H&E-SP ODS-TA column, 4.6 × 250 mm, s-5 μm; liquid phase conditions: mobile phase: a mixture of 45% methanol and 55% water, followed by the addition of 0.1% trifluoroacetic acid; detection time: 20 min; flow rate: 1 mL / min, column temperature: 35°C, injection volume: 20.0 μL; detection wavelength: 254 nm.

[0045] The results are as follows Figure 1 As shown, Figure 1 This is the high performance liquid chromatogram of compound AE-3-7 at a detection wavelength of 254 nm, with a retention time of 10.552 min.

[0046] The NMR spectra of compound AE-3-7 were recorded using a Bruker AvanceII-500 NMR spectrometer (Bruker, Switzerland). 1 H and 13 C NMR spectrum. 1 H and 13 The operating frequencies of C NMR were 500 MHz and 125 MHz, respectively. The sample was dissolved in 600 μL CD3OD and the experimental temperature was 300 K. The coupling constant ( J ) is expressed in Hz, and chemical shift is expressed in δ (ppm) and tetramethylsilane (TMS) was used as the internal standard. Figure 2 and Figure 3 .

[0047] 1 H-NMR (500 MHz, CD3OD) data are as follows: δ 7.73 (1H, d, J = 2.0 Hz, H-2'), 7.59(1H, dd, J = 8.4, 1.9 Hz, H-6'), 6.88 (1H, d, J = 8.5 Hz, H-5'), 6.37 (1H, s,H-8), 6.19 (1H, d, J = 2.0 Hz, H-6), 5.25 (1H, d, J = 7.5 Hz, Glc H-1"), 3.74(1H, dd, J= 11.9, 2.2 Hz, Glc H-6"a), 3.61 (1H, dd, J = 11.9, 5.3 Hz, Glc H-6"b), 3.49 (2H, dt, J = 17.8, 9.1 Hz, Glc H-2", 3"), 3.42 – 3.35 (1H, m, GlcH-5"), 3.26 (1H, ddd, J = 9.6, 5.2, 2.3 Hz, Glc H-4").

[0048] 13 C-NMR (125 MHz, CD3OD) data are as follows: δ 179.5 (C-4), 166.1 (C-7), 163.1 (C-5), 159.1 (C-9), 158.5 (C-2), 149.9 (C-4'), 146.0 (C-3'), 135.8 (C-3), 123.3(C-1'), 123.2 (C-6'), 117.8 (C-5'), 116.1 (C-2'), 105.8 (C-10), 104.57 (Glc,C-1"), 100.0 (C-6), 94.9 (C-8), 78.5 (Glc C-5"), 78.2 (Glc C-3"), 75.90 (GlcC-2"), 71.3 (GlcC-4"), 62.7 (Glc C-6").

[0049] Based on the NMR data and liquid chromatography data of compound AE-3-7 and compared with the literature, it is speculated that compound AE-3-7 contains isoquercitrin; Isoquercitrin, English name: Isoquercitrin; Molecular formula: C 21 H 20 O 12 ; Relative molecular weight: 464.3763; structure is as follows:

[0050] .

[0051] Determination of xanthine oxidase activity inhibition rate:

[0052] 1. Solution configuration:

[0053] PBS buffer (0.1 M, pH = 7.0): Accurately weigh 2.0660 g of disodium hydrogen phosphate, 0.6590 g of sodium dihydrogen phosphate, and 0.9000 g of NaCl, dissolve them in ultrapure water, and dilute to 100 mL.

[0054] 0.1 mM NaOH solution: Accurately weigh 0.0200 g of NaOH, dissolve it in ultrapure water, and dilute to volume in a 5 mL volumetric flask for later use.

[0055] 1 mM xanthine reaction solution: Accurately weigh 0.0075 g of xanthine powder, add 0.5 mL of 0.1 mM NaOH solution for dissolution, dilute to a 5 mL volumetric flask with PBS buffer, and refrigerate at 4 °C in the dark until use.

[0056] 0.1 U / mL xanthine oxidase solution: Dilute the 10 U / mL xanthine oxidase stock solution with PBS buffer to 0.1 U / mL and refrigerate at 4°C in the dark until use.

[0057] 80 μM allopurinol solution: Accurately weigh 0.0065 g of allopurinol and dilute to a 5 mL volumetric flask with PBS buffer to obtain a 10 mM allopurinol solution. Dilute to 80 μM allopurinol solution with PBS buffer and refrigerate at 4°C in the dark until used.

[0058] Sample solution: Accurately weigh 0.0050 g of isoquercetin (component AE-3-7), dissolve it in DMSO, and dilute it with PBS buffer, controlling the DMSO concentration within 3% to obtain a sample solution with a concentration of 1 mg / mL.

[0059] The addition amount was controlled according to the above method to obtain allopurinol and sample solutions of different concentrations.

[0060] 2. Xanthine oxidase inhibition test method:

[0061] 100 μL of PBS buffer, 25 μL of sample solution, and 25 μL of 0.1 U / mL xanthine oxidase were added to a 96-well plate, mixed by vortexing, and incubated at 37°C for 20 min. After incubation, 50 μL of 1 mM xanthine reaction solution was added, mixed by vortexing, and incubated at 37°C for 5 min. The absorbance of the sample wells was measured at 290 nm. In the blank control group, PBS buffer was used instead of the sample and xanthine reaction solution. In the complete reaction group, PBS buffer was used instead of the sample. In the sample control group, PBS buffer was used instead of xanthine oxidase. Allopurinol served as a positive control. The xanthine oxidase inhibition rate in the sample group was calculated using the following formula (Equation 1).

[0062]

[0063] Wherein, C1 is the absorbance value of the blank control group; C2 is the absorbance value of the complete reaction group; C3 is the absorbance value of the sample reaction group; and C4 is the absorbance value of the sample control group.

[0064] The inhibitory abilities of isoquercetin (component AE-3-7) and allopurinol obtained in the present invention on xanthine oxidase at different concentrations are shown in Table 1. In Table 1, Test 1, Test 2, and Test 3 are three parallel experiments respectively.

[0065]

[0066] 3. Conclusion

[0067] In the above enzyme activity assay, it was found that the component AE-3-7 (isoquercetin) obtained in the present invention can effectively reduce the activity of xanthine oxidase. At a concentration of 0.623 mg / mL, the inhibition rate can reach more than 98.5%.

[0068] The results are as follows Figure 4 , its IC 50 The value was 101.12 ± 1.66 μg / mL, which shows that the isoquercetin prepared in the present invention can effectively inhibit the activity of xanthine oxidase. 50 Half maximal inhibitory concentration refers to the half inhibitory concentration of the antagonist being measured.

[0069] In the description of this specification, reference to the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification.

[0070] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing isoquercetin that inhibits xanthine oxidase activity, characterized in that: The following steps are involved: (1) Using dried tangerine peel as raw material, after extraction with organic liquid, the extracts were combined and the solvent was extracted by rotary evaporation to obtain the organic layer extract AE; (2) dissolving the organic layer extract AE, and then separating and purifying it by column, isocratically eluting it, and collecting the fractions, which are fraction AE-3; (3) Dissolving the component AE-3 again, separating and purifying it, eluting it, and collecting the components to obtain component AE-3-7; nuclear magnetic resonance identification of the component AE-3-7 revealed that it contained isoquercetin; (4) Conducting enzyme activity inhibition experiments to detect the inhibition rate of component AE-3-7 on xanthine oxidase activity; In step (1), the extraction is carried out by soaking in ethyl acetate and accelerating the extraction with the assistance of ultrasound, the ultrasonic frequency is 20-50kHz, and the extraction is repeated 3 times; In step (2), the solution was dissolved in a 50% methanol-water solution, and the separation and purification was performed using a medium-low pressure reversed-phase C18 column with a flow rate of 30 mL / min. The elution was performed using a 30% methanol-water solution, and the collected components were those from 38 to 63 minutes. In step (3), pure methanol was used for dissolution, Sephadex LH-20 was used for separation and purification, the flow rate was set to 0.25 mL / min, methanol was used as the eluent, and the fractions collected were the fractions from 1635 to 1770 min; Isoquercetin is a flavonoid compound with C 21 H 20 O 12 The molecular formula and chemical structure are as follows: 。 2. The method for preparing isoquercetin for inhibiting xanthine oxidase activity according to claim 1, wherein: In step (4), the process of enzyme activity inhibition experiment includes: S1. Add 100 μL PBS buffer, 25 μL sample solution, and 25 μL 0.1 U / mL xanthine oxidase to a 96-well plate, shake well, and incubate at 37°C for 20 min. S2. After incubation, add 50 μL of 1 mM xanthine reaction solution, shake to mix, and incubate at 37°C for 5 min. S3. Then measure the absorbance of the sample well at 290 nm; S4. The blank control group consisted of PBS buffer instead of the sample and xanthine reaction solution. In the complete reaction group, PBS buffer was used instead of the sample. In the sample control group, PBS buffer was used instead of xanthine oxidase. Allopurinol served as a positive control. S5. The calculation formula for the inhibition rate of xanthine oxidase in the sample group is as follows: ， Wherein, C1 is the absorbance value of the blank control group; C2 is the absorbance value of the complete reaction group; C3 is the absorbance value of the sample reaction group; and C4 is the absorbance value of the sample control group.

3. The method for preparing isoquercetin for inhibiting xanthine oxidase activity according to claim 1, wherein: In step (4), isoquercetin inhibits the IC of xanthine oxidase 50 The value was 101.12 ± 1.66 μg / mL.

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