Application of abelmoschus manihot in preparation of psoriasis medicine, preparation method and preparation of abelmoschus manihot

Abstract

The invention relates to the field of traditional Chinese medicines, aims to disclose a medicine for treating psoriasis and application of the medicine, and particularly relates to application of abelmoschus manihot flower with an obvious treatment effect in preparation of a medicine for treating psoriasis and a preparation of the abelmoschus manihot flower.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine, and in particular to an application of hibiscus flower in preparing a psoriasis medicine, a preparation method and a preparation thereof. Background Art

[0002] Psoriasis is a chronic, systemic, immune-mediated disease characterized by excessive proliferation of keratinocytes and infiltrating immune cells. Clinically, it manifests primarily as skin erythema and scaling, and can affect the entire body. According to statistics, psoriasis affects approximately 1-2% of the world's population worldwide, with 7-8 million people in China suffering from the disease. The condition is recalcitrant and difficult to cure, severely impacting patients' physical and mental health and quality of life. Currently, numerous methods and medications are available for the clinical treatment of psoriasis, including topical glucocorticoids, retinoids, and immunosuppressants. However, these medications often present significant side effects, are prone to drug resistance, and are expensive. Therefore, developing a safe and effective psoriasis treatment is of great clinical significance.

[0003] Abelmoschl Manihot (L.) Medicinal, the dried corolla of Abelmoschus medicinalis, a plant of the Malvaceae family (Malvaceae), is an annual flowering herb with a long history of folk and medical use. Modern pharmaceutical research indicates that the entire plant of Abelmoschl contains flavonoids, making it one of the highest flavonoid extracts in plants, boasting dozens of times the amount found in commonly used flavonoid extracts, such as soybeans and ginkgo biloba. Flavonoids are natural phenolic compounds found in plants and can reduce free radicals in the body, providing antioxidant benefits.

[0004] According to current research, there are reports of the pharmacological effects of hibiscus flowers in antibacterial and anti-inflammatory effects, as well as the application of hibiscus flowers in the treatment of psoriasis. However, during the research process, the inventors discovered that not all purchased hibiscus flowers are effective in treating psoriasis, and there are obvious differences. The effects of some hibiscus flowers in treating psoriasis are not obvious. Summary of the Invention

[0005] The present invention aims to provide an application of hibiscus hibiscus flowers with obvious therapeutic effect in preparing psoriasis medicine and a preparation thereof.

[0006] The following preferred solutions of the present invention may be any preferred solution based on the basic technical solution, or may be a superposition of any several preferred solutions based on the basic technical solution.

[0007] The basic technical solution of the present invention is: use of hibiscus flower in preparing a psoriasis drug, the hibiscus flower is a hibiscus flower extract composition, the compounds in the composition include any several or a combination of hyperoside, rutin, isoquercetin, quercetin-3-O-β-d-pyranoglucoside, myricetin-3-O-glucoside, myricetin, myricetin, tiliacoside, rosella red-3-glucoside, and kaempferol-3-O-rutinoside, wherein the three compounds with the highest content in the composition are hyperoside, rutin, and isoquercetin.

[0008] Preferably, in addition to the basic regimen, the formulation of the drug includes hydrogel, ointment, liniment, spray, emulsion, lotion, paste, patch, spray, liposome, powder or gel.

[0009] Preferably, the total flavonoids concentration in the hydrogel formulation is higher than 0.6%.

[0010] Preferably, in the basic solution, the hibiscus flower in the medicine is a hibiscus flower extract.

[0011] Preferably, the total flavonoids concentration of the hibiscus flower extract is higher than 60% based on the basic solution.

[0012] Preferably, in addition to the basic scheme, the hibiscus flower is used to prepare a psoriasis drug for reducing the expression of TNF-α and IL-1β gene mRNA in skin tissue.

[0013] Preferably, in addition to the basic scheme, the hibiscus flower is used to prepare a psoriasis drug for inhibiting HaCaT cell proliferation.

[0014] Preferably, in addition to the basic scheme, the hibiscus flower is used to prepare a psoriasis drug that regulates and targets the IL-17A signaling pathway.

[0015] Preferably, in addition to the basic scheme, the hibiscus flower is used to prepare a psoriasis drug for reducing the mRNA expression levels of genes 1L-17A, IL-17F, S100A8, and S100A9 in the IL-17A signaling pathway.

[0016] In the course of research, the inventors collected hibiscus flowers for extraction to obtain an extract in order to clarify the phenomenon that some hibiscus flowers have no obvious effect in treating psoriasis while some have significant treatment effects. The extract was then detected using ultra-performance liquid chromatography-hybrid quadrupole orbitrap high resolution mass spectrometer (UHPLC-Q-Orbitrap HRMS) to detect the chemical components of traditional Chinese medicine in the extract sample. The components were qualitatively identified by comparison with a reference database and a theoretical database, combined with manual verification.

[0017] After research, it was found that the extract of hibiscus flowers obtained in Chongqing was more effective in treating psoriasis than that of hibiscus flowers obtained in other regions. Moreover, the extract obtained in Chongqing and the extract obtained in other regions showed obvious differences not only in medicinal effects and values, but also in component analysis. Therefore, it can be seen that the drug of the hibiscus extract composition of the present invention is more effective in treating psoriasis of the present invention.

[0018] The present invention uses a hydrogel as an example to experiment its effect in treating psoriasis. The results show that the hydrogel prepared from the hibiscus flower extract of the present invention can significantly inhibit the formation of skin scales in mice caused by imiquimod and reduce skin erythema; significantly inhibit the increase in the spleen / body weight ratio caused by imiquimod; significantly inhibit the thickening of mouse skin caused by imiquimod; significantly reduce the mRNA expression of inflammation-related genes TNF-α and IL-1β in mouse skin tissue after imiquimod induction; significantly reduce the m protein expression level of the inflammation-related factor TNF-α in mouse skin tissue after imiquimod induction; have the effect of inhibiting HaCaT cell proliferation; and significantly reduce the mRNA expression levels of key genes 1L-17A, IL-17F, S100A8, and S100A9 in the IL-17A signaling pathway.

[0019] The hydrogel of the present invention comprises raw materials of 0.6%, 1.2% or 2.4% total flavonoids of hibiscus flower extract, 1-6% sodium carboxymethyl cellulose, 0.1-0.5% poloxamer 188, 0-15% glycerol, 0.2% ethyl hydroxybenzoate, an appropriate amount of anhydrous ethanol, and the remainder being a solvent. When the hydrogel is prepared using 3% sodium carboxymethyl cellulose, 10% glycerol and 0.5% poloxamer 188, the obtained hydrogel is brown and translucent, fine, uniform in color, free of large particles, lumps and bubbles, comfortable to apply and has strong centrifugal stability.

[0020] When hydrogels prepared with total flavonoid concentrations of 0.6%, 1.2% or 2.4% were used to verify the efficacy of treating psoriasis, it was found that as long as the total flavonoid concentration reached above 0.6%, a significant effect could be achieved, and as the concentration increased, the difference in therapeutic effect was not obvious, indicating that a total flavonoid concentration of 0.6% can reach the optimal preparation concentration.

[0021] In addition, the inventors found that when extracting hibiscus flowers, different extraction methods could lead to differences in the compound content of the extract, namely the total flavonoid content. Plant extraction methods are often used to extract active ingredients from plant materials, remove ineffective or harmful ingredients, and thus improve the therapeutic effect. Traditional leaching methods often use water as the extraction solvent, which may cause the mucilage and water-soluble components in hibiscus flowers, such as polysaccharides and tannins, to leach out in large quantities. This not only increases the viscosity of the extract and makes filtration difficult, but also increases the impurity components in the extract and reduces the total flavonoid content. Even if alcoholic solvents are used, there are still problems such as long extraction time, low extraction efficiency, and high solvent consumption. Conventional reflux extraction methods increase the leaching of flavonoids and improve extraction efficiency by increasing the temperature and enlarging the contact area between the medicinal material powder and the solvent. However, higher temperature environments can also cause some flavonoids to decompose and deteriorate, resulting in the loss of active ingredients and safety concerns under industrial production. Enzymolysis can destroy plant tissue cells through enzymolysis, promote the dissolution and release of flavonoids, and improve extraction efficiency. However, this will also lead to the degradation of some flavonoids and the loss of effective monomer components, and the enzyme inactivation step of the enzymatic hydrolysis process may also lead to safety considerations under industrial production.

[0022] To this end, the inventors of the present invention utilized an extraction process previously developed by the inventors' team, namely, a Chinese invention patent application with publication number CN119386067A, entitled "A Method for Preparing a Total Flavonoid Extract from Abelmoschus Flower." Compared with traditional methods, this method has the advantages of fast extraction speed, high extraction yield, sufficient retention of active ingredients, and a simple process. The concentration of the extracted total flavonoids can reach above 60%.

[0023] The extraction method includes the steps of crushing, alcohol extraction and macroporous resin separation and purification.

[0024] Preferably, the extraction method comprises: in the alcohol extraction step, the alcohol solvent used is an ethanol solution with a concentration of 50% to 80%; the material-liquid ratio of the crushed hibiscus hollyhock powder to the ethanol solution is 1:25g / mL to 1:75g / mL; and the alcohol extraction process also includes high-speed homogenization and ultrasonic extraction steps.

[0025] Preferably, in the high-speed homogenization step, the homogenization speed is 5000rpm~7000rpm, and the homogenization time is 60min~90min; in the ultrasonic extraction step, the ultrasonic power is 300W~700W, the temperature is 50~70°C, and the extraction time is 60min~120min to obtain the extract.

[0026] Preferably, the macroporous resin separation and purification step includes a macroporous resin pretreatment step, and AB-8 type macroporous adsorption resin is used; the macroporous resin pretreatment step comprises: soaking the AB-8 type macroporous adsorption resin in 95% ethanol for 24 hours, washing the column bed with 95% ethanol, and then washing with distilled water until there is no alcohol smell, and then soaking in 5% hydrochloric acid and NaOH solution for 2 hours respectively, and then washing with distilled water until neutral, and finally washing with 95% ethanol until there is no white turbidity, and washing with water until there is no alcohol smell;

[0027] The macroporous resin separation and purification step further includes sample preparation: diluting the extract with water, with the concentration of the extract containing 0.2g of raw material per ml, and using acetic acid-sodium acetate buffer to control the pH to 4-6; in the macroporous resin separation and purification step, the volume ratio of the macroporous adsorption resin to the extract is 1:1-1:2, and the adsorption rate is 1BV / h; after the adsorption is completed, quickly rinsing with 5-6BV of pure water, and discarding the effluent; then quickly rinsing with 0.5BV of 5% ethanol solution, and discarding the effluent; and enriching the total flavonoids with 2-5BV of 40-90% ethanol solution, and the elution rate is 1-1.5BV / h.

[0028] The compound components in the extract composition of the present invention are analyzed based on the extracts obtained by the above-mentioned extraction method, and their content is higher. Of course, it also includes extracts obtained by other extraction methods, such as conventional water extraction, alcohol extraction, ultrasonic extraction, etc. The preparation method disclosed in the Chinese invention "A preparation method of a hibiscus flower extract and its application" and the Chinese invention "Application of a hibiscus flower alcohol extract" with publication number CN114082217A are different only in that the overall content of the extract is higher when the preparation method described in the present invention is used, and the total flavonoid concentration can reach more than 60%. The other preparation methods are not described here.

[0029] Another technical solution provided by the present invention utilizes the above-mentioned medicine, wherein the hibiscus flower is a hibiscus flower extract.

[0030] Preferably, the total flavonoids concentration of the hibiscus flower extract is greater than 60%; the pharmaceutical preparations include hydrogels, ointments, liniments, sprays, emulsions, lotions, pastes, patches, sprays, liposomes, powders or gels.

[0031] Preferably, the total flavonoids concentration in the hydrogel formulation is higher than 0.6%.

[0032] Preferably, the Hibiscus tiliaceus flower extract is a composition, and the compounds in the composition include any several or multiple combinations of hyperoside, rutin, isoquercetin, quercetin-3-O-β-d-pyranoglucoside, myricetin-3-O-glucoside, myricetin, myricetin, linalool, roselle red-3-glucoside, and kaempferol-3-O-rutinoside.

[0033] Preferably, the compound also includes astragaloside, ellagic acid, luteolin, quercetin, chlorogenic acid, kaempferol, quercetin, caffeic acid, quercetin, epicatechin, scopoletin, epigallocatechin, quercetin-3-O-sophoroside, luteolin, 6-methoxy-7-hydroxycoumarin, naringenin, typhalaenoside, p-coumaric acid, baicalein, ferulic acid, isoflurane, protocatechu Any one or more combinations of acid, catechin, salicin, sinapinic acid, puerarin, gardenia glycoside, tofu fruit glycoside, eleutheroside B, pinoresinol, deacetylcarbamate methyl ester, isovanillin, syringic acid, esculetin, 7-hydroxycoumarin, polydatin, protocatechuic aldehyde, isovanillic acid, 4'-hydroxyacetophenone, dihydroartemisinin, andrographolide, β-eudesmol, isocarpine lactone, and daphnetin.

[0034] Preferably, the compounds are arranged in descending order of content in the composition as follows: hyperoside, rutin / isoquercetin, quercetin-3-O-β-d-pyranoside, myricetin-3-O-glucoside, myricetin / myricetin / basin, and rosella red-3-glucoside / kaempferol-3-O-rutinoside.

[0035] Preferably, the total content of hyperoside, rutin, isoquercetin, quercetin-3-O-β-d-glucopyranoside, myricetin-3-O-glucoside, myricetin, myricetin, basilicin, rosella red-3-glucoside, and kaempferol-3-O-rutinoside in the compound is greater than 80%.

[0036] Preferably, the compound contains astragaloside, ellagic acid, luteolin, quercetin, chlorogenic acid, kaempferol, quercetin, caffeic acid, quercetin, epicatechin, scopoletin, epigallocatechin, quercetin-3-O-acacia glycoside, luteolin, 6-methoxy-7-hydroxycoumarin, naringenin, typhalaenoside, p-coumaric acid, baicalein, ferulic acid, isoflurane, protocatechuic acid , catechin, salicin, sinapinic acid, puerarin, gardenia glycoside, tofu fruit glycoside, acanthoside B, pinoresinol, deacetylcarbamate methyl ester, isovanillin, syringic acid, esculentol, 7-hydroxycoumarin, polydatin, protocatechuic aldehyde, isovanillic acid, 4'-hydroxyacetophenone, dihydroartemisinin, andrographolide, β-cineole, isocarpine lactone, and daphnetin, the total content is less than 5%.

[0037] The research results were able to express the relationship between the content of each compound in the extract. Although there were slight differences between the raw materials of each batch, the basic compound species and content relationship remained consistent, and the compound composition was different from that of hibiscus flower extracts from other regions. Specifically, the compounds in the composition were ranked from most to least: hyperoside, rutin / isoquercetin, quercetin-3-O-β-d-pyranoside, myricetin-3-O-glucoside, myricetin / myricetin / basin, and roselle-3-glucoside / kaempferol-3-O-rutinoside.

[0038] Because the content of each compound varies slightly, on the order of 1% or 0.1%, the rutin / isoquercetin content can be greater than rutin or greater than isoquercetin. The order of myricetin, myricetin, and basilisk from most to least can be myricetin, myricetin, basilisk; basilisk, myricetin, myricetin; myricetin, basilisk, myricetin; or myricetin, basilisk, myricetin. The content of roselle red-3-glucoside / kaempferol-3-O-rutinoside is arranged from large to small, and roselle red-3-glucoside may be greater than kaempferol-3-O-rutinoside; or kaempferol-3-O-rutinoside may be greater than roselle red-3-glucoside. There may be slight differences in the content ranking due to different product batches, but the overall content ranking is roughly the same, and the above compound combination accounts for more than 80% of the content of the extract composition.

[0039] The content of the compounds with a content of less than 5% in the composition is ranked as follows: compound astragaloside, ellagic acid, luteolin, quercetin, chlorogenic acid, kaempferol, quercetin, caffeic acid, quercetin, epicatechin, scopoletin, epigallocatechin, quercetin-3-O-sophoroside, luteolin, 6-methoxy-7-hydroxycoumarin, naringenin, typhalaenoside, p-coumaric acid, scutellaria baicalensis. Glucoside, ferulic acid, isoflurane, protocatechuic acid, catechin, salicin, sinapinic acid, puerarin, gardenia glycoside, tofu fruit glycoside, eleutheroside B, pinoresinol, deacetylcarbamate methyl ester, isovanillin, syringic acid, esculentol, 7-hydroxycoumarin, polydatin, protocatechuic aldehyde, isovanillic acid, 4'-hydroxyacetophenone, dihydroartemisinin, andrographolide, β-eudesmol, isocarpine lactone, daphnetin.

[0040] For compounds with a total content of less than 5%, the aforementioned content rankings do not exhibit significant differences, nor does the aforementioned ranking necessarily demonstrate preventive and therapeutic efficacy. Rather, according to the present invention, during experimental research, it was found that the compound content rankings of most extracts with significant efficacy exhibited the aforementioned trends. For example, for compounds with a total content of less than 5%, the differences between adjacent compounds in the content rankings may be as small as 0.01% or 0.001%. Therefore, the content rankings are merely one example in the examples, and the rankings may vary depending on the product batch.

[0041] The first major achievement of the present invention is that a hibiscus flower extract with more obvious effect in treating psoriasis was successfully screened out, and the compound combination of the hibiscus flower extract with more obvious effect was successfully identified and distinguished, which is significantly different from the existing compound combination of hibiscus flower extract; the second major achievement is that the action pathway and target of the hibiscus flower extract composition of the present invention in treating psoriasis were studied and obtained; the third major achievement is that total flavonoids with high anti-inflammatory activity in the hibiscus flower extract were successfully sorted out, and the concentration of total flavonoids was high, and hibiscus flower total flavonoids hydrogel was prepared using sodium carboxymethyl cellulose as a carrier, realizing the transdermal administration of total flavonoids, with high biocompatibility and pharmacological activity; and it was proved that the thickening of mouse skin and the release of inflammatory factors in mouse skin were inhibited, thereby alleviating the symptoms of psoriasis. The hydrogel preparation also has the advantages of simple and easy preparation process and low production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The figure is a standard curve of rutin according to Example 1 of the present invention;

[0043] Figure 2 Schematic diagram of the chemical analysis steps of the Hibiscus truncatum flower extract according to Example 2 of the present invention;

[0044] Figure 3 This is the base peak diagram of the positive ion mode detection of the hibiscus flower extract sample in Example 2 of the present invention;

[0045] Figure 4 This is the base peak diagram of the negative ion mode detection of the hibiscus flower extract sample in Example 2 of the present invention;

[0046] Figure 5 This is a diagram showing the therapeutic effect of total flavonoids from Abelmoschus fulva on psoriasis in Example 13 of the present invention;

[0047] Figure 6 This is a bar graph of spleen / body weight ratio in the pharmacodynamic study of total flavonoids from Abelmoschus fulva on psoriasis in Example 13 of the present invention;

[0048] Figure 7This is a HE staining image of mouse skin tissue from a pharmacodynamic study of total flavonoids from Abelmoschus fulva on psoriasis in Example 14 of the present invention;

[0049] Figure 8 This is a bar graph of inflammatory genes extracted from mouse skin tissue using RNA and qRT-PCR in Example 15 of the present invention;

[0050] Figure 9 This is a protein immunoblot image of mouse skin tissue in Example 16 of the present invention;

[0051] Figure 10 This is a CCK8 kit analysis diagram of mouse skin tissue in Example 17 of the present invention;

[0052] Figure 11 This is a graph showing transcriptome sequencing results of mouse skin tissue from Example 18 of the present invention;

[0053] Figure 12 This is a bar graph showing transcriptome sequencing analysis of mouse skin tissue in Example 18 of the present invention. DETAILED DESCRIPTION

[0054] This application involves the following terms and abbreviations:

[0055] The following is further described in detail through specific implementation methods:

[0056] BPI chromatogram: Base Peak Intensity is a graph commonly used in mass spectrometry analysis to show the relationship between the mass-to-charge ratio (m / z) and relative abundance of a molecule.

[0057] Total Ion Chromatogram (TIC) is an important graph in mass spectrometry analysis, which is used to reflect the change of the total intensity of all ions in the sample over time.

[0058] HE staining: Hematoxylin and eosin staining is a staining technique widely used in histology and pathology, primarily for observing the morphological structure of cells and tissues. Its principle is based on the selective staining of different cellular components by hematoxylin (a basic dye) and eosin (an acidic dye).

[0059] RNA extraction and qRT-PCR: RNA extraction and qRT-PCR are commonly used experimental techniques in molecular biology research to analyze gene expression levels.

[0060] Western Blot (WB) is a widely used experimental technique in molecular biology, biochemistry, and immunogenetics to detect and analyze the presence, size, and relative abundance of specific proteins. This technique combines the high resolution of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with the high specificity and sensitivity of solid-phase immunoassays, making it an important tool for studying protein expression and function.

[0061] CCK8 (Cell Counting Kit-8) is a kit used to detect cell proliferation and cytotoxicity. Its core principle is based on the reduction of WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazolium monosodium salt) by mitochondrial dehydrogenases to a water-soluble orange-yellow formazan product. The amount of formazan produced is proportional to the number of viable cells. The absorbance (OD) is measured at a wavelength of 450 nm using a colorimetric method, which indirectly reflects cell proliferation and cytotoxicity.

[0062] Example 1: Obtaining Hibiscus tiliaceus Flower Extract

[0063] 1. Raw material acquisition

[0064] The hibiscus flower extract is obtained from the hibiscus flowers grown in Chongqing, specifically in Wuxi County, Chongqing.

[0065] 2. Preparation of Hibiscus tiliaceus Flower Extract

[0066] 2.1 Raw material preparation:

[0067] Mature hibiscus flowers are harvested, dried, crushed into coarse powder, and sieved (50-mesh sieve) to obtain uniform flower powder as the extraction raw material.

[0068] 2.2 Alcohol extraction process:

[0069] The hibiscus flower powder is added to a 50% to 90% ethanol solution at a ratio of 1:25 to 1:75 (g / mL). High-speed homogenization (5000 to 7000 rpm for 60 to 90 minutes) combined with ultrasonic-assisted extraction (frequency 300 to 700 W, temperature 50 to 70° C. for 60 to 120 minutes) significantly improves the extraction efficiency of flavonoids.

[0070] 2.3 Filtration and concentration:

[0071] The extract was filtered to remove the residue, and then concentrated under reduced pressure (below 60° C., 0.09 MPa) to recover ethanol and obtain a concentrated extract.

[0072] 2.4 Macroporous resin separation and purification:

[0073] 2.4.1 Macroporous Resin Pretreatment: Soak the AB-8 macroporous adsorption resin in 95% ethanol for 24 hours to allow it to fully swell. Rinse the column bed with 95% ethanol and then with distilled water until the alcohol odor is gone. Soak the column bed in 5% hydrochloric acid and NaOH solutions for 2 hours, respectively, then rinse with distilled water until neutral. Finally, rinse with 95% ethanol until no white turbidity remains. Rinse with water until the alcohol odor is gone before use.

[0074] 2.4.2 Sample preparation: Dilute the extract with water to a concentration of 0.2 g of raw material per ml. Use acetic acid-sodium acetate buffer to control the pH to 4-6.

[0075] 2.4.3 Purification by macroporous adsorption resin column: the volume ratio of macroporous adsorption resin to upper column extract is 1:1~1:2, and the adsorption rate is 1BV / h; after the adsorption is completed, it is quickly rinsed with 5~6BV pure water, and the effluent is discarded; then it is quickly rinsed with 0.5BV 5% ethanol solution, and the effluent is discarded; 2~5BV 40~90% ethanol solution is used to enrich total flavonoids, and the elution rate is 1~1.5BV / h, and the eluate is collected.

[0076] 2.4.4 Drying: Collect the eluate, recover the ethanol under reduced pressure until there is no alcohol taste (below 60°C, 0.09 MPa), continue to concentrate to dryness, and dry in a vacuum drying oven until the moisture content is less than 5% to obtain a brown-yellow powder with a slightly bitter taste, which is the hibiscus flower extract, also known as the hibiscus flower total flavonoids extract, with a yield of 2.0% to 7.0%.

[0077] 3. Determination of total flavonoid concentration

[0078] 3.1 Reference solution

[0079] Take 0.00614 g of rutin reference substance, accurately weigh it, and add 50% ethanol to a 25 mL volumetric flask to prepare a reference substance solution containing 0.2456 mg of rutin per 1 mL.

[0080] 3.2 Extraction of flavonoids from the test sample Take 0.15 g of powder sample, weigh accurately, place in a 25 mL centrifuge tube, add appropriate amount of 50% ethanol, and sonicate for 15 min.

[0081] 3.3 Standard curve

[0082] Accurately pipette 1, 2, 3, 4, 5, and 6 mL of the reference solution under item "3.1" into 25 mL volumetric flasks respectively, add water to 6 mL each, add 1 mL of 5% sodium nitrite solution, mix well, and let it stand for 6 minutes, add 1 mL of 10% aluminum nitrate solution, shake well, and let it stand for 6 minutes, add 10 mL of 4% sodium hydroxide test solution, add water to the scale, shake well, and let it stand for 15 minutes. Using the corresponding reagent as the blank, measure the absorbance at a wavelength of 510 nm according to the UV-Vis spectrophotometry method (General Rule 0401), and draw a standard curve with absorbance as the ordinate and concentration as the abscissa.

[0083] 3.4 Determination of sample content Take three samples from the same batch and prepare the test solution according to the method under "3.2". Inject the sample for determination and calculate the sample content.

[0084] 3.5 Experimental Results

[0085] 3.5.1 Establishment of standard curve

[0086] According to the requirements of 3.3, the test results are as follows Figure 1 As shown in the figure, a linear regression was performed with rutin mass concentration (x, mg / mL) as the horizontal axis and absorbance (y) as the vertical axis, and the regression equation was y=11.753x-0.0156(R 2 =0.9998).

[0087] 3.5.2 Sample content determination

[0088] Accurately weigh the same batch of sunflower powder and determine the total flavonoids according to methods "3.2" and "3.4" respectively. Calculate the total flavonoids content. The results are shown in Table 1.

[0089] Table 1 Sample content determination results (n=3)

[0090]

[0091] 4. Conclusion

[0092] The total flavonoid content of the sample was determined by NaNO2-Al(NO3)3-NaOH colorimetry, and the absorbance was measured at 510 nm. The results showed that the rutin standard curve was y=11.753x-0.0156 (R2=0.9998), and the total flavonoid content of this sample was 68.37%.

[0093] Example 2: Component Analysis and Identification

[0094] Ultra-performance liquid chromatography-hybrid quadrupole orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was used to detect the chemical components of traditional Chinese medicine in the sample. The components of the hibiscus flower extract obtained in Example 1 were qualitatively analyzed by comparison with the reference database and theoretical database, combined with manual verification. The process is shown in FIG. Figure 2 :

[0095] 1. Medicinal taste information

[0096] Medicinal ingredients: Hibiscus tiliaceus

[0097] 2. Instruments and Reagents

[0098] 2.1 Instruments, as shown in Table 2

[0099] Table 2 Instruments

[0100]

[0101] 2.2 Reagents, as shown in Table 3

[0102] Table 3 Reagents

[0103] name Specification Item No. Manufacturer water LC-MS / 4L W6-4 Thermo Fisher Scientific Methanol LC-MS / 4L A456-4 Thermo Fisher Scientific Acetonitrile LC-MS / 4L A955-4 Thermo Fisher Scientific Formic acid LC-MS / 50mL A117-50 Thermo Fisher Scientific

[0104] 2.3 Experimental methods

[0105] 2.3.1 Sample preparation

[0106] Take 20.6 mg of a traditional Chinese medicine sample, add 1 mL of 50% methanol, and sonicate for 30 minutes. Place 0.5 mL of the solution in a 1.5 mL centrifuge tube and centrifuge at 4°C, 12,000 rpm, for 10 minutes. Transfer 100 μL of the solution to a sample vial for testing.

[0107] 2.3.2 Chromatographic methods

[0108] Chromatographic separation was performed using a Vanquish Flex UHPLC system (Thermo Fisher Scientific, Inc., Waltham, MA, USA) equipped with an ACQUITY UPLC HSS T3 reversed-phase column (2.1 mm (id) × 100 mm (length), 1.8 μm (particle size)) (Waters Corp., MA, USA). The mobile phase consisted of phase A (water + 0.1% formic acid) and phase B (acetonitrile). The elution gradient was as shown in Table 4, and the flow rate was 0.3 mL / min. The column temperature was 40°C, and the injection volume was 6.0 μL.

[0109] Table 4 Elution gradient

[0110]

[0111] 2.3.3 Mass spectrometry

[0112] The ion trap mass spectrometer (Q Exactive TM Mass spectrometry analysis was performed using a ThermoFisher Scientific, Inc., Waltham, MA, USA. The ion source voltages for positive and negative ions were 3.7 kV and 3.5 kV, respectively; the capillary heating temperature was 320°C; the sheath gas pressure was 30 psi, and the auxiliary gas pressure was 10 psi; the solvent heating evaporation temperature was 300°C; both the sheath and auxiliary gases were nitrogen; and the collision gas was nitrogen at a pressure of 1.5 mTorr.

[0113] Using Full scan / dd-MS 2 Mode data acquisition, Full scan parameters: resolution 70000, automatic gain control target 1×10 6 , maximum isolation time 50ms, mass-to-charge ratio scanning range 100–1500; dd-MS 2 Parameters: resolution 17500, automatic gain control target 1×10 5 , maximum isolation time 50ms, mass separation window 2, collision energy 10V, 30V, 60V, intensity limit 1×10 5 .

[0114] 2.4 Data Analysis

[0115] The acquired mass spectrometry data were processed using Progenesis QI 3.0 software (Waters Corp., MA, USA), following the steps of importing raw data, peak extraction, and deconvolution. A reference database (TCM Pro 2.0, Huijun Bio) and a theoretical database (constructed from literature and public databases) were searched. The identification results were analyzed through a multidimensional comprehensive assessment of reference retention time error, precursor ion mass error, secondary fragment matching, isotopic distribution, and peak intensity to yield the final results.

[0116] 3. Experimental Results

[0117] 3.1 Spectrum, such as Figure 3 、 Figure 4 As shown:

[0118] A BPI chromatogram is a continuous plot of the most abundant ion feature at each time point; a total ion current (TIC) chromatogram is a continuous plot of the sum of the abundances of all ion features at each time point. Compared to the TIC, the TIC offers advantages such as a lower baseline, higher abundance, more pronounced signal, and a more aesthetically pleasing appearance, and is therefore adopted in this paper.

[0119] 3.2 Composition results

[0120] The chemical composition of the composition of the hibiscus flower extract of the present invention is shown in Table 5.

[0121] Table 5

[0122]

[0123]

[0124] The results show that the content of each compound in the composition of the Hibiscus tiliaceus flower extract in the embodiment of the present invention is as follows from largest to smallest: hyperoside, rutin, isoquercetin, quercetin-3-O-β-d-pyranoside, myricetin-3-O-glucoside, myricetin, myricetin, tiliaoside, roselle red-3-glucoside, kaempferol-3-O-rutinoside, astragaloside, ellagic acid, luteolin, quercetin, chlorogenic acid, kaempferol, quercetin, caffeic acid, quercetin, epicatechin, scopoletin, epigallocatechin, quercetin, ru ... Quercetin-3-O-acacia glycoside, luteolin, 6-methoxy-7-hydroxycoumarin, naringenin, typhalaenoside, p-coumaric acid, baicalein, ferulic acid, isoflurane, protocatechuic acid, catechin, salicin, sinapinic acid, puerarin, gardenia glycoside, tofu fruit glycoside, eleutheroside B, pinoresinol, deacetylcarbamate methyl ester, isovanillin, syringic acid, aesculetin, 7-hydroxycoumarin, polydatin, protocatechuic aldehyde, isovanillic acid, 4'-hydroxyacetophenone, dihydroartemisinin, andrographolide, β-cineole, isoinulactone, and daphnetin.

[0125] Experimental studies have shown that the compounds in the composition of the hibiscus flower extract obtained in the present invention are significantly different from the compounds in the composition of the extract extracted from hibiscus flowers in other regions or the common hibiscus flowers currently available on the market. Experiments have confirmed that both the existing hibiscus flower extracts and the hibiscus flower extracts of the present invention have therapeutic effects on treating psoriasis, but the composition of the hibiscus flower extract of the present invention has a better therapeutic effect.

[0126] In the subsequent examples, the total flavonoids of hibiscus flowers are the hibiscus flower extract composition of the present invention.

[0127] Example 3: Preparation of hydrogel

[0128] The preparation method comprises the following steps:

[0129] S1: Weigh 1% sodium carboxymethyl cellulose and 15% glycerol, grind until uniform, add appropriate amount of purified water, heat to 40-50°C, and let stand overnight to allow full swelling to obtain a gel matrix;

[0130] S2: Dissolve 0.2% ethylparaben in a small amount of ethanol, and dissolve 0.1% poloxamer 188 and 0.6% / 1.2% / 2.4% total flavonoids from hibiscus flowers in appropriate amount of purified water;

[0131] S3: Slowly add the solution of S2 to the gel matrix of S1, then add purified water to 50g, stir evenly to obtain a transparent gel.

[0132] Example 4: Preparation of hydrogel

[0133] S1: Weigh 2% sodium carboxymethyl cellulose and 15% glycerol, grind until uniform, add appropriate amount of purified water, heat to 40-50°C, and let stand overnight to allow full swelling to obtain a gel matrix;

[0134] S2: Dissolve 0.2% ethylparaben in a small amount of ethanol, and dissolve 0.1% poloxamer 188 and 0.6% / 1.2% / 2.4% total flavonoids from hibiscus flowers in appropriate amount of purified water;

[0135] S3: Slowly add the solution of S2 to the gel matrix of S1, then add purified water to 50g, stir evenly to obtain a transparent gel.

[0136] Example 5: Preparation of hydrogel

[0137] S1: Weigh 6% sodium carboxymethyl cellulose and 15% glycerol, grind until uniform, add appropriate amount of purified water, heat to 40-50°C, and let stand overnight to allow full swelling to obtain a gel matrix;

[0138] S2: Dissolve 0.2% ethylparaben in a small amount of ethanol, and dissolve 0.1% poloxamer 188 and 0.6% / 1.2% / 2.4% total flavonoids from hibiscus flowers in appropriate amount of purified water;

[0139] S3: Slowly add the solution of S2 to the gel matrix of S1, then add purified water to 50g, stir evenly to obtain a transparent gel.

[0140] Example 6: Preparation of hydrogel

[0141] The only difference between this embodiment and embodiment 4 is that the weight ratio of glycerol is replaced by 10% in 15%.

[0142] Example 7: Preparation of hydrogel

[0143] The only difference between this embodiment and embodiment 4 is that the weight ratio of glycerol is replaced by 5% in 15%.

[0144] Example 8: Preparation of hydrogel

[0145] The only difference between this embodiment and embodiment 4 is that the weight ratio of 15% glycerol is replaced by 0%.

[0146] Example 9: Preparation of hydrogel

[0147] The only difference between this embodiment and embodiment 4 is that the weight ratio of poloxamer 188 is replaced by 0.2% in 0.1%.

[0148] Example 10: Preparation of hydrogel

[0149] The only difference between this embodiment and embodiment 4 is that the weight ratio of poloxamer 188 is replaced by 0.25%.

[0150] Example 11: Preparation of hydrogel

[0151] The only difference between this embodiment and embodiment 4 is that the weight ratio of poloxamer 188 is replaced by 0.5% with 0.1%.

[0152] Example 12: Evaluation of the hydrogels of Examples 3 to 11 of the present invention

[0153] 1. Establishment of hydrogel evaluation indicators

[0154] With reference to the "Chinese Pharmacopoeia (2020 Edition)", appearance, greasy feeling, uniformity, spreadability, centrifugal stability, cold storage stability, and thermal stability are used as evaluation indicators, and the scoring standard is 10 points, as shown in Table 6.

[0155] Table 6 Comprehensive scoring criteria for sensory indicators and test indicators of gels

[0156]

[0157] 2. The evaluation results of the hydrogels of Examples 3-11 of the present invention are shown in Table 7:

[0158] Table 7 Comprehensive scores of hydrogel sensory indicators and test indicators

[0159]

[0160]

[0161] By comparing the data of Examples 3, 4-6, 7-9, and 10-12, it is found that Examples 5, 7, 11, and 12 have higher comprehensive scores, indicating that 3% sodium carboxymethyl cellulose, 10% glycerol, and 0.5% poloxamer 188 can prepare a brown, translucent, delicate, uniform color, free of large particles, lumps, bubbles, comfortable application, and centrifugal stability.

[0162] Example 13: Construction of a mouse psoriasis model

[0163] 1. Construction method

[0164] 1.1 Group Processing

[0165] 1.1.1 Mice were randomly divided into the model group (IMQ group);

[0166] negative control group (IMQ+NC group);

[0167] low-dose administration group (IMQ+TFAM-0.6%);

[0168] medium-dose administration group (IMQ+TFAM-1.2%);

[0169] high-dose administration group (IMQ+TFAM-2.4%);

[0170] 1.1.2 Anesthetize the mouse with tribromoethanol, draw a 2 cm*3 cm area on the back of the mouse, and remove the hair on the back of the mouse to expose the skin.

[0171] 1.2 Establishment of a mouse psoriasis model by applying 5% imiquimod cream

[0172] 1.2.1 The model group and other groups were evenly smeared with 10 mg of 5% imiquimod cream, and the negative control group was treated with a blank hydrogel without the addition of total flavonoids from hibiscus flowers.

[0173] 1.2.2 Blank hydrogel and different concentrations of hibiscus flower hydrogel were applied to the backs of mice in each group. 5% imiquimod was then applied again 4 hours later for 6 days.

[0174] During the period 1.2.3, the back skin of the mice was photographed on days 2, 4, and 6 to record the erythema, scaling, and infiltration status.

[0175] 2. Tissue Collection and Processing

[0176] 2.1 On day 6, the mice were weighed and their body weights were recorded.

[0177] 2.2 Then, the exposed skin tissue of the mouse back was taken, one part was fixed in 4% paraformaldehyde, and the other part was quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator.

[0178] 2.3 Remove the mouse spleen, weigh it, and calculate the spleen weight to body weight ratio.

[0179] 3. Results

[0180] like Figure 5 As shown, after applying imiquimod on the skin surface of mice to construct a psoriasis model, hydrogel (containing 0.6%, 1.2%, and 2.4% total flavonoids from hibiscus flowers) was used for treatment. The results showed that the hydrogel of total flavonoids from hibiscus flowers inhibited the production of skin scales in mice caused by imiquimod and reduced skin erythema.

[0181] like Figure 6 As shown, after applying imiquimod on the skin surface of mice to construct a psoriasis model, hydrogel (containing 0.6%, 1.2%, and 2.4% total flavonoids from hibiscus flowers) was used for treatment. The results showed that the total flavonoids hydrogel from hibiscus flowers inhibited the increase in spleen / body weight ratio caused by imiquimod.

[0182] Example 14: HE staining

[0183] 1. Experimental Methods

[0184] 1.1 Mouse skin tissue was fixed in 4% paraformaldehyde for 24 hours, dehydrated using an automatic dehydrator, embedded in paraffin, and cut into 5 μM thick slices.

[0185] 1.2 Tissue sections of different components were selected, dewaxed with xylene and hydrated with gradient ethanol, and then stained with HE.

[0186] 1.3 Stain sections with eosin for 1 minute and hematoxylin for 30 seconds. Dehydrate in 95% and 100% ethanol, clear in xylene, and mount with a neutral resin. Flatten the slides and scan using brightfield scanning in an automated scanner.

[0187] 2. Experimental Results

[0188] like Figure 7 As shown, after applying imiquimod on the mouse skin surface to construct a psoriasis model, the hydrogel (containing 0.6%, 1.2%, and 2.4% total flavonoids from hibiscus flowers) was used for treatment. The results showed that the hibiscus flower total flavonoids hydrogel inhibited the thickening of the mouse skin caused by imiquimod.

[0189] Example 15: RNA extraction and qRT-PCR

[0190] 1. Experimental Methods

[0191] 1.1 Take mouse skin tissue, add 1 mL of TriZol, and grind in a tissue grinder. Remove the grinding tube, add 200 μL of chloroform, shake vigorously, and let it stand for 5 minutes.

[0192] 1.2 Centrifuge at 12,000 g for 10 minutes. Aspirate the upper aqueous phase and add an equal volume of isopropanol. Let stand at room temperature for 1 hour, then centrifuge at 12,000 g for 10 minutes at 4°C. Discard the supernatant and wash once with 75% ethanol (prepared with DEPC water). Centrifuge at 12,000 g for 10 minutes, discard the supernatant, invert the EP tube onto filter paper, remove excess water, and dissolve the RNA in DEPC water.

[0193] 1.3 After measuring RNA concentration using a NanoDrop, reverse transcription was performed. After obtaining cDNA, qRT-PCR was performed using the following protocol: 95°C for 5 min; 95°C for 20 s; 55°C for 20 s; and 72°C for 20 s, for 40 cycles. After obtaining the data, the ΔΔCT method was used for analysis.

[0194] 2. Experimental Results

[0195] like Figure 8As shown: After applying imiquimod on the mouse skin surface to establish a psoriasis model, hydrogel (containing 0.6%, 1.2%, and 2.4% total flavonoids from hibiscus flowers) was used for treatment. The results showed that the hibiscus flower total flavonoids hydrogel significantly reduced the mRNA expression of inflammation-related genes TNF-α and IL-1β in the mouse skin tissue after imiquimod induction, n=4.

[0196] Example 16: Western immunoblotting

[0197] 1. Experimental Methods

[0198] 1.1. Cut an appropriate amount of skin tissue, add RIPA lysis buffer containing protease inhibitors, and grind in a tissue grinder. Ultrasonicate three times and let it rest on ice for 5 minutes. Centrifuge at 13,500 rpm at 4°C for 20 minutes and aspirate the supernatant.

[0199] 1.2 Measure protein concentration using the BCA assay. Prepare the polyacrylamide gel according to the recipe and place it in an electrophoresis tank. Stir the electrophoresis buffer and then pour it into the tank until the gel is submerged. Add the protein samples according to their groupings and add varying amounts of protein marker to each end of the sample tank. Run the gel at 70V for 30 minutes or 110V for 90 minutes.

[0200] 1.3 Transfer the membrane using the semi-dry method. Block the membrane with 5% skim milk for 1 hour at room temperature. Incubate with the primary antibody at 4°C overnight and the secondary antibody the next day.

[0201] 2. Experimental Results

[0202] like Figure 9 As shown: After applying imiquimod on the surface of mouse skin to construct a psoriasis model, hydrogel (containing 0.6%, 1.2%, and 2.4% total flavonoids from hibiscus flowers) was used for treatment. The results showed that the hibiscus flower total flavonoids hydrogel significantly reduced the m protein expression level of TNF-α, an inflammatory-related factor, in the skin tissue of mice after imiquimod induction.

[0203] Example 17: CCK8

[0204] 1. Experimental Methods

[0205] 1.1 When the cells in the culture dish are growing well and in the logarithmic growth phase, they can be digested and subjected to the CCK 8 assay;

[0206] 1.2 Digest the cells, add culture medium, centrifuge and discard the supernatant, add 1 mL of complete culture medium again, pipette to mix, form a cell suspension, and count the cells; according to the number of cells required for plating, add the cell suspension to a 96-well plate, with a volume of 100 μL.

[0207] 1.3 On the day of detection, add 10 μL of CCK8 reagent to each well of the 96-well plate containing cells; continue to incubate the cells in the incubator and detect them after 2-4 hours. The OD value of the microplate reader is 450 mN.

[0208] 2. Experimental Results

[0209] like Figure 10 Figure: HaCaT cells were cultured and a cell proliferation model was established using IL-17a and TNF-α. Abelmoschus flavonoids were added to the cells at 14, 16, and 18 μg / mL. The results showed that flavonoids from Abelmoschus flavonoids inhibited HaCaT cell proliferation. **p<0.01 vs. control, ##p<0.01, ###p<0.001, ####p<0.0001 vs. IL-17a + TNF-α.

[0210] Example 18: Transcriptome Sequencing

[0211] 1. Experimental Methods

[0212] Skin tissues of mice were collected from the IMQ, IMQ+NC, and IMQ+TFAM-0.6% groups. Total RNA was extracted using TRIzol, and RNA integrity was assessed using a bioanalyzer. Poly(A)-tailed mRNA was captured using oligo(dT) magnetic beads, chemically fragmented to 200-300 bp, and double-stranded cDNA was synthesized using random hexamer primers. Sequencing adapters were then ligated and sequenced using Illumina. Data were analyzed using the BGISEQ-500 platform.

[0213] 2. Results

[0214] like Figure 11 As shown: Transcriptome sequencing was performed on mouse skin tissue, and bioinformatics analysis showed that total flavonoids from hibiscus flowers targeted the IL-17A signaling pathway to exert its anti-psoriasis effect.

[0215] like Figure 12 As shown: Based on the results of transcriptome sequencing analysis, qRT-PCR experiments were performed on the IL-17A signaling pathway in mouse skin tissue. The results showed that total flavonoids from hibiscus flowers can significantly reduce the mRNA expression levels of key genes 1L-17A, IL-17F, S100A8, and S100A9 in the IL-17A signaling pathway.

[0216] The above is only an embodiment of the present invention, and common knowledge such as the specific structure and characteristics of the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the structure of the present invention. These should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. The use of Hibiscus tiliaceus flower in preparing a psoriasis medicine, characterized in that: The hibiscus flower is a hibiscus flower extract composition, and the three compounds with the highest contents in the composition are hyperoside, rutin and isoquercetin.

2. The use according to claim 1, characterized in that: The drug preparation includes hydrogel, ointment, liniment, spray, emulsion, lotion, paste, patch, spray, liposome, powder or gel; Or, the hibiscus flower in the medicine is a hibiscus flower extract; Alternatively, the total flavonoids concentration of the hibiscus flower extract is higher than 60%.

3. The use according to claim 2, characterized in that: The total flavonoid concentration in the hydrogel preparation is higher than 0.6%, Or, the hibiscus flower is used to prepare a psoriasis drug for reducing the expression of TNF-α and IL-1β gene mRNA in skin tissue; Or, the hibiscus flower is used to prepare a psoriasis drug for inhibiting HaCaT cell proliferation; Or, the hibiscus flower is used to prepare a psoriasis drug that regulates and targets the IL-17A signaling pathway; Alternatively, the hibiscus flower is used to prepare a psoriasis drug for reducing the mRNA expression levels of genes 1L-17A, IL-17F, S100A8, and S100A9 in the IL-17A signaling pathway.

4. The drug for use according to any one of claims 1 to 3, characterized in that: The hibiscus flower is a hibiscus flower extract.

5. The drug according to claim 4, characterized in that: The total flavonoids concentration of the hibiscus flower extract is greater than 60%; The drug may be in the form of a hydrogel, ointment, liniment, spray, emulsion, lotion, paste, patch, spray, liposome, powder or gel.

6. The drug according to claim 5, characterized in that: The total flavonoids concentration in the hydrogel preparation is higher than 0.6%.

7. The drug according to claim 5, characterized in that: The hibiscus flower extract is a composition, and the compounds in the composition include any several or a combination of hyperoside, rutin, isoquercetin, quercetin-3-O-β-d-pyranoside, myricetin-3-O-glucoside, myricetin, myricetin, linalool, roselle red-3-glucoside, and kaempferol-3-O-rutinoside.

8. The drug according to claim 6, characterized in that: The compounds also include astragaloside, ellagic acid, luteolin, quercetin, chlorogenic acid, kaempferol, quercetin, caffeic acid, quercetin, epicatechin, scopoletin, epigallocatechin, quercetin-3-O-acacia glycoside, luteolin, 6-methoxy-7-hydroxycoumarin, naringenin, typhalaenoside, p-coumaric acid, baicalein, ferulic acid, isoflurane, protocatechuic acid, catechin, salicin, sinapinic acid, Any one or more combinations of puerarin, geniposide, tofu glycoside, eleutheroside B, pinoresinol, methyl deacetylcarbamate, isovanillin, syringic acid, aesculetin, 7-hydroxycoumarin, polydatin, protocatechuic aldehyde, isovanillic acid, 4'-hydroxyacetophenone, dihydroartemisinin, andrographolide, β-eudesmol, isoinulactone, and daphnetin.

9. The drug according to claim 7, characterized in that: The compounds are arranged in descending order of content in the composition as follows: hyperoside, rutin / isoquercetin, quercetin-3-O-β-d-pyranoside, myricetin-3-O-glucoside, myricetin / myricetin / basin, and roselle red-3-glucoside / kaempferol-3-O-rutinoside.

10. The drug according to claim 7, characterized in that: The total content of hyperoside, rutin, isoquercetin, quercetin-3-O-β-d-pyranoglucoside, myricetin-3-O-glucoside, myricetin, myricetin, basilicin, rosella red-3-glucoside, and kaempferol-3-O-rutinoside in the compound is greater than 80%.

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