Delinted wormwood extract gel patch as well as preparation method and application thereof

Abstract

The invention discloses a delinted wormwood extract gel patch as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing delinted wormwood powder and rice meal, and sterilizing to obtain a wormwood matrix; uniformly mixing the wormwood substrate with sterile water and an aspergillus niger seed solution, performing fermentation processing, and drying, crushing and sieving an obtained processed product to obtain processed product powder; extracting the processed product powder by using an ethanol solution to obtain a delinted wormwood extract; or mixing the processed product powder with cassia twig powder, galangal powder, pogostemon cablin powder, dalbergia wood powder, cyperus rotundus powder, radix angelicae powder, pericarpium citri reticulatae powder, salvia miltiorrhiza powder and unprocessed radix aconiti powder, and extracting with an ethanol solution to obtain the processed delinted wormwood extract. The content of polyphenol and flavone in the obtained delinted wormwood extract is increased, the activity of resisting xanthine oxidase is also improved, and the effect of relieving uric acid is remarkably improved. The delinted wormwood extract is prepared into the gel patch, so that the release process is gentle and stable, and the covered joint can be subjected to long-acting and continuous treatment.

Description

Technical Field

[0001] This invention belongs to the technical field of traditional Chinese medicine fermentation, and specifically relates to a de-flossed Artemisia argyi extract gel patch, its preparation method, and its application. Background Technology

[0002] Hyperuricemia is a metabolic disease caused by excessive purine intake, metabolic abnormalities, or reduced renal excretion. It is characterized by elevated blood uric acid levels and can lead to complications such as gout, hypertension, diabetes, and kidney damage, making it a significant public health issue. Clinical treatment primarily revolves around two strategies: reducing uric acid production and promoting uric acid excretion. Xanthine oxidase is a key enzyme in uric acid production, and inhibiting its activity is the core of treatment. Allopurinol, a commonly used inhibitor, can effectively alleviate uric acid levels, but it may cause adverse reactions such as gastrointestinal issues, rashes, and allergies. Therefore, developing safe and effective xanthine oxidase inhibitors from natural products has become a research hotspot in food science and pharmacy.

[0003] Artemisia argyi has been proven to be rich in polyphenols and flavonoids, which are active ingredients that can inhibit xanthine oxidase, and has the potential to be developed into a functional raw material. The waste or byproduct generated during the processing of Artemisia argyi into moxa wool is called de-linted Artemisia argyi. As a byproduct of Artemisia argyi processing into moxa wool, de-linted Artemisia argyi possesses active substances and effects comparable to moxa wool, but currently still suffers from extremely low resource utilization rates.

[0004] Existing technologies for utilizing de-fleshed Artemisia argyi are usually limited to simple physical extraction, which has obvious bottlenecks: First, the extraction efficiency is limited, and the cell wall structure hinders the full release of active ingredients; second, the composition and content of active ingredients in the extract are fixed, and its biological activity (such as the inhibition rate of xanthine oxidase) has reached its ceiling, making it difficult to achieve breakthrough improvement, resulting in low added value of the product and failing to support high-value utilization pathways.

[0005] Meanwhile, the main shortcomings of existing transdermal drug delivery technologies for Artemisia argyi lie in the difficulty of efficiently penetrating the skin barrier for its active ingredients, as well as the inadequacies in stable release and skin compatibility of related dosage forms. Future technological development needs to focus on novel delivery systems to overcome these bottlenecks.

[0006] Therefore, there is an urgent need to develop a method to increase the added value of delinted Artemisia argyi, especially a method to effectively inhibit xanthine oxidase activity and improve the uric acid-relieving activity of delinted Artemisia argyi. At the same time, there is an urgent need to develop a new type of gel patch to achieve a highly efficient, sustained and stable slow-release effect of the effective components of Artemisia argyi. Summary of the Invention

[0007] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for preparing de-fleshed Artemisia argyi extract.

[0008] Another object of the present invention is to provide a de-flossed Artemisia argyi extract obtained by the above preparation method.

[0009] Another object of the present invention is to provide a gel patch containing the above-mentioned delinted Artemisia argyi extract.

[0010] Another object of the present invention is to provide the application of the above-mentioned de-fleshed Artemisia argyi extract.

[0011] The objective of this invention is achieved through the following technical solution: a method for preparing de-flossed Artemisia argyi extract, comprising the following steps: (1) Mix the delinted mugwort powder and auxiliary materials, sterilize, and obtain mugwort matrix; (2) Mix the Artemisia argyi substrate obtained in step (1) with sterile water and Aspergillus niger seed liquid evenly, ferment, and obtain the processed product; (3) The processed product obtained in step (2) is dried, pulverized and sieved to obtain processed product powder; (4) Use ethanol solution to extract the processed product powder to obtain de-fleshed mugwort extract; or mix the processed product powder with cinnamon twig powder, galangal powder, patchouli powder, sandalwood powder, cyperus powder, angelica powder, tangerine peel powder, salvia powder and raw aconite powder, and then extract with ethanol solution to obtain processed de-fleshed mugwort extract.

[0012] The de-flossed mugwort mentioned in step (1) is a byproduct of the preparation of mugwort floss using mugwort.

[0013] The delinted mugwort powder mentioned in step (1) is preferably delinted mugwort powder that can pass through a 40-mesh sieve.

[0014] The auxiliary material mentioned in step (1) is at least one of rice flour, corn flour, corn syrup, yeast extract and peptone extract; more preferably rice flour.

[0015] The rice flour is preferably rice flour that can pass through a 40-mesh sieve.

[0016] The delinted mugwort powder and the rice powder mentioned in step (1) are preferably mixed in a mass ratio of 5:1 to 4; more preferably in a mass ratio of 5:2 to 4; and most preferably in a mass ratio of 5:2 to 3.

[0017] The amount of sterile water used in step (2) is preferably calculated according to a mass ratio of 6:3 to 8 of Artemisia argyi substrate and sterile water; more preferably, it is calculated according to a mass ratio of 6:6 of Artemisia argyi substrate and sterile water.

[0018] The Aspergillus niger mentioned in step (2) is preferably Aspergillus niger GDMCC No. 3.24.

[0019] The preparation process of Aspergillus niger seed liquid in step (2) includes strain activation and shake flask culture.

[0020] The bacterial strain activation was performed using PDA plates.

[0021] The culture medium used for the shake flask culture is potato glucose (PD) liquid medium.

[0022] The preferred conditions for the shake flask culture are 28–32 °C and 150–250 rpm for 24–72 h; more preferably, 30 °C and 180 rpm for 48 h.

[0023] The preferred dosage of Aspergillus niger in step (2) is 5 g of delinted Artemisia argyi powder in a ratio of 0.5 to 4 × 10⁻⁶. 8 CFU (Cellular Fusarium oxysporum) is preferred; more preferably, it is formulated with 5 g of delinted Artemisia argyi powder at a ratio of 1.0–2.0 × 10⁻⁶. 8 CFU (Cytocinon niger); the optimal ratio is 5 g of delinted Artemisia argyi powder at 2.0 × 10⁻⁶. 8 CFU (Cytosporum niger)

[0024] The fermentation conditions described in step (2) are preferably 28-32 ℃ for 4-10 days; more preferably 30 ℃ for 6-8 days.

[0025] The drying conditions described in step (3) are preferably 40-50 °C to constant weight; more preferably 45 °C to constant weight.

[0026] The sieving process described in step (3) is preferably sieved through a 40-mesh sieve.

[0027] In step (4), the processed product powder, cinnamon twig powder, galangal powder, patchouli powder, sandalwood powder, cyperus powder, angelica powder, tangerine peel powder, salvia powder, and raw aconite powder are mixed in a mass ratio of 20:1.0~1.5:1.0~1.5:0.4~0.6:1.5~2.0:0.4~0.6:0.8~1.2:0.4~0.6:0.4~0.6:7.0~8.0; preferably, in a mass ratio of 20:1.25:1.25:0.5:1.75:0.5:1:0.5:0.5:7.5.

[0028] The ethanol solution mentioned in step (4) is preferably an ethanol solution with a concentration of 65-75% v / v; more preferably an ethanol solution with a concentration of 70% v / v.

[0029] The preferred extraction method in step (4) is ultrasound.

[0030] The preferred conditions for the ultrasound are continuous ultrasound for 20 to 40 minutes at a power of 500 to 700 W and a frequency of 35 to 45 kHz; more preferably, continuous ultrasound for 30 minutes at a power of 600 W and a frequency of 40 kHz.

[0031] A de-fleshed Artemisia argyi extract was obtained by the above preparation method. It contains high levels of polyphenols and flavonoids, and exhibits higher anti-xanthine oxidase activity and antioxidant activity.

[0032] The application of the above-mentioned de-fleshed Artemisia argyi extract in the preparation of anti-xanthine oxidase active preparations.

[0033] The above-mentioned de-fleshed Artemisia argyi extract is used in the preparation of uric acid-relieving agents.

[0034] A gel patch containing the above-mentioned delinted Artemisia argyi extract; preferably prepared by the following steps: (5) Concentrate the de-fleshed Artemisia argyi extract, freeze-dry it, and obtain freeze-dried powder; (6) Using anhydrous ethanol as the dissolving medium, phospholipids, cholesterol, Tween-80 and the lyophilized powder obtained in step (5) are mixed and stirred to dissolve, evaporated under reduced pressure to form a lipid solution, and then the solvent is removed to obtain a flexible liposome film. (7) Mix the buffer solution and the flexible liposome membrane, hydrate them, and form a liposome suspension; (8) Filter the liposome suspension through a membrane, take the filtered liquid, and obtain the processed liposomes; (9) Mix sodium alginate with the liposomes processed in step (8) and add acrylamide, methylenebisacrylamide, ammonium persulfate and tetramethylethylenediamine in sequence. After mixing, pour into a mold, solidify and then soak in deionized water to remove unreacted components to obtain a gel patch.

[0035] The preferred concentration method described in step (5) is vacuum concentration.

[0036] The preferred conditions for vacuum concentration are as follows: operating temperature of 40-50 ℃ and vacuum degree of -0.1 to -0.09 MPa; more preferably, the operating temperature is 45 ℃ and the vacuum degree is -0.1 MPa.

[0037] The degree of concentration described in step (5) is preferably 25-35% of the original volume; more preferably 30% of the original volume.

[0038] The preferred conditions for freeze drying in step (5) are as follows: vacuum degree 1.3 Pa, pre-freezing temperature -40 ℃, and drying time 36 to 60 h.

[0039] The phospholipid mentioned in step (6) is preferably soybean lecithin.

[0040] The preferred amounts of each component in step (6) are as follows: phospholipids, cholesterol, Tween-80, and the lyophilized powder obtained in step (5) are calculated in the ratio of 0.2 g: 0.03-0.06 g: 50-200 μL: 5-20 mg; more preferably, phospholipids, cholesterol, Tween-80, and the lyophilized powder obtained in step (5) are calculated in the ratio of 0.2 g: 0.04 g: 200 μL: 15 mg.

[0041] The amount of anhydrous ethanol used in step (6) is preferably calculated as 5-20 mg of lyophilized powder to 40-60 mL of anhydrous ethanol; more preferably, it is calculated as 15 mg of lyophilized powder to 60 mL of anhydrous ethanol.

[0042] The conditions for vacuum evaporation described in step (6) are 35–45 °C, -0.1–-0.09 MPa, and 50–90 rpm; more preferably, 40 °C, -0.1 MPa, and 70 rpm.

[0043] The preferred method for removing the solvent in step (6) is vacuum drying.

[0044] The buffer solution mentioned in step (7) is preferably a phosphate buffer solution; more preferably a 0.01M phosphate buffer solution with pH=7.4.

[0045] The preferred hydration method described in step (7) is ultrasound.

[0046] The preferred conditions for ultrasound are continuous ultrasound at a power of 500-700 W and a frequency of 35-45 KHz for 40-80 min; more preferably, continuous ultrasound at a power of 600 W and a frequency of 40 KHz for 60 min.

[0047] The preferred steps for filtration in step (8) are as follows: first, use a 450 nm aqueous filtration membrane for filtration, and then use a 220 nm aqueous filtration membrane for filtration.

[0048] The preferred amounts of each component in step (9) are as follows: sodium alginate, liposomes treated in step (8), acrylamide, methylenebisacrylamide, ammonium persulfate and tetramethylethylenediamine are mixed in a ratio of 0.1-0.15 g: 10 mL: 1-2 g: 5 mg: 35 mg: 20 μL; more preferably, sodium alginate, liposomes treated in step (8), acrylamide, methylenebisacrylamide, ammonium persulfate and tetramethylethylenediamine are mixed in a ratio of 0.15 g: 10 mL: 1 g: 5 mg: 35 mg: 20 μL.

[0049] The solidification temperature in step (9) is preferably 30-40 °C; more preferably 35 °C.

[0050] The preferred specific operation for soaking in deionized water in step (9) is as follows: soak for 10 to 20 minutes each time, and repeat 2 to 3 times.

[0051] The above-mentioned gel patch has a highly efficient and sustained-release effect.

[0052] The above-mentioned gel patch is used in the preparation of topical agents for relieving uric acid.

[0053] The aforementioned topical agent is a joint patch.

[0054] The present invention has the following advantages and effects compared with the prior art: (1) This invention optimizes the types of microorganisms and fermentation conditions, and uses microbial fermentation technology to promote the dissolution of active ingredients from the cell wall and the transformation of active ingredients, thereby increasing the content of polyphenols and flavonoids in Artemisia argyi and providing an extract of Artemisia argyi rich in active ingredients, which solves the problems of low utilization of active ingredients and slow onset of action.

[0055] (2) The extract obtained by solid-state fermentation of Artemisia argyi provided by the present invention can be used to prevent and relieve hyperuricemia and gout by enhancing anti-xanthine oxidase activity and antioxidant activity, reducing uric acid production and inhibiting the body's oxidative stress response caused by excessive uric acid.

[0056] (3) The fermentation method of the present invention is simple to operate. It only requires mixing the raw materials and the inoculum evenly, without the need for complicated processes and expensive equipment. During the fermentation period, only the ambient temperature needs to be maintained, without the need for other operations or human supervision. It has good practical value and application prospects.

[0057] (4) The present invention scientifically combines the processed product with multiple Chinese medicines such as cinnamon twig and galangal and prepares them into nanoliposomes, which effectively improves the solubility and bioavailability of the drug. Furthermore, it loads the drug into an optimized hydrogel patch, achieving a smooth and stable release process, which can provide long-term and continuous treatment to the covered joint. Attached Figure Description

[0058] Figure 1 The figure shows the effect of different implementation methods on the inhibition rate of xanthine oxidase in Aspergillus niger fermentation of delinted Artemisia argyi.

[0059] Figure 2 The graph shows the effects of xanthine oxidase inhibition rate and polyphenol content on the fermentation of de-fleshed Artemisia argyi before and after fermentation.

[0060] Figure 3 The graph shows the effect of xanthine oxidase inhibition rate on the mixed extracts before and after fermentation.

[0061] Figure 4 The graph shows the effect of the ratio of sodium alginate to acrylamide on gel release efficiency. Detailed Implementation

[0062] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0063] The biological materials used in this experiment: Monascus purpureus WQ15, with accession number CGMCC No.10910, has been disclosed in Chinese invention patent CN201510449543.6 - A Monascus purpureus strain with high production of extracellular yellow pigment and its breeding method and application; Saccharomyces cerevisiae GDMCC No. 2.139 is available from Guangdong Provincial Microbial Culture Collection Center; Aspergillus cristatus BNCC146563, available from Beina Biotechnology; Cordyceps sinensis Cs-HK1 (Tolypocladium sinense), accession number CGMCCNo.6004, has been disclosed in Chinese patent CN202010185241.3; Lactobacillus plantarum BSe, with accession number GDMCC NO:63475, has been disclosed in Chinese invention patent CN202310884572.X-A strain of Lactobacillus plantarum and its application in the preparation of bio-nano selenium; Aspergillus niger GDMCC No. 3.24 is available from Guangdong Provincial Microbial Culture Collection Center.

[0064] Example 1 A method for preparing a de-fleshed Artemisia argyi extract, such as... Figure 1 As shown, it includes the following steps: (a) Raw material pretreatment The delinted mugwort and rice are crushed, ground, and sieved separately to obtain delinted mugwort powder and rice powder, respectively, for later use. (II) Seed liquid preparation 1. Activation of microbial strains: Freeze-dried powders of *Aspergillus niger*, *Monascus purpureus*, *Cordyceps sinensis*, and *Aspergillus cristatus* were inoculated onto PDA solid agar plates and incubated at 30 °C for 2 days. A piece of the mycelium was then inoculated onto a fresh PDA solid agar plate and subcultured once. The PDA solid agar plate formulation was: 3.0 g potato starch, 20.0 g glucose, and 14.0 g agar, diluted to 1.0 L with distilled water.

[0065] Saccharomyces cerevisiae and Lactobacillus plantarum lyophilized powder were inoculated onto LB agar plates and cultured at 37 °C for 2 days. A piece of the culture was then inoculated onto a fresh LB agar plate and passaged once. The LB agar plate formulation was: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and 14.0 g agar, diluted to 1.0 L with distilled water.

[0066] 2. Seed liquid preparation: One piece each of activated Aspergillus niger, Monascus purpureus, Cordyceps sinensis, and Aspergillus cristatus were transferred to potato dextrose broth medium and placed in a constant temperature shaker at 30 ℃ for 2 days at 180 rpm to obtain seed culture.

[0067] One piece of activated Saccharomyces cerevisiae and one piece of Lactobacillus plantarum were transferred to LB liquid medium and placed in a constant temperature shaker at 30 ℃ for 2 days at 180 rpm to obtain seed culture.

[0068] 3. Viable bacteria count: Seed cultures of Aspergillus niger, Monascus purpureus, Cordyceps sinensis, and Aspergillus cristatus were diluted to 10 μL. -7 Then, 0.1 mL was dropped onto the surface of a PDA solid plate culture medium, spread evenly with a spreader, and incubated at 30 ℃ for 2 days. The bacterial count was then checked to obtain the seed culture bacterial content. The seed culture bacterial contents of *Aspergillus niger*, *Monascus purpureus*, *Cordyceps sinensis*, and *Aspergillus cristatus* were adjusted to 1×10⁻⁶. 8 CFU / mL.

[0069] The seed cultures of brewer's yeast and lactobacillus plantarum were diluted to 10 μL respectively. -7 Then, 0.1 mL was dropped onto the surface of LB solid agar plate, spread evenly with a spreader, and incubated at 30 °C for 2 days. The bacterial count was then checked to obtain the seed culture concentration. The seed culture concentrations of *Saccharomyces cerevisiae* and *Lactobacillus plantarum* were adjusted to 1×10⁻⁶. 8 CFU / mL.

[0070] (III) Fermentation Take 5 g of de-fleshed mugwort powder, add 1 g of rice flour as an auxiliary material, add 6 mL of sterile water, and inoculate 1 mL each of Monascus purpureus, Saccharomyces cerevisiae, Aspergillus cristatus, Cordyceps sinensis, Lactobacillus plantarum and Aspergillus niger into the fermentation substrate. Control the fermentation time to 8 days, dry, pulverize and pass through a 40-mesh sieve to obtain de-fleshed mugwort processed products A to F. (iv) Extraction Extraction method: Accurately weigh 0.1 g each of the above-mentioned de-wooled Artemisia argyi processed products A to F, add 2.5 mL of 70% anhydrous ethanol solution to each, and sonicate continuously for 30 min at a power of 600 W and a frequency of 40 kHz. After sonication, centrifuge at 8000 rpm for 10 min and collect the supernatant. Repeat the extraction of the precipitate twice using the aforementioned method. Mix the supernatants after three centrifugations to obtain the de-wooled Artemisia argyi extract, and store at -4 ℃ for later use.

[0071] (v) Indicator Measurement The content of polyphenols and flavonoids in fermented Artemisia argyi and the inhibition rate of xanthine oxidase were determined. (1) Determination of xanthine oxidase inhibition rate: After diluting the *Artemisia argyi* extract by one-fold, 0.05 mL of the diluted extract and 0.05 mL of a xanthine oxidase solution with an activity unit of 0.5 U were incubated at 37 °C for 10 min. Then, 0.1 mL of a 0.1 mM xanthine solution was added, mixed thoroughly, and reacted for 4 min. The absorbance was measured at a wavelength of 290 nm. The formula for calculating the xanthine oxidase inhibition rate is shown in Formula I: Formula I.

[0072] In Formula I: A1 is the absorbance of the negative control experimental group, A0 is the absorbance of the negative control blank group, B1 is the absorbance of the sample experimental group, and B0 is the absorbance of the sample blank group.

[0073] (2) Polyphenol determination method: A standard curve was plotted using the Folin-Ciocalteu colorimetric method. A 0.1 mg / mL gallic acid standard solution was prepared, and 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the standard solution were placed in test tubes, and deionized water was added to bring the volume to 1 mL. 0.2 mL of gallic acid solution of different concentrations was added to 0.1 mL of 1 N Folin-Ciocalteu solution and 0.5 mL of 20% w / v Na₂CO₃ solution in centrifuge tubes, mixed thoroughly, and allowed to stand in the dark for 40 min. The supernatant was then centrifuged, and the absorbance was measured at 725 nm. The extract of Artemisia argyi was diluted 10 times and the absorbance was measured according to the standard curve method. The polyphenol content (mg / g) was then calculated.

[0074] (3) Flavonoid determination method: A standard curve was plotted using the rutin method. A rutin standard solution with a concentration of 4 mg / mL was prepared. 0.01, 0.04, 0.08, 0.12, 0.16, and 0.2 mL of the standard solution were placed in test tubes, and deionized water was added to bring the volume to 0.2 mL. 0.04 mL of 5% w / v NaNO2 solution was added, and the mixture was allowed to stand for 6 min. 0.04 mL of 10% w / v Al(NO3)3 solution was added, and the mixture was allowed to stand for 6 min. 0.4 mL of 4% w / v NaOH solution was added, and 0.32 mL of deionized water was added. After mixing thoroughly, the mixture was allowed to stand for 15 min, and the absorbance was measured at a wavelength of 510 nm. The absorbance of the Artemisia argyi extract was measured 10 times using the standard curve method, and the flavonoid content (mg / g) was calculated.

[0075] Each experiment was repeated in triplicate, and the results are shown in Table 1.

[0076] Table 1. Xanthine oxidase inhibition rate and polyphenol flavonoid content of Artemisia argyi fermented with different fermentation agents in solid-state fermentation

[0077] As shown in Table 1, when Aspergillus niger was used as the fermentation agent, the xanthine oxidase inhibition rate (i.e., the ability to alleviate uric acid) of solid-state fermented Artemisia argyi (48.271±1.701%), polyphenol content (33.210±1.541 mg / g), and flavonoid content (8.523±0.088 mg / g) were significantly higher than those of Monascus purpureus, Saccharomyces cerevisiae, Aspergillus cristatus, Cordyceps sinensis, and Lactobacillus plantarum. This indicates that Aspergillus niger is more suitable for solid-state fermentation of Artemisia argyi, which is beneficial to improving the polyphenol and flavonoid content and the ability to alleviate uric acid in Artemisia argyi.

[0078] Examples 2-7 Examples 2-7 each provide a method for preparing de-flossed Artemisia argyi extract, specifically including the following steps: (a) Raw material pretreatment Same as Example 1; (II) Seed liquid preparation The preparation of Aspergillus niger seed culture was the same as in Example 1, except that the bacterial count of the seed culture was adjusted to 1×10⁻⁶. 8 CFU / mL.

[0079] (III) Solid-state fermentation The effects of fermentation time of 6–10 days, auxiliary material addition of 2–4 g, and inoculum amount of 0.5–1.5 mL on the content of polyphenols and xanthine oxidase inhibition rate of fermented de-fleshed Artemisia argyi were investigated.

[0080] The specific differences between the above embodiments and Embodiment 1 are as follows: the fermentation time, the amount of auxiliary materials added, and the inoculation amount are different, as shown in Table 2.

[0081] Table 2. Fermentation time, amount of excipients added, and inoculum size in Examples 1-7

[0082] The processed products obtained in Examples 2-7 were dried in an oven at 45 ℃ to constant weight, pulverized and passed through a 40-mesh sieve, extracted with ethanol according to the method in Example 1, and then stored in a refrigerator at 4 ℃ for later use. The contents of polyphenols and flavonoids, as well as the xanthine oxidase inhibition rate, of the fermented Artemisia argyi were extracted and determined according to the method in Example 1. The results are as follows: Figure 1 As shown.

[0083] The fermentation conditions of Example 7 (i.e., fermentation time of 8 days, excipient addition of 2 g, and inoculum size of 1.5 mL) were verified again, with 9 parallel experiments set up, and the average value of the results was taken. The test results are shown below. Figure 2 As shown.

[0084] from Figure 1 As can be seen from the results, the polyphenol content and xanthine oxidase inhibition rate of the processed product obtained in Example 7 were both optimal.

[0085] from Figure 2 It can be seen from this: (i) Compared with the unfermented sample, fermentation increased the polyphenol content of the Artemisia argyi extract. Under the conditions of Example 7, the polyphenol content in the extract of the fermented sample reached 63.725±0.606 mg / g, which was 122.550% higher than the polyphenol content of 28.634±0.569 mg / g in the extract of the unfermented sample.

[0086] (ii) Fermentation improved the xanthine oxidase activity of the de-fleshed Artemisia argyi extract. The inhibition rate of xanthine oxidase by the processed product reached 76.194% under the conditions of Example 7, which was 245.034% higher than the xanthine oxidase inhibition rate of 22.083% of the extract of the unfermented sample.

[0087] (iii) The antixanthine oxidase activity of the extract is closely related to its polyphenol content.

[0088] In summary, Aspergillus niger fermentation can increase the polyphenol content of Artemisia argyi and enhance the xanthine oxidase inhibitory activity of the extract. The extract reduces uric acid production by increasing xanthine oxidase activity and alleviates oxidative stress caused by excessive uric acid by increasing antioxidant activity, thereby achieving the effect of uric acid relief.

[0089] Example 8 A method for preparing a de-linted Artemisia argyi extract gel patch specifically includes the following steps: (a) Raw material pretreatment The unfermented delinted mugwort and the delinted mugwort product obtained by fermentation according to Example 7 were dried, pulverized, ground, and sieved through a 40-mesh sieve for later use. Cinnamon twigs, galangal, patchouli, sandalwood, cyperus, angelica, tangerine peel, salvia miltiorrhiza, and raw aconite were pulverized, ground, and sieved through a 40-mesh sieve for later use.

[0090] (ii) Extraction 1. Extraction: The crushed and sieved delinted mugwort, cinnamon twigs, galangal, patchouli, sandalwood, cyperus, angelica, tangerine peel, salvia miltiorrhiza, and raw aconite were mixed in a mass ratio of 20:1.25:1.25:0.5:1.75:0.5:1:0.5:0.5:7.5 and extracted according to the extraction method in Example 1 to obtain the pre-fermentation mixed extract.

[0091] 2. Extraction: The fermented and delinted Artemisia argyi product after crushing and sieving, cinnamon twig, galangal, patchouli, sandalwood, cyperus rotundus, angelica dahurica, tangerine peel, salvia miltiorrhiza, and raw aconite root were mixed in a mass ratio of 20:1.25:1.25:0.5:1.75:0.5:1:0.5:0.5:7.5 and extracted according to the extraction method in Example 1 to obtain the fermented mixed extract.

[0092] (III) Concentration and vacuum freeze drying 1. Vacuum concentration: The operating temperature is 45 ℃, the vacuum degree is -0.1 MPa, and the concentration is carried out to 30% of the volume to obtain the concentrated solution.

[0093] 2. Vacuum freeze drying: vacuum degree 1.3 Pa, pre-freezing temperature -40 ℃, drying time 48 h, to obtain freeze-dried powder.

[0094] (iv) Liposome preparation 1. Weigh 0.2 g soybean lecithin, 0.04 g cholesterol, 200 μL Tween-80, and 15 mg of the above lyophilized powder into a pear-shaped flask, add 60 mL of anhydrous ethanol, and stir to dissolve in a 40℃ water bath to form a homogeneous lipid solution.

[0095] 2. Transfer the above lipid solution to a rotary evaporator and evaporate it under reduced pressure at 40 °C, -0.1 MPa, and 70 rpm to remove ethanol, allowing the lipid to form a uniform thin film on the inner wall of the flask. Then place the flask in a vacuum drying oven and dry it at room temperature for 2 hours to completely remove residual ethanol.

[0096] 3. Add 20 mL of phosphate buffer solution (0.01 M, pH=7.4) to the flask where the film is formed, and hydrate by sonication (600 W, frequency 40 KHz, continuous sonication for 60 min) for 1 h to fully hydrate the film and form a liposome suspension.

[0097] 4. Filter the liposome suspension sequentially through 450 nm and 220 nm aqueous membranes, and collect the filtered liquid to obtain the liposomes.

[0098] (V) Gel preparation Weigh 0.1–0.2 g of sodium alginate and add it to 10 mL of the above liposomes and stir to dissolve. Then add 1–2 g of acrylamide and stir to dissolve. Next, add 5 mg of methylenebisacrylamide, 35 mg of ammonium persulfate and 20 μL of tetramethylethylenediamine in sequence. Pour the solution into a mold and keep it at 35 °C for 12 h. After the gel is set, soak it in deionized water for 15 min. Repeat this process three times to remove uncrosslinked acrylamide monomers, and you will get de-fleshed Artemisia argyi extract gel patches with different synthesis ratios.

[0099] (vi) Performance testing 1. The pre-fermentation and post-fermentation mixed extracts were extracted and the xanthine oxidase inhibition rate was determined according to the method in Example 1. Each group of experiments was repeated in triplicate. The results are shown in the table below. Figure 3 As shown.

[0100] 2. Release Curve Determination: The prepared dewoolized Artemisia argyi extract gel patches were cut to obtain uniformly processed dewoolized Artemisia argyi extract gel patches. These patches were placed in 50 mL centrifuge tubes and immersed in 20 mL of phosphate buffer solution (0.01 M, pH=7.4), and incubated at 37℃ with shaking. Every 3 h, 1 mL of buffer solution was aspirated and an equal volume of phosphate buffer solution was added. The drug concentration in the buffer solution was measured at 324 nm, and drug release curves for hydrogels with different synthetic ratios were plotted. Each experiment was repeated in triplicate. The results are shown in the table below. Figure 4 As shown.

[0101] from Figure 3 The results showed that the enzyme inhibition rate of the product after mixed fermentation was significantly improved compared with that before fermentation, increasing from 37.155% to 89.172%, an increase of 140%. Compared with the xanthine oxidase inhibition rate of the unfermented mixed extract (76.194%), it was improved by 17.033%. This indicates that the processing of Artemisia argyi significantly enhances its ability to alleviate uric acid, making it more suitable for application in the field of uric acid relief.

[0102] from Figure 4The results showed that the gel synthesized with 0.15 g sodium alginate and 1 g acrylamide exhibited the best sustained-release effect. After 48 hours of continuous release, the drug release rate reached 81.382%, and the release process was relatively smooth, achieving stable and sustained drug release. This stable and sustained drug release also enables continuous treatment of the joints covered by the gel patch, achieving a highly efficient and stable sustained-release effect.

[0103] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a de-fleshed Artemisia argyi extract, characterized in that... Includes the following steps: (1) Mix the delinted mugwort powder and auxiliary materials, sterilize, and obtain mugwort matrix; (2) Mix the Artemisia argyi substrate obtained in step (1) with sterile water and Aspergillus niger seed liquid evenly, ferment, and obtain the processed product; (3) Dry and pulverize the processed product obtained in step (2) to obtain processed product powder; (4) Use ethanol solution to extract the processed product powder to obtain de-fleshed mugwort extract; or mix the processed product powder with cinnamon twig powder, galangal powder, patchouli powder, sandalwood powder, cyperus powder, angelica powder, tangerine peel powder, salvia powder and raw aconite powder, and then extract with ethanol solution to obtain processed de-fleshed mugwort extract.

2. The method for preparing the de-fleshed Artemisia argyi extract according to claim 1, characterized in that: The excipients mentioned in step (1) are at least one of rice flour, corn flour, corn syrup, yeast extract, and peptone. The Aspergillus niger mentioned in step (2) is Aspergillus niger GDMCC No. 3.

24.

3. The method for preparing the de-fleshed Artemisia argyi extract according to claim 1, characterized in that: The delinted mugwort powder and the rice flour mentioned in step (1) are mixed in a mass ratio of 5:1 to 4; The amount of sterile water used in step (2) is calculated based on a mass ratio of 6:3 to 8 between the Artemisia argyi substrate and sterile water. The amount of Aspergillus niger used in step (2) is 5 g of delinted Artemisia argyi powder with a ratio of 0.5 to 4 × 10⁻⁶. 8 CFU (Cytosporum niger) In step (4), the processed product powder, cinnamon twig powder, galangal powder, patchouli powder, sandalwood powder, cyperus powder, angelica powder, tangerine peel powder, salvia miltiorrhiza powder, and raw aconite powder are mixed in a mass ratio of 20:1.0~1.5:1.0~1.5:0.4~0.6:1.5~2.0:0.4~0.6:0.8~1.2:0.4~0.6:0.4~0.6:7.0~8.

0.

4. The method for preparing the de-fleshed Artemisia argyi extract according to claim 1, characterized in that: The fermentation conditions described in step (2) are 28–32 °C for 6–10 days; The ethanol solution mentioned in step (4) is an ethanol solution with a concentration of 65-75% v / v; The extraction method described in step (4) is ultrasound.

5. A delinted Artemisia argyi extract, characterized in that: It is obtained by the preparation method described in any one of claims 1 to 4.

6. The use of the dewool-removed Artemisia argyi extract according to claim 5 in the preparation of anti-xanthine oxidase active preparations or uric acid-relieving preparations.

7. A gel patch, characterized in that: It contains the delinted Artemisia argyi extract as described in claim 5.

8. The gel patch according to claim 7, characterized in that: It is prepared through the following steps: (5) Concentrate the de-flossed Artemisia argyi extract, freeze-dry or spray-dry it to obtain extract powder; (6) Using anhydrous ethanol as the dissolving medium, phospholipids, cholesterol, Tween-80 and the extract powder obtained in step (5) are mixed and stirred to dissolve, evaporated under reduced pressure to form a lipid solution, and then the solvent is removed to obtain a flexible liposome film. (7) Mix the buffer solution and the flexible liposome membrane, hydrate them, and form a liposome suspension; (8) Filter the liposome suspension through a membrane, and take the filtered liquid to obtain the processed liposomes; (9) Mix sodium alginate with the liposomes processed in step (8) and add acrylamide, methylenebisacrylamide, ammonium persulfate and tetramethylethylenediamine in sequence. After mixing, pour into a mold, solidify and then soak in deionized water to remove unreacted components to obtain a gel patch.

9. The gel patch according to claim 8, characterized in that: The concentration method described in step (5) is vacuum concentration; The degree of concentration mentioned in step (5) is to concentrate to 25-35% of the original volume; The phospholipid mentioned in step (6) is soybean lecithin; The dosage of each component in step (6) is as follows: phospholipids, cholesterol, Tween-80, and the extract powder obtained in step (5) are calculated according to the ratio of 0.2g: 0.03~0.06g: 50~200 μL: 5~20 mg; The amount of anhydrous ethanol used in step (6) is calculated by mixing 5-20 mg of lyophilized powder with 40-60 mL of anhydrous ethanol. The conditions for reduced pressure evaporation described in step (6) are 35–45 °C, -0.1–-0.09 MPa, and 50–90 rpm; The solvent removal process described in step (6) is vacuum drying or spray drying; The buffer solution mentioned in step (7) is a phosphate buffer solution; The hydration method described in step (7) is ultrasound; The filtration steps described in step (8) are as follows: first, use a 450 nm aqueous filter membrane for filtration, and then use a 220 nm aqueous filter membrane for filtration. The amounts of each component in step (9) are as follows: sodium alginate, liposomes treated in step (8), acrylamide, methylenebisacrylamide, ammonium persulfate and tetramethylethylenediamine are mixed in the ratio of 0.1-0.15 g: 10 mL: 1-2 g: 5 mg: 35 mg: 20 μL.

10. The use of the gel patch according to any one of claims 7 to 9 in the preparation of a topical agent for relieving uric acid.

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