Drug composition for treating pediatric pneumonia and its application

Abstract

The present invention belongs to the field of pharmaceutical technology, and particularly relates to a pharmaceutical composition for treating pneumonia and its application, especially a pharmaceutical composition for pediatric pneumonia. The pharmaceutical composition contains active ingredients arctiin and trichosanthesdiol, and the weight ratio of arctiin to trichosanthesdiol is 2.5-7.5:1; the pharmaceutical composition may also contain the active ingredient amygdalin. Pharmacological experiments show that the pharmaceutical composition of the present invention can significantly reduce the expression levels of IL-6 and IL-1β inflammatory factors, greatly improve the treatment effect of pediatric pneumonia, and the effect is significantly better than that of arctiin, trichosanthesdiol, and amygdalin used alone at the same dose. This indicates that after combining arctiin and trichosanthesdiol, or after combining arctiin, trichosanthesdiol, and amygdalin, there is a significant synergistic effect.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a pharmaceutical composition for treating pneumonia and its application, especially a pharmaceutical composition for treating pediatric pneumonia. Background Art

[0002] Pediatric pneumonia is one of the most common respiratory diseases in children, with common clinical manifestations including fever, cough, shortness of breath, dyspnea, and fixed moist rales in the lungs. It is one of the life-threatening diseases among common pediatric diseases. Pneumonia refers to lung inflammation caused by different pathogens or other factors. Among them, the TLR4 / NF-kB signaling pathway is closely related to the anti-inflammatory immune mechanism and plays a crucial role in the occurrence and development of inflammation. TLR4 is a bridge for the body to mediate the transition from innate immunity to acquired immunity, widely distributed in cells such as macrophages, neutrophils, endothelial cells, and smooth muscle cells. They participate in forming the first line of defense against invading pathogens and play a significant role in inflammation, immune cell regulation, survival, and proliferation. Nuclear transcription factor-kB (NF-kB) is located at the pivotal point downstream of the TLR4 signaling pathway. Usually, it binds to inhibitory proteins (such as IkBα) in the cytoplasm to form an inactive complex. When these inhibitory proteins are phosphorylated, NF-kB is activated and translocated from the cytoplasm to the nucleus to regulate the transcription of inflammatory genes, causing the expression of inflammatory cytokines such as IL-6 and IL-1β, and is considered an important initiating factor in the inflammatory cascade reaction.

[0003] Commonly used drugs for treating pediatric pneumonia clinically include western medicine antiviral drugs and traditional Chinese medicine preparations. While western medicine antiviral drugs play an antiviral role, they also have many adverse reactions. For example, they can cause side effects such as headache, fatigue, reduced levels of white blood cells and platelets, and elevated transaminases in patients. And clinically, it can be seen that some children with pneumonia still have symptoms such as cough and expectoration during the recovery period of pneumonia after symptomatic treatment, and are extremely prone to recurrence of pneumonia or suffering from other respiratory infectious diseases in the short term. The clearance of pathogenic microorganisms and epithelial repair usually requires a long process. The airway and alveolar tissues are easily transformed from acute inflammatory injury to chronic lung injury, increasing the risk of diseases such as asthma, pulmonary fibrosis, and bronchiolitis obliterans, causing children to have repeated respiratory tract infections, frequent hospitalizations, and reduced exercise endurance, seriously affecting their life and health and causing a huge economic and social burden. Therefore, developing new and effective drugs for treating pediatric pneumonia is still a technical problem that needs to be urgently solved by those skilled in the art.

[0004] Arctiin (Arc) is a lignan compound extracted and isolated from the dried ripe fruits of Arctium lappa L., a biennial herbaceous plant of the Compositae family. The molecular formula is C 27 H 34 O 11, with a molecular weight of 534.5523, its content reaches about 8.4% in Arctium lappa L., and there is also a small amount in Arctium lappa L. leaves. Modern pharmacological research shows that arctiin has physiological activities such as anti-inflammatory, antioxidant, antiviral, anti-tumor, and immune enhancement, which has attracted wide attention and has good application prospects in the future market.

[0005] Karumonidiol (Kar) is a triterpenoid compound extracted and isolated from the seeds of Trichosanthes kirilowii Maxim., a perennial climbing herbaceous vine of the Cucurbitaceae family. The molecular formula is C 30 H 48 O 2 , with a molecular weight of 440.70. Trichosanthes kirilowii Maxim. has high medicinal value. Its fruits, pericarp, and seeds are traditional Chinese medicines "Trichosanthes Root, Trichosanthes Fruit, Trichosanthes Pericarp, and Trichosanthes Seeds". Its root has the effects of clearing heat and promoting fluid production, detoxifying and reducing swelling. Moreover, trichosanthin in the root has an abortifacient effect and is a good contraceptive. The fruits, seeds, and pericarp have the effects of clearing heat and resolving phlegm, moistening the lungs and relieving cough, and lubricating the intestines.

[0006] Amygdalin (Amy) is an active ingredient of the traditional Chinese medicine bitter almond, widely present in the kernels and leaves of various Rosaceae plants such as bitter apricot, bitter almond, peach, nectarine, loquat, plum, apple, and black cherry, especially in bitter almond with a relatively high content of about 2%-3%. Amygdalin is a cyanide-containing glycoside compound with the molecular formula C 20 H 27 NO 11 , with a molecular weight of 457.428, having pharmacological effects such as relieving cough and asthma, lubricating the intestines and relieving constipation, anti-inflammatory, analgesic, anti-tumor, hypoglycemic, and hypolipidemic, and having relatively high medicinal value.

[0007] Through in-depth research, the present inventor unexpectedly found that by combining arctiin and karumonidiol, or by combining arctiin, karumonidiol and amygdalin, the curative effect of treating infantile pneumonia can be significantly enhanced, with small toxic and side effects and convenient administration. Summary of the Invention

[0008] The purpose of the present invention is to provide a pharmaceutical composition with a synergistic effect, good curative effect, small toxic and side effects, and convenient administration for treating infantile pneumonia and its application.

[0009] Specifically, the present invention is achieved through the following technical solutions:

[0010] In the first aspect, the present invention provides a pharmaceutical composition for treating pneumonia, the pharmaceutical composition contains the active ingredients arctiin and karumonidiol, and the weight ratio of arctiin to karumonidiol is 2.5-7.5:1.

[0011] In a preferred embodiment, the weight ratio of arctiin to trichosanthesdiol is 3 - 7:1; preferably 4 - 6:1; particularly preferably 5:1. By controlling the weight ratio of arctiin to trichosanthesdiol within the preferred range of the present invention, it helps to improve the therapeutic effect of the pharmaceutical composition on pediatric pneumonia.

[0012] In a preferred embodiment, the pharmaceutical composition consists of the active ingredients arctiin and trichosanthesdiol, and the weight ratio of arctiin to trichosanthesdiol is 2.5 - 7.5:1.

[0013] In a preferred embodiment, in the pharmaceutical composition, by using arctiin and trichosanthesdiol in combination, it can significantly enhance its therapeutic effect on pediatric pneumonia.

[0014] In a preferred embodiment, the pharmaceutical composition may further contain the active ingredient amygdalin.

[0015] In a preferred embodiment, the weight ratio of arctiin, trichosanthesdiol and amygdalin in the pharmaceutical composition is 2.5 - 7.5:1:25 - 75.

[0016] In a preferred embodiment, the weight ratio of arctiin, trichosanthesdiol and amygdalin is 3 - 7:1:30 - 70; preferably 4 - 6:1:40 - 60; particularly preferably 5:1:50. By controlling the weight ratio of arctiin, trichosanthesdiol and amygdalin within the preferred range of the present invention, it helps to improve the therapeutic effect of the pharmaceutical composition on pediatric pneumonia.

[0017] In a preferred embodiment, the pharmaceutical composition consists of the active ingredients arctiin, trichosanthesdiol and amygdalin, and the weight ratio of arctiin, trichosanthesdiol and amygdalin is 2.5 - 7.5:1:25 - 75.

[0018] In a preferred embodiment, in the pharmaceutical composition, by using arctiin, trichosanthesdiol and amygdalin in combination, it can significantly enhance its therapeutic effect on pediatric pneumonia.

[0019] The structural formula of arctiin of the present invention is as follows:

[0020]

[0021] The structural formula of trichosanthesdiol of the present invention is as follows:

[0022]

[0023] The structural formula of amygdalin of the present invention is as follows:

[0024]

[0025] In a second aspect, the present invention provides a pharmaceutical preparation, which comprises the pharmaceutical composition described in the first aspect above and a pharmaceutically acceptable carrier.

[0026] In a preferred embodiment, the dosage form of the pharmaceutical preparation is an oral dosage form.

[0027] In a preferred embodiment, the oral dosage form is tablets, capsules, granules, powders or syrups.

[0028] Preferably, the oral dosage form is syrup. By preparing the pharmaceutical preparation into syrup, it is more beneficial for pediatric patients to accept and can improve the compliance of patients.

[0029] In a third aspect, the present invention provides the use of the pharmaceutical composition described in the first aspect above or the pharmaceutical preparation described in the second aspect above in the preparation of a medicament for treating pneumonia.

[0030] In a fourth aspect, the present invention provides the use of the pharmaceutical composition described in the first aspect above or the pharmaceutical preparation described in the second aspect above in the preparation of a medicament for treating pediatric pneumonia.

[0031] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here.

[0032] The present invention has the following beneficial effects compared with the prior art:

[0033] Combining the advantages of China in the research of natural compounds, the present invention firstly discovers that the combined use of arctiin and trichosanthesdiol in a specific dosage ratio, or the combined use of arctiin, trichosanthesdiol and amygdalin, can significantly reduce the expression levels of IL-6 and IL-1β inflammatory factors and greatly improve the treatment effect of pneumonia. The effect is significantly better than the single use of arctiin, trichosanthesdiol and amygdalin at the same dose. This indicates that after the combined use of arctiin and trichosanthesdiol, or after the combined use of arctiin, trichosanthesdiol and amygdalin, there is a significant synergistic effect. The pharmaceutical composition of the present invention also has fewer toxic and side effects and is convenient to take, and has the prospect of being developed into a medicament for treating pneumonia, especially pediatric pneumonia, and has important social and economic benefits. Description of the Drawings

[0034] The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention, but do not constitute a limitation to the present invention. In the accompanying drawings:

[0035] Figure 1 are the cytotoxicities of arctiin, trichosantol, and amygdalin at different concentrations;

[0036] Figure 2 is the cytotoxicity of the combined use of arctiin, trichosantol, and amygdalin;

[0037] Figure 3 is the effect of the combined use of arctiin, trichosantol, and amygdalin on the protective effect against LPS-induced lung cell injury;

[0038] Figure 4 is the effect of the combined use of arctiin, trichosantol, and amygdalin on apoptosis caused by LPS-induced lung cell injury;

[0039] Figure 5 is the effect of arctiin, trichosantol, amygdalin and their combined use on the expression levels of IL-6 and IL-1β inflammatory factors;

[0040] Figure 6 is the effect of arctiin, trichosantol, amygdalin and their combined use on the mRNA expression levels of NF-kB and TLR4;

[0041] Figure 7 is the effect of arctiin, trichosantol, amygdalin and their combined use on the relative protein expression levels of phosphorylated NF-kB and TLR4. Detailed implementation manners

[0042] Through a large number of screenings, the present inventors first discovered that by using arctiin and trichosantol in combination, or by using arctiin, trichosantol and amygdalin in combination, the therapeutic effect of pediatric pneumonia can be significantly enhanced. Based on this, the present invention was completed.

[0043] As used herein, "pharmaceutically acceptable carrier" refers to any excipient that can be formulated with arctiin, trichosantol, and amygdalin into a pharmaceutical preparation and used for clinical treatment, and is selected from one or several of lubricants, fillers, binders, disintegrants, suspending agents, solvents, surfactants, wetting agents, pigments, flavoring agents, etc.

[0044] The above various dosage forms can be prepared according to the conventional processes in the field of pharmaceutical preparations.

[0045] In the pharmaceutical use described above, the administration time, administration frequency, and dosing frequency of the pharmaceutical composition of the present invention depend on the specific diagnosis results of the condition, which are within the technical scope mastered by those skilled in the art.

[0046] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the scope of the present invention.

[0047] For those not specifying specific techniques or conditions in the examples, they shall be in accordance with the techniques or conditions described in the literature in the art or in accordance with the product specifications. For those reagents or instruments not specifying the manufacturer, they are all conventional products that can be obtained through regular channels.

[0048] In the following experimental methods of the examples, unless otherwise specified, they are all conventional methods. For the test materials used in the following examples, unless otherwise specified, they are all commercially available products.

[0049] Unless otherwise stated, the percentages and parts involved in the present invention are all weight percentages and weight parts.

[0050] Example: Establishment and related research of an in vitro pneumonia cell model for treating pediatric pneumonia with the pharmaceutical composition of the present invention

[0051] 1. Experimental purpose

[0052] Construct an in vitro inflammatory cell model and study the biological effects and mechanisms of the sample in the cell model.

[0053] 2. Experimental materials and instruments

[0054] The brand and catalog number of arctiin are: MCE (HY-N0034); the brand and catalog number of trichosanthesdiol are: Macklin (K963182); the brand and catalog number of amygdalin are: MCE (HY-N0190); the brand and catalog number of A549 cells are: ATCC (XY-XB-1411); the brand and catalog number of human IL-6 ELISA KIT are: Solarbio (SEKH-0013); the brand and catalog number of human IL-1β ELISA KIT are: Solarbio (SEKH-0002); the brand and catalog number of p-NF-kB are: Affinity (AF2006); the brand and catalog number of TLR4 are: Affinity (AF7017).

[0055] 3. Experimental method

[0056] 3.1 Cell resuscitation

[0057] (1) Take out the centrifuge tube from liquid nitrogen, check the tightness of the centrifuge tube, and quickly put it into a 37°C water bath and shake to thaw;

[0058] (2) When thawing is almost complete, stop the water bath, transfer the cells to a 15 mL centrifuge tube containing 9 mL of culture medium prepared in advance, centrifuge at 1000 rpm for 5 minutes, aspirate the supernatant after centrifugation, add 5 mL of cell culture medium, transfer to a cell culture flask, and culture using a breathable cap.

[0059] 3.2 Cell Passage

[0060] (1) When the confluence of cells in the T25 flask reaches 80%, aspirate the original culture medium.

[0061] (2) Add about 2 mL of PBS, gently shake the culture flask to wash the cells, and aspirate and discard the PBS.

[0062] (3) Add about 1 mL of 0.25% trypsin solution (containing EDTA), gently shake the culture flask to wet all the cells.

[0063] (4) Place it in the incubator for digestion for 1 - 2 minutes. When the cells in the middle of the cell mass are obviously rounded, bright, and have gaps under the microscope, digestion can be terminated.

[0064] (5) Add 3 mL of serum-containing medium to terminate digestion, pipette the cells to detach them from the wall, and pipette repeatedly in the liquid to make the cells as single-cell suspension as possible.

[0065] (6) Collect the cell suspension and centrifuge at 1000 rpm for 5 minutes, aspirate the supernatant after centrifugation.

[0066] (7) Add fresh medium to the cell pellet, pipette a few times to mix the cells, and inoculate into a new culture flask or multi-well plate according to the recommended ratio.

[0067] 3.3 CCK8 Preliminary Experiment 1 (Experiment to Explore the Cytotoxic Concentration of Arctiin, Trichosanthesdiol, and Amygdalin)

[0068] (1) Cell Seeding: Resuspend the A549 cell suspension in the logarithmic growth phase with complete medium (89% DMEM / F12 + 10% FBS + 1% P / S), adjust the cell density to 3*10^4 cells / mL according to the counting results, inoculate into a 96-well plate, 100 μL of medium per well, with three replicates per group, and set a well with cell-free complete medium as the blank well (Blank); add sterile PBS buffer to the remaining wells and continue culturing for 24 h.

[0069] (2) Group treatment: Aspirate the culture medium in the original wells. Replace the normal culture medium in each well of the blank wells and the normal control group, and replace the culture medium containing different concentrations of drugs in the treatment groups. The drugs are as follows: arctiin at concentrations of 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 1 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 500 μg / mL; trichosantol at concentrations of 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 1 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL; amygdalin at concentrations of 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 500 μg / mL, 1000 μg / mL, 2000 μg / mL, 5000 μg / mL. Continue to incubate for 48 h;

[0070] (3) Absorbance measurement: After incubation at each time point, aspirate the original culture medium, add 100 μL of CCK8 working solution (90% DMEM / F12 + 10% CCK8 detection solution). The wells without cells are used as blank wells. After appropriate incubation, measure the absorbance at 450 nm on an enzyme-linked immunosorbent assay (ELISA) reader;

[0071] (4) Data analysis: Calculate the cell survival rate of each group and the differences between groups based on the absorbance data. The calculation formula is as follows:

[0072] Cell survival rate (%) = 100% * (OD 给药 - OD 空白 ) / (OD 正常 - OD 空白 )

[0073] 3.4 CCK8 pre-experiment 2 (Cytotoxicity detection of the combined use of arctiin, trichosantol and amygdalin)

[0074] (1) Cell seeding: Resuspend the A549 cell suspension in the logarithmic growth phase with complete culture medium (89% DMEM / F12 + 10% FBS + 1% P / S). Adjust the cell density to 3 * 10^4 cells / mL according to the counting results, and seed it in a 96-well plate, 100 μL of culture medium per well, with three replicates for each group. Set up wells with cell-free complete culture medium as blank wells (Blank); add sterile PBS buffer to the remaining wells and continue to culture for 24 h;

[0075] (2) Group treatment: Aspirate the culture medium in the original wells. Replace the normal culture medium in the blank wells and the normal control group with normal culture medium, and replace the treatment group with different concentrations of drug-containing culture medium, specifically 50 μg / mL Arc (arctiin), 10 μg / mL Kar (trichosanthesdiol), 500 μg / mL Amy (amygdalin), 50 μg / mL Arc + 10 μg / mL Kar, 50 μg / mL Arc + 10 μg / mL Kar + 500 μg / mL Amy. Continue to incubate for 48 h;

[0076] (3) Absorbance measurement: After incubation at each time point, aspirate the original culture medium, add 100 μL of CCK8 working solution (90% DMEM / F12 + 10% CCK8 detection solution). The wells without cells serve as blank wells. After appropriate incubation, measure the absorbance at 450 nm on an enzyme-linked immunosorbent assay (ELISA) reader;

[0077] (4) Data analysis: Calculate the cell survival rate and the differences between groups based on the absorbance data. The calculation formula is as follows:

[0078] Cell survival rate (%) = 100% * (OD 给药 - OD 空白 ) / (OD 正常 - OD 空白 )

[0079] 3.5 CCK8

[0080] (1) Cell seeding: Resuspend the A549 cell suspension in the logarithmic growth phase with complete culture medium (89% DMEM / F12 + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin (P / S)). Adjust the cell density to 3×10^4 cells / mL according to the counting results, and seed 100 μL of the culture medium into each well of a 96-well plate. Each group has three replicates, and a well with cell-free complete culture medium is set as the blank well (Blank); add sterile PBS buffer to the remaining wells and continue to culture for 24 h;

[0081] (2) Group treatment: Aspirate the culture medium in the original wells. Replace the culture medium in the blank wells and normal control group with normal culture medium per well, and replace the culture medium in the treatment group with different concentrations of drug-containing culture medium for pretreatment. Specifically, it is 50 μg / mL Arc arctiin, 10 μg / mL Kar trichosanthesdiol, 500 μg / mL Amy amygdalin, 560 μg / mL Arc arctiin, 560 μg / mL Kar trichosanthesdiol, 560 μg / mL Amy amygdalin, 50 μg / mL Arc arctiin + 10 μg / mL Kar trichosanthesdiol, 50 μg / mL Arc arctiin + 10 μg / mL Kar trichosanthesdiol + 500 μg / mL Amy amygdalin. Pretreat for 2 h. After the pretreatment, except for the control group, add the LPS stock solution to each well of the Model and treatment groups to a final concentration of 10 μg / mL, and continue to incubate for 48 h;

[0082] (3) Absorbance measurement: After the incubation, aspirate the original culture medium, add 100 μL of CCK8 working solution (90% DMEM / F12 + 10% CCK8 detection solution). The wells without cells are used as blank wells. After incubating for an appropriate time, measure the absorbance at 450 nm on an enzyme-linked immunosorbent assay (ELISA) reader;

[0083] (4) Data analysis: Calculate the cell survival rate of each group and the differences between groups based on the absorbance data; the calculation formula is as follows:

[0084] Cell survival rate (%) = 100% * (OD 给药 - OD 空白 ) / (OD 正常 - OD 空白 )

[0085] 3.6 Tunel

[0086] (1) Cell seeding: Resuspend the A549 cell suspension in the logarithmic growth phase with complete culture medium (89% DMEM / F12 + 10% FBS + 1% P / S). Adjust the cell density to 6 * 10^4 cells / mL according to the counting results, seed it in a 48-well plate, add 200 μL of culture medium to each well, and continue to culture for 24 h;

[0087] (2) Group treatment: Aspirate the culture medium in the original wells. Replace the normal culture medium in each well of the blank wells and the normal control group, and replace the culture medium containing drugs at different concentrations for pretreatment in the treatment group. Specifically, it is 50 μg / mL Arc (arctiin), 10 μg / mL Kar (trichosanthesdiol), 500 μg / mL Amy (amygdalin), 50 μg / mL Arc + 10 μg / mL Kar, 50 μg / mL Arc + 10 μg / mL Kar + 500 μg / mL Amy. Pretreat for 2 h. After the pretreatment, except for the control group, add the LPS mother solution to each well of the Model and treatment groups to a final concentration of 10 μg / mL, and continue to incubate for 48 h;

[0088] (3) Tunel assay: Wash once with PBS or HBSS. If the cells do not adhere firmly, the sample can be dried to make the cells adhere more firmly. Fix the cells with 4% paraformaldehyde for 30 minutes, wash once with PBS or HBSS, add PBS containing 0.3% Triton X-100, and incubate at room temperature for 5 minutes; Prepare the TUNEL detection solution and mix well: 5 μL of TdT enzyme + 45 μL of fluorescent labeling solution; Wash 2 times with PBS or HBSS, add 50 μL of TUNEL detection solution to the sample, incubate at 37 °C in the dark for 60 minutes, and wash 3 times with PBS or HBSS; After DAPI counterstains the cell nuclei, the available excitation wavelength range is 450 - 500 nm, and the emission wavelength range is 515 - 565 nm (green fluorescence).

[0089] 3.7 ELISA

[0090] (1) Transfer the cell culture medium after the treatment to a sterile centrifuge tube, centrifuge at 1000 × g for 10 min at 4 °C, and store at -20 °C or below for future measurement;

[0091] (2) Reagent warming: Place the reagent kit and the samples to be measured at room temperature 30 min before the experiment. If crystals appear in the concentrated washing solution, place it in a 37 °C water bath until all the crystals dissolve;

[0092] (3) Prepare the washing solution: Dilute the 20× concentrated washing solution with double-distilled water or deionized water to a 1× application solution;

[0093] (4) Standard product gradient dilution: Before opening the lid, ensure that all lyophilized standard products are at the bottom of the container. Add 1 mL of SR1 standard product / sample diluent to the lyophilized standard product (concentration: 2000 pg / mL). Let it stand for 10 - 30 min until completely dissolved, then gently mix well. Mix thoroughly before dilution and perform 2-fold dilution according to the following concentrations: IL-1β standard product: diluted to 1000, 500, 250, 125, 62.5, 31.25, 15.62 pg / mL; IL-6 standard product: diluted to 250, 125, 62.5, 31.25, 15.62, 7.81, 3.9 pg / mL.

[0094] (5) Dilute the 100× antibody concentrate to 1× application working solution with SR2 biotinylated antibody diluent;

[0095] (6) Dilute the 100× antibody concentrate to 1× application working solution with SR2 biotinylated antibody diluent;

[0096] (7) Dilute the 40× concentrated enzyme conjugate to 1× application working solution with SR3 enzyme conjugate diluent;

[0097] (8) Soak the enzyme-linked immunosorbent assay (ELISA) plate: Add 300 μL of 1× wash solution and let it stand for 30 seconds. After discarding the wash solution, pat the microplate dry on absorbent paper and repeat 2 times;

[0098] (9) Add standard products: Add 100 μL of the 2-fold serial diluted standard products to the standard product wells, and add 100 μL of standard product / sample diluent to well 0;

[0099] (10) Add samples: Add 100 μL of the sample to be tested to the sample wells, ensuring continuous sample addition in steps 4 and 5 without interruption; complete the sample addition process within 10 minutes;

[0100] (11) Incubation: Seal the plate with a new sealing film and incubate at 37°C for 90 minutes;

[0101] (12) Washing: Discard the liquid, add 300 μL of wash solution to each well to wash the plate 4 times. Pat dry on absorbent paper each time during washing;

[0102] (13) Add biotinylated detection antibody: Add 100 μL of biotinylated antibody working solution to the reaction wells;

[0103] (14) Incubation: Seal the plate with a new sealing film and incubate at 37°C for 60 minutes;

[0104] (15) Washing: Discard the liquid, add 300 μL of wash solution to each well to wash the plate 4 times; pat dry on absorbent paper each time during washing

[0105] (16) Incubation: Seal the plate with a new sealing film, and incubate it statically at 37°C for 30 min after sealing;

[0106] (17) Washing: Discard the liquid, add 300 μL of washing solution to each well to wash the plate, and wash 5 times; Each time the plate is washed, pat it dry on the absorbent paper;

[0107] (18) Adding substrate for color development: Add 100 μL of chromogenic substrate TMB to each well, avoid light, and develop color at 37°C in the dark for 15 min - 25 min; When an obvious gradient appears in the color of the standard wells (an obvious blue gradient appears in the first 4 wells of the standard wells, and it is not obvious in the last 3 - 4 wells), it can be terminated;

[0108] (19) Turn on the microplate reader 15 min in advance to preheat, and add stop solution: Add 50 μL of stop solution to each well, and the addition order of the stop solution should be as the same as that of the chromogenic substrate as much as possible

[0109] (20) Detection and reading: Within 5 min, use the microplate reader to measure the maximum absorption wavelength at 450 nm;

[0110] 3.8 PCR

[0111] 3.8.1 RNA Extraction

[0112] (1) Quickly add the processed cell samples in the 6-well plate to an appropriate amount of liquid nitrogen, grind them into powder, and collect them in a 1.5 mL RNase-free EP tube;

[0113] (2) Add 1 mL of Trizol and shake vigorously for 5 min;

[0114] (3) Add 200 μL of chloroform, tightly cover the centrifuge tube cap, shake forcefully until emulsified, and when the solution turns milky white, then let it stand at room temperature for 5 min. Centrifuge at 12000 g at 4°C for 15 min, take out the centrifuge tube from the centrifuge, and transfer the colorless supernatant to another new 1.5 mL centrifuge tube;

[0115] (4) Add an equal volume of isopropanol to the supernatant, invert the centrifuge tube up and down to mix well, and then let it stand at room temperature for 10 min.

[0116] Centrifuge at 12000 g at 4°C for 10 min;

[0117] (5) Carefully and thoroughly discard the supernatant, add 1 mL of 70% ethanol, gently invert the centrifuge tube up and down to wash it, until the precipitate block floats, then centrifuge at 12000 g at 4°C for 5 min, and carefully discard the ethanol;

[0118] (6) Air-dry the precipitate at room temperature for 2 min, and add an appropriate amount of RNA dissolution solution to dissolve the precipitate;

[0119] (7) Measure the concentration with a spectrophotometer.

[0120] 3.8.2 First-strand cDNA synthesis

[0121] (1) Synthesis of the first strand of cDNA: Reverse transcription reaction

[0122] Table 1

[0123]

[0124] (2) Incubate at 42 °C for 60 minutes using Oligo(dT)18 as the primer. Store the reverse transcription product at -20 °C for future use.

[0125] 3.8.3 RT-PCR

[0126] (1) Real-time PCR: Use the two-step method.

[0127] System

[0128] Table 2

[0129]

[0130] Procedure

[0131]

[0132] Melting curve step

[0133]

[0134] Table 3

[0135]

[0136] 3.9 Western Blot

[0137] (1) Sample treatment

[0138] 1) Collect the cells in each well, aspirate the culture medium, wash once with PBS, scrape the cells with a cell scraper, and centrifuge to collect the cell pellet;

[0139] 2) Add 200 μl of cell lysis buffer supplemented with PMSF and Triton X-100, mix well and incubate on ice for 40 - 60 min; Centrifuge at 12000 rpm for 15 min at 4 °C and aspirate the supernatant;

[0140] 3) Use a BCA protein concentration assay kit to adjust the protein concentration to the same total protein concentration among different groups according to the concentration measurement results;

[0141] 4) Dilute the 6X loading buffer with the sample to 1X loading buffer, boil for 5 min, and then store at -80 °C.

[0142] (2) Preparation of electrophoresis gel

[0143] 1) After washing and drying the glass plates, fix them on the gel maker and start preparing the separating gel. Pour 12% or 8% separating gel into the gap between the glass plates to an appropriate height (prepare 12% separating gel for β-actin and Bax, and 8% separating gel for p-gp). Cover the separating gel with absolute ethanol until the gel is completely polymerized.

[0144] 2) Pour out the absolute ethanol, gently rinse with double-distilled water, and then blot dry with filter paper. Then add the stacking gel to an appropriate height and insert the comb teeth. After the stacking gel is completely polymerized, remove the comb teeth.

[0145] 3) Preparation of separating gels with various concentrations:

[0146] Table 4

[0147]

[0148]

[0149] (3) Loading and electrophoresis

[0150] 1) Load 30 μg of protein per well, electrophorese at 80 V for 30 min, and then electrophorese at 100 V.

[0151] 2) Judge the position of the target protein according to the relative positions of the prestained Marker and the molecular weight of the target protein. Stop the electrophoretic separation when the target protein is about 1 / 3 below the separating gel surface or at the best resolution position.

[0152] (4) Transfer

[0153] 1) Soak the cut PVDF membrane in methanol for 2 min.

[0154] 2) Take out the gel, cut the separating gel according to the Marker, rinse with distilled water, cut a PVDF membrane of the same size as the PAGE gel, and soak the PVDF membrane and filter paper in the electrotransfer buffer together.

[0155] 3) Place them in order: black plate - fiber pad - filter paper - gel - PVDF membrane - filter paper - fiber pad - white plate, clamp the plates, put them into the transfer apparatus, and the black side of the black plate faces the black negative electrode. Fill the transfer tank with electrotransfer liquid and start the transfer; the transfer process is carried out at 4 °C, and the transfer conditions are 100 V / 400 mA.

[0156] (5) Immunoblotting and color development

[0157] 1) Soak the PVDF membrane in 3% BSA (blocking solution) and block it on a shaker at room temperature for 2 h;

[0158] 2) Dilute the corresponding primary antibodies (GAPDH: 1:3000, target protein: 1:1000) with the blocking solution, soak the PVDF membrane in the primary antibody incubation solution, and incubate it overnight at 4 °C; Wash the PVDF membrane thoroughly with TBST 5 - 6 times, 5 min each time;

[0159] 3) Dilute the HRP - labeled secondary antibody corresponding to the species at a dilution of 1:1000 with the blocking solution, soak the PVDF membrane in the secondary antibody incubation solution, incubate it on a shaker at 37 °C for 2 h, wash the PVDF membrane thoroughly with TBST 5 - 6 times, 5 min each time;

[0160] 4) Color development and exposure: Mix the enhancer solution in the ECL reagent and the stable peroxidase solution in a ratio of 1:1, soak the membrane in the working solution, wait until the fluorescent band is obvious, blot the excess substrate solution with filter paper, and expose and develop it with a chemiluminescence system; 4 Experimental results and analysis

[0161] 4.1 CCK8 preliminary experiment 1: Exploration of the cytotoxic concentration of arctiin, trichosanthesdiol, and amygdalin

[0162] The CCK8 results are as Figure 1 shown. Arctiin below 50 μg / ml, trichosanthesdiol below 10 μg / ml, and amygdalin below 500 μg / ml have no obvious cytotoxicity. In subsequent experiments, 50 μg / ml of arctiin, 10 μg / ml of trichosanthesdiol, 500 μg / ml of amygdalin, and their combined drugs will be selected for the cytotoxicity detection of the combined drugs.

[0163] 4.2 CCK8 preliminary experiment 2: Cytotoxicity detection of the combined use of arctiin, trichosanthesdiol, and amygdalin

[0164] The CCK8 results are as Figure 2As shown, compared with the normal control group, there were no significant differences in cell viability among the groups of 50 μg / ml Arctiin (Arc), 10 μg / ml Trichosanthesdiol (Kar), 500 μg / ml Amygdalin (Amy), 50 μg / ml Arc + 10 μg / ml Kar, and 50 μg / ml Arc + 10 μg / ml Kar + 500 μg / ml Amy, and there was no obvious cytotoxicity in each group. In subsequent experiments, the combined drug groups of 50 μg / ml Arc, 10 μg / ml Kar, 500 μg / ml Amy, 50 μg / ml Arc + 10 μg / ml Kar, and 50 μg / ml Arc + 10 μg / ml Kar + 500 μg / ml Amy were to be selected for the formal experiments of the cell model.

[0165] 4.3 CCK8

[0166] The results of CCK8 were as Figure 3 shown. Compared with the normal control group, the cell viability in the model group decreased significantly, and the difference was significant; pretreatment with appropriate concentrations of Arctiin, Trichosanthesdiol, Amygdalin, and their combined drug groups had a protective effect on LPS-treated A549 cells. Among them, the Arctiin + Trichosanthesdiol group significantly showed an obvious synergistic effect of the two herbs in protecting inflamed lung cells; the protective effect of the Arctiin + Trichosanthesdiol + Amygdalin group was significantly better than that of the single-herb groups with equal doses, and this difference in effect was statistically significant.

[0167] 4.4 Tunel

[0168] The results of Tunel were as Figure 4 shown. Compared with the normal control group, the number of TUNEL-stained positive cells increased in the model group, and obvious apoptosis occurred in the cells; the apoptosis of A549 cells in the Arctiin, Trichosanthesdiol, Amygdalin, and their combined drug groups was reduced compared with that in the Model group, and the results were different; pretreatment with Arctiin, Trichosanthesdiol, Amygdalin, and their combined drugs had a protective effect on LPS-treated A549 cells.

[0169] 4.5 Elisa

[0170] The results of ELISA were as Figure 5 shown. From Figure 5It can be seen that, compared with the normal control group, the expression levels of IL-6 and IL-1β inflammatory factors in the model group were significantly increased. While compared with the model group, the expression levels of IL-6 and IL-1β inflammatory factors in the arctiin group, trichosanthesdiol group, amygdalin group, arctiin + trichosanthesdiol group, and arctiin + trichosanthesdiol + amygdalin group decreased. And the arctiin + trichosanthesdiol group and the arctiin + trichosanthesdiol + amygdalin group had a significantly better effect on reducing the expression levels of IL-6 and IL-1β inflammatory factors than the arctiin, trichosanthesdiol, and amygdalin single-use groups. This indicates that when arctiin and trichosanthesdiol are used in combination, or when arctiin, trichosanthesdiol, and amygdalin are used in combination, the combined treatment group has a significantly better therapeutic effect on pediatric pneumonia than the arctiin, trichosanthesdiol, and amygdalin single-use groups at the same dose. This shows that after combining arctiin and trichosanthesdiol, or after combining arctiin, trichosanthesdiol, and amygdalin, there is a significant synergistic effect on the therapeutic effect of pediatric pneumonia.

[0171] 4.6 PCR

[0172] The results were as Figure 6 shown. Compared with the normal control group, the mRNA expressions of NF-kB and TLR4 in the model group increased, and the difference was significant; compared with the Model group, the mRNA expressions of NF-kB and TLR4 in the A549 cells of the arctiin group, trichosanthesdiol group, amygdalin group, and their combined drug groups decreased, and the results were significantly different.

[0173] 4.7 WB

[0174] The WB results were as Figure 7 shown. Compared with the normal control group, the relative protein expressions of phosphorylated NF-kB and TLR4 in the model group were significantly increased; compared with the model group, the arctiin group, trichosanthesdiol group, amygdalin group, arctiin + trichosanthesdiol group, and arctiin + trichosanthesdiol + amygdalin group could all reduce the relative protein expressions of phosphorylated NF-kB and TLR4; and the arctiin + trichosanthesdiol combined group and the arctiin + trichosanthesdiol + amygdalin combined group had a significantly better effect on reducing the relative protein expressions of phosphorylated NF-kB and TLR4 than the arctiin, trichosanthesdiol, and amygdalin single-use groups.

[0175] Overall, it can be seen that the pharmaceutical composition of the present invention can significantly improve the symptoms of pneumonia and greatly enhance the therapeutic effect of pneumonia. This indicates that after combining arctiin, trichosanthesdiol, and amygdalin, there is a significant synergistic effect. The pharmaceutical composition of the present invention also has fewer toxic and side effects, is convenient to take, has the prospect of being developed into a drug for treating pediatric pneumonia, and has important social and economic benefits.

[0176] Obviously, the above embodiments are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. Those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A pharmaceutical composition for treating pneumonia, characterized in that: The active ingredients of the pharmaceutical composition are composed of arctiin and trichosanthes diol, and the weight ratio of arctiin to trichosanthes diol is 5:

1.

2. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the pharmaceutical composition according to claim 1 and a pharmaceutically acceptable carrier.

3. The pharmaceutical preparation according to claim 2, characterized in that The dosage form of the pharmaceutical preparation is an oral dosage form.

4. The pharmaceutical preparation according to claim 3, characterized in that The oral dosage form is tablet, capsule, granule, powder or syrup.

5. Use of the pharmaceutical composition according to claim 1 or the pharmaceutical preparation according to any one of claims 2 to 4 in the preparation of a medicament for treating pneumonia, wherein the pneumonia is pediatric pneumonia.

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