Composition based on plant essential oil or active ingredients and application of composition in preparation of medicine for preventing and treating pulmonary edema

Abstract

The invention belongs to the technical field of biological medicines, and relates to a composition based on plant essential oil or active ingredients and application of the composition in preparation of medicines for preventing and treating pulmonary edema. Comprising at least three plant essential oils or plant effective components, the plant essential oil or the plant active ingredient has agonistic activity on aquaporin, and the plant essential oil or the plant active ingredient has agonistic activity on sodium-potassium ATP enzyme. The composition and the special preparation thereof provided by the invention not only have a good pulmonary edema prevention and treatment effect, but also can be applied to prevention and treatment of related lung diseases such as sputum hypersecretion, acute lung injury, acute respiratory distress syndrome, pulmonary fibrosis and chronic obstructive pulmonary disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to a composition based on plant essential oil or active ingredient and its application in the preparation of drugs for preventing and treating pulmonary edema. Background Art

[0002] Acute respiratory distress syndrome (ARDS) is one of the main complications of COVID-19 infection. The central link in the pathological process of ARDS is pulmonary edema, which is related to the increased permeability of alveolar capillaries and the reduced clearance of fluid in the alveoli. Inflammatory stress, oxidative stress, and immune imbalance are the main factors inducing pulmonary edema. The anti-inflammatory and antioxidant effects achieved by using anti-inflammatory drugs (such as the glucocorticoid dexamethasone) and antioxidant drugs (such as the antioxidants N-acetyl-L-cysteine (NAC) and lipoic acid) in the early stage of pulmonary edema are beyond doubt. However, the therapeutic effects on systemic oxygenation and mortality in late-stage pulmonary edema are very limited, which may be related to the toxicity of glucocorticoids, the choice of treatment regimens, and the anti-inflammatory limitations and compliance of antioxidants. Therefore, there is an urgent need to develop drugs with high efficiency and low toxicity to improve the prevention and treatment effects of pulmonary edema.

[0003] Previous studies have found that some plant essential oils, extracts, or active ingredients have various pharmacological effects such as bactericidal, anti-inflammatory, antioxidant, blood lipid regulation, blood pressure reduction, blood sugar reduction, liver protection, neuroprotection, immune regulation, and anti-tumor, and also show certain effects on acute lung injury (ALI) / acute respiratory distress syndrome (ARDS). However, studies have shown that when a single plant essential oil, extract, or active ingredient is used as the active ingredient, its anti-pulmonary edema effect is poor and it is difficult to achieve the expected results. Summary of the Invention

[0004] To solve the deficiencies of the prior art, the purpose of the present invention is to provide a composition based on plant essential oil or active ingredient and its application in the preparation of drugs for preventing and treating pulmonary edema. The multi-component composition provided by the present invention can not only be used for the prevention and treatment of pulmonary edema, but also for the prevention and treatment of respiratory diseases such as chronic obstructive pulmonary disease, pulmonary fibrosis, and acute respiratory distress syndrome.

[0005] To achieve the above purpose, the technical solution of the present invention is as follows: In the first aspect, a composition based on plant essential oil or active ingredient, comprising at least three plant essential oils or plant active ingredients; the plant essential oil or plant active ingredient has agonist activity on aquaporin, and the plant essential oil or plant active ingredient has agonist activity on sodium-potassium ATPase.

[0006] The clearance of fluid in the alveoli is an important link in the treatment of pulmonary edema. Alveolar epithelial cells are the main sites for the clearance of fluid in the alveoli. Sodium ions (Na +)Active transport is the main force for alveolar fluid clearance. Under normal circumstances, Na+ in the alveolar cavity enters the alveolar epithelial cells via the epithelial sodium channel (ENaC), and then, under the action of the sodium-potassium ATPase (Na + -K + -ATPase), Na + is pumped into the lung interstitium, thereby forming an osmotic gradient, and then water is transferred out through aquaporins (AQPs). Therefore, aquaporins and sodium-potassium ATPase of the present invention can be used as important drug targets for screening. It was unexpectedly found through experiments that some plant essential oils, extracts or active ingredients have the activity of promoting both aquaporins and sodium-potassium ATPase. However, further studies have shown that when these plant essential oils, extracts or active ingredients with the activity of promoting both aquaporins and sodium-potassium ATPase are used as single active ingredients, although they have the activity of promoting aquaporins and sodium-potassium ATPase, their therapeutic effect on pulmonary edema in animal models is limited. To solve this problem, the present invention continued the experiment and found that when three or more plant essential oils or plant active ingredients with the activity of promoting both aquaporins and sodium-potassium ATPase are combined, they have a synergistic effect and show a good therapeutic effect on pulmonary edema in animal models, and can also be used for the prevention and treatment of respiratory diseases such as chronic obstructive pulmonary disease, pulmonary fibrosis, and acute respiratory distress syndrome.

[0007] In some embodiments, the plants in the plant essential oils or plant active ingredients include but are not limited to garlic, moringa, mint, camphor leaf, dipterocarpus, cinnamon, clove, eucalyptus, gaultheria, thyme, vanilla, tea tree, etc. Among them, the garlic essential oil or its active ingredient is garlic oil and / or diallyl disulfide, etc.; the moringa essential oil or its active ingredient is moringa oil and / or moringa isothiocyanates; the mint essential oil or its active ingredient is peppermint oil and / or menthol, etc.; the camphor leaf essential oil or its active ingredient is 1,8-cineole, etc.; the dipterocarpus essential oil or its active ingredient is borneol oil and / or borneol, etc.; the cinnamon essential oil or its active ingredient is cinnamaldehyde and / or eugenol, etc.; the clove essential oil or its active ingredient is clove oil and / or eugenol, etc.; the eucalyptus essential oil or its active ingredient is eucalyptus oil and / or 1,8-cineole, etc.; the gaultheria essential oil or its active ingredient is methyl salicylate glycoside, etc.; the thyme essential oil or its active ingredient is thymol and / or carvacrol, etc.; the vanilla essential oil or its active ingredient is vanillyl alcohol, etc.; the tea tree essential oil or its active ingredient is tea tree essential oil and / or 1,8-cineole, etc.

[0008] Specifically, the plant essential oils or active ingredients of plants include, but are not limited to, garlic oil, moringa oil, peppermint oil, borneol oil, clove oil, eucalyptus oil, cinnamon essential oil, tea tree essential oil, diallyl disulfide, menthol, moringa isothiocyanate, borneol, cinnamaldehyde, eugenol, 1,8-cineole, methyl salicylate glycoside, thymol, carvacrol, vanillyl alcohol, etc.

[0009] In some embodiments, the plant essential oils or active ingredients of plants are ranked by upregulating the expression levels of aquaporin AQP1 and sodium-potassium ATPase-α mRNA genes in A549 cells with pulmonary edema, and three of the top 5 are selected to form a composition. The composition of the plant essential oils or active ingredients is from the screening of aquaporin and sodium-potassium ATPase agonists in pulmonary edema. The results show that the order of the plant essential oils upregulating the expression levels of aquaporin AQP1 and sodium-potassium ATPase-α mRNA genes in A549 cells with pulmonary edema is garlic oil, moringa oil, peppermint oil, eucalyptus oil, clove oil, borneol oil, cinnamon oil, etc., while the order of the plant active ingredients upregulating the expression levels of aquaporin AQP1 and sodium-potassium ATPase-α mRNA genes in A549 cells with pulmonary edema is diallyl disulfide, moringa isothiocyanate-1, menthol, 1,8-cineole, eugenol, borneol, cinnamaldehyde, etc. Research shows that the effect of the three-component composition of the above top 5 plant essential oils or active ingredients in upregulating the expression of aquaporin AQP1 and sodium-potassium ATPase-α mRNA genes in pulmonary edema is better than that of its two-component composition, indicating the superposition performance of each component in the composition of plant essential oils or active ingredients.

[0010] In some embodiments, at least one plant essential oil or active ingredient of plants contains sulfur components. The plant essential oils or active ingredients containing sulfur components can be garlic oil, moringa oil, diallyl disulfide, moringa isothiocyanate-1, etc.

[0011] Based on the above research results, the following embodiments are further proposed: In some embodiments, it is composed of garlic oil, peppermint oil, and eucalyptus oil in a mass ratio of 4-8:1-2:4-12, preferably a mass ratio of 4:1:4.

[0012] In some embodiments, it is composed of garlic oil, peppermint oil, and clove oil in a mass ratio of 4-8:1-2:12-36, preferably 4:1:12.

[0013] In some embodiments, it is composed of moringa oil, peppermint oil, and eucalyptus oil in a mass ratio of 4-8:1-2:4-12, preferably 4:1:4.

[0014] In some embodiments, it is composed of diallyl disulfide, L-menthol, and 1,8-cineole in a mass ratio of 4 - 8:1 - 2:4 - 12, preferably 4:1:4.

[0015] In some embodiments, it is composed of diallyl disulfide, L-menthol, and eugenol in a mass ratio of 4 - 8:1 - 2:12 - 36, preferably 4:1:12.

[0016] In some embodiments, it is composed of moringa isothiocyanate-1, L-menthol, and 1,8-cineole in a mass ratio of 4 - 8:1 - 2:4 - 12, preferably 4:1:4.

[0017] In order to further improve the volatility (odor), irritation, and stability of the composition preparation of plant essential oil or active ingredients, the following embodiments are further proposed: In some embodiments, the dosage form of the composition is one of injection liquid preparation, oral liquid preparation, microemulsion, nanoemulsion, liposome, nanopolymer preparation, soft capsule preparation, aerosol, nebulizer, powder aerosol, nasal spray, inclusion complex preparation, etc., preferably soft capsule preparation, nanoemulsion, or inclusion complex preparation. In some embodiments, the composition further includes pharmaceutical excipients. Specifically, the excipients include one or more of filler, emulsifier, co-emulsifier, capsule shell material, preservative, clathrant, etc.

[0018] The soft capsule preparation is a microemulsion-type soft capsule. Specifically, the excipients of the core material in the soft capsule preparation include oil-phase filler, emulsifier, and co-emulsifier. More specifically, plant organosulfides and taste-masking cooling system are used as active ingredients, and the mass ratio of active ingredients, oil-phase filler, emulsifier, and co-emulsifier is 1 - 10:1 - 10:1 - 10:1 - 5. More specifically, the oil-phase filler is medium-chain triglyceride. More specifically, the emulsifier is Tween-80. More specifically, the co-emulsifier is polyethylene glycol-400.

[0019] Specifically, the excipients of the nanoemulsion include emulsifier, co-emulsifier, and water. More specifically, plant organosulfides and taste-masking cooling system are used as active ingredients, and the mass ratio of active ingredients, emulsifier, co-emulsifier, and water is 2 - 6:3 - 9:1 - 3:10 - 20. More specifically, the emulsifier is polyoxyethylene hydrogenated castor oil RH-40 (Cremophor RH-40), Tween-80, Span-80, and their mixtures, and the co-emulsifier is preferably selected from one or more mixtures of ethanol, 1,2-propanediol, glycerol, diethylene glycol monoethyl ether, and polyethylene glycol-400. More specifically, the co-emulsifier is ethanol.

[0020] Specifically, the excipient of the inclusion complex preparation is a solubilizing inclusion material. More specifically, the mass ratio of the active ingredient to the solubilizing inclusion material is 1:10 - 30. More specifically, the solubilizing inclusion material is methyl-β-cyclodextrin.

[0021] On the other hand, an application of the above-mentioned composition based on plant essential oil or active ingredient in the preparation of a drug for preventing and treating pulmonary edema.

[0022] In the third aspect, an application of the above-mentioned composition based on plant essential oil or active ingredient in the preparation of a drug for preventing and treating high sputum secretion, acute lung injury, acute respiratory distress syndrome, pulmonary fibrosis or chronic obstructive pulmonary disease.

[0023] Pharmacodynamic studies have shown that the composition contains diallyl disulfide and the dosage form is a soft capsule preparation. At this time, oral administration of the composition has good therapeutic effects on anti-pulmonary fibrosis, alveolar neutrophils, inflammatory stress, oxidative stress, etc., and its therapeutic effect is significantly better than that of the two-component composition, and is also significantly better than that of the positive control N-acetyl-L-cysteine (NAC), showing a significant anti-pulmonary fibrosis therapeutic effect.

[0024] Pharmacodynamic studies have shown that the composition contains diallyl disulfide and the dosage form is a nanoemulsion, which has good therapeutic effects on anti-lung injury, inhibiting the high secretion of mucin, down-regulating mucin MUC5AC, anti-inflammatory stress, etc. And this therapeutic effect is significantly better than that of the two-component composition group, and is also significantly better than that of the positive control Myrtol, showing a significant therapeutic effect on inhibiting high sputum secretion and anti-lung injury. Description of the Drawings

[0025] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0026] Figure 1 It shows the influence of the soft capsule CPD0351A of the composition based on garlic oil in the embodiment of the present invention on the lung tissue destruction index of chronic obstructive pulmonary disease rats; Figure 2 It shows the influence of the soft capsule CPD0351A of the composition based on garlic oil in the embodiment of the present invention on the inflammatory factor TNF-α in the lung homogenate of chronic obstructive pulmonary disease rats; Figure 3 It shows the influence of the soft capsule CPD0351A of the composition based on garlic oil in the embodiment of the present invention on the antioxidant factor GSH in the lung tissue of chronic obstructive pulmonary disease rats; Figure 4 It shows the influence of the soft capsule CPD0351A of the composition based on garlic oil in the embodiment of the present invention on the spleen index of chronic obstructive pulmonary disease rats; Figure 5 Effect of the soft capsule CPD0351A of the garlic oil-based composition in the present invention's embodiment on relative pAkt in the lung tissue of chronic obstructive pulmonary disease rats; Figure 6 Effect of the soft capsule preparation CPD0353A of the moringa oil-based composition in the present invention's embodiment on the wet / dry weight ratio of pulmonary edema in ALI mice; Figure 7 Effect of the soft capsule preparation CPD0353A of the moringa oil-based composition in the present invention's embodiment on the inflammatory factor IL-6 in the lung tissue of pulmonary edema in ALI mice; Figure 8 Effect of the soft capsule CPD0354A of the diallyl disulfide-based composition in the present invention's embodiment on the pulmonary fibrosis area in fibrotic mice; Figure 9 Effect of the soft capsule CPD0354A of the diallyl disulfide-based composition in the present invention's embodiment on the number of neutrophils in the bronchoalveolar lavage fluid (BALF) of fibrotic mice; Figure 10 Effect of the soft capsule CPD0354A of the diallyl disulfide-based composition in the present invention's embodiment on the inflammatory factor IL-1β in the lung homogenate of fibrotic mice; Figure 11 Effect of the soft capsule CPD0354A of the diallyl disulfide-based composition in the present invention's embodiment on the antioxidant factor SOD1 in the lung homogenate of fibrotic mice; Figure 12 Anti-lung injury score of the soft capsule preparation CPD0356A of the moringin isothiocyanate-1-based composition in the present invention's embodiment; Figure 13 Effect of the soft capsule preparation CPD0356A of the moringin isothiocyanate-1-based composition in the present invention's embodiment on the inflammatory factor IL-1β in the lung homogenate of ALI rats; Figure 14 Effect of the nanoemulsion CPD0357A of the diallyl disulfide-based composition in the present invention's embodiment on lung injury in ARDS mice; Figure 15 Effect of the nanoemulsion CPD0357A of the diallyl disulfide-based composition in the present invention's embodiment on goblet cell formation and mucin secretion in the lung tissue of ARDS mice; Figure 16 Effect of the nanoemulsion CPD0357A of the diallyl disulfide-based composition in the present invention's embodiment on mucin MUC5AC in the lung tissue of ARDS mice; Figure 17Effect of the nanoemulsion CPD0357A of the composition based on diallyl disulfide on the inflammatory factor IL-1β in the lung homogenate of ARDS mice in the embodiments of the present invention; Figure 18 Effect of the inclusion complex preparation CPD0358A of the composition based on diallyl disulfide on lung injury in ALI mice in the embodiments of the present invention; Figure 19 Effect of the inclusion complex preparation CPD0358A of the composition based on diallyl disulfide on the wet / dry weight ratio of pulmonary edema in ALI mice; Figure 20 Effect of the inclusion complex preparation CPD0358A of the composition based on diallyl disulfide on the expression of aquaporin AQP3 mRNA in pulmonary edema of ALI mice; Figure 21 Effect of the inclusion complex preparation CPD0358A of the composition based on diallyl disulfide on the expression of sodium-potassium channel ATPase-α2 mRNA in pulmonary edema of ALI mice; Figure 22 Effect of the inclusion complex preparation CPD0358A of the composition based on diallyl disulfide on the inflammatory factor TNF-α in the serum of ALI mice. Detailed implementation manners

[0027] In order to enable those skilled in the art to more clearly understand the technical solutions of the present invention, the technical solutions of the present invention will be described in detail below in conjunction with specific embodiments.

[0028] Example 1: Activity determination of single plant essential oil and its active ingredient aquaporin agonist.

[0029] Through literature research and preliminary experiments, the preferred single plant essential oils include garlic oil, moringa oil, peppermint oil, eucalyptus oil, clove oil, borneol oil, cinnamon oil, etc.; the preferred single plant active ingredients contain diallyl disulfide, moringa isothiocyanate-1, menthol, 1,8-cineole, eugenol, borneol, cinnamaldehyde, etc.

[0030] Activity test of aquaporin agonist: (1) Pulmonary edema modeling: Select 20 μg / mL lipopolysaccharide (LPS) solution to stimulate 4×10 per well 5Establish an in vitro pulmonary edema model with A549 lung cancer cells of a certain density for 6 hours; (2) Drug administration and sample preparation: Add plant essential oil or its active ingredient to a 96-well plate containing the culture medium of LPS-stimulated pulmonary edema A549 cells at a concentration of 30 µg / mL, culture for 24 hours, and complete the preparation of relevant samples to be tested according to the experimental steps of RNA extraction, RNA reverse transcription, and RT-PCR reaction in the conventional RT-PCR detection technology; (3) Determination of the gene expression level of aquaporin APQ1 mRNA: According to the RT-PCR detection experimental steps, use RT-PCR technology to detect the gene expression level of aquaporin AQP1 mRNA in pulmonary edema A549 cells.

[0031] The order of plant essential oils upregulating the gene expression level of aquaporin AQP1 mRNA in pulmonary edema A549 cells is garlic oil, moringa oil, mentholatum, eucalyptus oil, clove oil, borneol oil, cinnamon oil, etc., while the order of plant essential oil active ingredients upregulating the gene expression level of aquaporin AQP1 mRNA in pulmonary edema A549 cells is diallyl disulfide, moringa isothiocyanate-1, menthol, 1,8-cineole, eugenol, borneol, cinnamaldehyde, etc.

[0032] Example 2: Activity determination of single plant essential oil and its active ingredient sodium-potassium ATPase agonist.

[0033] The single plant essential oil and its active ingredient preferred in this example are referred to Example 1.

[0034] Activity test of sodium-potassium ATPase agonist: Complete relevant experimental operations according to the experimental steps of pulmonary edema modeling, drug administration, and sample preparation to be tested in Example 1; Determination of sodium-potassium ATPase-α mRNA level: According to the RT-PCR detection experimental steps, use RT-PCR technology to detect the gene expression level of sodium-potassium ATPase-α mRNA in pulmonary edema A549 cells.

[0035] The order of plant essential oils upregulating the gene expression level of sodium-potassium ATPase-α mRNA in pulmonary edema A549 cells is garlic oil, moringa oil, mentholatum, eucalyptus oil, clove oil, borneol oil, cinnamon oil, etc., while the order of plant essential oil active ingredients upregulating the gene expression level of sodium-potassium ATPase-α mRNA in pulmonary edema A549 cells is diallyl disulfide, moringa isothiocyanate-1, menthol, 1,8-cineole, eugenol, borneol, cinnamaldehyde, etc.

[0036] Example 3: Determination of the superimposed performance of plant essential oil and its active ingredient multi-component composition aquaporin agonist.

[0037] Based on the activity determination results of single plant essential oils and their active ingredient aquaporin agonists in Example 1, garlic oil, moringa oil, peppermint oil, eucalyptus oil, and clove oil were established as the top 5 best aquaporin APQ1 agonists; with essential oils containing organosulfur compounds as the primary component, the following three-component (ratio 1:1:1) and two-component essential oil compositions (ratio 1:1) were optimized and combined: garlic oil + peppermint oil + eucalyptus oil, garlic oil + peppermint oil + clove oil, moringa oil + peppermint oil + eucalyptus oil, moringa oil + peppermint oil + clove oil, garlic oil + peppermint oil, garlic oil + eucalyptus oil, garlic oil + clove oil, moringa oil + peppermint oil, moringa oil + eucalyptus oil, moringa oil + clove oil; according to the experimental steps for the activity determination of aquaporin agonists in Example 1, the RT-PCR technique was used to detect the effects of the above three-component and two-component essential oil compositions on the expression level of aquaporin AQP1 mRNA gene in pulmonary edema A549 cells. The determination results showed that the activities of the above three-component compositions were all superior to the corresponding two-component compositions, indicating that the preferred multi-component essential oil compositions have a superimposed property in upregulating aquaporin AQP1.

[0038] Based on the activity determination results of single plant essential oils and their active ingredient aquaporin agonists in Example 1, the top 5 plant essential oil active ingredients include diallyl disulfide, moringa isothiocyanate-1, (-)-menthol, 1,8-cineole, and eugenol; with active ingredients containing organosulfur compounds as the primary component, the following three-component (ratio 1:1:1) and two-component active ingredient compositions (ratio 1:1) were optimized and combined: diallyl disulfide + (-)-menthol + 1,8-cineole, diallyl disulfide + (-)-menthol + eugenol, moringa isothiocyanate-1 + (-)-menthol + 1,8-cineole, moringa isothiocyanate-1 + (-)-menthol + eugenol, diallyl disulfide + (-)-menthol, diallyl disulfide + 1,8-cineole, diallyl disulfide + eugenol, moringa isothiocyanate-1 + (-)-menthol, moringa isothiocyanate-1 + 1,8-cineole, moringa isothiocyanate-1 + eugenol; according to the experimental steps for the activity determination of aquaporin agonists in Example 1, the RT-PCR technique was used to detect the effects of the above three-component and two-component active ingredient compositions on the expression level of aquaporin AQP1 mRNA gene in pulmonary edema A549 cells. The determination results showed that the activities of the above three-component compositions were all superior to the corresponding two-component compositions, indicating that the preferred multi-component active ingredient compositions have a superimposed property in upregulating aquaporin AQP1.

[0039] Example 4: Determination of the superimposed property of multi-component compositions of plant essential oils and their active ingredients as aquaporin agonists.

[0040] According to the experimental procedure for the activity determination of a single plant essential oil and its active ingredient, the sodium-potassium ATPase agonist, in Example 2, the superimposed performance of the plant essential oil or its active ingredient multi-component composition in Example 3 on the activity of the sodium-potassium ATPase-α agonist was evaluated. The measurement results showed that the activities of the above-mentioned three-component plant essential oil or its active ingredient composition were all superior to those of the corresponding two-component plant essential oil or its active ingredient composition, indicating that the preferred multi-component essential oil composition has a superimposed performance in upregulating sodium-potassium ATPase-α.

[0041] Example 5: Screening of the active ingredients and core material excipients of the soft capsule CPD0351 based on garlic oil.

[0042] Based on the screening results of aquaporin and sodium-potassium ATPase agonist and the pharmacological effects of related ingredients in the above Examples 1-4, taking into account the target indication of preventing and treating pulmonary edema of the soft capsule being studied, such as oxidative stress and inflammatory stress, the active ingredients of the formula of the soft capsule CPD0351 based on garlic oil were established as garlic oil, peppermint oil, and eucalyptus oil; according to the Chinese Pharmacopoeia, the recommended dosages of relevant traditional Chinese medicines (or extracts, etc.) in domestic and foreign studies, the contents of active ingredients, and their preliminary experiments, the mass ratio of the medicinal plant essential oils in the formula of the soft capsule CPD0351 was determined to be approximately 4-8:1-2:4-12.

[0043] Microemulsion soft capsules have relatively high requirements for the formulation. The excipients such as the oil phase, emulsifier, and co-emulsifier are required to be non-toxic, non-irritating, and have good biocompatibility. The types and proportions of the three may all affect the performance of the final product, such as droplet size, Zeta potential, and polydispersity index. The preferred ingredients and proportions are crucial for the formulation of soft capsules. The oil phase used in microemulsion soft capsules should have appropriate penetration and connection with the emulsifier molecules on the emulsion interfacial film and be prone to form an interfacial film with the emulsifier. Therefore, the molecular size of the oil phase is important for the formation of the microemulsion in soft capsules. The oil phases available for screening include soybean oil, corn oil, olive oil, rapeseed oil, peanut oil, isopropyl myristate, castor oil, isopropyl laurate, ethyl oleate, medium-chain triglycerides, etc. Classified according to the type of ions, emulsifiers include anionic, cationic, zwitterionic, non-ionic, etc. Considering safety and irritation in application, non-ionic emulsifiers are more widely used because they have the lowest toxicity, are less affected by changes in pH and ionic strength. At the same time, the hemolytic effect of the emulsifier also needs to be considered. The non-ionic emulsifiers available for screening include, for example, sorbitan fatty acids, polysorbates, polyoxyethylene fatty acid esters (trade name Myrj), polyoxyethylene fatty alcohol ethers (trade name Brij), polyoxyethylene-polyoxypropylene copolymers (polyether type), sucrose fatty acid esters, and glycerol monostearate, etc. The preferred emulsifiers include polysorbates (such as Tween-20, Tween-40, Tween-60, Tween-80), sorbitan fatty acids (such as Span-20, Span-40, Span-60, Span-80), polyoxyethylene castor oil (such as Kolliphor EL, Marlowet 40, Emulgin RO 40), polyoxyethylene hydrogenated castor oil (such as Kolliphor RH-40, Kolliphor RH-60), etc. Co-emulsifiers can improve the firmness and flexibility of the film, reduce the consumption of emulsifiers, and are beneficial to the formation of microemulsions. Co-emulsifiers should be selected from medicinal short-chain alcohols or non-ionic surfactants with appropriate hydrophilic-lipophilic balance (HLB) values. The co-emulsifiers available for screening include ethanol, ethylene glycol, propylene glycol, glycerol, n-butanol, diethylene glycol monoethyl ether, glycerin, polyethylene glycol (such as PEG 400, PEG 600), polyglycerol esters, etc.

[0044] The screening of the oil phase used in the soft capsule CPD0351 of the garlic oil-based composition is divided into two parts: (1) After the mixtures of the CPD0351 essential oil composition and different oil phases are placed for 48 hours, their miscibility is observed. No layering or color change occurs in oil phases such as soybean oil, corn oil, olive oil, rapeseed oil, peanut oil, castor oil, isopropyl myristate, isopropyl laurate, ethyl oleate, and medium-chain triglycerides; (2) After the mixtures of the CPD0351 essential oil composition, different oil phases, and emulsifiers Tween-80 or Span-80 are placed for 48 hours and observed, no color change or layering occurs in the Span-80 group, while the mixtures of the oil phases in (1) and Tween-80 show varying degrees of color change or layering except for ethyl oleate and medium-chain triglycerides.

[0045] The mixed oil phase, emulsifier, and co-emulsifier of the soft capsule CPD0351 based on garlic oil are shaken and mixed in a preliminary set ratio of 1:1:1 to form a clear and transparent solution. Take 0.1 g of the above mixed solution, add 50 mL of pure water preheated to 37 °C, shake, and record the combinations that can form a transparent solution; determine the types of the oil phase, emulsifier, and co-emulsifier through a pseudo-ternary phase diagram. The preferred emulsifiers are Tween-80, Span-80, Kolliphor RH-40, and their mixtures, while ethanol, diethylene glycol monoethyl ether, polyethylene glycol-400, and their mixtures are preferred co-emulsifiers.

[0046] Use the pseudo-ternary phase diagram to study the ideal formulations of the essential oil of the drug, filling oil phase, emulsifier, and co-emulsifier. The optimal ratio is determined to be 1-10:1-10:1-10:1-5. The preferred optimal filling oil phase, emulsifier, and co-emulsifier are medium-chain triglycerides, Tween-80, and polyethylene glycol-400, respectively.

[0047] Example 6: Screening and preparation of the capsule materials for the soft capsule CPD0351 series based on garlic oil.

[0048] According to the screening of Example 5, the mass ratio of the active ingredients of garlic oil, peppermint oil, and eucalyptus oil in the soft capsule CPD0351 based on garlic oil is 4:1:4, and the initial ratio of essential oil, medium-chain triglyceride in the oil phase, and emulsifier Tween-80 in the content is 9:4:12. Commonly used soft capsule shell materials include gelatin, plasticizer (glycerol), and water. The shell material skin is prepared according to the conventional ratio of 3:1:3 of the three. By conducting the compatibility study between the shell material skin and the content, gelatin from different sources and with different bloom strengths is screened. The specific feeding of CPD0351A is shown in Table 1. Cut the shell material skin into small pieces of 0.5 × 1 cm, put them into the injection vial, add 1 mL of the content of CPD0351, and place them under high temperature, high humidity, and light conditions according to the influencing factor test conditions. Separate pieces of gelatin are placed as blank controls under each condition. Samples are taken on the 5th and 10th days to observe the appearance changes and take photos for recording. There are three parallel samples in each group. The selected gelatin sources and bloom strengths include Fengyuan 150 bloom, Fengyuan 180 bloom, Pingda 150 bloom, and Pingda 180 bloom. By observing the appearance changes of the shell material skin, it can be seen that under each condition, none of the several shell material skins melted and deformed. The color of the shell material skin deepened under high temperature conditions, and the color change was small under high humidity and light conditions. Among them, Fengyuan 180 bloom had almost no change.

[0049] Table 1 Preparation Feeding Table of Soft Capsule CPD0351A Based on Garlic Oil

[0050] The preparation of soft capsules includes the following processes: (1) Preparation of glue solution: Dissolve methyl paraben, propyl paraben, and glycerol in 95% medicinal alcohol, dissolve them with boiling hot water under vacuum stirring, stir the mixed sol materials at a temperature of 75 - 78 °C and a pressure of 0.2 MPa for about 45 minutes, and discharge and weigh when the temperature drops to 58 - 62 °C.

[0051] (2) Preparation of capsule core material: Weigh the feeding amounts of garlic oil, peppermint oil, and eucalyptus oil, mix them evenly with a magnetic stirrer to obtain a compound raw material drug mixture, add the feeding amounts of medium-chain triglyceride, Tween 80, and PEG400 to the compound raw material drug mixture, and stir with a magnetic stirrer for 12 h to obtain the soft capsule core material.

[0052] (3)Soft capsule machine debugging: The prepared gelatin solution flows through the pipeline into the gelatin box of the soft capsule machine and is spread on the rubber film wheel to form the rubber film. The liquid medicine is injected from the nozzle through the feeding system to press the soft capsules, and the filling volume of the soft capsules is made to meet the requirements by adjusting the filling pump. Use the RJWJ-100G soft capsule filling machine of Wuxi Zhongyi. The cleanliness of the batching room is 100,000 class, the indoor temperature is controlled at 22-24 °C, and the relative humidity is ≤45-60%. Main control settings: The temperature of the gelatin tank is 46.5 °C, the temperatures of the left and right gelatin boxes are 55 °C, the temperature of the hopper is 40 °C, the main control speed is 2.6, the speed ratio of the left and right rubber film wheels is 0.5, the fan is 30 hz, and the filling volume of the soft capsules should be between 582-583 mg.

[0053] (4)Soft capsule pressing: The rotary pressing method with high degree of automation and small material loss is used to press the soft capsules.

[0054] (5)Capsule shaping: In order to remove the moisture in the capsule shell, usually first-stage rolling drying is used. It is required that the humidity of the hot air is low, and the drying time is 1.5-3 hours.

[0055] (6)Soft capsule washing: Wash the soft capsules with petroleum ether and blow dry.

[0056] (7)Secondary drying: Dry the soft capsules at 35±2 °C and a relative humidity of 35-45% for 5 hours.

[0057] (8)Inspect, sort and screen the prepared soft capsules by light.

[0058] Example 7: Formulation and preparation of soft capsules of the CPD0352 series of garlic oil-based compositions.

[0059] Based on the screening results of the taste masking agent / analgesic / anti-irritant and the pharmacological effects of related components in Examples 1-4 above, taking into account the target indications of preventing and treating pulmonary edema of the soft capsules studied such as oxidative stress and inflammatory stress, the effective ingredients of the formulation of the soft capsules of the CPD0352 series of garlic oil-based compositions are determined to be garlic oil, peppermint oil, and clove oil; according to the Chinese Pharmacopoeia, the recommended dosages of relevant traditional Chinese medicines (or extracts, etc.) in domestic and foreign research, the content of effective ingredients and their screening experiments, the mass ratio of plant essential oils in the formulation of the soft capsules of the CPD0352 series is about 4-8:1-2:12-36. The specific feeding of CPD0352A is shown in Table 2. The preparation process of the soft capsules refers to Example 6.

[0060] Table 2 Feeding table for the preparation of soft capsules of the CPD0352A series of garlic oil-based compositions

[0061] Example 8: Formulation and preparation of soft capsules of the CPD0353 series of moringa oil-based compositions.

[0062] Based on the screening results of aquaporin and sodium-potassium ATPase agonists and the pharmacological effects of related components in the above-mentioned Examples 1-4, taking into account the target indications of preventing and treating pulmonary edema of the soft capsules developed for oxidative stress and inflammatory stress, etc., the effective components of the formula of the soft capsules CPD0353 series based on moringa oil are determined to be moringa oil, menthol oil, and eucalyptus oil; according to the Chinese Pharmacopoeia, the recommended dosages of relevant traditional Chinese medicines (or extracts, etc.) in domestic and foreign studies, the content of effective components, and their screening experiments, the mass ratio of the essential oils in the formula of the soft capsules CPD0353 series is about 4-8:1-2:4-12. The specific feeding of CPD0353A is shown in Table 3. The preparation process of the soft capsules refers to Example 6.

[0063] Table 3 Preparation Feeding Table of Soft Capsules CPD0353A Based on Moringa Oil

[0064] Example 9: Formulation and Preparation of Soft Capsules CPD0354 Series Based on Diallyl Disulfide

[0065] Based on the screening results of aquaporin and sodium-potassium ATPase agonists and the pharmacological effects of related components in the above-mentioned Examples 1-4, taking into account the target indications of preventing and treating pulmonary edema of the soft capsules developed for oxidative stress and inflammatory stress, etc., the effective components of the formula of the soft capsules CPD0354 based on diallyl disulfide are determined to be diallyl disulfide, L-menthol, and 1,8-cineole; according to the Chinese Pharmacopoeia, the recommended dosages of relevant traditional Chinese medicines (or extracts, etc.) in domestic and foreign studies, the content of effective components, and their screening experiments, the mass ratio of the three effective components in the formula of the soft capsules CPD0354 is 4-8:1-2:4-12. The specific feeding of CPD0354A is shown in Table 4. The preparation process of the soft capsules refers to Example 6.

[0066] Table 4 Preparation Feeding Table of Soft Capsules CPD0354A Based on Diallyl Disulfide

[0067] Example 10: Formulation and Preparation of Soft Capsules CPD0355 Series Based on Diallyl Disulfide

[0068] Based on the screening results of aquaporin and sodium-potassium ATPase agonists and the pharmacological effects of related components in the above Examples 1-4, taking into account the target indication of preventing and treating pulmonary edema of the soft capsules studied for oxidative stress and inflammatory stress, etc., the effective components of the composition soft capsule CPD0355 based on diallyl disulfide are determined to be diallyl disulfide, L-menthol, and eugenol; according to the Chinese Pharmacopoeia, the recommended dosages of relevant traditional Chinese medicines (or extracts, etc.) in domestic and foreign studies, the contents of effective components, and their screening experiments, the mass ratio of the three effective components in the formula of soft capsule CPD0355 is clarified to be 4-8:1-2:12-36. The specific feeding amounts of CPD0355A are shown in Table 5. The preparation process of the soft capsule is shown in Example 6.

[0069] Table 5 Preparation feeding table of composition soft capsule CPD0355A based on diallyl disulfide

[0070] Example 11: Formulation and preparation of composition soft capsule CPD0356 series based on moringa isothiocyanate-1.

[0071] Based on the screening results of aquaporin and sodium-potassium ATPase agonists and the pharmacological effects of related components in the above Examples 1-4, taking into account the target indication of preventing and treating pulmonary edema of the soft capsules studied for oxidative stress and inflammatory stress, etc., the effective components of the composition soft capsule CPD0356 based on moringa isothiocyanate-1 are determined to be moringa isothiocyanate-1, L-menthol, and 1,8-cineole; according to the Chinese Pharmacopoeia, the recommended dosages of relevant traditional Chinese medicines (or extracts, etc.) in domestic and foreign studies, the contents of effective components, and their screening experiments, the mass ratio of the three essential oils in the formula of soft capsule CPD0356 is clarified to be 4-8:1-2:4-12. The specific feeding amounts of CPD0356A are shown in Table 6. The preparation process of the soft capsule is shown in Example 6.

[0072] Table 6 Preparation feeding table of composition soft capsule CPD0356A based on moringa isothiocyanate-1

[0073] Example 12: Formulation and preparation of composition nanoemulsion CPD0357 series based on diallyl disulfide.

[0074] Based on the screening results of aquaporin and sodium-potassium ATPase agonists and the pharmacological effects of related components in the above Examples 1-4, taking into account the prevention and treatment target indications of pulmonary edema of the soft capsules developed in terms of oxidative stress and inflammatory stress, the effective components of the composition nanoemulsion CPD0357 based on diallyl disulfide are determined to be diallyl disulfide, L-menthol, and 1,8-cineole; according to the Chinese Pharmacopoeia, the recommended dosages of relevant traditional Chinese medicines (or extracts, etc.) in domestic and foreign studies, the content of effective components, and the screening experiments, the mass ratio of the three effective components in the soft capsule CPD0357 formula is determined to be 4-8:1-2:4-12. The specific feeding of CPD0357A is shown in Table 7.

[0075] The reverse emulsification method was established as the preparation technology for nanoemulsions through preliminary experiments. The pseudo-ternary phase diagram was drawn by the dropping method, and the area and stability of the nanoemulsion region were used as the investigation indexes to screen the optimal prescription; the optimal preparation process was to weigh an appropriate amount of the effective components (medicinal essential oil) of CPD0357, mix them thoroughly with the emulsifier and co-emulsifier, and add distilled water dropwise while stirring until a uniform, clear and transparent nanoemulsion was formed. The screening research results showed that the emulsifiers in the optimal excipients of the CPD0357 nanoemulsion included polyoxyethylene hydrogenated castor oil RH-40 (Cremophor RH-40), Tween-80, Span-80 and their mixtures, and the co-emulsifiers were preferably selected from ethanol, 1,2-propanediol, glycerol, diethylene glycol monoethyl ether, polyethylene glycol-400 and their mixtures.

[0076] The pseudo-ternary phase diagram was used to study the ideal formula of the medicinal essential oil, emulsifier, co-emulsifier, and aqueous phase, and the optimal ratio was determined to be 2-6:3-9:1-3:10-20; the preferred optimal emulsifier and co-emulsifier were polyoxyethylene hydrogenated castor oil RH-40 (Cremophor RH-40) and ethanol, respectively.

[0077] Table 7 Preparation Feeding Table of the Composition Nanoemulsion CPD0357A Based on Diallyl Disulfide

[0078] Preparation process of the nanoemulsion CPD0357 series products: First, add diallyl disulfide, L-menthol, 1,8-cineole, polyoxyethylene hydrogenated castor oil RH-40, and ethanol to a suitable mixing tank, mix them evenly with a stirrer, continuously add purified water dropwise while stirring, stir to prepare the CPD0357 nanoemulsion, and transfer it to a high-speed centrifuge. Centrifuge at a speed of 4000 rpm for 20-40 minutes.

[0079] The characterization of the prepared nanoemulsion includes appearance, particle size, Zeta potential, drug loading, content, stability, etc. The particle size, Zeta potential, and drug loading of the prepared nanoemulsion CPD0357A are 32.77±0.38 nm, -2.65±0.25 mV, and 10.80% respectively; while the particle size, Zeta potential, and drug loading of the nanoemulsion CPD0357B are measured to be 35.50±0.58nm, -2.70±0.30 mV, and 11.10% respectively; after the nanoemulsions CPD0357A and CPD0357B are placed at 4°C and room temperature for 15 days, the appearance of the nanoemulsion is observed to be still clear and transparent, and the particle size fluctuation is small, indicating good stability.

[0080] Example 13: Formulation and preparation of the composition inclusion complex preparation CPD0358 series based on diallyl disulfide.

[0081] Based on the screening results of aquaporin and sodium-potassium ATPase agonists and the pharmacological effects of related components in the above Examples 1-4, taking into account the prevention and treatment target indications of pulmonary edema of the soft capsules studied such as oxidative stress and inflammatory stress, the effective components of the composition inclusion complex preparation CPD0358 based on diallyl disulfide are determined to be diallyl disulfide, L-menthol, and 1,8-cineole; according to the Chinese Pharmacopoeia, the recommended dosages of relevant traditional Chinese medicines (or extracts, etc.) in domestic and foreign research, the content of the active components, and their screening experiments, the mass ratio of the three active components in the soft capsule CPD0358 formulation is determined to be 4-8:1-2:4-12. The specific feeding of CPD0358A is shown in Table 8.

[0082] Using the phase solubility method, the solubilizing inclusion materials for the essential oil components of the drug are determined to be hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin, methyl-β-cyclodextrin, etc. The optimal inclusion ratio of the inclusion material to the essential oil of the drug is determined to be 10-30:1 (mass ratio) by single factor investigation experiments, and the preferred inclusion material is methyl-β-cyclodextrin.

[0083] Table 8 Preparation feeding table of the composition inclusion complex preparation CPD0358A based on diallyl disulfide

[0084] Preparation process of the inclusion complex preparation CPD03578 series products: The ultrasonic method is selected as the best preparation method from the ultrasonic method, stirring method, single-phase method, and grinding method by single factor investigation experiments; according to the above relevant feeding table, first dissolve methyl-β-cyclodextrin in pure water, add active ingredients such as diallyl disulfide, L-menthol, and 1,8-cineole, and then ultrasonicate for 40-60 minutes under key process conditions such as a temperature of 20°C and a power of 300 W to prepare the inclusion agent.

[0085] The quality evaluation study of the prepared complexing agent includes appearance, inclusion rate, content, stability, etc. The quality evaluation study shows that the quality and stability of the prepared complexing agent CPD0358A meet the requirements.

[0086] Example 14: Evaluation of the anti - COPD efficacy of soft capsules CPD0351A of a composition based on garlic oil.

[0087] A rat model of chronic obstructive pulmonary disease (COPD) was established by intraperitoneal injection of cigarette smoke extract solution (CSE). Male Sprague - Dawley rats (weighing 130 - 150 g) were selected as the research objects, and budesonide was used as the positive control drug. Twenty - eight male Sprague - Dawley rats were randomly divided into 4 groups of 7 rats each: (1) Blank control group (Control): When the rats in other groups received CSE injection to establish the model, the rats in the control group received an equal volume of phosphate injection (PBS); (2) COPD model group: On the 1st, 8th, 15th, and 22nd days of the experiment, 1 mL of CSE solution was injected intraperitoneally; (3) CPD0351A treatment group: 150 mg / kg (calculated based on the content of garlic oil) of soft capsules CPD0351A. The soft capsules were dispersed in an aqueous solution to prepare a suspension, and oral administration was started from the 2nd day, once a day; (4) Budesonide positive control group: 10 mg / kg of budesonide suspension, with the same administration time as CPD0351A. All animals were euthanized painlessly, and bronchoalveolar lavage fluid (BALF) and lung tissue were collected as samples for subsequent experimental studies.

[0088] The main anti - COPD efficacy evaluation studies include: (1) Evaluation of alveolar damage in lung tissue by hematoxylin - eosin (HE) staining; (2) Determination of inflammatory cytokines in the supernatant of lung tissue homogenate using ELISA according to the kit instructions of the supplier; (3) Determination of oxidative stress factors in lung (or liver) homogenate with reference to the BCA kit instructions of the supplier; (4) After the experiment, the animals were dissected, the spleen was removed, adipose tissue was removed, washed with ice - cold physiological saline to remove blood, and then the surface moisture was gently blotted with filter paper, weighed and recorded the weight of the spleen, and the spleen index was calculated to evaluate the immunomodulatory efficacy; (5) Detection of the relative expression of p - Akt, total - Akt, and mTOR in rats by western blotting to explore the feasibility of reversing budesonide resistance, etc. The effects of CPD0351A on anti - alveolar damage, anti - inflammatory stress, anti - oxidative stress, immunomodulation, and reversal of budesonide resistance are as Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 、 Figure 5As shown, these research results indicate that the antioxidant and immunomodulatory efficacy of CPD0351A is significantly superior to that of budesonide; in addition, CPD0351A reverses the resistance of budesonide by regulating the PI3K signaling pathway (ineffective against the overexpression of p-Akt in chronic obstructive pulmonary disease, Figure 5 ), suggesting that CPD0351A can be combined with hormonal drugs such as budesonide to achieve the special effect of enhancing efficacy and reducing toxicity.

[0089] Example 15: Evaluation of the anti-pulmonary edema effect of the inclusion complex preparation CPD0353A based on moringa oil.

[0090] An acute lung injury (ALI) model in male SD rats (weighing 180 - 220 g) was established by intratracheally instilling lipopolysaccharide (LPS) using a high-pressure spray needle. CPD0353A and the positive control Myrtol were administered by gavage. By recording the body weight changes, the therapeutic effects of CPD0353A in each group against lung tissue injury, pulmonary edema, oxidative stress, and inflammatory stress were investigated. The rats were randomly divided into a blank control group, a lung injury model group (ALI, 5 mg / kg LPS), a CPD0353A group (100 mg / kg, calculated based on moringa oil), and a Myrtol positive drug group (400 mg / kg). After anesthetizing the rats, a solution of sulfuric acid LPS (5 mg / kg) was instilled into the lungs of the rats through the high-pressure spray needle. After injection, the rats were kept sitting and gently shaken from side to side to ensure uniform distribution of LPS and normal saline in the lungs, thus establishing a rat pulmonary fibrosis model. First, gavage administration was carried out according to the above protocol, continuously administered once a day for 7 days. On the eighth day, LPS or normal saline was instilled into the trachea of the rats using a microsprayer. After 24 h of exposure to normal saline or LPS in each group of rats, euthanasia was performed. After 24 hours of drug withdrawal, all animals were painlessly sacrificed, and bronchoalveolar lavage fluid (BALF), serum, and lung tissue were collected as samples for subsequent experimental studies.

[0091] The main evaluations of the anti-lung injury and anti-pulmonary edema effects include: (1) A small piece of rat lung tissue surface was washed with normal saline to remove residual blood, and the excess moisture on the lung tissue surface was blotted with filter paper. The wet weight was obtained by weighing, and then the tissue was baked in an 80°C oven for 48 h until constant weight, and the dry weight was weighed to calculate the wet-to-dry weight ratio; (2) RT-PCR was used to detect inflammatory cytokines in the lung tissue.

[0092] The anti-pulmonary edema, anti-inflammatory stress, and other anti-lung injury and anti-pulmonary edema treatment effects of CPD0353A are respectively listed in Figure 6 and Figure 7 . As Figure 6 and Figure 7As shown, the therapeutic effects of CPD0353A with an oral administration dose of 100 mg / kg on anti-pulmonary edema and anti-inflammatory stress are significantly better than those of 400 mg / kg of Myrtol, showing significant efficacy in anti-pulmonary edema injury.

[0093] Example 16: Anti-pulmonary fibrosis evaluation of soft capsule CPD0354A based on diallyl disulfide composition.

[0094] A mouse pulmonary fibrosis model was established by intratracheally instilling bleomycin sulfate (BLM) into C57BL / 6 mice using a high-pressure spray needle. CPD0354A and the positive control N-acetylcysteine (NAC) were administered by gavage. By recording the body weight changes, the anti-pulmonary fibrosis conditions, lung tissue injury, pulmonary edema, cascade inflammation and other therapeutic effects of each group of CPD0354A were investigated. The mice were randomly divided into a blank control group (Ctrl), a BLM model group (BLM), a BLM + two-component composition group (100 mg / kg, DADS + MENT, diallyl disulfide + L-menthol), a BLM + CPD0354A group (100 mg / kg, CPD0354A), and a BLM + NAC positive drug group (600 mg / kg, NAC), a total of 5 groups. After anesthetizing the mice, a solution of BLM (5 mg / kg) was instilled into the lungs of the mice through the high-pressure spray needle. After injection, the mice were kept sitting and gently shaken left and right to make BLM and normal saline evenly distributed in the lungs to establish a mouse pulmonary fibrosis model. After 24 h, normal saline, DADS + MENT, CPD0354A and NAC were administered by gavage respectively. After continuous administration for several days, all animals were sacrificed painlessly, and bronchoalveolar lavage fluid (BALF) and lung tissue were collected as samples for subsequent experimental studies.

[0095] The main anti-pulmonary fibrosis efficacy evaluation studies included: (1) evaluating the pulmonary fibrosis area of lung tissue by Masson staining; (2) after the collection of BLAF, centrifuging and washing with phosphate buffer (PBS), and then using Wright-Giemsa staining to count neutrophils and total cells with a hemocytometer; (3) using ELISA to measure inflammatory cytokines in the supernatant of lung tissue homogenate according to the kit instructions of the supplier; (4) measuring oxidative stress factors in lung (or liver) homogenate with reference to the BCA kit instructions of the supplier.

[0096] The anti-pulmonary fibrosis treatment effects of CPD0354A on pulmonary fibrosis area, changes in the number of alveolar neutrophils, inflammation and oxidative stress in lung homogenate are respectively listed in Figure 8 、 Figure 9 、 Figure 10 、and Figure 11 . As Figures 8 - 11As shown, the therapeutic effects of orally administered CPD0354A at a dose of 100 mg / kg on anti-pulmonary fibrosis, alveolar neutrophils, inflammatory stress, oxidative stress, etc. were significantly better than those of the two-component composition group (100 mg / kg, DADS + MENT) and the positive control allyl cysteine (NAC) at 600 mg / kg, demonstrating significant anti-pulmonary fibrosis efficacy.

[0097] Example 17: Evaluation of the anti-lung injury effect of soft capsule CPD0356A based on moringa isothiocyanate-1.

[0098] An acute lung injury (ALI) model in male SD rats (weighing 180 - 220 g) was established by intratracheal instillation of lipopolysaccharide (LPS) using a high-pressure spray needle. CPD0356A and the positive control Myrtol were administered by gavage. By recording the body weight changes, the therapeutic effects of CPD0356A on anti-lung tissue injury, pulmonary edema, oxidative stress, inflammatory stress, etc. were investigated. The rats were randomly divided into a blank control group, a lung injury model group (ALI, 5 mg / kg LPS), a CPD0356A group (100 mg / kg, calculated as moringa isothiocyanate-1), and a Myrtol positive drug group (400 mg / kg). After anesthetizing the rats, a solution of sulfuric acid LPS (5 mg / kg) was instilled into the lungs of the rats through a high-pressure spray needle. After injection, the rats were kept in a sitting position and gently shaken left and right to evenly distribute LPS and normal saline in the lungs to establish a rat pulmonary fibrosis model. First, gavage administration was carried out according to the above protocol, and the administration was continued continuously for 7 days at a frequency of once a day. On the eighth day, LPS or normal saline was instilled into the trachea of the rats using a microsprayer. After 24 h of exposure to normal saline or LPS in each group of rats, euthanasia was performed. After 24 hours of drug withdrawal, all animals were painlessly sacrificed, and bronchoalveolar lavage fluid (BALF), serum, and lung tissue were collected as samples for subsequent experimental studies.

[0099] The main anti-lung injury efficacy evaluation studies included: (1) evaluating alveolar injury in lung tissue by hematoxylin-eosin (HE) staining; (2) detecting inflammatory cytokines in lung tissue by RT-PCR.

[0100] The anti-lung injury therapeutic effects of CPD0356A on anti-lung injury, anti-inflammatory stress, etc. are listed in Figure 12 and Figure 13 . As Figures 12 - 13 shown, the therapeutic effects of orally administered CPD0356A at a dose of 100 mg / kg on anti-lung injury, anti-inflammatory stress, etc. were significantly better than those of Myrtol at 400 mg / kg, demonstrating significant anti-injury efficacy.

[0101] Example 18: Evaluation of the expectorant and anti-lung injury effects of the nanoemulsion CPD0357A of the composition based on diallyl disulfide.

[0102] Twenty male BALB / c mice were randomly divided into 4 groups, with 6 mice in each group: blank control group (Ctrl), acute respiratory distress syndrome (ARDS) model group (ARDS), two-component composition group (100 mg / kg, DADS+MENT, diallyl disulfide + menthol), CPD0357A group (100 mg / kg, calculated as diallyl disulfide), and Myrtol positive drug group (300 mg / kg, Myrtol). The mice were anesthetized, and except for the blank control group, lipopolysaccharide (LPS, 5 mg / kg) solution was instilled into the lungs of the mice through intratracheal instillation to establish an ARDS mouse model. The same volume of normal saline was instilled into the blank control group in the same way. After 6 hours, normal saline, DADS+MENT, CPD0357A, and Myrtol were administered by gavage respectively. All the mice were euthanized 24 h later, and lung tissues and bronchoalveolar lavage fluid were taken for subsequent studies.

[0103] The main anti-lung injury and expectorant efficacy evaluation studies included: (1) evaluating alveolar injury in lung tissues by hematoxylin-eosin (HE) staining; (2) examining the inhibition of goblet cell production and mucin secretion in lung tissues using AB-PAS staining; (3) determining the expression of mucin MUC5AC in lung tissues by Western Blot; (4) using ELISA to measure inflammatory cytokines in the supernatant of lung tissue homogenates according to the kit instructions of the supplier.

[0104] The anti-lung injury and mucus hypersecretion treatment effects of CPD0357A on lung injury, the generation of goblet cells and the secretion of mucin in lung tissues, the expression of mucin MUC5AC in lung tissues, and inflammatory stress in the supernatant of lung tissue homogenates are respectively listed in Figure 14 、 Figure 15 、 Figure 16 、and Figure 17 。As Figures 14 - 17 shown, the treatment effects of orally administered CPD0357A at a dose of (100 mg / kg) on anti-lung injury, inhibiting mucus hypersecretion, downregulating mucin MUC5AC, and anti-inflammatory stress were significantly better than those of the two-component composition group (100 mg / kg, DADS+MENT) and the positive control Myrtol (300 mg / kg), showing significant efficacy in inhibiting high sputum secretion and anti-lung injury.

[0105] Example 19: Evaluation of the anti-pulmonary edema and anti-lung injury effects of the inclusion complex preparation CPD0358A of the composition based on diallyl disulfide.

[0106] An acute lung injury (ALI) model in male Sprague-Dawley rats (weighing 180 - 220 g) was established by instilling lipopolysaccharide (LPS) into the trachea using a high-pressure spray needle. CPD0358A and the positive control Myrtol were administered by gavage. By recording the body weight changes, the therapeutic effects of CPD0358A in each group against lung tissue injury, pulmonary edema, oxidative stress, and inflammatory stress were investigated. The rats were randomly divided into a blank control group, a lung injury model group (ALI, 5 mg / kg LPS), a CPD0358A group (80 mg / kg, calculated as diallyl disulfide), and a Myrtol positive drug group (400 mg / kg). After anesthetizing the rats, a solution of sulfuric acid LPS (5 mg / kg) was instilled into the lungs of the rats through the high-pressure spray needle. After injection, the rats were kept in a sitting position and gently shaken left and right to evenly distribute LPS and normal saline in the lungs, thus establishing a rat pulmonary fibrosis model. First, gavage administration was carried out according to the above protocol, and the administration was continued continuously for 7 days at a frequency of once a day. On the eighth day, LPS or normal saline was instilled into the tracheas of the rats using a microsprayer. After each group of rats was exposed to normal saline or LPS for 24 h, euthanasia was performed. After 24 hours of drug withdrawal, all animals were painlessly sacrificed, and bronchoalveolar lavage fluid (BALF), serum, and lung tissue were collected as samples for subsequent experimental studies.

[0107] The main evaluations of the anti-lung injury and anti-pulmonary edema effects included: (1) evaluating alveolar injury in lung tissue using hematoxylin-eosin (HE) staining; (2) washing away the residual blood on the surface of a small piece of rat lung tissue with normal saline and wiping off the excess moisture on the surface of the lung tissue with filter paper, then weighing to obtain the wet weight, and then baking in an 80°C oven for 48 h until constant weight, and weighing again to obtain the dry weight, and calculating the wet-to-dry weight ratio; (3) detecting the mRNA expression levels of related aquaporin AQP3 and Na + -K + channel ATPase-α2 in lung tissue using RT-PCR; (4) measuring the inflammatory cytokines in serum using ELISA according to the supplier's kit instructions.

[0108] The anti-lung injury and anti-pulmonary edema therapeutic effects of CPD0358A against lung injury, anti-pulmonary edema, upregulating aquaporin AQP3 and Na + -K + channel ATPase-α2 levels, and anti-inflammatory stress are respectively listed in Figure 18 、 Figure 19 、 Figure 20 、 Figure 21 and Figure 22 . As Figures 18 - 22 shown, CPD0358A at an oral administration dose of 80 mg / kg has anti-lung injury, anti-pulmonary edema, upregulates aquaporin AQP3 and Na+ -K + The therapeutic effects such as the level of channel ATPase-α2 and anti-inflammatory stress are significantly better than those of 400 mg / kg of Myrtol, showing significant curative effects against lung injury and pulmonary edema.

[0109] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A composition based on plant essential oils or active ingredients, characterized in that, Comprising at least three kinds of plant essential oils or plant active ingredients; the plant essential oils or plant active ingredients have agonist activity on aquaporin, and the plant essential oils or plant active ingredients have agonist activity on sodium-potassium ATPase.

2. The composition according to claim 1, characterized in that, The plant essential oils or active ingredients are selected from garlic oil, moringa oil, peppermint oil, eucalyptus oil, clove oil, borneol oil, cinnamon oil, diallyl disulfide, moringa isothiocyanate-1, menthol, 1,8-cineole, eugenol, borneol, cinnamaldehyde.

3. The composition according to claim 1, characterized in that, Sort the plant essential oils or plant active ingredients by up-regulating the expression levels of aquaporin AQP1 and sodium-potassium ATPase-α mRNA genes in A549 cells with pulmonary edema, and select three of the top 5 to form a composition.

4. The composition according to claim 1, characterized in that, At least one plant essential oil or plant active ingredient contains sulfur components.

5. The composition according to claim 1, characterized in that, Composed of garlic oil, peppermint oil, and eucalyptus oil in a mass ratio of 4-8:1-2:4-12; Or, composed of garlic oil, peppermint oil, and clove oil in a mass ratio of 4-8:1-2:12-36, Or, composed of moringa oil, peppermint oil, and eucalyptus oil in a mass ratio of 4-8:1-2:4-12; Or, composed of diallyl disulfide, L-menthol, and 1,8-cineole in a mass ratio of 4-8:1-2:4-12; Or, composed of diallyl disulfide, L-menthol, and eugenol in a mass ratio of 4-8:1-2:12-36; Or, composed of moringa isothiocyanate-1, L-menthol, and 1,8-cineole in a mass ratio of 4-8:1-2:4-12.

6. The composition according to claim 1, wherein The composition further comprises pharmaceutical excipients, and the excipients include one or more of fillers, emulsifiers, co-emulsifiers, capsule shell materials, preservatives, and complexing agents.

7. The composition according to claim 1, wherein The dosage form of the composition is an injection liquid preparation, an oral liquid preparation, a microemulsion, a nanoemulsion, a liposome, a nanopolymer preparation, a soft capsule preparation, an aerosol, an atomizing agent, a powder inhalant, a nasal spray, or an inclusion complex preparation.

8. The composition according to claim 7, characterized in that, The excipients of the core material in the soft capsule preparation include oil-phase fillers, emulsifiers, and co-emulsifiers; Or, the excipients of the nanoemulsion include emulsifiers, co-emulsifiers, and water; Or, the excipient of the inclusion complex preparation is a solubilizing complexing material.

9. Use of the composition based on plant essential oils or active ingredients according to any one of claims 1 to 8 in the preparation of a drug for preventing and treating pulmonary edema.

10. Use of the composition based on plant essential oils or active ingredients according to any one of claims 1 to 8 in the preparation of a drug for preventing and treating excessive sputum secretion, acute lung injury, acute respiratory distress syndrome, pulmonary fibrosis, or chronic obstructive pulmonary disease.

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