Method for extracting components for treating gout in sunflower disk

Abstract

The invention relates to a method for extracting components for treating gout in a sunflower disk, which improves the yield of a solid extract by improving the macroporous resin elution step, and in the extracted solid extract, the contents of effective components such as rutin, caffeic acid and 6-methylcoumarin for treating gout are obviously increased.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction, specifically to a method for extracting gout-treating components from sunflower heads. Background Technology

[0002] Gout is an inflammatory arthritis caused by the deposition of monosodium urate crystals in joints, tendons, bursae, and other tissues. Clinical features include hyperuricemia, recurrent acute arthritis, tophi deposition, tophaceous chronic arthritis and joint deformities, and uric acid urinary tract stones. It falls under the category of metabolic rheumatic diseases, and its incidence is increasing annually, posing a serious threat to patients' health and quality of life. The pathogenesis of gout is complex and closely related to uric acid metabolism disorders. When the uric acid level in the blood exceeds the saturation threshold, monosodium urate crystals deposit in the joints and surrounding tissues, causing severe pain, inflammation, and tissue damage. In recent years, the global incidence of gout has increased significantly, especially in developed regions. In my country, the incidence of gout in affluent urban areas is significantly higher than in rural areas, and gout has gradually become a disease of affluence. Currently, marketed anti-gout drugs are mainly Western medicines, primarily relieving pain and lowering uric acid levels. While they alleviate local symptoms of gout to some extent, they mostly treat the symptoms but not the root cause, and have many side effects, poor adherence, and are not conducive to long-term use. Some Western medicines treat gout by addressing uric acid production, but their mechanisms are not yet fully understood. They have low bioavailability, poor tolerability, numerous adverse reactions, and are expensive, placing a financial burden on gout patients. Therefore, the use of Western medicines in gout treatment is limited.

[0003] With the development of science and technology, research on traditional Chinese medicine (TCM) treatment for gout has become increasingly diversified. The efficacy and mechanisms of action of drugs are objectively explored from multiple perspectives, including molecular and cellular levels. TCM treatment for gout has continuously developed in theoretical and clinical practice research, demonstrating its unique advantages. Its anti-gout mechanisms include lowering uric acid, anti-inflammation, analgesia, and anti-oxidation, effectively relieving gout symptoms, reducing inflammatory markers, protecting liver and kidney function, reducing attacks, and improving patients' quality of life. However, clinical application requires drugs and prodrugs that can lower uric acid, reduce inflammation, and relieve analgesia while minimizing damage to organ function. However, truly effective TCM components with definite efficacy are difficult to enter the clinical trial stage; most remain only at the stage of exploring efficacy and animal experiments. There are many problems in how these basic research results can be truly translated into clinical applications, and the discovered effective components lack systematicity in basic research.

[0004] Sunflower disc (Helianthus annuus L.) refers to the sunflower head after the seeds are removed. The 2006 edition of the *Chinese Materia Medica* states that sunflower disc is sweet and cold in nature, enters the liver meridian, and functions to clear heat and calm the liver, relieve pain and stop bleeding; it is mainly used to treat headaches, dizziness, tinnitus, dysmenorrhea, hypertension, uterine bleeding, and sores. Studies have shown that total flavonoids from sunflower disc have antibacterial activity, with varying sensitivity to different bacterial groups. Sunflower disc contains effective substances for lowering blood pressure, and this has been confirmed by research from Nankai University. They found that the effective components of sunflower disc mainly exert their hypotensive effect peripherally, by dilating blood vessels, reducing peripheral resistance, and slowing the heart rate, thus reducing cardiac output and ultimately lowering blood pressure. Liu Xiaobo's research on sunflower disc granules found that sunflower disc powder not only has anti-inflammatory, anti-swelling, and analgesic effects, but can also lower blood uric acid concentration and dissolve sodium urate crystals. Further research on the extraction of active substances revealed that the alkaloids, flavonoids, and small molecule peptides in sunflower seed powder also have the same effects. These conclusions lay the foundation for research on the anti-gout effects of sunflower seed powder.

[0005] Traditional Chinese medicine (TCM) treatment, with its unique advantages of multi-target and multi-pathway synergistic regulation, has demonstrated significant characteristics in the treatment of chronic and complex diseases. In particular, many TCM herbs are derived from natural plants that are both food and medicine, exhibiting characteristics such as low toxicity and high safety, aligning with the modern development concept of "green medicine." However, the current domestic and international gout treatment drug market is almost entirely monopolized by chemical drugs, with a severe shortage of TCM-based anti-gout drugs, which is inconsistent with the traditional advantages of TCM in the treatment of metabolic diseases. Therefore, developing novel anti-gout drugs based on TCM theory not only has significant clinical value but also fills a market gap. In recent years, finding highly effective and low-toxicity active ingredients from natural plants has become an important direction in new drug development. Our previous research found that sunflower heads are rich in flavonoids, organic acids, polysaccharides, and other bioactive components, exhibiting significant uric acid-lowering, anti-inflammatory, and analgesic effects. Its mechanism of action may involve multiple aspects, such as inhibiting xanthine oxidase activity, regulating URAT1 transporter expression, and downregulating inflammatory factors, which highly coincides with several key targets in gout treatment. Of particular note is that the safety of sunflower heads, as a traditional edible plant, has been verified through long-term practical experience, providing an important guarantee for drug development. Based on these advantages, developing sunflower heads into novel anti-gout drugs has multiple important implications: First, it can provide a safer treatment option that aligns with the concept of "prevention is better than cure"; second, it can increase the added value of agricultural by-products and promote comprehensive resource utilization; and finally, it can provide a typical case for the modernization of traditional Chinese medicine research. Therefore, in-depth research on the isolation and identification of the active ingredients in sunflower heads for anti-gout, the study of their mechanisms of action, and the development of formulations is an important topic with both scientific value and social benefits.

[0006] CN112933126A discloses a composition for preventing and treating hyperuricemia, comprising the following active ingredients in weight percentages: 23.2% flavonoids, 22.9% coumarins, and 19.5% phenolic acids. The extraction method involves soaking sunflower heads (after seed removal) in water, decocting for 1.5 hours, filtering through a 300-mesh sieve to obtain a filtrate; concentrating the filtrate and passing it through a macroporous resin column, eluting sequentially with water and 70% ethanol solution, collecting the eluent of the ethanol solution to obtain the composition for preventing and treating hyperuricemia; the amounts of water and ethanol used are each 5 times the volume of the macroporous resin column. The solid yield of Example 1 is 1.45%.

[0007] CN112891386A discloses a composition for lowering uric acid, wherein the active ingredient of the composition is phenolic acid, and the mass of the phenolic acid is 37% of the mass of the composition. The extraction method involves soaking sunflower heads (after seed removal) in water, decocting for 2 hours, and then filtering the solution through a 300-mesh sieve to obtain the filtrate. The filtrate is concentrated and passed through a macroporous resin column, followed by elution with water and ethanol aqueous solutions with volume percentages of 10%, 20%, 60%, and 70%, respectively. The eluent from the ethanol aqueous solution is collected in fractions to obtain the composition for lowering uric acid. The amounts of water and ethanol aqueous solutions with volume percentages of 10%, 20%, 60%, and 70% are each three times the volume of the macroporous resin column. Table 2 of Example 1 shows that the solid mass extracted from 100g of sunflower heads is between 760-780mg, i.e., the solid yield is approximately 0.77%. Existing techniques result in low solid content and low levels of effective ingredients for treating gout when eluting with macroporous resins. Summary of the Invention

[0008] Therefore, the technical problem to be solved by the present invention is to overcome the defects in the prior art, thereby providing a method for extracting the gout-treating component from sunflower heads, comprising the following steps: (1) Obtain the sunflower disc decoction extract; (2) Concentrate the water-based extract and load it onto a macroporous resin column; (3) Wash away impurities with water and 3%-6% ethanol in sequence; (4) Elute with 60%-75% ethanol; (5) Collect the eluent from step (4), concentrate and dry it to obtain the gout treatment component.

[0009] Preferably, the macroporous resin column is a styrene polar macroporous resin, and more preferably HPD-500.

[0010] In step (3), it is preferable to wash away impurities with water and 4-5% ethanol in sequence.

[0011] In step (3), the preferred amount of ethanol is 1.1-1.3 times the column volume. The preferred flow rate is 1.3-1.6 times the column volume / h.

[0012] In step (4), it is preferred to use 70% ethanol for elution.

[0013] In step (4), the preferred amount of ethanol is 2 times the column volume. The preferred ethanol flow rate is 1.3-1.6 times the column volume / h.

[0014] The present invention also provides a sunflower disc extract, which is prepared using the above-described method.

[0015] It will be readily understood by those skilled in the art that, unless otherwise specified, the volume of ethanol mentioned in this application refers to the volume of the ethanol solution used, rather than the volume converted to pure ethanol.

[0016] The "old process" or "original process" referred to in this invention refers to the process in CN112933126A, which involves eluting directly with 70% ethanol after water washing, excluding the step of eluting with low-concentration ethanol.

[0017] The technical solution of this invention has the following advantages: This invention improves the macroporous resin elution process, thereby increasing the content of active ingredients in the product. Furthermore, the extracted product shows a significant increase in the content of rutin and caffeic acid, effective components for treating gout. Animal experiments also demonstrate that the product of this invention has an enhanced effect on reducing URAT1 protein expression. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 This is the rutin standard curve from Example 2; Figure 2 This is the standard curve for caffeic acid in Example 2; Figure 3 This is the coumarin standard curve from Example 2; Figure 4 This is the HPLC chromatogram of the standard. Figure 5 This is a comparison chart of the HPLC detection results of the products extracted by the present invention and existing technologies; Figure 6 This is a flowchart of the pilot-scale extraction process of Embodiment 3 of the present invention; Figure 7This is a process flow diagram for the purification of macroporous resin (CN112933126A). Figure 8 This is a process flow diagram of the pilot-scale macroporous resin purification in Example 3 of the present invention; Figure 9 This is a bar chart showing the uric acid-lowering effect of Experiment Example 1; Figure 10 This is a bar chart showing the analgesic effect of Experiment Example 1; Figure 11 This is a bar chart showing the anti-inflammatory effect of Experiment Example 1; Figure 12 This is the effect of Experiment 2 on the expression level of URAT1 protein. Detailed Implementation

[0020] Example 1.

[0021] Preparation and concentration of sunflower disc decoction extract 1. Pre-processing of medicinal materials Take 100g of sunflower discs, spread them on a cleaning workbench to remove impurities, sieve to remove mud and dust, and crush them into coarse powder.

[0022] 2. Water extraction First, add coarse sunflower seed powder to the extraction vessel, then add water equal to 10 times the total amount of medicinal materials. Soak for 30 minutes, then decoct for 2 hours. Filter the liquid through a 300-mesh sieve. Next, add water equal to 8 times the total amount of medicinal materials, decoct for 2 hours, and filter the liquid through a 200-mesh sieve. Combine the two decoctions.

[0023] 3. Concentration of aqueous extract Concentrate the medicinal solution until the relative density is 1.02~1.03 (20~30℃), then stop the concentration, filter, and obtain a clear extract.

[0024] Example 2.

[0025] Purification using macroporous resin.

[0026] 1. Column packing: Pack HPD-500 macroporous adsorption resin into the chromatography column, soak in 95% ethanol overnight, drain the 95% ethanol before loading the sample, and wash with water until there is no ethanol odor. At this time, the ratio of column volume to the mass of medicinal material should not be less than 1.2:1. 2. Sample loading: Slowly pump the drug solution into the column. The adsorption rate of the drug solution should not exceed 1.2 column volumes / h, and continue loading until the drug solution is completely loaded. 3. Washing The present invention first washes away impurities with water and low-concentration ethanol, and then elutes with 70% ethanol to obtain the product.

[0027] 3.1 Concentration study of low-concentration ethanol.

[0028] Before receiving 70% ethanol for elution, the present invention collects low-concentration ethanol eluents separately and investigates the process of eluting with 1.2 column volumes of low-concentration ethanol followed by 2 column volumes of 70% ethanol eluent. After the eluent is concentrated and dried into a solid, the content of the effective components ("three components", namely flavonoids, phenolic acids and coumarin compounds) in the eluent is detected and the solid yield is calculated. The experimental results are shown in Table 1.

[0029] Table 1 Results of different alcohol washing processes

[0030] .

[0031] By comparing the effective component content and total solids of the extract obtained by elution with 70% ethanol after adding different low-concentration ethanol eluents, this invention decided to use 3-6% ethanol, preferably 4-5% ethanol, and most preferably 5% ethanol to wash away impurities.

[0032] In Table 1, the method for detecting the content of the active ingredient is ultraviolet-visible spectrophotometry, and the specific operation is as follows: (1) Determination of total flavonoid content (a) Preparation of rutin reference solution Take an appropriate amount of rutin reference standard, accurately weigh it, add an appropriate amount of 60% ethanol, sonicate to dissolve it, cool it, add 60% ethanol to the mark, shake well, and prepare a solution containing 0.1 mg per 1 mL. (b) Preparation of standard curve.

[0033] Accurately pipette 1, 2, 3, 4, 5, and 6 mL of the reference solution into separate 25 mL volumetric flasks. Add 60% ethanol to each flask to a final volume of 6.0 mL. Add 1 mL of 5% sodium nitrite solution, mix well, and let stand for 6 minutes. Then add 1 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 minutes. Add 10 mL of 4% sodium hydroxide solution, then add water to the mark, shake well, and let stand for 15 minutes. Using the corresponding reagents as blanks, measure the absorbance at 500 nm using UV-Vis spectrophotometry (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0401). Plot a standard curve with absorbance on the ordinate and concentration on the abscissa. The measured absorbance results are shown in Table 2, and the standard curve is shown in Table 2. Figure 1 As shown, the standard curve is: y = 10.411x + 0.0041, R0 2 The value is 0.9993, indicating a good linear relationship in the standard curve, which can be used to calculate the total flavonoid content.

[0034] Table 2. Absorbance results of rutin at different concentrations .

[0035] (c) Preparation of the test solution Take 0.05g of sunflower disc extract and place it in a 50mL volumetric flask. Add 60% ethanol to the mark and shake well to prepare the test sample stock solution.

[0036] (d) Determination method Accurately measure 2 mL of the test solution and place it in a 25 mL volumetric flask. Add water to 6 mL, add 1 mL of 5% sodium nitrite solution, shake well, and let stand for 6 min. Add 1 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 min. Add 10 mL of 4% sodium hydroxide solution, then add water to the mark, shake well, and let stand for 15 min. Using the corresponding reagents as blanks (i.e., all reagents except the test solution), immediately use ultraviolet-visible spectrophotometry to measure the absorbance at a wavelength of 500 nm. Read the rutin content in the test solution from the standard curve and calculate the total flavonoid content.

[0037] (2) Determination of total phenolic acid content (a) Preparation of caffeic acid reference solution Take an appropriate amount of caffeic acid reference standard, accurately weigh it, and add anhydrous methanol to prepare a solution containing 90 μg per mL.

[0038] (b) Preparation of standard curve Accurately measure 0.25, 0.5, 1, 1.5, 2, and 2.5 mL of the reference solution into separate 25 mL volumetric flasks. Add anhydrous ethanol to bring the volume to 5 mL. Add 2 mL of 0.3% sodium dodecyl sulfate and 1 mL of a 1:0.9% ferric chloride-potassium ferricyanide mixed solution, mix well, and incubate in the dark for 5 min. Add 0.1 mol / L hydrochloric acid solution to the mark, shake well, and incubate in the dark for 20 min. Using the corresponding reagents as blanks, measure the absorbance at 700 nm using UV-Vis spectrophotometry. Plot a standard curve with absorbance on the ordinate and concentration on the abscissa. The measured absorbance results are shown in Table 3. The caffeic acid standard curve is shown in Table 3. Figure 2 As shown, the standard curve is: y = 193.83x + 0.3124, R0 2 The value is 0.9926, indicating a good linear relationship in the standard curve, which can be used to calculate the total phenolic acid content.

[0039] Table 3. Acid absorbance results of different coffee concentrations .

[0040] (c) Preparation of the test solution Place 0.05g of sunflower disc extract into a 50mL brown volumetric flask, add anhydrous methanol to the mark, and shake well (protect from light before use).

[0041] (d) Determination method Accurately measure 0.5 mL of the above test sample and place it in a 25 mL volumetric flask. Add anhydrous ethanol to bring the volume to 5.0 mL. Add 2.0 mL of 0.3% sodium dodecyl sulfate and 1.0 mL of a mixed solution of 0.6% ferric chloride and 0.9% potassium ferricyanide (1:0.9). Mix well and let stand in the dark for 5 min. Add 0.1 mol / L hydrochloric acid solution to the mark, shake well, and let stand in the dark for 20 min. Using the corresponding reagents as blanks (i.e., all reagents except the test sample solution), measure the absorbance at a wavelength of 700 nm using ultraviolet-visible spectrophotometry. Measure the absorbance and read the weight of caffeic acid in the test sample solution from the standard curve. Calculate the phenolic acid content.

[0042] (3) Determination of total coumarin content (a) Preparation of 6-methylcoumarin reference solution Accurately weigh 12.5 mg of 6-methylcoumarin reference standard, place it in a 25 mL volumetric flask, add 70% ethanol to the mark, and shake well to obtain the reference standard solution.

[0043] (b) Preparation of standard curve Accurately measure 0.1, 0.3, 0.4, 0.5, 0.6, 0.8, and 1.0 mL of the reference solution into separate 25 mL volumetric flasks. Add 70% ethanol to the mark, shake well, and use 70% ethanol as a blank. Measure the absorbance at 290 nm using UV-Vis spectrophotometry. Plot a standard curve with absorbance on the ordinate and concentration on the abscissa. The measured absorbance results are shown in Table 4, and the standard curve is shown below. Figure 3 As shown, the standard curve is: y = 44.307x + 0.049, R0 2 The value is 0.9945, indicating a good linear relationship in the standard curve, which can be used to calculate the total coumarin content.

[0044] Table 4. Absorbance results of different concentrations of 6-methylcoumarin .

[0045] (c) Preparation of the test solution Place 0.05g of sunflower seedpod extract into a 50ml volumetric flask, add 70% ethanol to the mark, and shake well. The extract is then ready.

[0046] (d) Determination method Accurately measure 1 mL of the above test solution, add 70% ethanol to dilute to a 25 mL volumetric flask, using the corresponding reagents as blanks (i.e., all reagents except the test solution), and measure the absorbance at a wavelength of 290 nm using ultraviolet-visible spectrophotometry. Measure the absorbance and read the weight of coumarin in the test solution from the standard curve. Calculate the total coumarin content.

[0047] The results of tests conducted on different elution stages are shown in Table 5 below: Table 5 Results of effective components and solid yield .

[0048] 3.2 Optimization of elution flow rate and elution volume The elution flow rate and elution volume were adjusted, and the solid yield and the proportion of the three components were examined. The results are shown in Table 6. In Table 6, the elution flow rate is expressed in column volume / h, and the elution volume is expressed in column volume.

[0049] Experimental results show that the optimal elution rate for 5% ethanol elution (1.5 column volumes / h, 1.2 column volumes) and the optimal elution rate for 70% ethanol elution (2 column volumes) yield the best extract content and solid yield.

[0050] Table 6 Results of Elution Flow Rate and Elution Volume .

[0051] 3.3 Comparison of old and new processes for sunflower disc extract 3.3.1 Comparison of components in sunflower disc extract processed with old and new methods Table 7 Comparison of content of extracts obtained from new and old extraction and purification processes

[0052] As shown in Table 7, the solid yield of the new process is comparable to that of the old process, but the content of active ingredients is significantly increased, with the total content of total flavonoids, total phenolic acids, and total coumarins increasing by about 19%.

[0053] To further clarify the differences in the content of active ingredients in the extracts, this invention used HPLC to determine the content of rutin, 6-methylcoumarin, and caffeic acid, three components in the sunflower disc extract. The determination method is as follows: (1) Chromatographic conditions Chromatographic column: 5µL, 250×4.6mm column; mobile phase: 0.2% phosphoric acid aqueous solution (A)-methanol solution (B); flow rate: 1mL / min; detection wavelength: 320nm; column temperature: 40℃; injection volume: 10µL, gradient elution sequence as follows: 0-30min: 90%~80% (A), 30~80min: 80%~50% (A).

[0054] 2) Preparation of mixed reference solution Accurately weigh appropriate amounts of rutin, caffeic acid, and 6-methylcoumarin standards, and add 50% methanol to prepare a mixed reference solution with a concentration of 0.04 mg / mL for each standard.

[0055] (3) Preparation of the test solution Accurately weigh 0.2g of sunflower disc extract, add 50% methanol to a 50mL volumetric flask, shake well, filter, and collect the filtrate.

[0056] (4) Determination method Accurately pipette 10 µL each of the mixed reference solution and the test solution into the liquid chromatograph, determine the concentrations, and record the chromatograms. The relative retention times of rutin, 6-methylcoumarin, and caffeic acid in the mixed standard and extracts from both the old and new processes are shown in Table 8. Figure 4 As shown.

[0057] Table 8. Relative retention times of the three components .

[0058] The results of the analysis of sunflower disc extract are shown in Table 9 below. The high-performance liquid chromatogram is shown in the instruction manual. Figure 5 As shown, Figure 5 In the diagram, S2 is the liquid chromatogram of the new process, and S1 is the liquid chromatogram of the old process.

[0059] Table 9 Comparison of Single Component Content in Extracts from New and Old Processes .

[0060] Rutin, caffeic acid, and 6-methylcoumarin are representative of flavonoids, phenolic acids, and coumarins, and the efficacy of rutin and caffeic acid in treating gout and lowering blood uric acid has been documented in numerous studies. Experimental results show that by optimizing the process and increasing elution with 5% ethanol, the content of representative compounds of these three classes in the obtained sunflower disc extract was increased, with a significant increase in the content of the active ingredients rutin and caffeic acid.

[0061] Example 3.

[0062] Scale-up pilot production and granulation.

[0063] The pilot-scale experiment was commissioned to Changchun People's Pharmaceutical Factory. The scale-up experiment was conducted according to the methods in Examples 1 and 2, and the procedure is as follows: Figure 6 and Figure 8 The old water extraction process flow is as follows: Figure 6 The purification process of macroporous resin is as follows: Figure 7 .

[0064] The extract was powdered and granulated; see Table 10 for the formulation.

[0065] Table 10 Granule Prescription .

[0066] Weigh the sunflower disc extract powder and pregelatinized starch according to the prescription, mix them evenly, add an appropriate amount of 95% ethanol solution, make a soft mass, granulate it through an 18-mesh sieve, dry it at 60℃, granulate it through a 20-mesh sieve, add the prescribed amount of magnesium stearate, mix evenly, and the product is ready.

[0067] Experimental Example 1.

[0068] 1. Experimental Objective This invention utilizes a mouse model of hyperuricemia established using yeast combined with potassium oxonate to conduct uric acid-lowering experiments, employs acetic acid to induce writhing in mice for analgesia experiments, and utilizes lipopolysaccharide (LPS) to induce inflammation in mice for anti-inflammatory experiments. The test sample, sunflower disc extract granules, was administered by gavage, and its analgesic, anti-inflammatory, and uric acid-lowering effects were observed.

[0069] 2. Laboratory animals and sample types (1) Uric acid lowering experiment Strain: C57BL / 6 mice, age: 6 weeks, sex: male, quantity: 70 (n=10) Samples: Mouse kidneys, mouse serum (2) Analgesia experiment Strain: ICR mice, age: 6 weeks, sex: half male and half female, quantity: 35 females (n=5), 35 males (n=5) (3) Anti-inflammatory experiment Strain: Kunming mice, age: 6 weeks, sex: half male and half female, quantity: 35 females (n=5), 35 males (n=5) 3. Experimental Materials (1) Medicines Test samples: Sunflower disc extract granules (pilot product of Example 3), potassium oxonate (Sigma-Aldrich, Lot: STBH8632); yeast powder (OXOID, Lot: 2746246-02); benzbromarone (Excella GmbH & Co. KG, Germany), packaged by Kunshan Longdeng Ruide Pharmaceutical Co., Ltd., production batch number: 1806602, national drug approval number J20180056, expiry date: May 22, 2023); uric acid assay kit (uric acid enzymatic method), Sinopharm Group Beijing Enterprises Biotechnology Co., Ltd., batch number: 190902; ibuprofen dispersible tablets (Chifeng Weikang Biochemical Pharmaceutical Co., Ltd.); indomethacin (Kunming Pharmaceutical Group Co., Ltd.); Tween 80 (Soleprosy, Inc.); acetic acid (glacial acetic acid), Beijing Chemical Plant; uric acid (Sigma-Aldrich).

[0070] (2) Instruments Synergyhio microplate reader, manufactured by Biotek; centrifuge, Kubota Corporation, model KUBOTA5922; Sartorius BP211D electronic balance for quantitative weighing of trace substances in laboratory 210; Sartorius BSA3202S-CW electronic balance (serial numbers 24790283, 24790266, 36892266).

[0071] (3) Dosage, method of administration and drug preparation Yeast 0.2 mL / 10 g (body weight) was administered by gavage; benzbromarone 0.1 mL / 10 g (body weight) was administered by gavage; ibuprofen 0.1 mL / 10 g (body weight) was administered by gavage; indomethacin 0.1 mL / 10 g (body weight) was administered by gavage; test sample 0.1 mL / 10 g (body weight) was administered by gavage; potassium oxonate 0.1 mL / 10 g (body weight) was administered by intraperitoneal injection; lipopolysaccharide (LPS) 0.1 mL / 10 g (body weight) was administered by intraperitoneal injection.

[0072] 1) Yeast solution Weigh 50g of yeast powder, dissolve it in 50mL of physiological saline, stir well, and prepare a yeast physiological saline solution of 1g / mL. Prepare and use immediately.

[0073] 2) Potassium oxazine solution Weigh 1.2g of potassium oxonate powder, dissolve it in 40mL of physiological saline, stir and mix well to prepare a 300mg / mL potassium oxonate physiological saline solution, and use it immediately.

[0074] 3) Benzbromarone solution (50 mg / day orally for adults) 50mg / 70kg×70kg×0.0026 / 0.02kg=6.5mg / kg Weigh 19.5 mg of benzbromarone, dissolve it in 30 mL of physiological saline, stir well, and prepare a 0.65 mg / mL benzbromarone physiological saline solution. Prepare and use immediately.

[0075] 4) Ibuprofen: 0.4g / day 0.4g / 70kg×70kg×0.0026 / 0.02kg=52mg / kg Weigh 156 mg of ibuprofen, dissolve it in 30 mL of physiological saline, stir well, and prepare a 5.2 mg / mL benzbromarone saline solution. Prepare and use immediately.

[0076] 5) Lipopolysaccharide (LPS) solution 38.5mg / 70kg×70kg×0.0026 / 0.02kg=5.0mg / kg Weigh 15.0 mg of ibuprofen, dissolve it in 30 mL of physiological saline, stir well, and prepare a 0.5 mg / mL benzbromarone saline solution. Prepare and use immediately.

[0077] 6) Indomethacin solution 25mg / 70kg×70kg×0.0026 / 0.02kg=3.25mg / kg Weigh out 9.75 mg of indomethacin, dissolve it in 30 mL of physiological saline, stir well, and prepare a 0.325 mg / mL benzbromarone saline solution. Prepare and use immediately.

[0078] 7) Test solution Dosage calculation: Based on daily human administration of 3g, 6g, and 12g, and considering the gavage volume in mice, the required gavage solution concentration is calculated as follows: Low dose: 3g / 70kg × 70kg × 0.0026 / 0.02kg = 0.39g / kg Medium dose: 6g / 70kg × 70kg × 0.0026 / 0.02kg = 0.78g / kg High dose: 12g / 70kg × 70kg × 0.0026 / 0.02kg = 1.56g / kg 390 mg / kg low-dose solution of test sample Weigh 390 mg of the test sample, dissolve it in 8 mL of physiological saline, and make up to 10 mL to prepare a solution with a concentration of 390 mg / kg (3 g / day). Mix well and set aside for use.

[0079] 780 mg / kg medium-dose solution of test sample Weigh 780 mg of the test sample, dissolve it in 8 mL of physiological saline, and make up to 10 mL to prepare a solution with a concentration of 780 mg / kg (6 g / day). Mix well and set aside for use.

[0080] 1.56 g / kg high-dose solution of test sample Weigh 1.56g of the test sample, dissolve it in 8mL of physiological saline, and make up to 10mL to prepare a solution with a concentration of 1.56g / kg (12g / day). Mix well and set aside for use.

[0081] Verification of the efficacy of sunflower disc extract granules in lowering uric acid, relieving pain, and reducing inflammation.

[0082] The experimental procedure is as follows: 1. Uric acid-lowering experiment - mouse model of hyperuricemia Six-week-old male C57BL / 6N mice, weighing 18-22g, were used. Before modeling, the mice were randomly grouped according to their body weight. During the experiment, the drugs were administered by gavage and intraperitoneal injection. The gavage administration volume was 0.2mL / 10g (body weight), and the intraperitoneal injection volume was 0.1mL / 10g (body weight). The drugs were administered once a day.

[0083] Phase 1 of the experiment: Except for the control group, all other groups of animals were administered 15 g / kg of yeast by gavage and 300 mg / kg of potassium oxonate by intraperitoneal injection to establish the uric acid model. The modeling period was from day 1 to day 5 at the beginning of the experiment. Two hours after the modeling on day 5, blood was collected from the jaw vein of the mice, and the serum was separated and the serum uric acid level was measured. The mice were then randomly assigned to groups according to the uric acid level.

[0084] The second stage of the experiment: Except for the blank group and the model group, all other groups of animals were administered 15 g / kg yeast by gavage and 300 mg / kg potassium oxonate by intraperitoneal injection for continuous modeling. One hour later, the treatment groups were given different doses (low, medium, and high doses) of sunflower disc extract granules. The treatment group received continuous administration from day 6 to day 12 of the experiment. On day 12, 2 hours after administration, the mice were weighed and sacrificed. Blood was collected, and after standing overnight at 4°C, the blood samples were centrifuged at 3500 r / min for 10 min. The supernatant serum was collected, and the serum uric acid concentration was detected using a uricase kit. The experimental results are shown in Table 11 below. Figure 9 As shown.

[0085] Table 11 Effects of sunflower disc extract particles on serum uric acid levels in mice with hyperuricemia ( ±SD)

[0086] Note: Compared with the blank control group, ###p<0.001. Compared with the model group, , , .

[0087] From Table 11, Figure 9The results showed that the serum uric acid level in model mice induced by yeast combined with potassium oxonate (model group) was significantly increased, with a statistically significant difference compared with the blank group (p<0.001), indicating that the yeast combined with potassium oxonate successfully established a mouse model of hyperuricemia. The positive control drug benzbromarone could reduce serum uric acid, with a statistically significant difference compared with the model group (p<0.05). After administration of low, medium, and high doses (0.36 g / kg, 0.78 g / kg, and 1.56 g / kg) of sunflower disc extract granules, the experimental animals... Serum uric acid levels in the mice were significantly reduced, with statistically significant differences compared to the model control group (p<0.01, p<0.001, p<0.001). Furthermore, the reduction in serum uric acid levels in the low-dose group was similar to that of the positive control drug benzbromarone, while the reduction in serum uric acid levels in the medium- and high-dose groups was superior to that of benzbromarone. This indicates that at doses of 0.36 g / kg, 0.78 g / kg, and 1.56 g / kg, the combination of yeast and potassium oxonate significantly reduced uric acid in the mouse model of hyperuricemia.

[0088] 2. Analgesia test - Mouse acetic acid writhing test Seventy healthy ICR mice, half male and half female, weighing 17-20g, were selected. After acclimatization, the experimental animals were randomly divided into five groups according to their weight: the model group, the positive control drug ibuprofen dispersible tablet group, and the test sample group (0.039g / kg), with 10 mice in each group, half male and half female, and housed separately.

[0089] During the experiment, the drug was administered via gavage (ig) at a volume of 0.1 mL / 10 g (body weight), once daily for 3 consecutive days. The model group received the same volume of physiological saline via gavage. One hour after the last ig administration of the test drug, mice were intraperitoneally injected (ip) with a 0.6%-0.7% glacial acetic acid solution (an analgesic) prepared with physiological saline at a volume of 0.1 mL / 10 g (body weight). The number of writhing movements (abdominal contraction and concavity, trunk and hind limb extension, and body twisting) per mouse within 20 minutes after administration of the analgesic was observed and recorded. The number of writhing movements was used to determine the analgesic effect of the test drug, and intergroup comparisons were performed. The experimental results are shown in Table 12. Figure 10 As shown.

[0090] Table 12 Effect of sunflower disc extract particles on the number of writhing movements induced by acetic acid in mice ( ±SD) .

[0091] Note: Compared with the model group, , .

[0092] From Table 12, Figure 10The results showed that ibuprofen, the positive control drug, reduced the number of writhing movements in mice induced by acetic acid, and the difference was statistically significant compared with the model group (p<0.001). After administration of low, medium, and high doses (0.36 g / kg, 0.78 g / kg, and 1.56 g / kg) of sunflower disc extract granules, the number of writhing movements in experimental animals was significantly reduced, and the difference was statistically significant compared with the model control group (p<0.001, p<0.001, p<0.001). Furthermore, the reduction in serum uric acid levels in the high-dose group was similar to that of ibuprofen, indicating that it has a significant analgesic effect on writhing movements in mice induced by acetic acid at doses of 0.36 g / kg, 0.78 g / kg, and 1.56 g / kg.

[0093] 3. Anti-inflammatory effect experiment - mouse acute systemic inflammation model Mice preparation: Fasted for 4 hours (to reduce gastrointestinal irritation). LPS injection: Mice were immobilized, and the left lower abdomen was disinfected with alcohol. The needle was inserted into the abdominal cavity at a 45° angle. After aspiration to remove any fluid, LPS solution was injected (injection volume: 200 μL / 20g). Control group: An equal volume of sterile saline was injected. One hour after injection, the positive control group and the treatment group were administered indomethacin and different doses of sunflower disc extract granules by gavage. Two hours after administration, blood samples were collected to detect the level of TNF-α anti-inflammatory factor, and statistical analysis was performed. The experimental results are shown in Table 13. Figure 11 As shown.

[0094] Table 13. Validation of TNF-α inflammatory factor levels in lipopolysaccharide-induced inflammatory mice by sunflower disc extract particles ( ±SD)

[0095] Note: Compared with the blank control group, p < 0.001. Compared with the model group, ... , .

[0096] From Table 13, Figure 11The results showed that the TNF-α inflammatory factor level in mice induced by lipopolysaccharide (LPS) was significantly increased, with a statistically significant difference compared to the control group (p<0.001), indicating that LPS successfully established a mouse systemic inflammation model. The positive control drug indomethacin reduced the TNF-α inflammatory factor level in mice, with a statistically significant difference compared to the model group (p<0.05). After administration of sunflower disc extract granules at low, medium, and high doses (0.36 g / kg, 0.78 g / kg, and 1.56 g / kg), the TNF-α inflammatory factor level in the experimental animals was significantly reduced, with statistically significant differences compared to the model control group (p<0.01, p<0.001, p<0.001). Furthermore, the TNF-α inflammatory factor levels in the low, medium, and high dose groups were superior to those of indomethacin, indicating that LPS has a significant anti-inflammatory effect on the mouse systemic inflammation model at doses of 0.36 g / kg, 0.78 g / kg, and 1.56 g / kg.

[0097] Experimental Example 2.

[0098] The effects of sunflower disc extract obtained from new and old extraction processes on URAT1 protein expression were compared.

[0099] URAT1 (urate transporter 1) is a key urate reabsorption transporter located in the proximal tubular epithelial cells of the kidney, belonging to the solute transporter family (SLC22A12). It mediates the entry of uric acid from the renal tubules into the bloodstream, maintaining uric acid homeostasis. URAT1 dysfunction is closely associated with hyperuricemia and gout, with specific mechanisms including: 1) functionally gain-enhancing mutations leading to excessive uric acid reabsorption, accounting for approximately 40% of cases of hereditary hyperuricemia; 2) acquired dysfunction is common in patients with metabolic syndrome, often related to transcriptional regulatory abnormalities caused by insulin resistance; 3) drug inhibition of this transporter can significantly promote uric acid excretion (increasing it by 60%–70%), such as benzbromarone, which exerts its uric acid-lowering effect by competitively inhibiting URAT1. Therefore, drugs targeting URAT1 can effectively reduce serum uric acid levels by inhibiting its activity, reducing uric acid reabsorption, and promoting uric acid excretion. Compared to traditional uricosuric drugs (such as probenecid), these drugs have higher selectivity.

[0100] Against this background, to evaluate the efficacy of sunflower disc extract, this study employed in vitro cell experiments to compare the effects of sunflower disc extracts obtained from new and old extraction processes on URAT1 protein expression. The specific experimental methods are as follows: 1. Experimental Objective The expression levels of relevant proteins in the samples were verified using Western blotting in cell experiments.

[0101] 2. Experimental Materials (1) Main materials and reagents for the experiment Table 14 Main Materials and Reagents .

[0102] (2) Main experimental instruments Table 15 Main Experimental Instruments

[0103] 3 Experimental Methods (1) Experimental grouping This experiment was divided into four groups: control group, model group, original process group of sunflower disc extract, and new process group of sunflower disc extract.

[0104] (2) Cell treatment 1) 18 hours before transfection, seed approximately 500,000 cells per well into a 6-well plate and culture at 37°C with 5% CO2 until the cell density reaches approximately 90% the next day; 2) Replace 2 mL of fresh culture medium (containing serum, but without antibiotics) in each well of the 6-well plate containing cells; 3) For each well of the 6-well plate to be transfected, take two clean, sterile centrifuge tubes and add 125 µL of MEM culture medium (without antibiotics and serum) to each tube. Then, add 2.5 µg of plasmid DNA to one tube and gently mix by pipetting; 4) Add 5 µL of Lipo6000™ transfection reagent to the other tube and gently mix by pipetting. After standing at room temperature for 5 minutes, gently add the culture medium containing DNA to the culture medium containing Lipo6000™ transfection reagent by pipetting, gently invert the centrifuge tube or gently mix by pipetting, and let stand at room temperature for 5 minutes; 5) Continue culturing for approximately 24 hours, then replace with complete culture medium; treat different groups with the corresponding concentration of sample for 24 hours.

[0105] (3) Sample preparation 1) Add PMSF and Triton X-100 to the basic lysis buffer; 2) Add lysis buffer and operate on ice, shaking on ice for 60 min; 3) Centrifuge at 12000 rpm for 15 min at 4℃; 4) Determine protein concentration: Take an appropriate amount of 25 mg / mL protein standard and dilute to a final concentration of 0.5 mg / mL. Prepare an appropriate amount of BCA working solution by adding 1 volume of BCA reagent B to 50 volumes of BCA reagent A (50:1) and mix thoroughly. Add 0, 1, 2, 4, 8, 12, 16, and 20 µL of standard solution to the wells of a 96-well plate, and add standard diluent to bring the total to 20 µL. Add 2 µL of sample to the wells of the 96-well plate. Add standard diluent to bring the total to 20 µL. Add 200 µL of BCA working solution to each well and incubate at 37ºC for 30 min. Measure the absorbance at a wavelength of 595 nm using a microplate reader. Calculate the protein concentration of the sample based on the standard curve and the sample volume used; 5) Dilute 5× loading buffer to 1× loading buffer with the sample, boil for 10 min, and then store at -20℃.

[0106] (4) SDS-PAGE electrophoresis 1) Preparation of electrophoretic gel After washing and drying the glass plate, fix it on the gel casting machine. Begin preparing the separating gel by pouring it into the gaps in the glass plate to an appropriate height. Cover the separating gel with anhydrous ethanol until it is completely polymerized. Pour out the anhydrous ethanol, gently rinse with double-distilled water, and then blot dry with filter paper. Next, add the stacking gel to the appropriate height and insert the comb teeth. Remove the comb teeth after the stacking gel has completely polymerized.

[0107] Table 16 Preparation of 12% Concentration Adhesive .

[0108] Table 17 Preparation of 5% Concentration Adhesive

[0109] .

[0110] 2) Sample loading and electrophoresis Load 50 μg of total protein into each well. Perform electrophoresis with a constant current of 15 mA per gel. Determine the position of the target protein based on the relative position of the pre-stained marker and the molecular weight of the target protein. Stop electrophoresis when the target protein is in the optimal resolvable position in the lower 1 / 3 of the separating gel surface. (5) Transfer membrane Soak the cut PVDF membrane in methanol for 2 minutes; remove the gel and cut the target band according to the marker, rinse with distilled water, and cut PVDF membrane and filter paper of the same size as the PAGE gel. Soak the PVDF membrane and filter paper together in the electrotransfer buffer. Arrange the plates in the following order: black plate - fiber pad - filter paper - gel - PVDF membrane - filter paper - fiber pad - white plate, clamp the plates and place them in the transfer apparatus, with the black plate facing the black negative electrode. Fill the transfer tank with electrotransfer buffer and begin the transfer process; the transfer process is carried out at 4℃ and the transfer conditions are 100V / 100mA. (6) Immunoblot staining (ECL staining system) 1) Sealing: Soak the PVDF membrane in a solution containing 3% BSA (sealing solution) and seal it on a shaker at room temperature for 2 hours; 2) Dilute the corresponding primary antibody with blocking solution, immerse the PVDF membrane in the primary antibody incubation solution, and incubate overnight at 4°C.

[0111] 3) Wash away excess primary antibody: Wash the PVDF membrane thoroughly with TBST 5-6 times, 5 min / time; 4) Secondary antibody: Dilute the HRP-labeled secondary antibody with blocking buffer—1:1000 dilution, immerse the PVDF membrane in the secondary antibody incubation solution, and incubate at 37°C on a shaker for 2 hours; 5) Wash away excess secondary antibody: Wash the PVDF membrane thoroughly with TBST 5-6 times, 5 min / time; 6) Colorimetric Exposure: Mix the enhancement solution and stable peroxidase solution in the ECL reagent at a 1:1 ratio, add the working solution to the PVDF membrane, react for several minutes, and after the fluorescent bands are clear, blot off the excess substrate solution with filter paper, and develop and photograph the membrane using a chemiluminescence analyzer. Quantitative analysis is performed based on the grayscale values ​​of each protein band.

[0112] 4. Evaluation results of the efficacy of extracts from sunflower discs using new and old extraction and purification processes The effects of the old and new extraction and purification processes on the expression level of the protein URAT1 were as follows: Figure 12 As shown.

[0113] Depend on Figure 12 The results showed that the URAT1 protein level in the model group was significantly increased, showing a significant upregulation compared with the blank group; after administration of the sunflower disc extracts from both the old and new extraction processes (0.8 mg / mL, 0.8 mg / mL), the URAT1 protein level decreased significantly, and the differences between the old and new extraction processes were statistically significant compared with the model group. This represents p < 0.001. The results (p<0.01) indicate that the extracts from both the new and old processes significantly inhibited the expression of URAT1 protein. Furthermore, the inhibitory effect of the extract from the new process on URAT1 protein was statistically significant compared to that from the extract from the old process (&& represents p<0.01). The protein expression level results show that the inhibitory effect of the extract from the new process on URAT1 protein is significantly better than that from the old process.

[0114] In summary, the sunflower disc extract obtained by the new purification process exhibited stronger URAT1 inhibitory activity in in vitro cell models compared to the product obtained by the old process. This finding corroborates its significant uric acid-lowering, analgesic, and anti-inflammatory effects in whole animal models, suggesting that its therapeutic efficacy for gout stems not only from promoting uric acid excretion but may also involve multi-target effects. Therefore, the sunflower disc extract particles prepared in this study are promising candidates for gout treatment and warrant further investigation.

[0115] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for extracting gout-treating components from sunflower heads, characterized in that, Includes the following steps: (1) Obtain the sunflower disc decoction extract; (2) Concentrate the water-based extract and load it onto a macroporous resin column; (3) Wash away impurities with water and 3%-6% ethanol in sequence; (4) Elute with 60%-75% ethanol; (5) Collect the eluent from step (4), concentrate and dry it to obtain the gout treatment component.

2. The method according to claim 1, characterized in that, The macroporous resin column is a styrene polar macroporous resin.

3. The method according to claim 2, characterized in that, The macroporous resin column is HPD-500.

4. The method according to claim 1, characterized in that, In step (3), impurities are washed away sequentially with water and 4-5% ethanol.

5. The method according to claim 4, characterized in that, In step (3), the amount of ethanol used is 1.1-1.3 times the column volume.

6. The method according to claim 4, characterized in that, In step (3), the ethanol flow rate is 1.3-1.6 times column volume / h.

7. The method according to claim 1, characterized in that, In step (4), elution is performed using 70% ethanol.

8. The method according to claim 7, characterized in that, In step (4), the amount of ethanol used is twice the column volume.

9. The method according to claim 7, characterized in that, In step (4), the ethanol flow rate is 1.3-1.6 times column volume / h.

10. A sunflower disc extract, characterized in that, Prepared by the method according to any one of claims 1-9.

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