LU502598B1

LU502598B1 - Composition for preventing and treating hyperuricemia, and preparation method and application thereof

Info

Publication number: LU502598B1
Application number: LU502598A
Authority: LU
Inventors: Qinghe Zhao; Li Liu; Miyi Yang; Shi Yang; Hai Ma; Yuqing Tan; Guojun Han
Original assignee: Beijing Kino Biotechnology Co Ltd; Shi Yang
Priority date: 2022-07-28
Filing date: 2022-07-28
Publication date: 2024-02-01

Abstract

The present disclosure relates to the field of pharmacological treatment, in particular to a composition for preventing and treating hyperuricemia, and a preparation method and application thereof.

Description

BL-5541

COMPOSITION FOR PREVENTING AND TREATING HYPERURICEMIA, AND LU502598

PREPARATION METHOD AND APPLICATION THEREOF

TECHNICAL FIELD

[01] The present disclosure relates to the field of pharmacological treatment, in particular to a composition for preventing and treating hyperuricemia, and a preparation method and application thereof.

BACKGROUND ART

[02] There are fewer studies or no large-scale studies on compounds of traditional Chinese medicine for the prevention and treatment of hyperuricemia (HUA), fewer compounds can be really and widely applied clinically, and almost no traditional Chinese medicine are available for treating gout on the current market. Thus, search of novel anti-HUA drugs with high efficiency and low toxicity is still a hot spot in current research.

SUMMARY

[03] To solve the above problem, the present disclosure provides a composition for preventing and treating hyperuricemia, and a preparation method and application thereof. The composition of the present disclosure is capable of achieving the effect of reducing the level of uric acid in blood, and meanwhile basically has no toxic or side effects.

[04] In order to achieve the above purpose, the present disclosure provides the following technical solution:

[05] The present disclosure provides the composition for preventing and treating the hyperuricemia, and the composition includes the following effective components in percentage by mass: 23.2% of flavones, 22.9% of coumarins and 19.5% of phenolic acids.

[06] The present disclosure provides application of the composition or the composition prepared by the preparation method in preparation of drugs for preventing and treating the hyperuricemia.

[07] Beneficial effects: the present disclosure provides the composition for preventing and treating the hyperuricemia, and the composition includes the effective components: 23.2% of flavones, 22.9% of coumarins and 19.5% of phenolic acids. The composition of the present disclosure is capable of reducing the level of uric acid in blood, and basically has no toxic or side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

[08] FIG. 1 is a standard curve of rutin;

[09] FIG. 2 is a standard curve of caffeic acids; and

[10] FIG. 3 is a standard curve of coumarins.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiment 1

[11] 1. Preparation of composition for preventing and treating hyperuricemia 1

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[12] (1) 100 g of sunflower calathide was cut into 2-3 cm pieces, and put into a 2 L round- bottom flask, water with a mass of 15 times that of the sunflower calathide was added for LU502598 soaking for 30 min, then the round-bottom flask was heated for decocting the sunflower calathide for 1.5 h, and the sunflower calathide was filtered to collect first filtrates and first residues; the first residues were mixed with water with a mass of 15 times that of the sunflower calathide, then decocted for 1.5 h, and filtered, to obtain second filtrates and second residues; the first filtrates and the second filtrates were combined, put into a water bath, and concentrated at 80°C to 700 mL for later use, 50 mL of concentrated solutions were taken and put into an evaporating dish, and evaporated to dryness, and a calculated solid yield was 50.6%.

[13] (2) Macroporous resin with a model of HPD500 was washed with 95% ethanol, water,

IN HCI, water, IN NaOH, water and 95% ethanol in sequence, then the properly treated macroporous resin was packed into columns, a column volume was 120 mL, a loading liquid amount was 700 mL, the concentrated solutions were centrifuged (centrifugal machine: 4000 / min, centrifuging: 2 min), then supernatants were poured into the columns along column walls slowly, and then washed with water in a volume of 5 times that of the columns and 70% ethanol in a volume of 5 times that of the columns respectively, eluants of the 70% ethanol were collected, concentrated and dried in a 80°C water bath to solids, to obtain the composition for preventing and treating the hyperuricemia, and the calculated (solid) yield of the composition for preventing and treating the hyperuricemia was 1.45%.

[14] 2. Determination of contents of flavones, coumarins and phenolic acids in composition

[15] 1. Determination of content of flavones

[16] (a) Preparation of rutin reference solutions

[17] 2.5 mg of rutin reference substances were weighed precisely, and put into a 50 mL volumetric flask, a proper amount of absolute ethanol was added, ultrasonic treatment was performed for dissolving the rutin reference substances, the solutions were placed for cooling, the absolute ethanol was added to the scale, and shaken up to obtain the rutin reference solutions (anhydrous rutin concentration: 0.05 mg/mL).

[18] (b) Preparation of a standard curve

[19] O05 mL, 1 mL, 2 mL, 5 mL, 8 mL, 10 mL and 12 mL of reference solutions were weighed precisely, and put into 25 mL volumetric flasks respectively, 1 mL of 5% sodium nitrite solutions were added, shaken up, and placed for 6 min; 1 mL of 10% aluminum nitrate solutions were added, shaken up, and placed for 6 min; 10 mL of sodium hydroxide solutions were added, water was added to the scale, the solutions were shaken up, and placed for 15 min, and the absorbance was immediately determined at a 500 nm wavelength with an ultraviolet-visible spectrophotometry by taking corresponding reagents as blank, namely all reagents except for the test solutions; the standard curve was drawn by taking the absorbance as ordinates and the content as abscissas, the determined absorbance results were shown in Table 1, the standard curve was shown in FIG. 1, the standard curve was: y=0.7699x-0.0178, R2 was 0.9999, and the standard curve had a good linear relationship, and can be used for calculating the content of the flavones.

Table 1 Absorbance results of rutin of different concentrations [emma [ows | 005 | 01 | 025 | 04 | 05 [06

[20] (c) Preparation of test solutions 2

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[21] 200 mg of composition (solid) for preventing and treating the hyperuricemia was weighed, a small amount of 70% ethanol was added for dissolving the composition, the LU502598 solutions were transferred to the 50 mL volumetric flask, 70% ethanol was added to the scale again, and shaken up, to obtain the reference solutions.

[22] (d) Determination method

[23] 2 mL of test solutions were weighed precisely, and put into the 25 mL volumetric flask, water was added to 6 mL, 1 mL of 5% sodium nitrite solutions were added, shaken up, and placed for 6 min, 1 mL of 10% aluminum nitrate solutions were added, shaken up, and placed for 6 min, 10 mL of sodium hydroxide test solutions were added, water was added to the scale again, and the solutions were shaken up, and placed for 15 min; the absorbance was immediately determined at the 500 nm wavelength with the ultraviolet-visible spectrophotometry by taking the corresponding reagents as blank, namely all reagents except for the test solutions, the content of the rutin in the test solutions was read from the standard curve, and the content of the flavones was obtained by calculation.

[24] (2) Determination of content of phenolic acids

[25] (a) Preparation of caffeic acid reference solutions

[26] A proper amount of caffeic acid reference substances were taken and weighed precisely, and absolute methanol was added to prepare 90 ng/mL solutions, to obtain the caffeic acid reference solutions.

[27] (b) Preparation of a standard curve

[28] 025 mL, 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL and 2.5 mL of reference solutions were weighed precisely, and put into 25 mL volumetric flasks respectively, water was added to 6 mL, 1 mL of 5% sodium nitrite solutions were added, shaken up, and placed for 6 min; 1 mL of 10% aluminum nitrate solutions were added, shaken up, and placed for 6 min; 10 mL of sodium hydroxide test solutions were added, water was added to the scale again, the solutions were shaken up, and were placed for 15 min, and the absorbance was immediately determined at the 500 nm wavelength with the ultraviolet-visible spectrophotometry by taking the corresponding reagents as blank, namely all reagents except for the test solutions; the standard curve was drawn by taking the absorbance as the ordinates and the concentration as the abscissas, the determined absorbance results were shown in Table 2, the caffeic acid standard curve was shown in FIG. 2, the standard curve was: y=11.169x-0.2483, R? was 0.9902, and the standard curve had a good linear relationship, and can be used for calculating the content of the phenolic acids.

Table 2 Absorbance results of phenolic acids with different concentrations

Cones | 0025 | 005 | ow | om | ow | om

[29] (©) Preparation of test solutions

[30] 200 mg of the dried composition (solid) for preventing and treating the hyperuricemia was weighed precisely, a small amount of 70% ethanol was added for dissolving the composition, the solutions were transferred to the 50 mL volumetric flask, 70% ethanol was added to the scale again, and shaken up, to obtain the test solutions.

[31] (d) Determination method 3

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[32] 0.5 mL of the test articles were weighed precisely, and put into the 25 mL volumetric flask, absolute ethanol was added to 5.0 mL, 2.0 mL of 0.3% sodium dodecyl sulfate and 1.0 LU502598 mL of mixed solutions of 0.6% ferric chloride and 0.9% potassium ferricyanide (1:0.9) were added, shaken up, and placed for 5 min in the dark, and 0.1 mol/L of hydrochloric acid solutions were added to the scale, shaken up, and placed for 20 min in the dark; the absorbance was determined at a 700 nm wavelength with the ultraviolet-visible spectrophotometry by taking the corresponding reagents as blank, namely all reagents except for the test solutions, the weight of the caffeic acids in the test solutions was read from the standard curve, and the content of the phenolic acids was obtained by calculation.

[33] (3) Determination of content of coumarins

[34] (a) Preparation of coumarin reference solutions

[35] 2.0 mg/mL reference stock solutions were prepared with methanol, and then diluted into 200 pg/mL coumarin standard solutions for later use.

[36] (B) Selection of wavelength for determination

[37] 0.3 mL of coumarin standard solutions were weighed precisely, and put into a 10 mL volumetric flask, the methanol was added to the scale, and shaken up, and scanning was performed within the wavelength range from 200 to 400 nm; additionally, 0.3 mL of sunflower calathide extract test solutions were weighed precisely, and put into the 10 mL volumetric flask, the methanol was added to the scale, and shaken up, scanning was performed within the wavelength range from 200 to 400 nm (or determination was directly conducted with the test solutions), and the determined maximum absorption wavelength was 287 nm.

[38] (c) Preparation of a standard curve

[39] 0.1 mL, 03 mL, 04 mL, 0.5 mL, 0.6 mL, 0.8 mL and 1 mL of 200 pg/mL reference solutions were precisely weighed respectively, and put into 10 mL volumetric flasks, methyl alcohol was added to the scale with constant volume, and the absorbance was determined at a 287 nm wavelength with the ultraviolet-visible spectrophotometry by taking the corresponding reagents as blank, namely all reagents except for the test solutions; the standard curve was drawn by taking the absorbance as ordinates and the content as abscissas, the determined absorbance results were shown in Table 3, the standard curve was shown in FIG. 3, the standard curve was: y=2.1748x+0.0291, R? was 0.9954, and the standard curve had a good linear relationship, and can be used for calculating the content of the coumarins.

Table 3 Absorbance results of coumarins of different concentrations

[40] (c) Preparation of test solutions

[41] 200mg of the dried composition (solid) for preventing and treating the hyperuricemia was weighed precisely, a small amount of 70% ethanol was added for dissolving the composition, the solutions were transferred to the 50 mL volumetric flask, 70% ethanol was added to the scale again, and shaken up, to obtain the test solutions.

[42] (d) Determination method

[43] 1 mL of the test solutions were weighed precisely, and 70% ethanol was added to the 4

BL-5541 constant volume in the 25 mL volumetric flask, the absorbance was determined at the 287 nm wavelength with the ultraviolet-visible spectrophotometry by taking the corresponding reagents LU502598 as blank, namely all reagents except for the test solutions, the weight of the coumarins in the test solutions was read from the standard curve, and the content of the coumarins was obtained by calculation.

[44] The above-mentioned preparation process of the composition for preventing and treating the hyperuricemia was repeated for 3 times, the composition for reducing uric acid obtained by each extraction was determined, and experimental results were shown in Table 4.

Table 4 Determination results of contents of flavones, coumarins and phenolic acids in composition for preventing and treating hyperuricemia

Total

Flavones | Phenolic acids | Coumarins | amount of | Solid mass 0

No. Percentage % (mg) (mg) (mg) components (2) (2) 328.0 271.4 342.8 0.942 1.448 336.1 282.0 332.0 0.951 1.448 314.2 265.2 314.8 0.892 1.420

[45] It can be known from the data recorded in Table 4 that the extraction process of the present disclosure was stable, and the percentages of three effective components were between 63% and 65.1%.

Embodiment 2

[46] Optimization of macroporous resin columns

[47] Macroporous resin columns with different models of HPD500 HPD750, HPD826 and

HPD-BJQH were adopted, the rest steps were the same as those in the embodiment 1; the percentages of the contents of the flavones, the coumarins and the phenolic acids in purified substances serve as objects of investigation in this experiment, the models of macroporous resin was optimally screened, and optimization results were shown in Table 5.

Table 5 Optimization results of macroporous resin columns

Phenolic ; Total ; 1.

Flavones ; Coumarins | amount of Solid Percentage | Solid yield

Model acids ° 0 (mg) (mg) components | mass (g) (%) (%) (mg) (e) 8

HPD500 | 2257 | 2398 | 2702 1.188 | 619%

HPD750 | 185.1 | 2103 | 2345 | 06 | L112 | 566%

HPD826 | 1928 | 2005 | 2392 | 06 | 0997 | 634%

HPD-BJQH | 1933 | 2249 246,3 1.072 62.0%

[48] It can be known from data recorded in Table 5 that, two factors of the solid yield and percentage were comprehensively considered, and experimental results showed that the macroporous resin with the model of HPD500 was optimal.

Embodiment 3

[49] In this experiment, three factors of a macroporous resin column packing volume, a loading liquid amount and an eluant concentration, which affect the contents of the effective

BL-5541 components, were studied, and experimental results were shown in Table 6.

LU502598

Table 6 Optimization results of macroporous resin column packing volume, loading liquid amount and eluant concentration

Colu | Loadi Phenol Total ; mn ng Ethanol Flavon ; Coum | Solid amount

Experim 7 . IC ; Percenta volum | liquid | Concentrati es ; arins | mass of ° ent No. 0 acids ge (%) e amoun on (%) (mg) (me) (mg) (g) compone (mL) | t (mL) 5 nts (g)

Table 7 Analysis results of orthogonal test data 0.659 0.602 0.549 0.567 0.597 0.611 0.560 0.587 0.626 0.099 0.015 0.077

[50] Note: 1 represents the macroporous resin column packing volume, 2 represents the loading liquid amount, and 3 represents the eluant concentration. [S51] It can be known from the experimental data recorded in Table 6 and Table 7, orthogonal experimental results and variance analysis were used to conclude that, in the optimal macroporous resin purification process, the macroporous resin column packing volume was 120 mL, the loading liquid amount was 700 mL, 70% ethanol solutions were eluants, and the factors affecting the effective components was the packing volume, the eluant concentration and the loading liquid amount, which are ranked in percentages.

Comparative Example 1

[52] 1. Preparation of sunflower calathide water extract

[53] Experimental operation was the same as the step (1) in item 1 of the embodiment 1.

[54] 2. Determination of contents of flavones, coumarins and phenolic acids in composition

[55] Experimental operation was the same as the content determination steps in item 2 of the embodiment 1.

[56] The step (1) in item 1 of the embodiment 1 was repeated for 3 times, the composition obtained by each extraction was determined, and experimental results were shown in Table 8. 6

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Table 8 Determination results of contents of flavones, coumarins and phenolic acids in composition obtained by water extraction process LU502598

Total amount

No Flavones | Phenolic acids | Coumarins of Solid mass | Percentage ; (mg) (mg) (mg) components (g) (%) (8)

[57] It can be known from the data recorded in Table 8 that, the process of the comparative example 1 was adopted, and the percentages of three effective components were 2% respectively.

Comparative Example 2

[58] 1. Sunflower calathide water extraction and alcohol precipitation process

[59] (1) Preparation of sunflower calathide water extract

[60] Experimental operation was the same as the step (1) in item 1 of the embodiment 1, to obtain extracting solutions.

[61] (2) Preparation of sunflower calathide water extract alcohol sediment

[62] The obtained extracting solutions were concentrated to a density of 1.2 g/mL, then 95% ethanol was added such that the content of the ethanol was 70%, the solutions stood for 24 h, and then were filtered, and the filtrates were dried to obtain the composition, so that the sunflower calathide water extract alcohol sediment was obtained.

[63] 2. Determination of contents of flavones, coumarins and phenolic acids in composition

[64] Experimental operation was the same as the content determination steps in item 2 of the embodiment 1.

[65] The step (1) and the step (2) in 1 of the comparative example 1 were repeated for 3 times, the composition obtained by each extraction was determined, and experimental results were shown in Table 9.

Table 9 Determination results of contents of flavones, coumarins and phenolic acids in composition obtained by water extraction and alcohol precipitation process

Total

Flavones | Phenolic acids | Coumarins | amount of | Solid mass 0

No. Percentage % (mg) (mg) (mg) components (2) (g)

[66] It can be known from the data recorded in Table 9 that, the process of the comparative example 2 was adopted, and the percentages ofthree effective components were between 14.1% and 15%. 7

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Comparative Example 3

LU502598

[67] 1. Sunflower calathide water extract alcohol sediment passing macroporous resin process

[68] (1) Preparation of sunflower calathide water extract alcohol sediment

[69] Experimental operation was the same as the experimental steps (1) and (2) in item 1 of the comparative example 2.

[70] (2) Sunflower calathide water extract alcohol sediment passing macroporous resin

[71] Experimental operation was the same as the step (2) in item 1 of the embodiment 1.

[72] Experiment 2: Determination of contents of flavones, coumarins and phenolic acids in composition

[73] Experimental operation was the same as the content determination steps in the experiment 2 of the embodiment 1.

[74] The step (1) and the step (2) in 1 of the comparative examples were repeated for 3 times, the composition obtained by each extraction was determined, and experimental results were shown in Table 10.

Table 10 Determination results of contents of flavones, coumarins and phenolic acids in composition obtained by water extraction and alcohol extraction and passing macroporous resin process

Total

Flavones Phenolic acids | Coumarins | amount of | Solid mass 0

No. Percentage % (mg) (mg) (mg) components (2) (2)

It can be known from the data recorded in Table 10 that, the process of the comparative example 3 was adopted, and the percentages of three effective components were between 37.1% and 38.4%. 8

Claims

BL-5541 CLAIMS LU502598

1. A composition for preventing and treating hyperuricemia, characterized in that the composition comprises the following effective components in percentage by mass: 23.2% of flavones, 22.9% of coumarins and 19.5% of phenolic acids.

2. A preparation method of the composition according to claim 1, characterized by comprising the following steps: soaking a sunflower calathide with seeds removed after mixing it with water, decocting it for 1.5 h, and filtering the mixtures with a 300-mesh filter sieve, to obtain filtrates; and concentrating the filtrates, then enabling the filtrates to pass through macroporous resin columns, conducting elution with water and 70% ethanol solutions in sequence, collecting eluants of the ethanol solutions, to obtain the composition for preventing and treating the hyperuricemia, wherein the amounts of the water and the ethanol solutions are both 5 times the volume of the macroporous resin columns.

3. The preparation method according to claim 2, characterized by further comprising: cutting up the sunflower calathide before soaking, wherein a particle size of the chopped sunflower calathide is 2 to 3 cm.

4. The preparation method according to claim 2, characterized in that the decoction and filtration are conducted for 2 times; and a mass of the water for each decoction is 15 times that of the sunflower calathide.

5. The preparation method according to claim 2, characterized in that the concentration temperature is 80°C.

6. The preparation method according to claim 5, characterized in that a density of concentrated solutions obtained after concentration is 1.033 g/mL.

7. The preparation method according to claim 2, characterized in that the model of the macroporous resin columns is HPD500; and a column packing volume of the macroporous resin columns is 120 mL.

8. The preparation method of claim 7, characterized in that a loading liquid amount of the macroporous resin columns is 700 mL.

9. The preparation method according to claim 2 or 5 or 6, characterized by further comprising: centrifuging after concentration, wherein the rotating speed of centrifuging is 4000 rpm, and the time is 2 min.

10. Application of the composition according to claim 1 or the composition prepared by the preparation method according to any one of claims 2-9 in preparation of drugs for preventing and treating the hyperuricemia. 9