Traditional Chinese medicine compound, preparation method, verification method and application

Abstract

The present disclosure provides a traditional Chinese medicine compound, a preparation method, a verification method and an application, and belongs to the technical field of traditional Chinese medicine. The traditional Chinese medicine compound is used for treating respiratory syncytial virus, and comprises the following components: platycodon grandiflorum, lonicera japonica, radix angelicae dahuricae, prunus mume, saposhnikovia divaricata, glycyrrhiza uralensis, mulberry root-bark and radix scrophulariae. The traditional Chinese medicine compound can inhibit the replication of RSV infection and reduce the inflammatory response in the lungs.

Description

Technical Field

[0001] This disclosure belongs to the field of traditional Chinese medicine technology, and specifically relates to a compound of traditional Chinese medicine, its preparation method, verification method and application. Background Technology

[0002] Respiratory syncytial virus (RSV) is one of the most important viral pathogens causing respiratory infections in infants, the elderly, and immunocompromised individuals, posing a significant disease burden worldwide each year. The most common clinical symptom of RSV infection is upper respiratory tract infection, but in young children, RSV often manifests as lower respiratory tract bronchiolitis due to small airway obstruction. A small percentage of cases progress to pneumonia, respiratory failure, apnea, and death.

[0003] Regarding the treatment of RSV, there is currently no definitive cure. Supportive therapy is considered the primary treatment. Ribavirin and palilizumab are two FDA (Food and Drug Administration) approved drugs for treating severe RSV infections. However, the high toxicity risks associated with ribavirin and the high cost of palilizumab limit their use. Summary of the Invention

[0004] This disclosure provides a traditional Chinese medicine compound, its preparation method, verification method, and application, which can inhibit RSV replication in vivo and reduce pulmonary inflammatory response, and can be widely used. The technical solution is as follows:

[0005] This disclosure provides a traditional Chinese medicine compound for treating respiratory syncytial virus (RSV). The compound comprises the following components by weight: 9%-15% Platycodon grandiflorus, 9%-15% Magnolia biondii, 5%-10% Angelica dahurica, 20%-40% Prunus mume, 2%-15% Saposhnikovia divaricata, 5%-15% Glycyrrhiza uralensis, 5%-15% Morus alba root bark, and 9%-15% Scutellaria baicalensis.

[0006] In another implementation of this disclosure, the Platycodon grandiflorus, Magnolia biondii, Angelica dahurica, Prunus mume, Saposhnikovia divaricata, Glycyrrhiza uralensis, Morus alba root bark, and Scutellaria baicalensis are all granules or herbal medicines.

[0007] In another implementation of this disclosure, the equivalent ratios between the granules and herbs of the Platycodon grandiflorus, Magnolia biondii, Angelica dahurica, Prunus mume, Saposhnikovia divaricata, Glycyrrhiza uralensis, Morus alba root bark, and Scutellaria baicalensis are 5:1, 6:1, 12:1, 4:1, 20:1, 6:1, 10:1, and 6.7:1, respectively.

[0008] In another implementation of this disclosure, the granules corresponding to Platycodon grandiflorus, Magnolia biondii, Angelica dahurica, Prunus mume, Saposhnikovia divaricata, Glycyrrhiza uralensis, Morus alba root bark, and Scutellaria baicalensis are 1.8 g, 1.5 g, 0.75 g, 5 g, 0.45 g, 1 g, 0.9 g, and 1.34 g, respectively.

[0009] In another implementation of this disclosure, the traditional Chinese medicine compound contains an active ingredient that can inhibit cholesterol synthesis to inhibit respiratory syncytial virus.

[0010] In another implementation of this disclosure, the active ingredients are β-sitosterol and stigmasterol.

[0011] In another implementation of this disclosure, this embodiment also discloses a method for verifying the inhibition of respiratory syncytial virus (RSV) by a traditional Chinese medicine compound, wherein the traditional Chinese medicine compound is the one described above, and the verification method includes: determining that inhibiting the cholesterol synthesis pathway is a target for anti-RSV replication; determining, based on the target for anti-RSV replication, that the traditional Chinese medicine compound contains an active ingredient capable of affecting the target for anti-RSV replication; and verifying the inhibitory effect of the active ingredient on cholesterol synthesis.

[0012] In another implementation of this disclosure, this embodiment also discloses a method for preparing a traditional Chinese medicine compound compound, wherein the traditional Chinese medicine compound compound is the one described above, and the preparation method includes: combining multiple traditional Chinese medicine components in the following weight ratio, wherein the multiple traditional Chinese medicine components include 9%-15% Platycodon grandiflorus, 9%-15% Magnolia biondii, 5%-10% Angelica dahurica, 20%-40% Prunus mume, 2%-15% Saposhnikovia divaricata, 5%-15% Glycyrrhiza uralensis, 5%-15% Morus alba root bark, and 9%-15% Scutellaria baicalensis.

[0013] In another implementation of this disclosure, the preparation method further includes: preparing the traditional Chinese medicine ingredients into herbal medicine materials or granules; mixing the herbs corresponding to each of the traditional Chinese medicine ingredients according to the following ratio: 9 grams of Platycodon grandiflorus, 9 grams of Magnolia biondii, 9 grams of Angelica dahurica, 20 grams of Prunus mume, 9 grams of Saposhnikovia divaricata, 6 grams of Glycyrrhiza uralensis, 9 grams of Morus alba root bark, and 9 grams of Scutellaria baicalensis.

[0014] Alternatively, the granules corresponding to each of the aforementioned Chinese herbal ingredients can be mixed in the following proportions: 1.8g of Platycodon grandiflorus, 1.5g of Magnolia biondii, 0.75g of Angelica dahurica, 5g of Prunus mume, 0.45g of Saposhnikovia divaricata, 1g of Glycyrrhiza uralensis, 0.9g of Morus alba root bark, and 1.34g of Scutellaria baicalensis.

[0015] In another implementation of this disclosure, this embodiment also discloses the application of a traditional Chinese medicine compound in the treatment of respiratory syncytial virus, wherein the traditional Chinese medicine compound is the one described above.

[0016] The beneficial effects of the technical solutions provided in this disclosure are:

[0017] The traditional Chinese medicine compound provided in this embodiment includes Platycodon grandiflorus, Magnolia biondii, Angelica dahurica, Prunus mume, Saposhnikovia divaricata, Glycyrrhiza uralensis, Morus alba root bark, and Scutellaria baicalensis. These are all traditional Chinese medicine ingredients, and traditional Chinese medicine is not only highly safe but also inexpensive and cost-effective in treating respiratory syncytial virus (RSV). Furthermore, the combination of Platycodon grandiflorus, Angelica dahurica, and Magnolia biondii can astringe lung qi and promote the production of body fluids to quench thirst. Prunus mume can also astringe lung qi and promote the production of body fluids to quench thirst. Morus alba root bark, when combined with Scutellaria baicalensis, can clear lung heat. Platycodon grandiflorus is an adjuvant and can be combined with other traditional Chinese medicines to help relieve exterior syndromes. Saposhnikovia divaricata has the effects of dispelling wind and relieving exterior syndromes, eliminating dampness and relieving pain, and stopping spasms. Therefore, the combination of these traditional Chinese medicines can both relieve exterior syndromes and clear lung heat, both promote lung function and resolve phlegm, and astringe lung qi. In this way, exterior syndromes are dispersed, lung heat is cleared, phlegm and dampness are resolved, lung qi is promoted and descended, and lung yin is astringed, making it an effective formula for treating RSV. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this disclosure, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 These are liquid chromatograms of different Chinese herbal components in a compound Chinese medicine formula;

[0020] Figure 2 This is a gas chromatogram of Platycodon grandiflorus, a compound in traditional Chinese medicine.

[0021] Figure 3 This is a flowchart of a method for preparing a compound from a traditional Chinese medicine formula;

[0022] Figure 4 This is a flowchart of a method for validating the inhibition of respiratory syncytial virus by compound traditional Chinese medicine;

[0023] Figure 5 This is a diagram showing the experimental groupings;

[0024] Figure 6 It is a pathological analysis diagram of tissue sections;

[0025] Figure 7 This is a test diagram of cytokines in lung tissue;

[0026] Figure 8 This is a test graph for determining RSV titer by RT-PCR;

[0027] Figure 9 This is a test diagram of key genes involved in cholesterol synthesis;

[0028] Figure 10 This is a test diagram of cholesterol synthesis intermediates;

[0029] Figure 11 This is a graph showing the inhibition of RSV by lovastatin in in vitro experiments;

[0030] Figure 12 This is a graph showing the test results of various pro-inflammatory cytokines after lovastatin treatment in an in vitro experiment.

[0031] Figure 13 This is a graph showing the inhibition of RSV by the active ingredient in an in vitro experiment;

[0032] Figure 14 This is a graph showing the inhibition of key genes in cholesterol synthesis by the active ingredient in in vitro experiments.

[0033] Figure 15 This is a schematic diagram illustrating the mechanism by which compound traditional Chinese medicine inhibits RSV replication. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this disclosure clearer, the embodiments of this disclosure will be described in further detail below with reference to the accompanying drawings.

[0035] This disclosure provides a traditional Chinese medicine compound for treating respiratory syncytial virus. The traditional Chinese medicine compound comprises the following components by weight ratio: 9%-15% Platycodon grandiflorus, 9%-15% Magnolia biondii, 5%-10% Angelica dahurica, 20%-40% Prunus mume, 2%-15% Saposhnikovia divaricata, 5%-15% Glycyrrhiza uralensis, 5%-15% Morus alba root bark, and 9%-15% Scutellaria baicalensis.

[0036] The traditional Chinese medicine compound provided in this embodiment includes Platycodon grandiflorus, Magnolia biondii, Angelica dahurica, Prunus mume, Saposhnikovia divaricata, Glycyrrhiza uralensis, Morus alba root bark, and Scutellaria baicalensis. These are all traditional Chinese medicine ingredients, and traditional Chinese medicine is not only highly safe but also inexpensive and cost-effective in treating respiratory syncytial virus (RSV). Furthermore, the combination of Platycodon grandiflorus, Angelica dahurica, and Magnolia biondii can astringe lung qi and promote the production of body fluids to quench thirst. Prunus mume can also astringe lung qi and promote the production of body fluids to quench thirst. Morus alba root bark, when combined with Scutellaria baicalensis, can clear lung heat. Platycodon grandiflorus is an adjuvant and can be combined with other traditional Chinese medicines to help relieve exterior syndromes. Saposhnikovia divaricata has the effects of dispelling wind and relieving exterior syndromes, eliminating dampness and relieving pain, and stopping spasms. Therefore, the combination of these traditional Chinese medicines can both relieve exterior syndromes and clear lung heat, both promote lung function and resolve phlegm, and astringe lung qi. In this way, exterior syndromes are dispersed, lung heat is cleared, phlegm and dampness are resolved, lung qi is promoted and descended, and lung yin is astringed, making it an effective formula for treating RSV.

[0037] Optionally, Platycodon grandiflorus, Magnolia biondii, Angelica dahurica, Prunus mume, Saposhnikovia divaricata, Glycyrrhiza uralensis, Morus alba root bark, and Scutellaria baicalensis can all be granules or all be herbal medicines.

[0038] Of course, if all the above-mentioned Chinese medicine ingredients are herbal medicines, they must be decocted into a soup (i.e., a decoction) before consumption. The herbal medicines mentioned here are authentic medicinal materials, and when used, they are processed according to different parts (roots, stems, leaves, etc.) and different methods before being used in medicine.

[0039] The granules mentioned above are granules formed after standardized extraction and concentration of traditional Chinese medicine. Compared with the decoction of herbal medicines, granules have a stable drug content, are convenient to carry, and can be taken orally, making them more suitable for clinical applications. Therefore, the traditional Chinese medicine compound used in the experiments mentioned below in this disclosure is actually a traditional Chinese medicine compound formed by mixing the granules of the above-mentioned components.

[0040] In this embodiment, the above-mentioned Platycodon grandiflorus granules, Magnolia biondii granules, Angelica dahurica granules, Prunus mume granules, Saposhnikovia divaricata granules, Glycyrrhiza uralensis granules, Morus alba root bark granules, and Scutellaria baicalensis granules produced by Sanjiu Company in Shenzhen, China, can be selected as granules of the Chinese herbal ingredients in the above-mentioned compound Chinese medicine formula.

[0041] Optionally, the equivalent ratios between the herbal materials and granules of Platycodon grandiflorus, Magnolia biondii, Angelica dahurica, Prunus mume, Saposhnikovia divaricata, Glycyrrhiza uralensis, Morus alba root bark, and Scutellaria baicalensis are 5:1, 6:1, 12:1, 4:1, 20:1, 6:1, 10:1, and 6.7:1, respectively.

[0042] Since granules are also made from herbs, and the manufacturing process requires the extraction and purification of the herbs, the conversion between herbs and granules can be done using the stoichiometric ratio mentioned above. The stoichiometric ratio refers to the ratio of the stoichiometric coefficients of two chemical substances in a reaction.

[0043] Optionally, the granules corresponding to Platycodon grandiflorus, Magnolia biondii, Angelica dahurica, Prunus mume, Saposhnikovia divaricata, Glycyrrhiza uralensis, Morus alba root bark, and Scutellaria baicalensis are 1.8g, 1.5g, 0.75g, 5g, 0.45g, 1g, 0.9g, and 1.34g, respectively.

[0044] By combining the components according to the above weights, this traditional Chinese medicine compound can exert its maximum effect in inhibiting the replication of respiratory syncytial virus, while reducing the inflammatory response during infection.

[0045] Furthermore, if all the above-mentioned Chinese herbal ingredients are in granule form, the dosage for human consumption is 0.51g / kg per day. That is, for a 50kg adult, the dosage would be 25.48g, which is equivalent to the two compound Chinese herbal formulas mentioned above: 3.6g Platycodon grandiflorus, 3g Magnolia biondii, 1.5g Angelica dahurica, 10g Prunus mume, 0.9g Saposhnikovia divaricata, 2g Glycyrrhiza uralensis, 1.8g Morus alba root bark, and 2.68g Scutellaria baicalensis.

[0046] If the above-mentioned Chinese medicine ingredients are all herbal medicines, when consuming them, an adult weighing 50kg should directly prepare a prescription of 9g Platycodon grandiflorus, 9g Magnolia biondii, 9g Angelica dahurica, 20g Prunus mume, 9g Saposhnikovia divaricata, 6g Glycyrrhiza uralensis, 9g Morus alba root bark, and 9g Scutellaria baicalensis, and decoct them before use.

[0047] For ease of recording, the components of traditional Chinese medicine compound compounds can be found in Table 1 below.

[0048] Table 1. Components of the compound in the traditional Chinese medicine formula

[0049]

[0050]

[0051] In addition, to further confirm that the granules corresponding to each component of the traditional Chinese medicine compound meet the relevant requirements of the Chinese Pharmacopoeia, the embodiments of this disclosure use ultra-high performance liquid chromatography (UPLC) and gas chromatography to perform quality identification analysis on the granules of each component of the above traditional Chinese medicine compound.

[0052] Table 2 Information on identification standards for each component of traditional Chinese medicine compound preparations.

[0053]

[0054] The analysis was performed using an ultra-high performance liquid chromatography (UPLC) system. The chromatographic column in the UPLC system was a WATERS water-resistant C18 (HSS T3 1.8m × 100mm × 2.1mm) column. The column temperature was set at 35℃ during analysis.

[0055] The mobile phase used for elution was an aqueous solution of acetonitrile and 1% phosphoric acid, and a gradient elution was employed. Elution was carried out at a constant flow rate of 0.3 mL / min.

[0056] The chromatogram was monitored at a wavelength of 277 nm during the full scan, with the scan range from 210 nm to 600 nm.

[0057] A comparison of the chromatograms of the granules of each component of the traditional Chinese medicine compound with those of the standard showed that the granules of each component exhibited peaks at their corresponding retention times, indicating that the granules of each component of the traditional Chinese medicine compound meet the requirements of the Chinese Pharmacopoeia and are suitable for clinical treatment and research.

[0058] for example, Figure 1 The upper half of the graph shows the chromatograms of the standards corresponding to each component in the granule form, while the lower half shows the chromatograms of the granule form itself. From... Figure 1 As can be seen, peaks were observed below the corresponding time points for each standard, indicating that the compound compounds of traditional Chinese medicine meet the requirements of the Chinese Pharmacopoeia and are suitable for clinical treatment and research.

[0059] For platycodin D, due to its high volatility, liquid chromatography cannot be used; therefore, gas chromatography is employed in this application. Analysis was performed using an evaporative light scattering detector (ELSD) at a gas flow rate of 3.0 mL / min, while maintaining the temperature at 50 °C.

[0060] Figure 2 This is the chromatogram of platycodon saponin D, the corresponding standard of platycodon. According to... Figure 2 According to the information provided, the standard for platycodon saponin D elutes around 24 minutes. Figure 2 The peak corresponding to the solid line b) is shown here, and the Platycodon grandiflorus granules in the above compounds also have a corresponding peak here. Figure 2 The peak corresponding to the dashed line a indicates that the Platycodon grandiflorus granules in the compound meet the requirements of the Chinese Pharmacopoeia and are suitable for clinical treatment and research.

[0061] In this embodiment, the traditional Chinese medicine compound inhibits respiratory syncytial virus (RSV) replication by inhibiting cholesterol synthesis. In other words, the above-mentioned traditional Chinese medicine compound can inhibit cholesterol synthesis, thereby inhibiting RSV replication.

[0062] Optionally, the compound of traditional Chinese medicine contains active ingredients that inhibit cholesterol synthesis. The active ingredients are β-sitosterol and stigmasterol.

[0063] Experiments have shown that β-sitosterol and stigmasterol have a higher docking ability with the INSIG-SCAP complex than the intracellular ligand cholesterol. Therefore, β-sitosterol and stigmasterol can competitively inhibit the binding of the INSIG-SCAP complex, stably maintaining the SCAP-INSIG complex on the endoplasmic reticulum, thereby inhibiting the expression and cleavage maturation of SREBF2, and thus inhibiting cholesterol synthesis (for details, please refer to (2.4) of the experimental method in the instruction manual).

[0064] Furthermore, in vitro experiments confirmed that β-sitosterol and stigmasterol strongly inhibited the expression and cleavage maturation of SREBF2 in HEp-2 cells. For details, please refer to section (2.4) of the experimental methods in the instruction manual. Figure 14 The corresponding content.

[0065] On the other hand, this disclosure also provides a method for preparing a traditional Chinese medicine compound, such as... Figure 3 As shown, the traditional Chinese medicine compound is the same as the traditional Chinese medicine compound mentioned above, and its preparation method includes:

[0066] S301: A combination of various Chinese herbal ingredients in the following weight ratios, including 9%-15% Platycodon grandiflorus, 9%-15% Magnolia biondii, 5%-10% Angelica dahurica, 20%-40% Prunus mume, 2%-15% Saposhnikovia divaricata, 5%-15% Glycyrrhiza uralensis, 5%-15% Morus alba root bark, and 9%-15% Scutellaria baicalensis.

[0067] The above preparation methods have the same beneficial effects as those mentioned above, and will not be repeated here.

[0068] Optionally, the preparation method also includes: processing the traditional Chinese medicine ingredients into herbal medicines or granules.

[0069] If the Chinese medicine ingredients are herbal materials, then it shall be prepared according to 3011. If the Chinese medicine ingredients are granules, then it shall be prepared according to 3012.

[0070] 3011: Mix the corresponding herbal materials of each Chinese medicine ingredient according to the following ratio: 9g Platycodon grandiflorus, 9g Magnolia biondii, 9g Angelica dahurica, 20g Prunus mume, 9g Saposhnikovia divaricata, 6g Glycyrrhiza uralensis, 9g Morus alba root bark, and 9g Scutellaria baicalensis.

[0071] 3012: Mix the corresponding granules of each Chinese herbal medicine ingredient according to the following ratio: 1.8g Platycodon grandiflorus, 1.5g Magnolia biondii, 0.75g Angelica dahurica, 5g Prunus mume, 0.45g Saposhnikovia divaricata, 1g Glycyrrhiza uralensis, 0.9g Morus alba root bark, and 1.34g Scutellaria baicalensis.

[0072] When preparing the above-mentioned compound traditional Chinese medicine formulas, the granules of each component can be mixed in the manner described above, or the traditional Chinese medicine components can be mixed together. As mentioned earlier, for the convenience of clinical use, the compound traditional Chinese medicine formulas are generally formed according to step 3012. At this time, the compound traditional Chinese medicine formula formed is also known as compound traditional Chinese medicine granules.

[0073] Furthermore, this disclosure also provides an application of a traditional Chinese medicine compound in the treatment of respiratory syncytial virus. The traditional Chinese medicine compound is the one mentioned above, which includes Platycodon grandiflorus, Magnolia biondii, Angelica dahurica, Prunus mume, Saposhnikovia divaricata, Glycyrrhiza uralensis, Morus alba root bark, and Scutellaria baicalensis.

[0074] The above applications have the same beneficial effects as those mentioned earlier, and will not be repeated here.

[0075] Furthermore, this disclosure also provides a method for verifying the inhibitory effect of traditional Chinese medicine compound on respiratory syncytial virus, such as... Figure 4 As shown, the verification method includes:

[0076] S401: Inhibition of the cholesterol synthesis pathway has been identified as a target for inhibiting respiratory syncytial virus replication.

[0077] The cholesterol mentioned above refers to total cholesterol.

[0078] In this embodiment, experiments confirmed that cholesterol synthesis affects RSV infection and replication. Therefore, the cholesterol synthesis pathway became the focus of the study.

[0079] By comparing changes in gene expression in the lung tissue of experimental subjects in the infection model control group and the drug treatment group, the target sites in the cholesterol synthesis pathway can be identified. Specifically, the expression and cleavage maturation of SREBF2 are inhibited by traditional Chinese medicine compound compounds, thereby inhibiting cholesterol synthesis. The traditional Chinese medicine compound compounds lock the key activation step of SREBF2, namely INSIG-SCAP, which is bound by the active ingredient, preventing SREBF2 from entering the cell nucleus and activating the cholesterol synthesis pathway.

[0080] Therefore, inhibiting the expression and cleavage maturation of SREBF2 protein is a key step in inhibiting cholesterol synthesis.

[0081] S402: Based on the target of anti-respiratory syncytial virus replication, the active ingredients in traditional Chinese medicine compound compounds that can affect the target of anti-respiratory syncytial virus replication were identified.

[0082] Through network pharmacology studies, active ingredients that bind to INSIG-SCAP were screened.

[0083] As described above, the active ingredients are β-sitosterol and stigmasterol.

[0084] S403: Verify the inhibitory effect of the active ingredient on cholesterol synthesis.

[0085] In this embodiment, the inhibitory effect of the active ingredient on cholesterol synthesis was verified at the cellular level. In vitro experiments confirmed that the active ingredient can reduce cholesterol synthesis and inhibit RSV infection replication.

[0086] The experimental procedure for the above verification method can be found in the experimental section (2.4) of the instruction manual.

[0087] To further illustrate the effectiveness of the above-mentioned traditional Chinese medicine compound in treating respiratory syncytial virus (RSV), the following experiments are conducted:

[0088] 1. Experimental Groups:

[0089] The experimental subjects were 6-week-old BALB / c (albino lab mice) of SPF (Specific Pathogen Free) grade.

[0090] Group A: Normal control group;

[0091] Group B: Infection model control group for 1 day (samples were taken 1 day after viral challenge, i.e., 1 day after nasal instillation); Group D: Infection model control group for 4 days (samples were taken 4 days after viral challenge); Group F: Infection model control group for 6 days (samples were taken 6 days after viral challenge).

[0092] Group C: Samples were taken 1 day after viral challenge in the drug treatment group; Group E: Samples were taken 4 days after viral challenge in the drug treatment group; Group G: Samples were taken 6 days after viral challenge in the drug treatment group.

[0093] The above groupings can be found in [reference]. Figure 5 As shown.

[0094] After feeding BALB / c for one week, animals from groups B to G were placed in a P3 biosafety laboratory. After anesthetizing the animals with ether inhalation, 50 μL of purified RSV virus (1 x 10⁻⁶) was collected. 6 Group A received nasal drops of PFU (phosphoric acid / animal), while Group B received an equal volume of PBS (phosphate-buffered saline). Groups C, E, and G received traditional Chinese medicine via gavage (note: traditional Chinese medicine gavage administration 6 hours after challenge). Groups A, B, D, and F received an equal volume of aqueous solution simultaneously. The drug dosage was 4.64 g / kg. Animals were sacrificed on day 1 (24 hours after challenge), day 4 (96 hours after challenge), and day 6 (144 hours after challenge), and lung tissue and lung homogenate were collected for analysis.

[0095] When administering the drug to mice, the drug concentration was 0.51 * 9 = 4.64 g / kg (the conversion ratio between human and mouse drug dosage is 1:9), and the mouse weight was 16-20 g. Using 20 g (equivalent to 0.02 kg) as a baseline, the dosage for mice was: 4.64 g / kg * 0.02 kg = 0.0928 g / mouse / day.

[0096] 2. Experimental methods:

[0097] (2.1) Histological analysis of the therapeutic effect of traditional Chinese medicine compound on RSV infection:

[0098] Whole lung tissue samples were collected from mice in each of the above groups at specific time points, then preserved in 4% formaldehyde, and subsequently embedded in paraffin for sectioning. These tissue sections were fixed on glass slides and stained with hematoxylin and eosin (H&E), see [link to relevant documentation]. Figure 6 ( Figure 6 Group A consists of two parts, which are essentially the same; the difference is only for layout purposes.

[0099] According to the relevant content of histopathological analysis, the more white areas in the diagram, the milder the infection symptoms. Figure 6 As can be seen, mice in the infection model control group (group B) showed mild to moderate inflammation in the alveoli, bronchi, and blood vessels one day after RSV infection, characterized by significant thickening of the alveolar and blood vessel walls (no white area in the figure, indicating that alveoli were not formed and the alveolar walls were thickened), and a certain number of lymphocyte infiltrations. However, at 4 and 6 days post-infection, lung tissue damage in mice in groups D and F was reduced, and the inflammation was milder than on day 1. The pathological damage in the drug treatment groups (groups C, E, and G) was less severe compared to the infection model control groups (groups B, D, and F) at the same time point.

[0100] according to Figure 6 Pathological images showed that on the first day of RSV infection, the alveolar and bronchial walls in the control group of the infection model were significantly thickened, gradually recovering over the following week. In contrast, the alveolar and bronchial walls of mice in the drug treatment group remained normal throughout the validation period.

[0101] (2.2) Quantitative analysis of the therapeutic effects of traditional Chinese medicine compound compounds on RSV infection:

[0102] To further evaluate the therapeutic effect of the above-mentioned traditional Chinese medicine compound on RSV infection, the levels of cytokines and RSV viral titers in lung tissue homogenate were measured in this embodiment.

[0103] (2.2.1) Detection of cytokine levels.

[0104] This disclosure describes the determination of cytokine expression levels in lung tissue using an enzyme-linked immunosorbent assay (ELISA). The cytokine content in lung tissue was detected according to a pre-established ELISA procedure. Lung samples were collected at specific time intervals. These samples were then homogenized and centrifuged. Cytokines, including IL-6, IL-8, and IL-17A, in the homogenized lung tissue were quantitatively assessed using commercial ELISA kits provided by BioLegend and Biotechnology.

[0105] See Figure 7 Interleukin-6 (IL-6) in mice (see Figure 7 (Top left of the middle), interleukin-8 (IL-8) (see...) Figure 8 (lower left in the middle) and interleukin-17A (IL-17A) (see...) Figure 7 Pro-inflammatory factors, including those in the lower right corner of the graph, peaked on the first day of infection (the bar corresponding to group B in each figure is the tallest), and gradually decreased in the following days (the bar corresponding to groups D and F in each figure is shorter than that of group B). The trend of pathological changes was consistent with that of inflammatory factors in serum. Meanwhile, drug treatment significantly reduced the levels of these indicators (in each figure, the relative expression of pro-inflammatory factors in group C was lower than that in group B, the relative expression of pro-inflammatory factors in group E was lower than that in group D, and the relative expression of pro-inflammatory factors in group G was lower than that in group F). It is evident that, compared with the control group of the infection model, the drug treatment group significantly reduced the expression of inflammatory cytokines in lung tissue and reduced the inflammatory response in the mouse lungs after administration. This indicates that the above-mentioned traditional Chinese medicine compound can significantly improve lung inflammation caused by RSV infection.

[0106] (2.2.2) RSV virus titer determination.

[0107] To detect the viral titer in mice after RSV infection, lung tissue was collected from mice in the above-mentioned groups, and RNA was extracted after grinding. The copy number of the RSV N gene in the mouse lung tissue was detected by RT-PCR (reverse transcription PCR) and quantitative real-time PCR. PCR is a polymerase chain reaction.

[0108] RT-PCR results showed that mice in the infection model control group showed RSV infection on days 1, 4, and 6 (corresponding to...) Figure 8 The RSV N gene was detected in mice in groups B, D, and F, while in the drug-treated group, the presence of the RSV N gene was detected 1 day after infection (corresponding to...). Figure 8In group C, a weak RSV N gene was detected, with a copy number slightly higher than that in normal group A (above the dotted line; below the dotted line corresponds to false amplification in the normal control group of group A). ​​However, the RSV N gene was not detected at days 4 and 6 (corresponding to...). Figure 8 In groups E and G, the RSV N gene copy numbers in groups E and G were roughly equivalent to those in group A (near the dotted line). These results indicate that the compound of this traditional Chinese medicine can significantly reduce RSV virus replication in the lung tissue of infected mice, thereby rapidly clearing the RSV virus from the mouse lung tissue.

[0109] (2.3) Verify the inhibitory effect of the drug on cholesterol synthesis:

[0110] Existing literature reports that RSV infection induces increased cholesterol synthesis in organisms. Furthermore, cholesterol-rich lipid raft structures play a crucial role in RSV invasion and subsequent replication. Therefore, the antiviral mechanism of the above-mentioned traditional Chinese medicine compound may rely on inhibiting cholesterol synthesis.

[0111] (2.3.1) Analysis of the effects of drugs on key genes involved in cholesterol synthesis in mouse lung tissue.

[0112] To confirm that the compound of traditional Chinese medicine inhibits RSV replication by inhibiting cholesterol synthesis, the embodiments of this disclosure first use qPCR (quantitative polymerase chain reaction) to verify the changes in the transcriptional level of key genes involved in cholesterol synthesis in lung tissue.

[0113] Key genes involved in cholesterol synthesis include Hmgcr, Hmgcs1, Sqle, Dhcr24, Srebf2, and Mvd. In this embodiment, the effect of the drug on cholesterol synthesis was further confirmed by detecting the copy number of the mRNA corresponding to the key genes (Hmgcr, Hmgcs1, Sqle, Dhcr24, Srebf2, and Mvd).

[0114] During the experiment, from Figure 5Total RNA was extracted from the lung tissue of mice in groups A, B, and C, and the extracted RNA was diluted and dissolved using RNA Pure reagent (Aidlab, China). The RNA was converted to complementary DNA (cDNA) using a reverse transcription kit provided by the manufacturer (Toyoba, Japan). The mRNA copy number was detected using a 2×SYBR green master mix (Novoprotein, China) and a 7500 Real-Time PCR System analyzer (Applied Biosystems, USA). Figure 9 The corresponding results.

[0115] Figure 9 In the figures, the horizontal axis represents the group (A, B, C), and the vertical axis represents the relative expression level of the mRNA of each key gene. Based on... Figure 9 As can be seen from the graphs, compared to group A, the relative expression level of mRNA in group B is increased, indicating that the key genes in group B are replicating extensively one day after viral infection, thus inducing cholesterol synthesis. Conversely, compared to group B, the relative expression level of mRNA in group C is decreased, indicating that after medication, the replication of the key genes involved in cholesterol synthesis is inhibited, thus suppressing cholesterol synthesis. Therefore, the above-mentioned traditional Chinese medicine compound compounds, after treatment with RSV, reversed (i.e., inhibited) the RSV-induced upregulation of cholesterol synthesis (the copy number of the mRNA of all six key genes decreased after medication).

[0116] (2.3.2) Analysis of the effects of drugs on intermediates involved in cholesterol synthesis in mouse lung tissue.

[0117] Furthermore, the measurement of cholesterol synthesis intermediates (including HMG-CoA and Mevalonate) by ELISA further confirms that RSV-induced cholesterol synthesis is inhibited by the above-mentioned compound Chinese medicine.

[0118] For example, see Figure 10 , Figure 10 The horizontal axis represents the cholesterol synthesis intermediates HMG-CoA and Mevalonate corresponding to different groups, while the vertical axis represents the content of the intermediates.

[0119] according to Figure 10 As can be seen, in both groups, the number of intermediates in group B (1 day after viral infection) was significantly increased compared to group A. However, in group C (1 day after viral infection and treated with the drug), the number of intermediates was significantly reduced compared to group B. This further demonstrates that the drug can inhibit cholesterol synthesis.

[0120] (2.3.3) Hep-2 cell in vitro experiments were used to verify that inhibiting cholesterol synthesis can inhibit RSV replication.

[0121] As previously stated, the above-mentioned traditional Chinese medicine compound can inhibit cholesterol synthesis. To further verify the conclusion that the reason why the traditional Chinese medicine compound can inhibit the virus is because it can inhibit cholesterol synthesis, in this embodiment, the cholesterol synthesis inhibitor lovastatin was used to treat RSV-infected Hep-2 cells (human laryngeal epidermoid carcinoma cells, an important cell type of the upper respiratory tract infected with RSV, commercially available) in vitro to evaluate RSV replication and pro-inflammatory cytokine secretion in RSV-infected Hep-2 cells.

[0122] In other words, the above experiments can further confirm whether lovastatin can also inhibit RSV.

[0123] See Figure 11 In RSV-infected Hep-2 cells, after treatment with lovastatin (corresponding to...) Figure 11 As shown in the curve below, intracellular cholesterol synthesis is inhibited (because lovastatin can inhibit cholesterol synthesis), and the RSV viral titer in Hep-2 cells is significantly reduced. This indicates that lovastatin can significantly inhibit RSV replication, further demonstrating that lovastatin treatment, while inhibiting cholesterol synthesis, can also correspondingly inhibit RSV replication.

[0124] See Figure 12 The horizontal axis represents the number of days of infection, and the vertical axis represents the number of mRNA molecules. From... Figure 12 As can be seen, compared to 1 day after RSV infection, Hep-2 cells in group a (RSV-infected cell model group, i.e., Hep-2 cells inoculated with RSV after culture) showed a significant increase in all pro-inflammatory cytokines 4 days after infection. Group b (Hep-2 cells inoculated with RSV and treated with lovastatin, i.e., the lovastatin-treated RSV-infected cell model) showed significantly lower levels of inflammatory factors in group b compared to group a at the same time point. Lovastatin treatment significantly reduced the levels of various pro-inflammatory cytokines within Hep-2 cells.

[0125] Based on the above experimental conclusions, it can be concluded that after Hep-2 cells are infected with RSV and treated with lovastatin, the viral titer in Hep-2 cells is significantly reduced, indicating that lovastatin can inhibit RSV replication while inhibiting cholesterol. Combining this with the content in sections 2.3.1 and 2.3.2 above, we know that the traditional Chinese medicine compound can inhibit RSV replication by inhibiting cholesterol synthesis. Furthermore, since the above experiments were conducted in vitro, RSV is not eliminated by clearance systems such as macrophages, neutrophils, or lymphocytes. Therefore, the results of the above in vitro experiments show that inhibiting cholesterol synthesis can inhibit RSV. This also indicates that the traditional Chinese medicine compound can inhibit RSV replication by inhibiting cholesterol synthesis, thereby reducing the expression of inflammatory factors associated with RSV infection, and is unrelated to the immune system. In other words, the above traditional Chinese medicine compound is confirmed to inhibit RSV by inhibiting cholesterol synthesis.

[0126] (2.4) Analysis of the mechanism by which compound traditional Chinese medicines inhibit cholesterol synthesis:

[0127] To further clarify the mechanism by which compound traditional Chinese medicines inhibit cholesterol synthesis, the following terms related to cholesterol synthesis will be explained:

[0128] SREBF2 (Sterol Regulatory Element-Binding Factor 2), SCAP (SREBPCleavage-Activating Protein), and INSIG (Insulin-Induced Gene) play important roles in cholesterol homeostasis and metabolic regulation. SREBF2 is a key transcription factor in cholesterol metabolism. When cells sense a decrease in cholesterol levels, the precursor SREBF2 protein is cleaved, matures, enters the nucleus, and promotes increased expression of cholesterol synthesis genes. This initiates the cholesterol synthesis pathway, thereby promoting intracellular cholesterol synthesis.

[0129] SCAP is a chaperone protein of SREBF2, forming a complex with SREBF2. SCAP senses cholesterol levels within the cell and regulates SREBF2 activity according to the cell's cholesterol requirements. When cholesterol levels decrease, the SCAP-SREBF2 complex dissociates from INSIG, leaves the endoplasmic reticulum, and translocates to the Golgi membrane. Subsequently, SREBF2 is cleaved by proteases, matures, and eventually enters the nucleus to increase cholesterol synthesis and related gene transcription. Conversely, when cholesterol levels increase, the SCAP-SREBF2 complex binds to INSIG, allowing it to remain stably on the endoplasmic reticulum and preventing excessive cholesterol synthesis.

[0130] In summary, SREBF2, SCAP, and INSIG work together to regulate cholesterol synthesis, ensuring that intracellular cholesterol levels remain within an appropriate range to meet the physiological needs of the cell.

[0131] In summary, SCAP and INSIG regulate SREBF2 activity by controlling its protease cleavage and nuclear translocation, thereby adjusting cholesterol metabolism according to cellular cholesterol requirements. This complex regulatory mechanism helps maintain intracellular cholesterol needs to adjust cholesterol metabolism.

[0132] In the experiment, it was found that β-sitosterol and stigmasterol in the traditional Chinese medicine compound more readily bind to SCAP and INSIG, causing the SCAP-SREBF2 complex to remain on the endoplasmic reticulum. The stable SCAP-SREBF2 complex cannot enter the Golgi apparatus, preventing SREBF2 from being cleaved by proteases. This results in a decrease in the amount of SREBF2 in the cell nucleus, reduced cholesterol synthesis, and inhibition of RSV replication.

[0133] (2.4.1) It was determined that inhibiting the cholesterol synthesis pathway is a target for inhibiting respiratory syncytial virus replication.

[0134] In this embodiment, by comparing the gene expression of the cholesterol synthesis pathway in the lung tissue of mice in the infection model control group and the drug treatment group, no expression of key enzymes and two regulatory transcription factors in the cholesterol synthesis process was found in the lung tissue of mice in the drug treatment group. This indicates that the compound of traditional Chinese medicine inhibited the expression of all 11 key enzymes and 2 regulatory transcription factors in the cholesterol synthesis process. Therefore, inhibiting the cholesterol synthesis pathway is a target for resisting respiratory syncytial virus replication.

[0135] Subsequently, predictive analysis of common transcription factors of differentially expressed genes was performed based on the ChEA3 and HTFtargets databases. Among the candidate transcription factors, SREBF2 showed a high correlation with cholesterol synthesis transcription levels. In other words, SREBF2 exhibited the strongest correlation with the expression levels of these differentially expressed genes. This indicates that the key target of the cholesterol synthesis pathway is the activity of SREBF2. Therefore, regulating SREBF2 activity is a target for combating respiratory syncytial virus replication.

[0136] Furthermore, according to the cholesterol synthesis mechanism described on page 12 of the instruction manual, the activity of SREBF2 is related to the cholesterol content in the endoplasmic reticulum. At low cholesterol concentrations, the SCAP-SREBF2 complex is transported to the Golgi apparatus for cleavage and maturation.

[0137] (2.4.2) Identify active ingredients in traditional Chinese medicine compound compounds that can affect RSV replication targets.

[0138] According to common knowledge in the field, the functional activity of SREBF2 is affected by phytosterols. Therefore, the above-mentioned drugs should contain phytosterols.

[0139] To further confirm whether the above-mentioned drugs contain phytosterols, based on network pharmacology results, gas chromatography-mass spectrometry (GC-MS) was used to identify the compounds in the traditional Chinese medicine compound. It was found that the compound contained two phytosterols, β-sitosterol and stigmasterol.

[0140] The gas chromatograms showed that stigmasterol and sitosterol had elution times of 20.0 min and 21.6 min, respectively. Subsequent mass spectrometry also revealed characteristic fragment ions of stigmasterol at M / Z 484, 469, 394, 379, and 355, and characteristic fragment ions of β-sitosterol at M / Z 486, 471, 396, 381, and 357. Based on the corresponding peak times of the characteristic fragment ions in the standard substance chromatograms, the presence of stigmasterol and sitosterol in the traditional Chinese medicine compound was confirmed. The data from the standard separated compounds were validated against data in the NIST 2020 library.

[0141] In addition, molecular docking experiments showed that β-sitosterol and stigmasterol have a strong docking ability with the INSIG-SCAP conjugate, even stronger than cholesterol.

[0142] (2.4.3) Verify the inhibitory effect of the active ingredient on cholesterol synthesis.

[0143] In this embodiment, in vitro experiments verified that β-sitosterol and stigmasterol exhibited dose-dependent inhibitory effects on the expression and cleavage maturation of SREBF2 in HEp-2 cells.

[0144] In the experiment, HEp-2 cells were used to test for β-sitosterol and stigmasterol. 50 μM of 2,5-hydroxycholesterol (i.e., total cholesterol in serum), 50 μM of β-sitosterol, and 50 μM of stigmasterol were sequentially injected into different groups of HEp-2 cells containing RSV. The RSV titer (viral titer, i.e., viral virulence) in each HEp-2 cell was then measured. See [link to relevant documentation]. Figure 13 .

[0145] Figure 13The horizontal axis represents, in descending order: HEp-2 cells (normal control group), HEp-2 cells infected with RSV (RVS infection model group), HEp-2 cells cultured for 24 hours in 50 μM 25-hydroxycholesterol medium before RSV infection (25-hydroxycholesterol + RSV group), HEp-2 cells cultured for 24 hours in 50 μM β-sitosterol medium before RSV infection (β-sitosterol + RSV group), and HEp-2 cells cultured for 24 hours in 50 μM stigmasterol medium before RSV infection (stigmasterol + RSV group). The vertical axis represents the RSV viral copy number.

[0146] according to Figure 13 As can be seen, intervention with β-sitosterol and stigmasterol significantly reduced RSV titers in the cell model, comparable to the effect of the 25-hydroxycholesterol intervention group. This indicates that β-sitosterol and stigmasterol interventions have the same interventional effect as 25-hydroxycholesterol, both inhibiting cholesterol synthesis and thus suppressing RSV viral titers. In the 25-hydroxycholesterol intervention group, the decrease in RSV was due to an increase in intracellular total cholesterol, which inhibited cholesterol synthesis and consequently suppressed RSV.

[0147] Furthermore, in vitro experiments were conducted to confirm the inhibitory effect of the active ingredient on key genes involved in cholesterol synthesis. In the experiment, HEp-2 cells were sequentially treated with 50 μM 25-hydroxycholesterol, 50 μM β-sitosterol, and 50 μM stigmasterol, respectively, before being infected with RSV. The mRNA expression of key genes in the cholesterol synthesis pathway (HMGCR, SREBF2, SQLE, HMGCS1) in each group of cells was detected. The results are as follows: Figure 14 .

[0148] See Figure 14 The x-axis represents the expression of key genes in the cholesterol synthesis pathway in each group. The y-axis represents the mRNA copy number. Groups a, b, c, d, and e represent the blank control group, RSV infection group, 25-hydroxycholesterol + RSV group, β-sitosterol + RSV group, and stigmasterol + RSV group, respectively. Compared with group a, group b showed increased mRNA expression of the four key genes. However, compared with group b, the groups treated with 25-hydroxycholesterol, β-sitosterol, and stigmasterol showed decreased mRNA expression of all four key genes. This indicates that intervention with β-sitosterol and stigmasterol can inhibit cholesterol synthesis, thereby inhibiting RSV viral replication.

[0149] In other words, the mechanism by which the above-mentioned traditional Chinese medicine compound compounds inhibit RSV viral replication can be summarized as follows: Figure 15The mechanism is as follows: Phytosterols are present in the compounds of traditional Chinese medicine. Because phytosterols bind more readily to SCAP and INSIG, the SCAP-SREBF2 complex remains on the endoplasmic reticulum. The stable SCAP-SREBF2 complex cannot enter the Golgi apparatus, and SREBF2 cannot be cleaved by proteases into the nucleus, thereby inhibiting the cholesterol synthesis pathway and thus suppressing RSV viral replication.

[0150] In summary, the above experiments identified that the compound in this traditional Chinese medicine formula contains effective active compounds that inhibit key transcription factors in cholesterol synthesis, with SREBF2 playing a central role in the inhibition of the cholesterol synthesis pathway. These findings reveal the mechanism by which these drugs affect cholesterol synthesis and contribute to a deeper understanding of their therapeutic potential.

[0151] The above description is merely an optional embodiment of this disclosure and is not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.

Claims

1. A compound of traditional Chinese medicine, characterized in that, The traditional Chinese medicine compound is used to treat respiratory syncytial virus. The traditional Chinese medicine compound is composed of the following herbal ingredients: 9g Platycodon grandiflorus, 9g Magnolia biondii, 9g Angelica dahurica, 20g Prunus mume, 9g Saposhnikovia divaricata, 6g Glycyrrhiza uralensis, 9g Morus alba root bark, and 9g Scutellaria baicalensis. The compound of traditional Chinese medicine contains active ingredients that can inhibit the synthesis of cholesterol to inhibit the replication of respiratory syncytial virus. The active ingredients are β-sitosterol and stigmasterol.

2. A method for preparing a compound of traditional Chinese medicine, characterized in that, The traditional Chinese medicine compound is the traditional Chinese medicine compound according to claim 1, and the preparation method includes: Mix the following herbs: 9g Platycodon grandiflorus, 9g Magnolia biondii, 9g Angelica dahurica, 20g Prunus mume, 9g Saposhnikovia divaricata, 6g Glycyrrhiza uralensis, 9g Morus alba root bark, and 9g Scutellaria baicalensis.

3. The application of a traditional Chinese medicine compound in the preparation of a drug for treating respiratory syncytial virus, characterized in that, The traditional Chinese medicine compound is the traditional Chinese medicine compound according to claim 1.

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