Application of Abelmoschus flower in preparation of lung injury medicine, preparation method and preparation thereof

Abstract

The application relates to the field of traditional Chinese medicines, and discloses a medicine for treating acute lung injury and application of the medicine, in particular to application of Abelmoschus flower in preparation of a medicine for preventing, treating and / or relieving acute lung injury.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, specifically to a drug for acute lung injury and its application. Background Technology

[0002] Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are common critical illnesses in clinical practice. ARDS affects approximately 3 million patients annually, accounting for 10% of intensive care unit admissions. The core pathology of ALI / ARDS is damage to the alveolar-capillary barrier, leading to increased vascular permeability and non-cardiac pulmonary edema. Current treatments have significant limitations; while steroids and mechanical ventilation can provide temporary support, their efficacy is limited and they often involve side effects. Given the limitations of current treatments and the high mortality rate, developing new and effective therapies is particularly urgent and important.

[0003] Hibiscus rosa-sinensis, a plant belonging to the Malvaceae family and the genus Hibiscus, is a traditional medicinal plant. Modern research has confirmed that the entire plant is rich in flavonoids, making it a highly valuable source of flavonoid extraction. Total flavonoids extracted from its dried flowers possess potent antioxidant properties, significantly scavenging free radicals, reducing oxidative stress, and protecting cells. They can also alleviate chronic inflammation by inhibiting inflammatory signaling pathways such as COX-2 and NF-κB. Our study further elucidates the role and mechanism of total flavonoids from Hibiscus rosa-sinensis flowers in the treatment of acute lung injury, providing a scientific basis for the development of safe and reliable drugs against acute lung injury. Summary of the Invention

[0004] The purpose of this invention is to provide a drug that can prevent, treat and / or alleviate acute lung injury and its application.

[0005] The basic technical solution of this invention is the application of okra flowers in the preparation of drugs for the prevention, treatment and / or relief of acute lung injury.

[0006] Preferably, the herbaceous hibiscus flower is a herbaceous hibiscus flower extract composition, and the three compounds with the highest content in the composition are, in descending order: hyperoside, rutin, and isoquercitrin.

[0007] Preferably, okra flower is a drug for restoring the mitochondrial morphology of Beas-2b cells.

[0008] Preferably, okra flower is a drug that increases the mitochondrial membrane potential of Beas-2b cells; Preferably, okra flower is a drug that reduces the accumulation of reactive oxygen species in Beas-2b cells; Preferably, okra flower is a drug that regulates ESR1 in thyroid hormone signaling.

[0009] Experimental results showed that okra flowers may regulate acute lung injury through ESR1 in the thyroid hormone signaling pathway; they can significantly restore the normal morphology of mitochondria in Beas-2b cells after LPS induction; they can significantly increase the membrane potential of mitochondria in Beas-2b cells after LPS induction; and they can significantly reduce the accumulation of reactive oxygen species in Beas-2b cells after LPS induction.

[0010] The results of HE staining experiments showed that total flavonoids from Hibiscus syriacus flowers can significantly improve the structural remodeling of mouse lung tissue and reduce the infiltration of inflammatory cells; significantly alleviate LPS-induced collagen deposition in mouse lung tissue; and significantly reduce the mRNA expression levels of LPS-induced inflammatory factors TNF-α and IL-1β, as well as the wet / dry weight ratio of mouse lung tissue, thus having a significant therapeutic effect on acute lung injury.

[0011] Another technical solution provided by the present invention utilizes the above-mentioned drug, wherein the herbaceous hibiscus flower is a herbaceous hibiscus flower extract.

[0012] Preferably, the extract of Hibiscus syriacus is a composition, and the compounds in the composition 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.

[0013] Preferably, 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, isozymine, 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.

[0014] Preferably, the compounds in the composition, in descending order of content, are: hyperoside, rutin / isoquercetin, quercetin-3-O-β-d-glucopyranoside, myricetin-3-O-glucoside, myricetin / myricetin / linosin, rosé-3-glucoside / kaempferol-3-O-rutin.

[0015] Preferably, the total content 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 glycoside in the compound is greater than 80%.

[0016] Preferably, the compounds 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, 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%.

[0017] Previously, during their research on the medicinal application of Hibiscus syriacus flower extract, the inventors of this application discovered that the medicinal effects of different batches of Hibiscus syriacus flower extract showed significant differences. Therefore, the inventors collected Hibiscus syriacus flowers for extraction to obtain extracts, and used ultra-performance liquid chromatography-hybrid quadrupole orbitrap high resolution mass spectrometer (UHPLC-Q-Orbitrap HRMS) to detect the traditional Chinese medicine chemical components in the extract samples. The components were qualitatively identified by comparing with reference standard databases and theoretical databases, combined with manual verification.

[0018] Furthermore, the inventors' team's research on the components of different okra plants revealed significant differences in the composition of extracts from different batches of okra grown in Chongqing compared to those from other regions. Additionally, the inventors' team discovered differences in the breadth of therapeutic applications of okra extracts from Chongqing. To clarify the reasons behind these differences, the inventors' team conducted a study on the components of okra extracts from Chongqing compared to those from other regions.

[0019] The study results show the content relationships of various compounds in the extract. While there were slight differences between batches of raw materials, the basic compound types and content relationships remained consistent, and the compound combinations differed from those of Hibiscus syriacus flower extracts from other regions. Specifically, the compounds in the composition, from most abundant to least abundant, are: hyperoside, rutin / isoquercetin, quercetin-3-O-β-d-glucopyranoside, myricetin-3-O-glucoside, myricetin / myricetin / linaloside, rosé-3-glucoside / kaempferol-3-O-rutin.

[0020] Because the content differences among the compounds are small, ranging from 1% to 0.1%, the content of rutin / isoquercetin can be either rutin more than isoquercetin, or isoquercetin more than rutin. The order of myricetin, myricetin, and timbolic acid can also be from most to least: myricetin, myricetin, timbolic acid; timbolic acid, myricetin, myricetin; myricetin, timbolic acid, myricetin; or myricetin, timbolic acid, myricetin. The content of rosé-3-glucoside / kaempferol-3-O-rutinoside is ordered from highest to lowest. It can be that rosé-3-glucoside is more than kaempferol-3-O-rutinoside, or kaempferol-3-O-rutinoside is more than rosé-3-glucoside. The content order may vary slightly due to different product batches, but the overall content order is roughly the same, and the above compound combination accounts for more than 80% of the content of the extract composition.

[0021] The compounds with a content of less than 5% in the composition are listed in the following order: astragaloside, 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, 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.

[0022] For compounds with a total content of less than 5%, the above content ranking does not show significant differences, nor is it only when the content follows the above ranking that preventive and therapeutic effects are observed. Rather, it is because, during experimental research according to this invention, it was found that the content ranking of compounds in most extracts with significant medicinal effects exhibits the above trend. For example, in compounds with a total content of less than 5%, the content of adjacent compounds may differ by only 0.01% or 0.001%. Therefore, the content ranking is only one example in the embodiments, and the ranking may change depending on the product batch.

[0023] Furthermore, the inventors discovered that different extraction methods lead to variations in the compound content of the extract. Plant extraction methods are commonly used to extract active ingredients from plant materials, removing ineffective or harmful components to improve therapeutic efficacy. Traditional maceration methods often use water as the extraction solvent, which may result in the leaching of large amounts of mucilage and water-soluble components such as polysaccharides and tannins from *Abelmoschus manihot*, increasing the viscosity of the extract, making filtration difficult, increasing impurities in the extract, and decreasing 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 the contact area between the herbal powder and the solvent. However, higher temperatures can also cause the decomposition and deterioration of some flavonoids, leading to the loss of active ingredients 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 monomer components. Furthermore, the enzyme inactivation step in the enzymatic hydrolysis process may pose safety concerns under industrial production conditions.

[0024] Therefore, the inventors of this invention utilize the extraction process previously obtained by their team, namely, 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 retention of effective components, and simple process.

[0025] The extraction method includes the steps of pulverization, alcohol extraction, and separation and purification with macroporous resin.

[0026] Preferably, 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.

[0027] Preferably, 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.

[0028] 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 includes: 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, then 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 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.

[0029] The compound components in the extract composition of this invention have a higher content than those obtained by the extraction method described above. This also includes extracts obtained by other methods, such as conventional water extraction, alcohol extraction, and ultrasonic extraction, which can also yield the extract composition of this invention. For example, the preparation method disclosed in Chinese invention CN114082217A (a method for preparing and applying Hibiscus syriacus flower extract) and the application of Hibiscus syriacus flower alcohol extract disclosed in Chinese invention CN106176840A differ only in that the overall content of the extract is higher using the preparation method described in this invention, but the proportions of the compounds remain consistent. Other preparation methods will not be described here. Attached Figure Description

[0030] Figure 1 This is a standard curve diagram of rutin from Example 1; Figure 2 This is a schematic diagram of the chemical analysis steps for the okra flower extract in Example 2; Figure 3 This is the baseline peak diagram of the positive ion mode detection of the okra flower extract sample in Example 2; Figure 4 This is the base peak diagram of negative ion mode detection for the okra flower extract sample in Example 2; Figure 5 The CCK8 experimental results are shown for the compositions of Hibiscus syriacus flower extracts from different origins in Experiment 2. Figure 6 HE staining image of Experiment 1 (inflammatory cells); Figure 7 This is an HE staining image (collagen deposition) from Experiment 1. Figure 8 This is a graph showing the wet / dry weight ratio of mouse lung tissue in the qRT-PCR experiment of Experiment 2. Figure 9 This is a diagram showing the network pharmacology results of Experiment 3; Figure 10 This is a diagram showing the detection of thyroid hormone signaling pathways in Experiment 4. Figure 11 MitoTracker staining experiment of Beas-2b cells for Experiment 5; Figure 12 The membrane potential diagram of reactive oxygen species in Beas-2b cells for the ROS staining experiment in Experiment 6; Figure 13 This is a graph showing the accumulation of reactive oxygen species in Beas-2b cells during the ROS staining experiment in Experiment 7. Detailed Implementation

[0031] This application involves the following terms and abbreviations: Acute lung injury: Acute lung injury; JHK: Total flavonoids from Hibiscus syriacus, i.e., the Hibiscus syriacus extract composition of the present invention; LPS: Lipopolysaccharide.

[0032] The following detailed description illustrates the specific implementation method: Example 1: Obtaining Hibiscus Flower Extract 1. Raw material acquisition The extract of okra flower is obtained from okra flowers grown in Chongqing, specifically in Wuxi County, Chongqing.

[0033] 2. Preparation of Hibiscus Flower Extract 2.1 Raw material preparation: Mature okra flowers are harvested, dried, pulverized into coarse powder, and sieved (using a 50-mesh sieve) to obtain uniform flower powder, which is then used as the extraction raw material.

[0034] 2.2 Alcohol Extraction Process: Take the above-mentioned Hibiscus syriacus flower powder and add it to a 50%–90% ethanol solution at a ratio of 1:25–1:75 (g / mL). High-speed homogenization (5000–7000 rpm, 60–90 minutes) combined with ultrasonic-assisted extraction (300–700 W, 50–70℃, 60–120 minutes) significantly improves the extraction efficiency of flavonoids.

[0035] 2.3 Filtration and Concentration: The extract was filtered to remove residue, and then concentrated under reduced pressure (below 60℃, 0.09MPa) to recover ethanol and obtain concentrated extract.

[0036] 2.4 Macroporous resin separation and purification: 2.4.1 Pretreatment of macroporous resin: AB-8 type 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 then rinsed with distilled water until no alcohol odor was observed. After soaking in 5% hydrochloric acid and NaOH solution for 2 hours respectively, it was rinsed with distilled water until neutral, and finally washed with 95% ethanol until no white turbidity was observed. It was then washed with water until no alcohol odor was observed before use.

[0037] 2.4.2 Sample preparation: Dilute the extract with water to a concentration of 0.2 g of raw material per ml, and control the pH to 4-6 using an acetate-sodium acetate buffer solution.

[0038] 2.4.3 Macroporous adsorption resin column purification: The volume ratio of macroporous adsorption resin to the extract on the column is 1:1 to 1:2, and the adsorption rate is 1 BV / h. After adsorption, the column is quickly rinsed with 5 to 6 BV of pure water, and the eluent is discarded. Then, it is quickly rinsed with 0.5 BV of 5% ethanol solution, and the eluent is discarded. The total flavonoids are 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 is collected.

[0039] 2.4.4 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 extract of Hibiscus syriacus flower, also known as the total flavonoid extract of Hibiscus syriacus flower, with a yield of 2.0% to 7.0%.

[0040] 3. Determination of total flavonoid concentration 3.1 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.

[0041] 3.2 Flavonoid Extraction from Test Sample 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.

[0042] 3.3 Standard Curve Accurately pipette 1, 2, 3, 4, 5, and 6 mL of the reference solution under section "3.1" 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.

[0043] 3.4 Determination of Sample Content 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.

[0044] 3.5 Experimental Results 3.5.1 Establishment of the standard curve According to requirement 3.3, the test results are as follows: Figure 1 As shown, a linear regression was performed with rutin concentration (x, mg / mL) on the x-axis and absorbance (y) on the y-axis, yielding the regression equation y = 11.753x - 0.0156 (R²). 2 =0.9998).

[0045] 3.5.2 Determination of Sample Content Accurately weigh the same batch of sunflower powder and determine its total flavonoid mass fraction according to methods "3.2" and "3.4". The results are shown in Table 1.

[0046] Table 1. Results of sample content determination (n=3)

[0047] 4. 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%.

[0048] Example 2: 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 comparison with reference standard databases and theoretical databases, combined with manual verification. The procedure 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

[0049] 2.2 Reagents, as shown in Table 3 Table 3 Reagents

[0050] 2.3 Experimental Methods 2.3.1 Sample Preparation Take 20.6 mg of the traditional Chinese medicine 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.

[0051] 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 4. The flow rate was 0.3 mL / min. The column temperature was 40°C. The injection volume was 6.0 µL.

[0052] Table 4 Elution gradient table

[0053] 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.

[0054] 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 .

[0055] 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.

[0056] 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.

[0057] 3.2 Composition Results Component analysis revealed that the chemical composition of the okra flower extract composition of this invention is shown in Table 5: Table 5

[0058]

[0059] Research revealed that, in the composition of the okra flower extract in the embodiments of the present invention, the contents of each compound, from largest to smallest, are as follows: 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- Sophoroside, luteolin, 6-methoxy-7-hydroxycoumarin, naringenin, typhain, p-coumaric acid, baicalin, ferulic acid, isozymidine, protocatechuic acid, catechin, salicin, sinapic acid, puerarin, geniposide, tau guar glycoside, eleutheroside B, pinoresinol, methyl desacetylated succinate, isovalin, eugenol, fraxinol, 7-hydroxycoumarin, polygalactoside, protocatechuic aldehyde, isovalin, 4'-hydroxyacetophenone, dihydroartemisinin, andrographolide, β-eudesminol, isocarboxylic acid, daphne, 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.

[0060] Experimental Verification 1: Compound identification of extracts from Hibiscus syriacus flowers from different origins (Hubei, Hebei, Anhui, Jiangsu). Note that components below the limit of quantitation and those with extremely low concentrations (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 normalized percentages of each component, with three decimal places, to show the relative order of the components.

[0061] Experimental Verification 3: Verifying the therapeutic effect of combinations of okra flower extracts from different origins on LPS-induced acute lung injury. The experimental methods involved are consistent with those in Experimental Verification 3.

[0062] Experimental Verification 3: Verifying the therapeutic effect of a combination of okra flower extracts on LPS-induced acute lung injury. Male C57BL / 6 mice aged 6-8 weeks and weighing 20-25g were selected. An acute lung injury model was established by intratracheal instillation of LPS. For the first two days after model establishment, mice were administered an intraperitoneal injection of a combination of Hibiscus syriacus flower extract (100mg / Kg) once daily. Two days after model establishment, mice were sacrificed and lung tissue was collected. A portion of the lung tissue was fixed in 4% paraformaldehyde or flash-frozen in liquid nitrogen and stored at -80℃. The fixed lung tissue was dehydrated, embedded in paraffin, and cut into 5μm thick slices, followed by HE staining to measure epidermal thickness. The lung tissue stored at -80℃ was cut into small pieces, and total RNA was extracted using TriZol reagent. The RNA was reverse transcribed into cDNA, and qRT-PCR was performed to detect inflammatory factor-related genes. Total protein was extracted using RIPA lysis buffer and a protease inhibitor, and Western blotting was used to detect changes in the protein expression levels of inflammatory factors.

[0063] In the following experiments, the total flavonoids from Hibiscus syriacus flowers were the Hibiscus syriacus flower extract composition of the present invention.

[0064] Experimental methods: ① Establishment of a mouse model of acute lung injury Male C57BL / 6 mice weighing 20-22g were selected. Mice were anesthetized with tribromoethanol, and the trachea was exposed by incision of the neck skin. An acute lung injury model was established by instilling LPS (10mg / Kg) into the trachea. Subsequently, mice were injected intraperitoneally with physiological saline, blank control, and total flavonoids from Hibiscus syriacus.

[0065] Groups: Saline control group; LPS lipopolysaccharide model group; LPS administration group.

[0066] ② Lung tissue processing Forty-eight hours after establishing the LPS-induced lung injury model in mice, the mice were sacrificed, and lung tissue was removed and fixed in 4% paraformaldehyde. Another portion was flash-frozen in liquid nitrogen and stored in a -80°C freezer.

[0067] ③ HE staining Lung tissue preserved in 4% paraformaldehyde was removed, dehydrated, embedded, and cut into 5 μm thick sections. Sections from different groups were dewaxed and hydrated, then stained with eosin and hematoxylin, respectively. After rapid dehydration and clearing, the sections were mounted with neutral resin and scanned under a panoramic scanner.

[0068] ④Masson staining Lung tissue sections from different groups were dewaxed and hydrated. Hematoxylin was used to stain cell nuclei for 5-10 minutes. After a brief rinse with running water, the sections were rapidly differentiated using 1% hydrochloric acid-alcohol solution, followed by thorough blue re-staining with running water until the nuclei appeared a clear blue-black. Subsequently, ponceau red and fuchsin acidic staining was applied for 5-10 minutes to initially stain muscle fibers and cellulose. The sections were then treated with phosphomolybdic acid solution for 5-10 minutes for color separation and mordanting until collagen fibers faded to a pale pink under the microscope. Immediately afterward, aniline blue was used for counterstaining for 5 minutes to ensure specific blue binding of collagen fibers. After counterstaining, the sections were rinsed again with 1% glacial acetic acid aqueous solution for 1-2 minutes to enhance contrast. Finally, the sections were rapidly dehydrated with a series of ethanol solutions, cleared with xylene, and mounted with neutral resin. A panoramic scanner was then used for scanning.

[0069] ⑤ qRT-PCR A suitable amount of mouse lung tissue was excised, and 1 mL of TRIZOL reagent was added. The tissue was homogenized thoroughly in a tissue homogenizer, and then 200 μL of chloroform was added. The mixture was vortexed vigorously for 15 seconds and allowed to stand at room temperature for 2-3 minutes. The tissue was then centrifuged at 12,000 g for 15 minutes at 4°C. The aqueous phase was then aspirated and an equal volume of isopropanol was added. The mixture was gently inverted and allowed to stand at room temperature for 30 minutes. Afterward, the tissue was centrifuged at 12,000 g for 10 minutes at 4°C, the supernatant was discarded, and the tissue was washed with 75% ethanol. The tissue was then centrifuged at 12,000 g for 10 minutes, and an appropriate amount of DEPC water was added to dissolve the RNA. Reverse transcription was performed after measuring the RNA concentration, and the transcription was repeated for 40 cycles at three different temperatures: 95°C for 5 min; 95°C for 20 s; 55°C for 20 s; and 72°C for 20 s. After obtaining the data, Experiment 2... -ΔΔCT The method is used for analysis.

[0070] ⑥ Network pharmacology analysis First, active ingredients of traditional Chinese medicine were screened using databases such as TCMSP combined with ADME parameters (e.g., OB≥30%, DL≥0.18), and their targets were predicted using SwissTargetPrediction and PharmMapper platforms. Then, disease-related targets were obtained from disease databases such as DisGeNET and GeneCards, and potential therapeutic targets were obtained by taking the intersection. Next, a protein-protein interaction network was constructed using the STRING database, and topological analysis was performed using Cytoscape to screen core targets. Finally, GO function and KEGG pathway enrichment analysis were performed on the core targets using DAVID or Metascape platforms to preliminarily elucidate the synergistic mechanism of the drug's multi-component, multi-target, and multi-pathway interactions.

[0071] ⑦ MitoTracker staining Beas-2B cells were cultured with LPS or a combination of LPS and total flavonoids from Hibiscus syriacus flowers, and divided into Control, LPS, and LPS+TFAM groups. MitoTracker staining solution was then prepared at a ratio of 1:1000. The culture medium was discarded, and the staining solution was added. The cells were incubated at 37°C for 1 hour, then the MitoTracker staining solution was discarded, and Hoechst 33342 was added and incubated for 10 minutes. The cells were then observed and photographed under an inverted fluorescence microscope.

[0072] ⑧ JC-1 staining Beas-2B cells were cultured with LPS or a combination of LPS and total flavonoids from Hibiscus syriacus. Cells were divided into Control, LPS, and LPS+TFAM groups. 0.5 mL of JC-1 working solution was added per 500,000-1,000,000 cells, and the cells were incubated at 37°C in the dark for 30 minutes. Hoechst 33342 was then added, and the cells were incubated for another 10 minutes. Observations and photographs were taken under an inverted fluorescence microscope using red and green fluorescence, respectively.

[0073] 9. ROS staining Beas-2B cells were cultured with LPS or a combination of LPS and total flavonoids from Hibiscus syriacus, and divided into Control, LPS, and LPS+TFAM groups. A DCFH-DA probe (10 μM) was prepared and incubated at 37°C in the dark for 30 minutes, followed by incubation with Hoechst 33342 for 10 minutes. The cells were then observed and photographed under an inverted fluorescence microscope.

[0074] ⑩ Data Statistics All data were analyzed using GraphPad Prism 8.0 and expressed as mean ± SEM. Student's t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups, with Bonferroni test correction. P A value <0.05 is considered statistically significant.

[0075] Experimental results: Experimental Result 1: Identification of the Chemical Composition of Total Flavonoids from Hibiscus Flowers from Different Origins The methods for obtaining the extract of Hibiscus syriacus, determining the concentration of total flavonoids, and analyzing and identifying its chemical components are consistent with those in Examples 1 and 2.

[0076] Table 6 shows the comparison of chemical composition between Chongqing and other regions:

[0077]

[0078]

[0079] It is evident that the okra flower extract composition of the present invention differs significantly from okra flower extracts from other regions.

[0080] Experimental Result 2: The therapeutic effect of combinations of okra flower extracts from different origins on LPS-induced acute lung injury. This invention uses LPS to construct a Beas-2B cell damage model, then adds total flavonoids from Hibiscus syriacus flowers from Hubei, Hebei, Anhui, Jiangsu, and Chongqing, and uses the CCK8 assay to detect changes in cell viability.

[0081] The results are as follows Figure 5 As shown in the CCK8 experiment, the total flavonoids from Abelmoschus manihot in Hubei, Jiangsu and Chongqing can significantly increase the viability of Beas-2B cells after LPS induction. Among them, the total flavonoids from Abelmoschus manihot in Chongqing have the most significant effect on restoring cell viability. Therefore, the subsequent experimental verification three selected the total flavonoids from Abelmoschus manihot in Chongqing (hereinafter referred to as total flavonoids from Abelmoschus manihot flowers) for the experiment.

[0082] Experimental Result 3: Experiment 1: HE staining like Figure 6 As shown, HE staining results indicate that total flavonoids from Hibiscus syriacus flowers can significantly improve the structural remodeling of mouse lung tissue and reduce the infiltration of inflammatory cells; like Figure 7 As shown, total flavonoids from Hibiscus syriacus flowers can significantly reduce LPS-induced collagen deposition in mouse lung tissue.

[0083] Experiment 2: qRT-PCR Experiment like Figure 8 As shown, total flavonoids from Hibiscus syriacus flowers can significantly reduce the mRNA expression levels of LPS-induced inflammatory factors TNF-α and IL-1β, as well as the wet / dry weight ratio of mouse lung tissue.

[0084] Experiment 3: Network Pharmacology like Figure 9 As shown, the network pharmacology results screened out 5 flavonoids with high biological activity and good drug-like properties, as well as their targets. By intersecting the targets of flavonoids and the targets of acute lung injury, 33 common genes were obtained.

[0085] Experiment 4: GO analysis of intersecting genes, followed by qRT-PCR experiment. like Figure 10As shown, GO analysis of the intersection genes revealed significantly differential expression in the thyroid hormone signaling pathway. Subsequent qRT-PCR detection of genes within the thyroid hormone signaling pathway revealed the most significant difference in ESR1 expression. This suggests that total flavonoids from Hibiscus mutabilis flowers may regulate acute lung injury through ESR1 in the thyroid hormone signaling pathway.

[0086] Experiment 5: MitoTracker staining like Figure 11 As shown, Beas-2b cells were cultured, and a cell damage model was constructed using LPS. Subsequently, total flavonoids from Hibiscus syriacus flowers were added. The results of the MitoTracker experiment showed that total flavonoids from Hibiscus syriacus flowers could significantly restore the normal morphology of mitochondria in Beas-2b cells after LPS induction.

[0087] Experiment 6: JC-1 staining like Figure 12 As shown, Beas-2b cells were cultured, and a cell damage model was constructed using LPS, followed by the addition of total flavonoids from Hibiscus syriacus flowers. The results of the JC-1 experiment indicated that total flavonoids from Hibiscus syriacus flowers significantly increased the mitochondrial membrane potential of Beas-2b cells after LPS induction.

[0088] Experiment 7: ROS staining like Figure 13 As shown, Beas-2b cells were cultured, and a cell damage model was constructed using LPS. Subsequently, total flavonoids from Hibiscus syriacus flowers were added. ROS staining results indicated that total flavonoids from Hibiscus syriacus flowers significantly reduced the accumulation of reactive oxygen species in Beas-2b cells after LPS induction.

[0089] Therefore, it can be seen that the composition of the okra flower extract of the present invention differs from the prior art not only in the chemical composition of the composition, but also in the effect of treating acute lung injury.

[0090] 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. Use of Hibiscus flowers in the preparation of drugs for the prevention, treatment and / or relief of acute lung injury.

2. The application according to claim 1, characterized in that: The aforementioned okra flower is an extract composition of okra flower, and the three compounds with the highest content in the composition are, in order: hyperoside, rutin, and isoquercitrin.

3. The application according to claim 1, characterized in that: The aforementioned okra flower is a drug for restoring the mitochondrial morphology of Beas-2b cells.

4. The application according to claim 1, characterized in that: The aforementioned okra flower is a drug that increases the mitochondrial membrane potential of Beas-2b cells; The aforementioned okra flower is a drug that reduces the accumulation of reactive oxygen species in Beas-2b cells; The aforementioned okra flower is a drug that regulates ESR1 in thyroid hormone signaling.

5. The medicament used according to any one of claims 1 to 4, characterized in that: The aforementioned okra flower is an extract composition of okra flowers.

6. The drug according to claim 5, characterized in that: The okra flower extract is a composition, and the compounds in the composition 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.

7. The drug according to claim 6, characterized in that: 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, isopyridine, protocatechuic acid, catechin, salicin, 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.

8. The drug according to claim 6, characterized in that: The compounds in the composition, in descending order of content, are: hyperoside, rutin / isoquercetin, quercetin-3-O-β-d-glucopyranoside, myricetin-3-O-glucoside, myricetin / myricetin / titanin, lycopene-3-glucoside / kaempferol-3-O-rutin.

9. The drug according to claim 6, characterized in that: The total content of the compounds mentioned is greater than 80% including hyperoside, rutin, isoquercitrin, quercetin-3-O-β-d-glucopyranoside, myricetin-3-O-glucoside, myricetin, myricetin, lindenin, lycopene-3-glucoside, and kaempferol-3-O-rutin.

10. The medicament according to claim 7, 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%.

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