An abelmoschus flower extract composition

Abstract

This invention relates to the field of traditional Chinese medicine and discloses a composition of okra flower extract. The purpose of this invention is to provide an okra extract with significant medicinal effects and high medicinal value.

Description

Technical Field

[0001] This invention relates to the field of plant extraction and pharmaceutical applications, specifically to an extract of Abelmoschus manihot. Background Technology

[0002] Yellow hibiscus, also known as golden hibiscus, belongs to the Malvaceae family and is an annual herbaceous flowering plant. Its flowers are used medicinally, serving as both food and medicine, and possessing health benefits. It is a plant with extremely rich medicinal value, hailed by the biological community as the "plant panda" and "life-saving herb." Historically, it has been cultivated and used. The Compendium of Materia Medica records its effects of clearing heat and dampness, reducing inflammation and relieving pain; internally, it is used to treat urinary tract infections and edema; externally, it is used to treat burns. Wild resources were on the verge of extinction in the 1980s, but were later discovered and cultivated by researchers in remote mountains. Currently, artificial cultivation sites are mainly distributed in Hubei, Jiangsu, Anhui, Hebei, Guizhou, and Chongqing.

[0003] Modern medical research has found that okra is rich in total bioflavonoids, vitamin E, unsaturated fatty acids, dietary fiber, collagen, and various beneficial trace elements. It possesses anti-inflammatory, analgesic, anti-fatigue, anti-aging, anti-cancer, and lipid-lowering effects, making it a very valuable medicinal and food ingredient. Among more than 200 okra plants, it has the most edible, medicinal, and health-promoting functions and high utilization value.

[0004] Total flavonoids are natural phenolic substances widely found in plants in nature. In vivo and in vitro studies in humans and animals have shown that flavonoids have many potential health-promoting effects, including anti-allergic, anti-inflammatory, antiviral, anti-cancer, cancer-preventive, blood sugar-lowering, blood lipid-lowering, and antioxidant effects. As an antioxidant, it can prevent cytotoxicity and tissue damage caused by oxygen free radicals and hydrides in various human diseases. The entire plant of *Abelmoschus manihot* contains flavonoid compounds, making it one of the plants with the highest flavonoid content, exceeding that of commonly used raw materials in flavonoid production such as soybeans and ginkgo by dozens of times. The total flavonoid content varies in different parts of the plant, with the highest content in the flowers, reaching as high as 3.97% in dried flowers. Literature research indicates that *Abelmoschus manihot* flowers contain a rich variety of flavonoids. High-performance liquid chromatography (HPLC) can determine and separate 63 flavonoid peaks, including known rutin, hyperoside, vitexin rhamnoside, and quercetin, as well as several unknown flavonoids with relative contents exceeding 1%.

[0005] To effectively utilize these active ingredients, plant extraction methods are commonly used to extract the effective components from plant raw materials and remove ineffective or harmful components, thereby improving therapeutic efficacy. Traditional maceration methods often use water as the extraction solvent, which may lead to the leaching of large amounts of mucilage and water-soluble components such as polysaccharides and tannins from okra. This increases the viscosity of the extract, makes filtration difficult, increases impurities in the extract, and decreases the total flavonoid content. Even with alcohol solvents, 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 increasing the contact area between the herbal powder and the solvent. However, higher temperatures can also cause the decomposition and deterioration of some flavonoids, resulting in the loss of effective components and raising safety concerns in industrial production. Enzymatic hydrolysis can destroy plant tissue cells through enzymatic action, promoting the dissolution and release of flavonoids and improving extraction efficiency. However, this can also lead to the degradation of some flavonoids, resulting in the loss of effective monomeric components, and the enzyme inactivation step in the enzymatic hydrolysis process may also pose safety concerns in industrial production.

[0006] To this end, after multiple studies, the inventors of this invention obtained a Chinese invention patent application, publication number CN119386067A, entitled "A Method for Preparing Total Flavonoid Extract from Hibiscus Flower". Compared with traditional methods, this method has the advantages of fast extraction speed, high extraction yield, sufficient extraction and retention of effective components, and simple process.

[0007] However, the inventors' team discovered that in the process of treating diseases and researching drugs, some okra flowers had poor medicinal effects, while others had significant effects. Therefore, how to obtain okra and okra extracts with significant medicinal effects and high medicinal value is a problem that the inventors' team urgently needs to solve. Summary of the Invention

[0008] The purpose of this invention is to provide a composition of okra flower extract with significant medicinal effects and high medicinal value, and the same as the drug thereof.

[0009] The basic technical solution of the present invention is: a composition of okra flower extract, the compounds comprising any combination of several or more of hyperoside, rutin, isoquercitrin, quercetin-3-O-β-d-glucopyranoside, myricetin-3-O-glucoside, myricetin, myricetin, lindenin, lycopene-3-glucoside, and kaempferol-3-O-rutin. The CAS numbers for each compound are as follows: CAS number 482-36-0 Hyperoside, CAS number 153-18-4 Rutin, CAS number 21637-25-2 Isoquercetin, CAS number 22688-79-5 Quercetin-3-O-β-d-glucopyranoside, CAS number 19833-12-6 Myricetin-3-O-glucoside, CAS number 17912-87-7 Myricetin, CAS number 529-44-2 Myricetin, CAS number 20316-62-5 Lindin, CAS number 482-34-8 Roselle-3-glucoside, and CAS number 17650-84-9 Kaempferol-3-O-rutin.

[0010] Furthermore, the compounds also include astragaloside, ellagic acid, luteolin, fraxin, chlorogenic acid, kaempferol, fraxin, caffeic acid, fraxin A, epicatechin, scopolamine, epigallocatechin, quercetin-3-O-sophoroside, luteolin, 6-methoxy-7-hydroxycoumarin, naringenin, typhain, p-coumaric acid, baicalin, ferulic acid, isozymidine, protocatechuic acid, catechin, salicin, sinapic acid, and sinapic acid. The following are any combination of several or more of the following: puerarin, geniposide, tofu glycoside, eleutheroside B, pinoresinol, methyl desacetyl succinate, isovanillin, eugenol, fraxinol, 7-hydroxycoumarin, polygalactoside, protocatechuic aldehyde, isovanillic acid, 4'-hydroxyacetophenone, dihydroartemisinin, andrographolide, β-eucalyptol, isocarboxylic acid lactone, and daphne. The CAS numbers for each compound are as follows: CAS number 480-10-4 Astragaloside, CAS number 476-66-4 Ellagic acid, CAS number 5373-11-5 Luteolin, CAS number 574-84-5 Fraxinol, CAS number 327-97-9 Chlorogenic acid, CAS number 520-18-3 Kaempferol, CAS number 524-30-1 Fraxinol, CAS number 331-39-5 Caffeic acid, CAS number 531-75-9 Fraxinoside A, CAS number 490-46-0 Epicatechin, CAS number 92-61-5 Scopolamine, CAS number 970-74-1 Epigallocatechin, CAS number 52525-35-6 Quercetin-3-O-sophoroside, CAS No. 491-70-3; Luteolin, CAS No. 92-61-56; methoxy-7-hydroxycoumarin, CAS No. 480-41-1; Naringin, CAS No. 104472-68-6; Typhain, CAS No. 501-98-4; p-Coumaric acid, CAS No. 491-67-8; Baicalein, CAS No. 1135-24-6; Ferulic acid, CAS No. 486-21-5; Isocyanidin, CAS No. 99-50-3; Protocatechuic acid, CAS No. 18829-70-4; Catechin, CAS No. 138-52-3; Salicylic acid, CAS No. 530-59-6; Sinapic acid, CAS No. 3681-99-0 Puerarin (CAS No. 24512-63-8), Gentianoside (CAS No. 80154-34-3), Tocopheryl glycoside (CAS No. 118-34-3), Acanthopanaxadione B (CAS No. 487-36-5), Pinoresinol (CAS No. 52525-35-6), Quercetin 3-O-Sophoroside (CAS No. 52613-28-2), Deacetylated methyl ester (CAS No. 621-59-0), Isovalin (CAS No. 530-57-4), Syringic acid (CAS No. 305-01-1), Fraxinol (CAS No. 93-35-6)7-Hydroxycoumarin (CAS No. 27208-80-6), Polygonum cuspidatum glycoside (CAS No. 139-85-5), Protocatechuic aldehyde (CAS No. 645-08-9), Isovaleric acid (CAS No. 99-93-4), 4'-Hydroxyacetophenone (CAS No. 71939-50-9), Dihydroartemisinin (CAS No. 5508-58-7), Andrographolide (CAS No. 51317-08-9), β-Cephalotaxelol (CAS No. 470-17-7), Isothomyl lactone (CAS No. 486-35-1), and any combination of several or more of the following: scopolamine (CAS No. 92-61-5) and 6-methoxy-7-hydroxycoumarin (CAS No. 92-61-5) are different names and forms of the same substance; therefore, they are combined under CAS No. 92-61-5. Scopolamine lactone.

[0011] The inventors' team, through Chinese invention patent application (publication number CN119386067A), entitled "A Method for Preparing Total Flavonoid Extract from Hibiscus Flower," extracted total flavonoids from collected Hibiscus flowers to obtain the extract. Ultra-performance liquid chromatography-hybrid quadrupole orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was used to detect the herbal chemical components in the extract sample. The components were qualitatively identified by comparison with reference standard databases and theoretical databases, combined with manual verification. The research found that Hibiscus flowers obtained in Chongqing, after extraction, yielded extracts with broader medicinal value and more significant efficacy. Furthermore, the extracts obtained in Chongqing showed significant differences from those obtained in other regions not only in medicinal effects and value but also in component analysis. Thus, the inventors obtained a combination of Hibiscus extracts with higher medicinal value, providing a strong foundation for further expanding its medicinal value.

[0012] The study results can express the content relationship of each compound in the extract. There are slight differences between batches of raw materials, but the basic compound varieties and content relationships remain consistent, and the compound combination is different from that of okra flower extracts from other regions.

[0013] The results show that the compounds in the composition, ranked from highest to lowest content, are: hyperoside, rutin, isoquercitrin, quercetin-3-O-β-d-glucopyranoside, myricetin-3-O-glucoside, myricetin, myricetin, lindenin, lycopene-3-glucoside, and kaempferol-3-O-rutin. Furthermore, the total content of these compounds accounts for more than 80% of the extract composition.

[0014] The compounds whose total content is less than 5% are listed in the composition from highest to lowest content as follows: astragaloside, ellagic acid, luteolin, fraxin, chlorogenic acid, scopolamine, kaempferol, fraxin, caffeic acid, fraxin A, epicatechin, epigallocatechin, quercetin-3-O-sophoroside, luteolin, naringenin, typhain, p-coumaric acid, baicalin, ferulic acid, isozymidine, protocatechuic acid, catechin, salicin, sinapic acid. Puerarin, geniposide, tofu glycoside, eleutheroside B, pinoresinol, methyl desacetyl succinate, isovanillin, eugenol, fraxin, 7-hydroxycoumarin, polygalactoside, protocatechuic aldehyde, isovanillic acid, 4'-hydroxyacetophenone, dihydroartemisinin, andrographolide, β-eucalyptol, isocarboxylic acid lactone, daphne.

[0015] The above composition can be used to prepare a medicine, comprising an effective amount of the above composition and one or more pharmaceutically acceptable carriers, excipients, or diluents. For example, it can be prepared into dosage forms such as capsules, pills, tablets, and granules.

[0016] The above composition is used in the preparation of pharmaceuticals, including any one of the following: prevention and / or treatment of stroke, psoriasis, zebrafish tail fin injury, lung injury, and liver injury.

[0017] The composition, the extraction method of which includes the steps of pulverization, alcohol extraction, and separation and purification with macroporous resin.

[0018] The compound components in the extract composition of this invention are obtained by analyzing the extract obtained based on the above-described extraction method. Of course, this also includes extracts obtained by other extraction methods that can also yield the extract composition of this invention, such as conventional water extraction, alcohol extraction, ultrasonic extraction, etc. The preparation method disclosed in Chinese invention with publication number CN114082217A is a method for preparing and applying Hibiscus syriacus flower extract, and the application of Hibiscus syriacus flower alcohol extract disclosed in Chinese invention with publication number CN106176840A are different only in that the preparation method described in this invention is used, and the overall content of the extract is higher, but the proportion of each compound remains consistent. Other preparation methods will not be described here.

[0019] The extraction method used in this invention is the Chinese invention application with publication number CN119386067A, entitled "A Method for Preparing Total Flavonoid Extract from Hibiscus Flower".

[0020] Furthermore, the extraction method includes: in the alcohol extraction step, the alcohol solvent used is an ethanol solution with a concentration of 50% to 80%; the ratio of the powder of Okra pulverized to the ethanol solution is 1:25g / mL to 1:75g / mL; the alcohol extraction process also includes high-speed homogenization and ultrasonic extraction steps.

[0021] Furthermore, in the high-speed homogenization step, the homogenization speed is 5000 rpm to 7000 rpm and the homogenization time is 60 min to 90 min; in the ultrasonic extraction step, the ultrasonic power is 300 W to 700 W, the temperature is 50 to 70 °C, and the extraction time is 60 min to 120 min, to obtain the extract.

[0022] Furthermore, 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 includes: AB-8 type macroporous adsorption resin is soaked in 95% ethanol for 24 hours, the column bed is washed with 95% ethanol, and then washed with distilled water until there is no alcohol odor, then soaked in 5% hydrochloric acid and NaOH solution for 2 hours respectively, then washed with distilled water until neutral, and finally washed with 95% ethanol until there is no white turbidity, and washed with water until there is no alcohol odor; The macroporous resin separation and purification step further includes sample preparation: the extract is diluted with water to a concentration of 0.2 g of raw material per ml, and the pH is controlled to 4-6 using an acetate-sodium acetate buffer solution; in the macroporous resin separation and purification step, the volume ratio of macroporous adsorption resin to extract is 1:1 to 1:2, and the adsorption rate is 1 BV / h; after adsorption, the sample is quickly rinsed with 5-6 BV of pure water, and the effluent is discarded; then, it is quickly rinsed with 0.5 BV of 5% ethanol solution, and the effluent is discarded; the total flavonoids are enriched using 2-5 BV of 40-90% ethanol solution, and the elution rate is 1-1.5 BV / h.

[0023] Studies have shown that the okra flower extract composition of this invention has more significant efficacy, broader and higher medicinal value compared to other existing okra flower extract compositions. For example, in the treatment of psoriasis, inflammation, zebrafish tail fin injury, diabetic nephropathy or other types of kidney disease, cosmetics, and tumors (cancer), the effects in Chongqing are more significant and superior compared to other regions. Currently, there are no reports on the treatment of certain diseases with okra flower extract, including ischemic stroke, non-alcoholic liver injury, and lung injury. Okra flowers from Chongqing have shown positive medicinal effects in these areas. Experiments of this invention have verified that the therapeutic effects of this invention are far superior to those of other regions.

[0024] Currently, experimental studies have shown that the composition of the Hibiscus syriacus flower extract of this invention has significant effects in treating non-alcoholic liver injury, especially non-alcoholic fatty liver injury; in preventing and / or treating stroke, especially ischemic stroke; and in treating and / or preventing diabetic liver injury. Furthermore, it has also shown very significant positive effects in anti-inflammation, such as in the treatment of psoriasis and zebrafish disease. A broader range of medicinal effects of Hibiscus syriacus flower extract has been successfully screened, and compound combinations with more pronounced effects have been successfully identified and distinguished, showing significant differences from existing compound combinations of Hibiscus syriacus flower extracts. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the chemical analysis steps of the okra flower extract of the present invention; Figure 2 This is the baseline peak diagram of the positive ion mode detection of the okra flower extract sample according to the present invention; Figure 3 This is the base peak diagram of negative ion mode detection of the okra flower extract sample of the present invention; Figure 4 This is a graph showing the changes in cell viability detected by the CCK8 assay in Experiment 1 of this invention, which was used to construct a HaCaT cell proliferation model. Figure 5 This is a graph showing the changes in cell proliferation capacity detected by EdU staining experiment in Experiment 1 of this invention to construct a HaCaT cell proliferation model. Figure 6 This is a diagram illustrating the therapeutic effect of the experimental combination of two types of hollyhock flowers of the present invention on psoriasis. Figure 7 This is a bar chart showing the spleen / body weight ratio in the pharmacodynamic study of the two hollyhock flower compositions of the present invention on psoriasis. Figure 8 This is a diagram illustrating the repairing effect of extracts from various regions of Hibiscus syriacus flower composition on the caudal fin of zebrafish in Experiment 3 of this invention. Figure 9 Image J analysis of the caudal fin area of ​​the compositions of okra flower extracts from various origins in Experiment 3 of this invention; Figure 10 This is a graph showing the effect of the composition of extracts of okra flowers from different producing areas in Experiment 4 of this invention on the upregulation of expression levels of RNA related to tail fin repair. Figure 11 This is a graph showing the expression levels of collagen-related genes in Experiment 4 of this invention. Figure 12 This is a graph showing the mRNA expression detection results of various pro-inflammatory / anti-inflammatory factors in Experiment 5 of this invention; Figure 13 This is a graph showing the changes in mRNA expression of anti-inflammatory and repair-related factors in the compositions of Hibiscus rosa-sinensis flower extracts from different origins in Experiment 5 of this invention. Figure 14 This is a graph showing the change in body weight during the pharmacodynamic study of total flavonoids from *Hippophae rhamnoides* flower on non-alcoholic fatty liver disease in Experiment 6 of this invention. Figure 15 The liver index diagram is from the pharmacodynamic study of total flavonoids from *Hippophae rhamnoides* flower on non-alcoholic fatty liver disease in Experiment 6 of this invention. Figure 16 This is a graph showing glucose metabolism indicators from the pharmacodynamic study of total flavonoids from *Hippophae rhamnoides* flowers on non-alcoholic fatty liver disease in Experiment 6 of this invention. Detailed Implementation

[0026] The okra flowers of this invention are from Chongqing. The preparation method of the extract adopts alcohol solvent extraction, high-speed homogenization-ultrasound-assisted liquid phase extraction, and macroporous adsorption resin purification to remove impurities, thereby separating and enriching the total flavonoids of okra.

[0027] I. The detailed preparation method of the okra flower extract of this invention is as follows: 1. Crushing Dried okra flowers from Chongqing were crushed and sieved (using a No. 3 sieve with a mesh size of 50) to obtain a dark yellow powder.

[0028] 2. Alcohol solvent-high-speed homogenization-ultrasound-assisted liquid phase extraction 2.1 Preparation of extract: Prepare an ethanol solution with a concentration of 50% to 80%, and mix the medicinal powder and solution according to the material-liquid ratio of 1:25g / mL to 1:75g / mL.

[0029] 2.2 High-speed homogenization: Homogenization speed 5000rpm~7000rpm, homogenization time 60min~90min.

[0030] 2.3 Ultrasonic-assisted extraction: Ultrasonic power 300W~700W, temperature 50~70℃, extraction time 60min~120min.

[0031] 2.4 Filtration: Filter the extract to remove solids, and combine the filtrates.

[0032] 2.5 Concentration: The filtrate is concentrated under reduced pressure (below 60℃, 0.09MPa) to recover all ethanol.

[0033] 3. Macroporous resin separation and purification 3.1 Macroporous Resin Pretreatment: AB-8 macroporous adsorption resin was soaked in 95% ethanol for 24 hours to allow it to fully swell. The column bed was then rinsed with 95% ethanol and followed by rinsing with distilled water until no alcohol odor remained. The resin was then soaked in 5% hydrochloric acid and NaOH solution for 2 hours each, rinsed with distilled water until neutral, and finally washed with 95% ethanol until no white turbidity was observed. The resin was then rinsed with water until no alcohol odor remained before use.

[0034] 3.2 Sample preparation: The extract was diluted with water to a concentration of 0.2 g of raw material per ml. The pH was controlled to 4-6 using an acetate-sodium acetate buffer solution.

[0035] 3.3 Macroporous adsorption resin column purification: The volume ratio of macroporous adsorption resin to the extract on the column was 1:1 to 1:2, and the adsorption rate was 1 BV / h. After adsorption, the column was quickly rinsed with 5 to 6 BV of pure water, and the eluent was discarded. Then, the column was quickly rinsed with 0.5 BV of 5% ethanol solution, and the eluent was discarded. The total flavonoids were enriched with 2 to 5 BV of 40 to 90% ethanol solution at an elution rate of 1 to 1.5 BV / h, and the eluent was collected.

[0036] 3.4 Drying: Collect the eluent, recover the ethanol under reduced pressure until there is no alcohol odor (below 60℃, 0.09MPa), continue to concentrate to dryness, and dry in a vacuum drying oven until the moisture content is less than 5%, to obtain a brownish-yellow powder with a slightly bitter taste, which is the total flavonoid extract, with a yield of 2.0% to 7.0%.

[0037] 4. Determination of total flavonoid concentration 4.1 Reference solution Accurately weigh 0.00614 g of rutin reference standard, add 50% ethanol to a 25 mL volumetric flask, and prepare a reference solution containing 0.2456 mg of rutin per mL.

[0038] 4.2 Flavonoid Extraction from Test Samples Take 0.15 g of powder sample, accurately weigh it, place it in a 25 mL centrifuge tube, add an appropriate amount of 50% ethanol, and sonicate for 15 min.

[0039] 4.3 Standard Curve Accurately pipette 1, 2, 3, 4, 5, and 6 mL of the reference solution under section “4.1” into separate 25 mL volumetric flasks. Add water to each flask to a final volume of 6 mL. Add 1 mL of 5% sodium nitrite solution, mix well, and let stand for 6 minutes. Add 1 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 minutes. Add 10 mL of 4% sodium hydroxide solution, then add water to the mark, shake well, and let stand for 15 minutes. Using the corresponding reagents as blanks, measure the absorbance at a wavelength of 510 nm using ultraviolet-visible spectrophotometry (General Rule 0401). Plot a standard curve with absorbance as the ordinate and concentration as the abscissa.

[0040] 4.4 Sample content determination Take three portions of the same batch of samples, prepare test solutions according to the method in section "3.2", inject and determine the sample content.

[0041] 4.5 Experimental Results 4.5.1 Establishment of the Standard Curve According to requirement 4.3, the determination results were used to perform a linear regression with rutin concentration (x, mg / mL) as the abscissa and absorbance (y) as the ordinate. The regression equation was y = 11.753x - 0.0156 (R²). 2 =0.9998).

[0042] 4.5.2 Determination of Sample Content Accurately weigh the same batch of sunflower powder and determine its total flavonoid mass fraction according to methods "4.2" and "4.4". The results are shown in Table 1.

[0043] Table 1. Results of sample content determination (n=3)

[0044] 5. Conclusion The total flavonoid content of the sample was determined by the NaNO2-Al(NO3)3-NaOH colorimetric method. 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 the sample was 68.37%.

[0045] II. Component Analysis and Identification 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 samples. The components were qualitatively identified by comparing them with reference standard databases and theoretical databases, combined with manual verification. The project workflow is as follows: Figure 1 : 1. Medicinal Flavor Information Medicinal ingredients: okra 2. Instruments and Reagents 2.1 Instruments, as shown in Table 2 Table 2 Instruments

[0046] 2.2 Reagents, as shown in Table 3 Table 3 Reagents

[0047] 2.3 Experimental Methods 2.3.1 Sample Preparation Take 20.6 mg of sample, add 1 mL of 50% methanol, and sonicate for 30 min. Take 0.5 mL of the solution, place it in a 1.5 mL centrifuge tube, and centrifuge at 4℃ and 12000 rpm for 10 min. Take 100 μL of the solution, place it in a sample vial, and wait for analysis.

[0048] 2.3.2 Chromatographic Methods Chromatographic separation was performed using a Vanquish Flex UHPLC (Thermo Fisher Scientific, Inc., Waltham, MA, USA) ultra-high performance liquid chromatography system equipped with an ACQUITY UPLC HSS T3 (2.1 mm (inner diameter) × 100 mm (length), 1.8 μm (particle size)) reversed-phase column (Waters Corp., MA, USA). The mobile phase consisted of phase A (water + 0.1% formic acid) and phase B (acetonitrile), with the elution gradient shown in Table 3. The flow rate was 0.3 mL / min. The column temperature was 40°C. The injection volume was 6.0 µL.

[0049] Table 4 Elution gradient table

[0050] 2.3.3 Mass Spectrometry Methods Mass spectrometry analysis was performed using a quadrupole orbital ion trap mass spectrometer equipped with a thermospray ion source (Q Exactive™, 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 and evaporation temperature was 300°C; both the sheath gas and the auxiliary gas were nitrogen; the collision gas was nitrogen at a pressure of 1.5 mTorr.

[0051] Full scan / dd-MS 2 Data acquisition mode, Full scan parameters: resolution 70000, automatic gain control target 1×10⁻⁶. 6 Maximum isolation time 50 ms, mass-to-charge ratio scan range 100 – 1500; dd-MS 2 Parameters: Resolution 17500, Automatic Gain Control target 1×10 5 Maximum isolation time 50 ms, mass separation window 2, collision energy 10 V, 30 V, 60 V, intensity limit 1×10 5 .

[0052] 2.4 Data Analysis The acquired mass spectrometry data were processed using Progenesis QI 3.0 software (Waters Corp., MA, USA). The steps were as follows: importing raw data, peak extraction, and deconvolution. Reference databases (TCM Pro 2.0, Huijun Biotechnology) and theoretical databases (constructed through literature and public databases) were searched. The identification results were comprehensively analyzed using multiple dimensions, including reference retention time error, precursor ion mass error, secondary fragment matching, isotope distribution, and peak intensity, to obtain the final results.

[0053] 3. Experimental Results 3.1 Spectrum, such as Figure 2 , Figure 3 As shown: BPI chromatograms are continuously plotted from the ion features with the highest abundance at each time point; total ion chromatograms (TIC) chromatograms are continuously plotted from the sum of the abundance of all ion features at each time point. Compared to the latter, the former has advantages such as lower baseline, higher abundance, more significant signal, and more aesthetically pleasing results, and is therefore adopted in this invention.

[0054] 3.2 Composition Results According to the component analysis, the chemical composition of the composition of the okra flower extract of the present invention is shown in Table 5. In the okra flower extract composition of the present invention, components below the limit of quantitation and those with extremely small proportions (content less than 0.01%) are not included. Only chemical components with a content greater than 0.01% are disclosed. The proportions listed in the table are the content percentages of each component after normalization, and are retained to 3 decimal places to show the relative content order of each component.

[0055] Table 5 Chemical composition of Hibiscus flower extract compositions in Chongqing area

[0056]

[0057] Research revealed that the compounds in the composition of the okra flower extract of this invention, in descending order of content, are: hyperoside, rutin, isoquercitrin, quercetin-3-O-β-d-glucopyranoside, myricetin-3-O-glucoside, myricetin, myricetin, linaloside, rosé-3-glucoside, kaempferol-3-O-rutin, astragaloside, ellagic acid, luteolin, fraxin, chlorogenic acid, kaempferol, fraxin, caffeic acid, fraxin A, epicatechin, scopolamine, epigallocatechin, quercetin-3-O-acacia. Glycosides, luteolin, 6-methoxy-7-hydroxycoumarin, naringenin, typhain, p-coumaric acid, baicalin, ferulic acid, isozymidine, protocatechuic acid, catechin, salicin, sinapic acid, puerarin, geniposide, taurin, eleutheroside B, pinoresinol, desacetylated methyl ester, isovalin, eugenol, fraxinol, 7-hydroxycoumarin, polygalactoside, protocatechuic aldehyde, isovalin, 4'-hydroxyacetophenone, dihydroartemisinin, andrographolide, β-eudesminol, isocarboxylic acid, daphnetin, due to CAS number 92-61-5 Scopolamine and 6-methoxy-7-hydroxycoumarin (CAS number 92-61-5) are different names for the same substance in different forms. Therefore, they are combined into scopolamine (CAS number 92-61-5). As a result, the content of scopolamine (CAS number 92-61-5) has increased from between epicatechin and epigallocatechin to between chlorogenic acid and kaempferol.

[0058] III. Comparative Analysis of Hibiscus Flower Extract Components with those from Other Regions The preparation methods, total flavonoid concentration determination, and component analysis methods for the extracts of Hibiscus syriacus in other regions are consistent with the methods used in Chongqing. The component determination results are shown below. Tables 6 to 9 show the main component data for Hubei, Hebei, Anhui, and Jiangsu, respectively. Table 10 compares the determination results of Chongqing with those of the above regions. The CAS number, molecular formula, and English name of the compound are all corresponding to those in Example 1.

[0059] Table 6. Component Analysis of Hubei Region

[0060] Table 7. Component Analysis of Hebei Region

[0061] Table 8. Component Analysis of Anhui Region

[0062] Table 9. Component Analysis of Jiangsu Region

[0063] Table 10 Comparison of chemical composition in Chongqing with other regions

[0064] The above analysis shows that: The compounds in the composition of the extract of Hibiscus syriacus obtained in Chongqing are present in other extract compositions of Hibiscus syriacus from other regions, and there are significant differences between them. The main chemical components in Chongqing include hyperoside, rutin, isoquercitrin, quercetin-3-O-β-d-glucopyranoside, myricetin-3-O-glucoside, myricetin, lindenin, lycopene-3-glucoside, and kaempferol-3-O-rutin (content higher than 1%), and the content is significantly higher than in other regions. In Chongqing, the content of these ingredients was less than 1%, while in other regions they were either undetectable or present in extremely low concentrations. These ingredients include astragaloside, ellagic acid, luteolin, fraxin, chlorogenic acid, kaempferol, and fraxin. Caffeic acid, fraxin, epicatechin, scopolamine lactone, epigallocatechin, quercetin-3-O-sophoroside, luteolin, naringenin, typhain, p-coumaric acid, baicalin, ferulic acid, isopyridine, protocatechuic acid, catechin, salicin, sinapic acid, puerarin, geniposide, tocopheryl glycoside, eleutheroside B, pinoresinol, deacetylated methyl ester, isovanillin, eugenol, fraxin, 7-hydroxycoumarin, polygalactoside, protocatechuic aldehyde, isovanillic acid, 4'-hydroxyacetophenone, dihydroartemisinin, andrographolide, β-eucalyptol, isocarboxylic acid, daphnetin; The highest content of hyperoside in Chongqing was 23.083%, while the highest content of the same component in other regions was around 15% to 18%, far lower than the highest content in Chongqing. Furthermore, Chongqing had four components with chemical contents above 10% but below the highest content, while Hubei had two, Hebei had two, Anhui had three, and Jiangsu had three, all with lower contents than Chongqing. Chongqing had five components with contents between 1% and 10%, Hubei had nine, Hebei had eleven, Anhui had twelve, and Jiangsu had twelve. This shows that the main components in Chongqing are relatively concentrated, while the main components in other regions are more dispersed, and the total content of the main components in Chongqing is higher than in other regions. The total content of major components in Chongqing reaches 98%, while that in Hubei and Hebei is around 88%, in Anhui it reaches around 91%, and in Jiangsu it reaches 92%. It can be seen that the total content of major components in Chongqing is much higher than that in other regions, while the content of trace components is lower than that in other regions.

[0065] Total flavonoids were extracted from Hibiscus flowers sourced from Hubei, Hebei, Anhui, Jiangsu, and Chongqing regions according to the method described in Example 1. The flavonoids were dissolved in pure water to prepare a 10 mg / mL solution.

[0066] 1. Experimental Methods 1.1 CCK8 1.1.1 Culture HaCaT cells until they are in good growth condition and in the logarithmic growth phase, then digest them for CCK 8 assay. 1.1.2 Digest cells, add culture medium, centrifuge and discard supernatant, add 1 mL of complete culture medium again, pipette and mix well to form a cell suspension, and count cells, seeding about 5000 cells per well; 1.1.3 Adding solution Incubate the 96-well plates overnight (or 4-24 hours) in an incubator until the cells have fully adhered and grown. Discard the old culture medium and dilute the flavonoid solution of the extracts from various regions of Hibiscus mutabilis flowers into complete culture medium containing TNF-α and IL-17A. Add 100 μL to each well of the 96-well plate. A control group without medication and blank wells without cells are also included. Incubate the 96-well plates for another 24 hours. 1.1.4 Dilute the CCK8 reagent at a ratio of 1:10 into the complete culture medium and add it to the control wells, test wells, and blank wells; continue to incubate the cells in an incubator, and perform detection after 2-4 hours. The OD value of the microplate reader is 450 nm.

[0067] 1.2 EdU 1.2.1 HaCaT cells were cultured. After constructing a cell proliferation model using TNF-α and IL-17a, flavonoid solutions extracted from Hibiscus syriacus flowers from various regions were added. After 24 hours, EdU working solution of appropriate concentration was prepared using complete culture medium and added to the cell culture wells to replace the original culture medium.

[0068] 1.2.2 Incubate at 37°C in a 5% CO2 incubator for 4 hours. Remove the EdU medium and gently wash the cells 1-2 times with PBS. Add 4% paraformaldehyde and fix at room temperature in the dark for 15-30 minutes.

[0069] 1.2.3 Add 0.5% Triton X-100 permeabilization buffer and incubate at room temperature in the dark for 10-20 minutes. Prepare the click chemistry reaction solution. Add the reaction solution to the fixed and permeabilized cell sample, ensuring complete coverage, and incubate at room temperature in the dark for 30 minutes. Add DNA dye and incubate at room temperature in the dark for 10 minutes. Observe and photograph under a fluorescence microscope.

[0070] 2. Experimental Results 2.1 As Figure 4 As shown, after constructing a HaCaT cell proliferation model using TNF-α and IL-17A, HaCaT cell viability significantly increased. However, the use of the flavonoids in the pharmaceutical composition of this invention significantly reduced cell viability. Among them, the total flavonoids from Hibiscus syriacus flowers from Chongqing had the strongest inhibitory effect on cell viability induced by TNF-α and IL-17A, compared with the effects of total flavonoids from Hibiscus syriacus flowers from Hubei, Hebei, Anhui, and Jiangsu provinces.

[0071] 2.2 such as Figure 5 As shown, after TNF-α and IL-17A induced HaCaT cells, the proliferation level of HaCaT cells increased significantly, while the proliferation capacity of HaCaT cells decreased significantly after the use of total flavonoids from Hibiscus syriacus. Among them, the total flavonoids from Hibiscus syriacus produced in Chongqing had the strongest inhibitory effect on the cell proliferation capacity induced by TNF-α and IL-17A, indicating that the anti-inflammatory effect of the combination of Hibiscus syriacus extract from Chongqing was the best.

[0072] Experiment 2: Verification of the therapeutic effect on psoriasis 1. Preparation of hydrogels 1.1 Weigh 3% sodium carboxymethyl cellulose and 15% glycerol, grind them evenly, add an appropriate amount of purified water, heat to 40~50℃, and let stand overnight to allow them to fully swell, thus obtaining a gel matrix; 1.1 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 syriacus in an appropriate amount of purified water; 1.1 Slowly add the solution of S2 to the gel matrix of S1, then add purified water to 50 g, stir well to obtain a transparent gel.

[0073] 1.1 The hydrogel prepared by this experiment is brownish-brown and translucent, fine, uniform in color, free of large particles, clumps, and bubbles, comfortable to apply, and has good centrifugal stability.

[0074] 2. Constructing a mouse model of psoriasis 2.1 Construction Method 2.1.1 Grouping treatment: Mice were randomly divided into the model group (IMQ group). Negative control group (IMQ+NC group); Low-dose administration group (IMQ+TFAM-0.6%); Medium-dose administration group (IMQ+TFAM-1.2%); High-dose administration group (IMQ+TFAM-2.4%); 2.1.2 Anesthetize mice with tribromoethanol, draw a 2cm * 3cm area on the back of the mouse, and remove the hair on the back of the mouse to expose the skin.

[0075] 2.1.2 A mouse psoriasis model was established by applying 5% imiquimod cream. 2.1.3 The model group and other groups were uniformly coated with 10 mg of 5% imiquimod cream, while the negative control group used a blank hydrogel without total flavonoids from Hibiscus syriacus. 2.1.4 Blank hydrogel and different concentrations of okra flower hydrogel were applied to the backs of mice in each group. Four hours later, 5% imiquimod was applied again, and this was continued for 6 days.

[0076] During period 2.1.5, the skin on the back of the mice was photographed and the erythema, scaling and infiltration were recorded on days 2, 4 and 6 respectively.

[0077] 2.2 Tissue sampling and processing 2.2.1 On day 6, the mice were weighed and their weight was recorded.

[0078] 2.2.2 Subsequently, bare skin tissue from the back of mice was taken, a portion of which was fixed in 4% paraformaldehyde, and the other portion was flash-frozen in liquid nitrogen and stored in a -80°C freezer.

[0079] 2.2.3 Remove the mouse spleen, weigh it, and calculate the spleen weight to body weight ratio.

[0080] 3. Results like Figure 6 As shown, after establishing a psoriasis model by applying imiquimod to the skin surface of mice, treatment with hydrogels (containing 0.6%, 1.2%, and 2.4% total flavonoids from Hibiscus syriacus) showed that the hydrogels containing total flavonoids from Hibiscus syriacus inhibited the production of imiquimod-induced skin scaling in mice and reduced skin erythema.

[0081] like Figure 7 As shown, after establishing a psoriasis model by applying imiquimod to the skin surface of mice, treatment with hydrogels (containing 0.6%, 1.2%, and 2.4% total flavonoids from Hibiscus syriacus) showed that the hydrogel containing total flavonoids from Hibiscus syriacus could effectively inhibit the increase in spleen / body weight ratio induced by imiquimod.

[0082] Experiment 3: Verification of the regenerative and repairing effects of total flavonoids from Hibiscus syriacus flowers in different regions on the caudal fins of zebrafish. 1. Experimental system: Wild-type AB strain zebrafish Zebrafish age: 3 days post-fertilization (3 dpf) 2. Experimental Methods 2.1: Collecting fish eggs The night before the experiment, select adult fish that are in good condition and have not laid eggs within 7 days. Place two male and one female fish in each mating box and separate them with a baffle. The next morning, remove the baffle and fertilized eggs can be obtained within 30 minutes. Collect the fish eggs and transfer them into a culture dish containing embryo culture medium.

[0083] 2.2: Establishing a zebrafish tissue regeneration model by surgically severing the caudal fin. Fish eggs at 3 days post-fertilization (3 dpf) were transferred to 24-well plates, with 20 fish per well, and treated according to the following grouping: Normal control group: No caudal fin removal surgery, 2 mL of fresh zebrafish culture water; Model control group: caudal fin removed, 2 mL of fresh zebrafish culture water; Drug experimental group: The zebrafish culture water was replaced with 2 mL of total flavonoid extract from Hibiscus syriacus flowers from different origins at a concentration of 0.2 mg / mL.

[0084] The zebrafish were cultured in an incubator at 28°C until 2 days post-fertilization (3 days after fertilization). Ten zebrafish were randomly selected from each experimental group and photographed under a stereomicroscope. The data were analyzed and collected using advanced image processing software, and the caudal fin area (A) of the zebrafish was analyzed.

[0085] 3. Experimental Results like Figure 8 , Figure 9 As shown, the caudal fin area of ​​zebrafish in the okra flower extract group was significantly increased compared with that in the model control group, revealing that the sample has a caudal fin repair function, and the repair effect of the total flavonoid extract of okra flowers from Chongqing is better than that of other producing areas.

[0086] Experiment 4: Anti-wrinkle effect of total flavonoids from Hibiscus flowers in different regions on zebrafish skin 1. Experimental system: Wild-type AB strain zebrafish Zebrafish age: 4 days post-fertilization (3 dpf) Sample size per group: 30 tails (N=3) 2. Experimental Procedure 2.1 Collecting fish eggs The night before the experiment, select adult fish that are in good condition and have not laid eggs within 7 days. Place two male and one female fish in each mating box and separate them with a baffle. The next morning, remove the baffle and fertilized eggs can be obtained within 30 minutes. Collect the fish eggs and transfer them into a culture dish containing embryo culture medium.

[0087] 2.2 Experimental Methods and Procedures 2.2.1 Randomly select zebrafish into a 6-well plate, with 30 fish per well.

[0088] 2.2.2 Water-soluble samples were administered, with a normal control group included. Each well contained 3 mL. Three biological replicates were performed.

[0089] 2.2.3 Incubate at 28.5℃ in the dark for 24 h. 2.2.4 Total RNA was extracted from zebrafish in each experimental group, cDNA was synthesized, and the gene expression of β-actin and the target gene was detected by q-PCR.

[0090] 2.2.5 Using β-actin as an internal reference for gene expression, the relative RNA expression level of the target gene was calculated. Relative RNA expression level = 2ΔΔC(t) (1) △△C(t) = △C(t) normal control group - △C(t) sample group (2) △C(t) = C(t)target gene C(t)β-actin (3) 3. Experimental Results 3.1 Statistical analysis of differentially expressed genes: To obtain the differential gene expression in a zebrafish caudal fin injury model treated with total flavonoid extract of Hibiscus syriacus, the criteria for screening differentially expressed genes were FC≥1 and FDR<0.05.

[0091] In the control group vs. the model group, a total of 268 differentially expressed genes were found, including 68 downregulated genes (tmem269, pde6ha, pdk2b, calcoco2, ccdc136a, si:ch211-13f8.1, otoa, hamp, fabp6, cyp24a1, LOC100330746, lrit1a, guca1c, cabp5b, zgc:194659, armc9, irx4b, camkk). 1b, ccdc120b, etc.), upregulated 200 genes (rhbg, col11a2, col12a1b, matn1, elnb, rhcga, fbn2b, col6a2, myh9b, acanb, fbln1, col6a1, mthfd1l, col6a4a, c4b, rab25b, tg, flncb, lpar3, etc.); in the drug group vs. the model group, 37 genes were upregulated (acanb, afap1, etc.). l1a, ccdc80, ceacam1, col8a2, elnb, fbln1, fbxo44.9, fmn1, fmodb, ftr31, il13ra2, itpr2, LOC101883 994, LOC571124, lpar3, mfsd10, mhc2dhb, or80a5, otol1a, ptger1b, s1pr5a, sdr42e1, si:ch211-106n1 3.3, si:ch73-380l 3.2, si:dkey-165e 2 4.1, si:dkey-178e 1 7.3, si:dkey-187j 1 4.4, si:dkey-22i 1 6.7, si:dkey-8l 1 3.5, slc14a2, spdef, sphk1, stim1b, thbs2b, tnxba, zgc:123217), downregulated 1 gene (zgc:172065). The intersection of the control group vs. model group and the drug group vs. model group yielded 17 target genes (elnb, acanb, fbln1, lpar3, si:dkey-22i16.7, itpr2, tnxba, thbs2b, ceacam1, fmodb, slc14a2, stim1b, si:ch211-106n13.3, il13ra2, si:ch73-380l3.2, ccdc80, col8a2). Genes related to the extracellular matrix and structure were screened from these genes using GO enrichment analysis, as shown in Table 11. Table 11 Genes related to extracellular matrix and structure

[0092] 3.2 The differentially expressed genes were verified by qPCR.

[0093] The results show that: Figure 10 , Figure 11 It can be seen that, compared with the model group, the expression of ccdc80, col8a2 and fbln1 in the control group and the drug group was significantly upregulated.

[0094] The relative expression levels of Col1a1a, Col1a1b, and Col1a2 genes in zebrafish from the total flavonoid extract sample group produced in Chongqing were significantly different from those in the normal control group, revealing that the sample has anti-wrinkle effects and is superior to those from other producing areas.

[0095] 1. Constructing an experimental animal model of ischemic stroke Eight-week-old male C57 mice, weighing 25–30 g, were used in the experiment. The rearing environment was controlled at a temperature of 23°C and a humidity of 60%, using a 12-hour light / 12-hour dark light cycle. The mice were acclimatized for one week before the experiment.

[0096] Sham group: control group, consisting of healthy mice.

[0097] MCAO Group: The ischemic stroke model was simulated using the middle cerebral artery occlusion (MCAO) model. Preoperative anesthesia was administered with 3% sodium pentobarbital, and body temperature was maintained at 37±0.5℃. A midline incision was made in the neck, and the common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were separated. After ligation of the ECA, a suture plug was inserted at the CCA incision site and advanced along the ICA to the origin of the middle cerebral artery (approximately 9±1 mm). Blood flow was blocked for 2 hours, after which the suture plug was removed, and the incision was sutured. Postoperatively, the mice were placed back in a temperature-controlled incubator, and changes in neurological function and behavior were observed. This model is used to study the mechanisms of ischemic brain injury and potential treatment strategies.

[0098] MCAO+TFA group: One week before modeling, mice were injected intraperitoneally with 125 mg / kg of the total flavors of Abelmoschus manihot TFA of this invention, once a day. The normal control group was injected intraperitoneally with an equal volume of physiological saline at the same time interval.

[0099] 2. Test methods: 2.1 Real-time quantitative PCR (qPCR) Total RNA was extracted from brain tissues of the experimental and control groups using the TRIZOL method. After treatment with DNase, the RNA was reverse transcribed into cDNA using a reverse transcription kit. Specific primers were designed based on the target gene and internal reference gene. The qPCR reaction system included cDNA template, SYBR Green qPCR Master Mix, forward and reverse primers, and RNase-free water. PCR amplification conditions were set as follows: 95℃ pre-denaturation for 30 s, followed by 95℃ denaturation for 5 s, 60℃ annealing, and extension for 30 s, for a total of 40 cycles. Melting curve analysis was performed after the experiment (95℃ 15 s, 60℃ 60 s, 95℃ 15 s). The relative gene expression levels were calculated using the 2^−ΔΔCt method, and statistical analysis was performed.

[0100] 3. Experimental Results qPCR test results as follows Figure 12 As shown, compared with the Sham group, the mRNA expression of pro-inflammatory factors IL-6, IL-1β, and TNF-α was significantly upregulated in the MCAO group (P<0.001), while the expression of anti-inflammatory factors IL-10 and TGF-β was decreased (P<0.05, P<0.01). After intervention with Hibiscus syriacus flower extract, the expression levels of pro-inflammatory factors IL-6, IL-1β, and TNF-α were significantly reduced (##P<0.01, ###P<0.001), while the expression levels of anti-inflammatory factors IL-10 and TGF-β were increased (##P<0.01, ###P<0.001).

[0101] 3.1 Real-time quantitative PCR (qPCR) experiments in various regions 3.1.1 The test method is the same as in 2.1, and the results are as follows: Figure 13 As shown, compared with the sham group, the expression of TGF-β, IL-6, and IL-10 mRNA in the brain tissue of the MCAO group all showed an increased trend, indicating that the inflammatory response was activated after cerebral ischemia-injury, and the body also initiated certain anti-inflammatory feedback and tissue repair responses. Among them, IL-6, as a typical pro-inflammatory factor, was significantly increased in the MCAO group, indicating that the inflammatory damage to brain tissue after MCAO was more severe. After intervention with golden hibiscus from different origins, the expression of IL-6 mRNA decreased to varying degrees, with the Chongqing group showing the most significant decrease, and the expression level was close to that of the sham group, suggesting that golden hibiscus from Chongqing may have a good effect in inhibiting the pro-inflammatory response after cerebral ischemia.

[0102] 3.1.2 Regarding anti-inflammatory and repair-related factors, both TGF-β and IL-10 were elevated after MCAO compared to the sham group, reflecting the body's compensatory initiation of anti-inflammatory regulation and tissue repair processes after cerebral ischemia-reperfusion injury. Compared with the MCAO group, the expression changes of TGF-β and IL-10 varied in most treatment groups, with the Chongqing group showing higher levels of both TGF-β and IL-10 expression, especially IL-10. IL-10, as an important anti-inflammatory factor, can participate in inhibiting excessive inflammatory responses; TGF-β is closely related to anti-inflammatory regulation, tissue repair, and post-injury remodeling. Therefore, the Chongqing group, while significantly reducing IL-6, maintained or enhanced the expression of anti-inflammatory and repair-related factors such as IL-10 and TGF-β, suggesting that it may not simply inhibit all inflammatory factors, but rather has a certain regulatory effect on the inflammatory response and repair process after cerebral ischemia.

[0103] Experiment 6: Pharmacodynamic study of total flavonoids from Hibiscus syriacus flowers on non-alcoholic fatty liver disease 1. Experimental subjects: Grouping animals for modeling Sixty 6-week-old male C57BL / 6 mice (weighing 25g-30g) were selected and fed a high-fat diet to establish a NAFLD model. They were randomly divided into a normal control group (Control group), a high-fat model group (Model group), and a total flavonoids treatment group (TFA group).

[0104] Normal control group (Control group): fed with normal feed and administered an equal volume of physiological saline by gavage; The high-fat model group (Model group) was fed a high-fat diet and administered an equal volume of physiological saline by gavage daily; Total flavonoids from Hibiscus flowers (TFA group): fed a high-fat diet and administered total flavonoids from Hibiscus flowers (250 mg / kg) by gavage daily for 16 consecutive weeks.

[0105] 2. Management and observation during the experiment During the experiment, the temperature in the animal room was maintained at 22±2℃, with a 12-hour day-night cycle, and the mice had free access to food and water. The mice were weighed using an electronic balance at fixed times each week, and the weight changes were recorded.

[0106] 3. Experiment Content Sixteen weeks later, the mice were euthanized, the livers were quickly dissected, the surface liquid was blotted dry with filter paper, the wet weight of the livers was measured, and the ratio of liver weight to body weight (%) was calculated.

[0107] Simultaneously, fasting blood samples were collected from mice to detect fasting blood glucose (FBG) and fasting insulin (FINS) levels, and HOMA-IR was calculated according to the following formula: HOMA-IR = FBG (mmol / L) × FINS (mU / L) / 22.5.

[0108] During the period, after fasting for 6 hours, the participants were given a 2g / kg glucose solution by gavage. Blood glucose was measured by tail vein sampling at 0, 30, 60, 90 and 120 minutes. The blood glucose curve was plotted and the area under the curve (AUC) was calculated to conduct an oral glucose tolerance test.

[0109] Fasting insulin levels were measured by collecting blood from the orbital venous plexus after a 6-hour fast and detecting serum insulin levels using enzyme-linked immunosorbent assay (ELISA).

[0110] 4. Experimental Results like Figure 14 As shown: In terms of weight change, the weight of the Model group mice was significantly higher than that of the normal control group starting from week 5, while the weight of the TFA treatment group was significantly lower than that of the Model group after 8 weeks of intervention.

[0111] like Figure 15 As shown, the liver quality index decreased significantly after TFA intervention.

[0112] like Figure 16 As shown, TFA significantly improves glucose metabolism. Through the intraperitoneal insulin tolerance test (IPITT) and intraperitoneal glucose tolerance test (IPGTT), the inventors of this application found that TFA can significantly improve insulin resistance in NAFLD mice.

[0113] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness of the invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A composition of okra flower extract, characterized in that: The compounds include any combination of several or more of the following: hyperoside, rutin, isoquercitrin, quercetin-3-O-β-d-glucopyranoside, myricetin-3-O-glucoside, myricetin, myricetin, lindenin, lycopene-3-glucoside, and kaempferol-3-O-rutin.

2. The composition according to claim 1, characterized in that: The compounds also include any combination of several of the following: astragaloside, ellagic acid, luteolin, fraxin, chlorogenic acid, kaempferol, fraxin, caffeic acid, fraxin A, epicatechin, scopolamine, epigallocatechin, quercetin-3-O-sophoroside, luteolin, naringenin, typhain, p-coumaric acid, baicalin, ferulic acid, isopyridine, protocatechuic acid, catechin, salicin, sinapic acid, puerarin, geniposide, tocopheryl glycoside, eleutheroside B, pinoresinol, methyl desacetylated succinate, isovanillin, eugenol, fraxin B, 7-hydroxycoumarin, polygalactoside, protocatechualdehyde, isovanillic acid, 4'-hydroxyacetophenone, dihydroartemisinin, andrographolide, β-eudesminol, isocarboxylic acid, and daphne.

3. The composition according to claim 1, characterized in that: The compounds in the composition, in descending order of content, are: hyperoside, rutin, isoquercitrin, quercetin-3-O-β-d-glucopyranoside, myricetin-3-O-glucoside, myricetin, myricetin, lindenin, lycopene-3-glucoside, and kaempferol-3-O-rutin.

4. The composition according to claim 1, characterized in that: The total content of the compounds hyperoside, rutin, isoquercitrin, quercetin-3-O-β-d-glucopyranoside, myricetin-3-O-glucoside, myricetin, myricetin, lindenin, lycopene-3-glucoside, and kaempferol-3-O-rutin glycoside is greater than 80%.

5. The composition according to claim 2, characterized in that: The compounds mentioned include astragaloside, ellagic acid, luteolin, fraxin, chlorogenic acid, kaempferol, fraxin, caffeic acid, fraxin A, epicatechin, scopolamine, epigallocatechin, quercetin-3-O-sophoroside, luteolin, 6-methoxy-7-hydroxycoumarin, naringenin, typhain, p-coumaric acid, baicalin, ferulic acid, isopyridine, protocatechuic acid, catechin, salicin, and sinapic acid. The total content of puerarin, geniposide, douchiside, eleutheroside B, pinoresinol, methyl desacetylated succinate, isovanillin, eugenol, fraxin, 7-hydroxycoumarin, polygalactoside, protocatechualdehyde, isovanillic acid, 4'-hydroxyacetophenone, dihydroartemisinin, andrographolide, β-eucalyptol, isocarboxol, and daphne is present in less than 5%.

6. The composition according to claim 2, characterized in that: The compounds, listed in descending order of their content in the composition, are as follows: cloverin, ellagic acid, luteolin, fraxin, chlorogenic acid, kaempferol, fraxin, caffeic acid, fraxin A, epicatechin, scopolamine lactone, epigallocatechin, quercetin-3-O-sophoroside, luteolin, 6-methoxy-7-hydroxycoumarin, naringenin, typhain, p-coumaric acid, baicalin, ferulic acid, isopyridine, protocatechuic acid, catechin, salicin, and sinapic acid. Puerarin, geniposide, tofu glycoside, eleutheroside B, pinoresinol, methyl desacetyl succinate, isovanillin, eugenol, fraxin, 7-hydroxycoumarin, polygalactoside, protocatechuic aldehyde, isovanillic acid, 4'-hydroxyacetophenone, dihydroartemisinin, andrographolide, β-eucalyptol, isocarboxylic acid lactone, daphne.

7. A medicament prepared using the composition according to any one of claims 1 to 6, characterized in that: It includes an effective amount of the above composition, and one or more pharmaceutically acceptable carriers, excipients or diluents.

8. The use of the composition according to any one of claims 1 to 6 in the preparation of a pharmaceutical product, characterized in that: The drugs mentioned include any one of the following: prevention and / or treatment of stroke, psoriasis, zebrafish tail fin injury, lung injury, and liver injury.

9. A method for preparing the composition according to any one of claims 1 to 6, characterized in that: The extraction method includes the steps of pulverization, alcohol extraction, and separation and purification with macroporous resin.

10. The method for preparing the composition according to claim 9, characterized in that: Its extraction methods include: In the alcohol extraction step, the alcohol solvent used is an ethanol solution with a concentration of 50% to 80%; the ratio of the powdered okra to the ethanol solution is 1:25 g / mL to 1:75 g / mL. The alcohol extraction process also includes high-speed homogenization and ultrasonic extraction steps; In the high-speed homogenization step, the homogenization speed is 5000 rpm to 7000 rpm and the homogenization time is 60 min to 90 min. In the ultrasonic extraction step, the ultrasonic power is 300W-700W, the temperature is 50-70℃, and the extraction time is 60min-120min to obtain the extract. 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 steps include: soaking AB-8 type macroporous adsorption resin in 95% ethanol for 24 hours, rinsing the column bed with 95% ethanol, rinsing with distilled water until there is no alcohol odor, soaking in 5% hydrochloric acid and NaOH solution for 2 hours respectively, rinsing 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 odor. The macroporous resin separation and purification step also includes sample preparation: the extract is diluted with water, the concentration of the extract is 0.2g of raw material per ml, and the pH is controlled to 4-6 using an acetate-sodium acetate buffer. In the macroporous resin separation and purification step, the volume ratio of macroporous adsorption resin to extraction liquid is 1:1 to 1:2, and the adsorption rate is 1 BV / h. After adsorption, the solution is quickly rinsed with 5 to 6 BV of pure water, and the effluent is discarded. Then, the solution is quickly rinsed with 0.5 BV of 5% ethanol solution, and the effluent is discarded. The total flavonoids are enriched using 2 to 5 BV of 40 to 90% ethanol solution, and the elution rate is 1 to 1.5 BV / h.

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