Novel menthol-eucalyptus oil antitussive spray and use thereof

Abstract

A novel menthol-eucalyptus oil antitussive spray and the use thereof in the preparation of a medicine for treating cough. Said spray uses two natural plant medicines menthol and eucalyptus oil as effective components to synergistically function, does not contain alcohol, and is directly administrated in a spray form via the airway, thus achieving a small dosage, rapid production of desired effects and high safety.

Description

A novel menthol eucalyptus oil antitussive spray and its application

[0001] This application claims priority to the Chinese patent application filed with the State Intellectual Property Office / China Patent Office on June 30, 2023, with application number CN202310803538.5 and invention name “A new type of menthol eucalyptus oil cough suppressant spray and its application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application belongs to the field of respiratory disease treatment drugs and pharmaceuticals. Specifically, the present application provides a novel menthol eucalyptus oil antitussive spray and its application. Background Art

[0003] Menthol, also known as menthol, has the chemical name l-1-methyl-4-isopropylcyclohexan-3-ol. It is obtained from the fresh stems and leaves of the mint plant (Mentha haplocalyx Briq) of the Lamiaceae family through steam distillation, freezing, and recrystallization.

[0004] Menthol has a wide range of uses in the pharmaceutical, health, food, cosmetics and other industries. For example, it is used as a flavoring agent in the production of beverages, candies, toothpaste, perfumes and other foods and light industrial products. In medicine, it can be used to cool, relieve itching and analgesic effects on the skin or mucous membranes, or for the treatment of inflammation of the nose, pharynx and throat, as well as headaches, neuralgia and other symptoms. It can also be taken internally as a wind-repellent and other purposes.

[0005] In terms of pharmacological effects, menthol is known to have cooling, antipruritic, analgesic, antiseptic, anesthetic, and certain antibacterial and antiviral effects. The cooling effect of menthol is induced by the receptor TRPM (Transient receptor potential melastatin) 8 [1]; its cold sensing channel TRPM8 is a member of the TRP (Transient Receptor Potential) family. When activated by cooling compounds, it can easily lead to membrane potential depolarization and Ca 2+Ion influx and facilitates channel opening. Because TRPM8 is expressed in the cold-responsive sensory neurons of the dorsal root ganglion and trigeminal nerve, cold stimulation and menthol can stimulate the afferent vagus nerve of the lung bronchi. TRPM8 is expressed to varying degrees in the mesenteric vagal ganglion, as well as in the stomach, vascular smooth muscle, liver, and bladder epithelium [1,2]. Another member of the TRP family, TRPV1, is a capsaicin receptor and is primarily expressed in sensory nerves. TRPV1-positive nerves innervate the entire respiratory tract, including the upper respiratory tract (nose, larynx, trachea) and lung parenchyma (alveoli, smooth muscle, and blood vessels). Similar TRPV1 agonists, such as capsaicin and citric acid, can induce cough in humans and guinea pigs. The animal experiments in this application and literature reports have shown that menthol can significantly inhibit the cough-inducing effect of capsaicin or citric acid [3,4]. Although the exact mechanism of menthol's antitussive effect is not very clear, it is clinically supported that it can increase the cough-inducing threshold of capsaicin or citric acid. Menthol's cough suppressant effect has also been widely demonstrated in human trials (including adults and children) [5-9]. Unlike its cough suppressant effect, menthol can stimulate swallowing, which may alleviate concerns about its throat or anesthetic effect. Furthermore, studies have shown that menthol also has expectorant properties.

[0006] Menthol's cough-relieving properties are not documented in the Chinese Pharmacopoeia, but it is used as an adjuvant in various cough suppressant formulations and mouthwashes. Internationally, menthol is used in over-the-counter cough drops (such as Halls Cough Drops, Ricola Natural Herb Cough Drops, Honees Cough Drops, and CVS Cough Drops) and syrups (such as NatraBio Adult Cough Syrup).

[0007] Eucalyptus oil is a volatile oil obtained by steam distillation from Eucalyptus globulus Labill (Myrtaceae), Cinnamomum camphora (L.) Sieb (Lauraceae), or other plants of the same genera in these two families. According to the Chinese Pharmacopoeia standard, this product contains no less than 70.0% (g / g) of eucalyptol (or 1,8-cineol).

[0008] Eucalyptus oil has anti-inflammatory and analgesic properties and is used for itchy skin, neuralgia, and other conditions. It is also used abroad to treat upper respiratory tract infections, coughs, and bronchitis. Eucalyptol, the main component of eucalyptus oil, has been shown in clinical trials to have anti-inflammatory and antitussive properties.

[0009] Summary of the Invention

[0010] To this end, the present application introduces a new type of menthol eucalyptus oil antitussive spray, which can not only superimpose or synergize the antitussive effects of two single drugs to exert significantly enhanced antitussive efficacy, but also quickly exert an antitussive effect through a non-traditional route of local direct administration of the respiratory mucosa. There are currently no real spray-type antitussive drugs on the market, and the so-called "cough sprays" searched online are basically traditional Chinese medicine preparations for the treatment of pharyngitis, and their instructions do not indicate the "cough-relieving" effect. The 2020 edition of the Chinese Pharmacopoeia does not include antitussive drugs in spray dosage forms, and the CFDA has not approved the production and sale of such drugs.

[0011] The antitussive mechanism and corresponding receptors of menthol are relatively clear (see the background technology section). In animal experiments, it can effectively inhibit coughs induced by capsaicin and citric acid. The antitussive mechanism of eucalyptus oil is not very clear, although the antitussive effect of its main component, eucalyptol, has been confirmed in clinical trials [10-12]. Eucalyptus oil has no obvious antitussive effect in the guinea pig capsaicin model [3], but both menthol and eucalyptus oil have antitussive effects in the guinea pig citric acid model. The research of this application further proves this fact (see animal experiment 1). The experiment showed that the cough suppression rate of 2% menthol was 37.39%, and the cough suppression rate of 3.6% eucalyptus oil was 33.88%; while the cough suppression rate of the combination of the two (2% menthol + 3.6% eucalyptus oil) was 68.11%, indicating that the combined use of the two can significantly enhance the antitussive effect (see animal experiment 1). In this model, the concentration ratio of menthol and eucalyptus oil to achieve the same antitussive effect when used alone is approximately 1:2.

[0012] Eucalyptus oil is a potent anti-inflammatory drug, confirmed in animal models and human studies [12-14]. Its anti-inflammatory effects are primarily achieved by inhibiting the production of inflammatory substances such as leukotrienes (LTB4), prostaglandins (PGE2), and various cytokines (TNF-α, IL-1β, IL-4, IL-5, IL-6, IL-8, and IL-10). In clinical trials for asthma, eucalyptus oil can partially replace the use of glucocorticoids. Eucalyptus oil also has potent antibacterial properties [15,16]. Therefore, eucalyptus oil likely exerts its antitussive effects through its anti-inflammatory properties, but existing antitussive animal models are unable to verify the antitussive effects of drugs on inflammatory coughs. Therefore, this application establishes an antitussive animal model for inflammatory coughs for the first time, incorporating an LPS pretreatment step into the original citrate guinea pig model to induce inflammatory coughs (see Animal Experiment 2). Pretreatment with LPS can significantly increase the sensitivity of guinea pigs to cough induced by sodium citrate. Only half the dose of sodium citrate in the original model is needed to induce coughing in a considerable number of times.

[0013] The antitussive results of the new model showed that 4% menthol suppressed cough by 39.78%, while 0.15% eucalyptus oil suppressed cough by 36.47% (see Animal Experiment 2). The concentration ratio of menthol to eucalyptus oil, when used alone, was approximately 20:1. This suggests that the antitussive effect of menthol on inflammatory coughs is reduced, while the antitussive effect of eucalyptus oil is significantly enhanced. Based on the experimental results of the original model described above, the menthol concentration required to achieve a 37.39% cough suppression rate was 2%. Compared to the 4% concentration that achieved similar cough suppression in the new model (see Animal Experiment 1, Table 2; Animal Experiment 2, Tables 4 and 6), the antitussive effect of menthol on inflammatory coughs is approximately two-fold lower. Because the sodium citrate concentration used to induce cough in the new model was 0.2 M, only half the concentration used in the original model, the actual antitussive effect of menthol on inflammatory coughs was approximately four-fold lower. In stark contrast, eucalyptus oil achieved a 33.88% cough suppression rate at a concentration of 3.6% in the original model, but achieved a 36.47% cough suppression rate at a concentration of 0.15% in the new inflammatory cough model (see Animal Experiment 1, Table 2; Animal Experiment 2, Tables 4 and 6), representing a 24-fold increase in antitussive efficacy. Considering that the sodium citrate concentration used to induce cough in the new model was only half that used in the original model, the actual antitussive efficacy of eucalyptus oil against inflammatory cough was at least 12-fold higher than in the original model. Such a significant effect was completely unexpected. Furthermore, the combination of 1% menthol and 0.15% eucalyptus oil achieved a 67.57% cough suppression rate in the new model (see Animal Experiment 2, Tables 4 and 6). Compared to the 68.11% cough suppression rate achieved with 2% menthol and 3.6% eucalyptus oil in the original model, this demonstrates a significant reduction in the dosage required for inflammatory cough.

[0014] The above animal experiments once again show that the antitussive mechanisms of menthol and eucalyptus oil are different. The former is superior to the latter for non-inflammatory coughs, while the latter is significantly superior to the former for inflammatory coughs. In other words, menthol is more suitable for the treatment of subacute, chronic, refractory or stubborn coughs, while eucalyptus oil has a particularly significant therapeutic advantage for inflammatory coughs caused by acute upper respiratory tract infections, bronchitis, pneumonia, etc. The compound preparation composed of the two drugs can achieve complementary advantages and synergize with each other in terms of mechanism of action, significantly expanding the spectrum of treatable diseases to cover various types of coughs, and can obviously exert a better synergistic antitussive effect clinically. Moreover, menthol and eucalyptus oil are both natural botanicals, and the preparations do not contain alcohol. They can be directly administered to the airway in the form of a spray, with a small dosage, rapid effect, high safety, and suitable for all populations, including children. Currently, there is no spray dosage form of antitussive drugs on the market, and there is no antitussive drug preparation with menthol or eucalyptus oil as the main ingredients.

[0015] The new menthol eucalyptus oil cough suppressant spray is an oil-in-water emulsion composed of an oil phase, an emulsifier, and an aqueous phase. Menthol and eucalyptus oil are first dissolved in vegetable oil, then the appropriate proportions of emulsifier and water are added. The spray is fully emulsified using an emulsifier and finally diluted with water to the desired concentration. This preparation process does not require ethanol as a solubilizing agent, making it an ethanol-free formulation.

[0016] The vegetable oil of the oil phase is at least one selected from olive oil, soybean oil, corn oil, avocado oil, sunflower oil, peanut oil, tea seed oil, sesame oil or refined rapeseed oil.

[0017] The emulsifier is soybean lecithin, and may be replaced by other lecithins, or at least one of phospholipids, gelatin, gum arabic, tragacanth gum, sorbitan fatty acid, polysorbate, Pluronic F-68, polyoxyethylene fatty acid esters, polyoxyethylene fatty acid alcohol ethers, and polyoxyethylene polyoxypropylene copolymers.

[0018] Co-emulsifiers include Tween (such as Tween-80, 60, 40, 20), methylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, sodium alginate, tragacanth gum, gum arabic, cetyl alcohol, glyceryl monostearate, stearic acid, stearic acid alcohol, bile acid, bile salt, oleic acid or oleate.

[0019] Antioxidants, including vitamin E, sulfites, metabisulfites, sodium thiosulfate, methionine, glutathione, ascorbic acid, ascorbyl palmitate, thiourea, propyl gallate, butylated hydroxyanisole, or butylated cresol.

[0020] Preservatives include sorbic acid and its salts (such as potassium sorbate), benzoic acid and its salts (such as sodium benzoate), dehydroacetic acid and its sodium salts, parabens, etc.

[0021] Flavorings include sugars (such as sucrose, glucose, fructose, isomerized sugars, maltitol, etc.), non-sugar sweeteners (such as saccharin sodium, cyclamate, acesulfame potassium, aspartame, etc.), natural sweeteners (such as licorice, stevia, monk fruit, etc.), fruit flavors, etc.

[0022] The eucalyptus oil in this application can be replaced by eucalyptol / cineole / 1,8-Cineol to achieve similar effects.

[0023] The pharmaceutical composition of the present application does not exclude the addition of other antitussive and expectorant Chinese and Western medicinal ingredients to further enhance the therapeutic effect. These ingredients include but are not limited to dextromethorphan, carbocisteine, ambroxol, bromhexine, loquat, pinellia, fritillaria, platycodon, snow pear, ophiopogon, lily, almond, trichosanthes, mulberry bark, anemarrhena, radix stemonae, etc., and Chinese herbal extracts with antitussive and expectorant effects.

[0024] On the one hand, the present application provides a novel menthol eucalyptus oil antitussive spray, characterized in that the novel menthol eucalyptus oil antitussive spray contains menthol and eucalyptus oil or eucalyptol as active ingredients.

[0025] Furthermore, the concentration of menthol in the novel menthol eucalyptus oil antitussive spray is 0.01-20% (w / v) and the concentration of eucalyptus oil or eucalyptol is 0.01-20% (v / v).

[0026] Furthermore, the novel menthol eucalyptus oil antitussive spray contains 0.25%-1% menthol and 0.05-0.15% eucalyptus oil or eucalyptol; or it contains 1%-3% menthol and 2.5%-5% eucalyptus oil or eucalyptol, preferably 2% menthol and 3.6% eucalyptus oil or eucalyptol.

[0027] Furthermore, the novel menthol eucalyptus oil antitussive spray is an oil-in-water emulsion, which further comprises:

[0028] The oil phase is selected from at least one of olive oil, soybean oil, corn oil, avocado oil, tea seed oil, sunflower oil, peanut oil, sesame oil or refined rapeseed oil; the emulsifier is selected from at least one of lecithin, phospholipids, gelatin, gum arabic, tragacanth gum, sorbitan fatty acid, polysorbate, Pluronic F-68, polyoxyethylene fatty acid ester, polyoxyethylene fatty acid alcohol ether, and polyoxyethylene polyoxypropylene copolymer.

[0029] Furthermore, the novel menthol eucalyptus oil antitussive spray further comprises one or more of the following ingredients: an emulsifier selected from at least one of Tween, such as Tween-80 / 60 / 40 / 20, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl cellulose, sodium alginate, tragacanth gum, gum arabic, cetyl alcohol, glyceryl monostearate, stearic acid, stearic acid alcohol, bile acid, bile salt, oleic acid or oleate; an antioxidant selected from at least one of vitamin E, sulfites, metabisulfites, sodium thiosulfate, methionine, At least one of glutathione, ascorbic acid, ascorbyl palmitate, thiourea, propyl gallate, butylated 4-hydroxyanisole or di-tert-butylated 4-cresol; a preservative selected from at least one of sorbic acid and its salts (such as potassium sorbate), benzoic acid and its salts (such as sodium benzoate); a flavoring agent selected from at least one of sugars such as sucrose, glucose, fructose, isomerized sugar, maltitol, non-sugar sweeteners such as saccharin sodium, cyclamate, acesulfame potassium, aspartame, natural sweeteners, licorice, stevia, monk fruit and fruit flavoring.

[0030] Furthermore, the novel menthol eucalyptus oil antitussive spray also contains other antitussive and expectorant Chinese and Western medicinal ingredients selected from at least one of dextromethorphan, carbocisteine, ambroxol, bromhexine, loquat, pinellia, fritillaria, platycodon, snow pear, ophiopogon, lily, almond, trichosanthes, mulberry bark, anemarrhena and radix stemonae, and Chinese medicine extracts.

[0031] On the other hand, the present application provides the use of the novel menthol eucalyptus oil antitussive spray in the preparation of medicines or preparations for treating various types of coughs, asthma, upper respiratory tract infections, pharyngitis, rhinitis, and oral hygiene.

[0032] On the other hand, the present application provides the use of any one of menthol, eucalyptus oil, and eucalyptol as the main active ingredient in the preparation of medicines or preparations for treating various types of coughs, asthma, upper respiratory tract infections, pharyngitis, rhinitis, and oral hygiene.

[0033] On the other hand, the present application provides the use of eucalyptus oil, eucalyptol or 1,8-cineol in combination with menthol in the preparation of medicines or preparations for treating various types of coughs, asthma, upper respiratory tract infections, pharyngitis, rhinitis, and oral hygiene, wherein the preparation contains active ingredients other than eucalyptol / 1,8-cineol and menthol.

[0034] Furthermore, the cough is an inflammation-related cough caused by pathogen infection or other causes.

[0035] Furthermore, the spray has a clear antitussive effect, and directly exerts the antitussive effect through a new route of local administration in the respiratory tract, and has the characteristics and advantages of small dosage, rapid effect, and small toxic and side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is the experimental flow chart of animal experiment 1;

[0037] Figure 2 shows the effects of menthol and eucalyptus oil on the latency time and frequency of cough induced by sodium citrate in animal experiment 1 (##, P < 0.01 vs control group; **, P < 0.01 vs solvent group; $$, P < 0.01);

[0038] Figure 3 is the experimental flow chart of animal experiment 2;

[0039] Figure 4 shows the effects of menthol and eucalyptus oil on the latency and frequency of cough induced by LPS combined with citric acid in animal experiment 2 (##, P < 0.01 vs control group; *, P < 0.05, **, P < 0.01 vs model group);

[0040] Figure 5 Comparative effects of combined use of menthol and eucalyptus oil on the number of coughs induced by LPS combined with citric acid in animal experiment 2 ($, P < 0.01 vs control group; +, P < 0.05, ++, P < 0.01); DETAILED DESCRIPTION

[0041] The present application is described in detail below with reference to specific embodiments. The following embodiments are for illustration only, and the scope of protection of the present application is defined by the claims and is not limited to the following embodiments.

[0042] Example 1 Novel Menthol Eucalyptus Oil Antitussive Spray

[0043] [Formula] The novel menthol eucalyptus oil antitussive spray described in this embodiment comprises the following components: 2 g of menthol, 4 mL of eucalyptus oil, 10 mL of olive oil, 5 g of soybean lecithin, and 20 mL of distilled water.

[0044] Preparation Method: Dissolve 2g of peppermint and 4mL of eucalyptus globulus oil in 10mL of olive oil to prepare the oil phase. Then, add 5g of soy lecithin and 20mL of distilled water. Emulsify thoroughly in an emulsifier to prepare an oil-in-water emulsion. Finally, mix the emulsion with distilled water and prepare a 2% menthol (g / v) and 4% eucalyptus oil (v / v) spray or other spray concentrations, depending on the desired spray concentration.

[0045] Example 2 Novel Menthol Eucalyptus Oil Antitussive Spray

[0046] [Formula] The novel menthol eucalyptus oil antitussive spray described in this embodiment comprises the following components: 4 g of menthol, 2 mL of eucalyptus oil, 10 mL of refined soybean oil, 5 g of soybean lecithin, and 20 mL of distilled water.

[0047] Preparation Method: Dissolve 4g of peppermint and 2mL of eucalyptus cerifera oil in 10mL of refined soybean oil to prepare the oil phase. Then, add 5g of soybean lecithin and 20mL of distilled water. Emulsify thoroughly in an emulsifier to prepare an oil-in-water emulsion. Finally, mix the emulsion with distilled water and prepare a 4% menthol (g / v) and 2% eucalyptus oil (v / v) spray or other spray concentrations, depending on the desired spray concentration.

[0048] Example 3 Novel Menthol Eucalyptus Oil Antitussive Spray

[0049] [Formula] The novel menthol eucalyptus oil antitussive spray described in this embodiment comprises the following components: 4 g of menthol, 1 mL of eucalyptus oil, 10 mL of refined corn oil, 5 g of soybean lecithin, 1.2 g of vitamin E, and 20 mL of distilled water.

[0050] Preparation Method: 4g of peppermint, 1mL of eucalyptus cerifera oil, and 1.2g of vitamin E were dissolved in 10mL of refined corn oil to prepare an oil phase. 5g of soybean lecithin and 20mL of distilled water were then added and fully emulsified using an emulsifier to prepare an oil-in-water emulsion. Finally, the emulsion was mixed with distilled water to prepare a 4% menthol (g / v) and 1% eucalyptus oil (v / v) spray, or other spray concentrations, depending on the desired spray concentration.

[0051] Example 4 Novel Menthol Eucalyptus Oil Antitussive Spray

[0052] [Formula] The novel menthol eucalyptus oil antitussive spray described in this embodiment comprises the following components: 10 g of menthol, 2 mL of eucalyptus oil, 30 mL of refined soybean oil, 10 g of egg yolk lecithin, 0.75 g of ascorbic acid, and 20 mL of distilled water.

[0053] Preparation Method: 10g of menthol and 2mL of eucalyptus cerifera oil were dissolved in 30mL of refined soybean oil to prepare an oil phase. 10g of egg yolk lecithin, 0.75g of ascorbic acid, and 20mL of distilled water were then added and fully emulsified using an emulsifier to prepare an oil-in-water submicron emulsion. Finally, the emulsion was mixed with distilled water to prepare a 2.5% menthol (g / v) and 0.5% eucalyptus oil (v / v) spray, or other spray concentrations, depending on the desired spray concentration.

[0054] Example 5 Novel Menthol Eucalyptus Oil Antitussive Spray

[0055] [Formula] The menthol eucalyptus oil antitussive spray formula described in this embodiment includes the following components: menthol 10g, eucalyptus oil 1mL, olive oil 30mL, soy lecithin 10g, Tween 80 1g, and distilled water 20mL.

[0056] Preparation Method: 10g of menthol and 1mL of eucalyptus globulus oil are dissolved in 30mL of olive oil to prepare the oil phase. 10g of soy lecithin, 1g of Tween 80, and 20mL of distilled water are then added and fully emulsified using an emulsifier to prepare an oil-in-water submicron emulsion. Finally, the emulsion is mixed with distilled water to prepare a 2% menthol (g / v) and 0.2% eucalyptus oil (v / v) spray, or other concentrations, depending on the desired spray concentration.

[0057] Example 6 Novel Menthol Eucalyptus Oil Antitussive Spray

[0058] [Formula] The novel menthol eucalyptus oil antitussive spray described in this embodiment comprises the following components: menthol 8g, eucalyptus oil 2mL, refined corn oil 30mL, soybean lecithin 8g, Tween 80 0.5g, vitamin E 1g, and distilled water 20mL.

[0059] Preparation Method: 8g of menthol and 2mL of eucalyptus globulus oil were dissolved in 30mL of refined corn oil to prepare the oil phase. 8g of soy lecithin, 0.5g of Tween 80, 1g of vitamin E, and 20mL of distilled water were then added and fully emulsified using an emulsifier to prepare an oil-in-water submicron emulsion. Finally, the emulsion was mixed with distilled water to prepare a 4% menthol (g / v) and 1% eucalyptus oil (v / v) spray, or other spray concentrations, depending on the desired spray concentration.

[0060] Example 7 Novel Menthol Eucalyptus Oil Antitussive Spray

[0061] [Formula] The novel menthol eucalyptus oil antitussive spray described in this embodiment comprises the following components: 1 g of menthol, 2 mL of eucalyptus oil, 4 mL of sunflower oil, 1 g of Tween 60, 1 g of tragacanth gum, and 40 mL of distilled water.

[0062] Preparation Method: 1g of menthol and 2mL of eucalyptus oil are dissolved in 4mL of sunflower oil to prepare an oil phase. 1g of Tween 60, 1g of tragacanth gum, and 40mL of distilled water are then added and fully emulsified using an emulsifier to prepare an oil-in-water submicron emulsion. Finally, the submicron emulsion is mixed with physiological saline to prepare a spray containing 1% menthol (g / v) and 2% eucalyptus oil (v / v) or other concentrations.

[0063] Example 8 Novel Menthol Eucalyptus Oil Antitussive Spray

[0064] [Formula] The novel menthol eucalyptus oil antitussive spray described in this embodiment comprises the following components: 10 g of menthol, 2 mL of eucalyptus oil, 40 mL of refined rapeseed oil, 2 g of sodium alginate, 10 g of polyoxyethylene fatty acid ester, and 50 mL of distilled water.

[0065] Preparation Method: 10g of menthol and 2mL of eucalyptus oil are dissolved in 40mL of refined rapeseed oil to prepare an oil phase. 2g of sodium alginate, 10g of polyoxyethylene fatty acid ester, and 50mL of distilled water are then added and fully emulsified using an emulsifier to prepare an oil-in-water submicron emulsion. Finally, the submicron emulsion is mixed with physiological saline to prepare a spray containing 2.5% menthol (g / v) and 0.5% eucalyptus oil (v / v) or other concentrations.

[0066] Example 9 Novel Menthol Eucalyptus Oil Antitussive Spray

[0067] [Formula] The menthol cough spray formula described in this embodiment includes the following components: 50g of menthol, 30mL of eucalyptus oil, 120mL of refined soybean oil, 40g of polysorbate, 0.1g of methylcellulose, and 100mL of distilled water.

[0068] Preparation Method: The menthol and eucalyptus oil are dissolved in an oil phase, and then the emulsifier, co-emulsifier, and distilled water are added. The mixture is fully emulsified using an emulsifier to produce an oil-in-water microemulsion. Finally, the microemulsion is mixed with physiological saline to produce a spray containing 5% menthol (g / v) and 3% eucalyptus oil (v / v), or other concentrations.

[0069] Example 10 Novel Menthol Eucalyptus Oil Antitussive Spray

[0070] [Formula] The novel menthol eucalyptus oil antitussive spray formula described in this embodiment includes the following components: menthol 80g, eucalyptus oil 10mL, refined tea seed oil 240mL, gelatin 80g, sodium alginate 10g, vitamin E 4g, and distilled water 180mL.

[0071] Preparation Method: The menthol and eucalyptus oil are dissolved in an oil phase, and then the emulsifier, co-emulsifier, antioxidant, and distilled water are added. The mixture is fully emulsified using an emulsifier to produce an oil-in-water emulsion. Finally, the emulsion is mixed with a phosphate buffer solution to produce a spray containing 4% menthol (g / v) and 0.5% eucalyptus oil (v / v) or other concentrations.

[0072] Example 11 Novel Menthol Eucalyptus Oil Antitussive Spray

[0073] [Formula] The novel menthol eucalyptus oil antitussive spray formula described in this embodiment includes the following components: menthol 3g, eucalyptus oil 1mL, refined sesame oil 9mL, tragacanth gum 2g, Tween 0.1g, ascorbic acid 0.5g, and distilled water 5mL.

[0074] Preparation Method: The menthol and eucalyptus oil are dissolved in an oil phase, and then the emulsifier, co-emulsifier, antioxidant, and distilled water are added. The mixture is fully emulsified using an emulsifier to produce an oil-in-water submicron emulsion. Finally, the emulsion is mixed with physiological saline to produce a spray containing 1.5% menthol (g / v) and 0.5% eucalyptus oil (v / v) or other concentrations.

[0075] Example 12 Novel Menthol Eucalyptus Oil Antitussive Spray

[0076] [Formula] The novel menthol eucalyptus oil antitussive spray described in this embodiment comprises the following components: 100 g of menthol, 5 mL of eucalyptus oil, 300 mL of avocado oil, 100 g of soybean lecithin, 2 g of oleic acid, 1 g of propyl gallate, and 20 mL of distilled water.

[0077] Preparation Method: The menthol and eucalyptus oil are dissolved in an oil phase, and then the emulsifier, co-emulsifier, antioxidant, and distilled water are added. The mixture is fully emulsified using an emulsifier to produce an oil-in-water microemulsion. Finally, the microemulsion is mixed with water to produce a spray containing 5% menthol (g / v) and 0.25% eucalyptus oil (v / v) or other concentrations.

[0078] Example 13 Novel Menthol Eucalyptus Oil Antitussive Spray

[0079] [Formula] The novel menthol eucalyptus oil antitussive spray described in this embodiment comprises the following components: 5 g of menthol, 2.5 mL of eucalyptus oil, 15 mL of avocado oil, 5 g of egg yolk lecithin, 1 g of gum arabic, 1 g of vitamin E, 0.5 g of potassium sorbate, 5 g of sucrose, an appropriate amount of fruit flavoring agent, and 100 mL of distilled water.

[0080] Preparation Method: The menthol and eucalyptus oil are dissolved in an oil phase, and then the emulsifier, co-emulsifier, antioxidant, preservative, sucrose, fruit flavoring agent, and distilled water are added. The mixture is fully emulsified using an emulsifier to produce an oil-in-water submicron emulsion. Finally, the submicron emulsion is mixed with water to produce a spray containing 5% menthol (g / v) and 2.5% eucalyptus oil (v / v) or other concentrations.

[0081] Example 14 Novel Menthol Eucalyptus Oil Antitussive Spray

[0082] [Formula] The novel menthol eucalyptus oil antitussive spray described in this embodiment comprises the following components: 60 g of menthol, 15 mL of eucalyptus oil, 180 mL of avocado oil, 60 g of lecithin, 5 g of Tween-80, 10 g of glutathione, 6 g of sodium benzoate, 0.5 g of sweetener, 0.5 g of fruit flavor, and 120 mL of distilled water.

[0083] Preparation Method: The menthol and eucalyptus oil are dissolved in an oil phase, and then the emulsifier, co-emulsifier, antioxidant, preservative, sweetener, fruit flavoring agent, and distilled water are added. The mixture is fully emulsified using an emulsifier to produce an oil-in-water emulsion. Finally, the emulsion is mixed with water to produce a spray containing 4% menthol (g / v) and 1% eucalyptus oil (v / v) or other concentrations.

[0084] Example 15 Novel Menthol Eucalyptus Oil Antitussive Spray

[0085] [Formula] The novel menthol eucalyptus oil antitussive spray described in this embodiment includes the following components: 5 g of menthol, 6 mL of eucalyptol (also known as 1,8-cineole or eucalyptol), 15 mL of avocado oil, 5 g of soybean lecithin, 1 g of gum arabic, 1 g of vitamin E, 0.5 g of potassium sorbate, 5 g of sucrose, an appropriate amount of fruit flavoring agent, and 100 mL of distilled water.

[0086] Preparation Method: The menthol and eucalyptol are dissolved in an oil phase, and then the emulsifier, co-emulsifier, antioxidant, preservative, sucrose, fruit flavoring agent, and distilled water are added. The mixture is fully emulsified using an emulsifier to produce an oil-in-water emulsion. Finally, the emulsion is mixed with water to produce a spray containing 5% menthol (g / v) and 6% eucalyptol (v / v), or other concentrations.

[0087] Example 16 Novel Menthol Eucalyptus Oil Antitussive Spray

[0088] [Formula] The novel menthol eucalyptus oil antitussive spray described in this embodiment comprises the following components: menthol 5g, eucalyptus oil 1.5mL, avocado oil 16mL, lecithin 6g, Tween-80 0.5g, ambroxol hydrochloride 1g, glutathione 0.2g, sodium benzoate 0.5g, sweetener 2g, fruit flavor 0.5g, and distilled water 100mL.

[0089] Preparation method: The menthol and eucalyptus oil are dissolved in an oil phase, and then the emulsifier, co-emulsifier, expectorant, antioxidant, preservative, sweetener, fruit flavoring agent, and distilled water are added. The mixture is fully emulsified using an emulsifier to prepare an oil-in-water emulsion. Finally, the emulsion is mixed with water to prepare a spray containing 5% menthol (g / v), 1.5% eucalyptus oil (v / v), and 1% ambroxol hydrochloride or other concentrations.

[0090] Example 17 Novel Menthol Eucalyptus Oil Antitussive Spray

[0091] [Formula] The novel menthol eucalyptus oil antitussive spray described in this embodiment comprises the following components: menthol 4g, eucalyptol 5mL, olive oil 12mL, soy lecithin 4g, Tween-20 0.5g, carbocisteine ​​2g, vitamin E 0.5g, potassium sorbate 0.2g, sucrose 3g, fruit flavor 0.1g, and distilled water 100mL.

[0092] Preparation Method: The menthol and eucalyptol are dissolved in an oil phase, and then the emulsifier, co-emulsifier, expectorant, antioxidant, preservative, sucrose, fruit flavoring agent, and distilled water are added. The mixture is fully emulsified using an emulsifier to produce an oil-in-water emulsion. Finally, the emulsion is mixed with water to produce a spray containing 4% menthol (g / v) and 5% eucalyptol (v / v) or other concentrations.

[0093] Example 18 Novel Menthol Eucalyptus Oil Antitussive Spray

[0094] [Formula] The novel menthol eucalyptus oil antitussive spray described in this embodiment comprises the following components: 2 g of menthol, 4 mL of eucalyptus oil, 10 mL of tea seed oil, 2 g of soybean lecithin, 0.2 g of dextromethorphan hydrobromide, 0.5 g of oleic acid, 0.5 g of glutathione, and 100 mL of distilled water.

[0095] Preparation Method: The menthol, eucalyptus oil, and dextromethorphan hydrobromide oil are dissolved in an oil phase. The emulsifier, co-emulsifier, antioxidant, and an appropriate amount of distilled water are then added and fully emulsified using an emulsifier to produce an oil-in-water microemulsion. Finally, the microemulsion is mixed with water to produce a spray containing 2% menthol (g / v), 4% eucalyptus oil (v / v), and 0.2% dextromethorphan hydrobromide or other concentrations.

[0096] Example 19 Novel Menthol Eucalyptus Oil Antitussive Spray

[0097] [Formula] The novel menthol eucalyptus oil antitussive spray formula described in this embodiment includes the following components: menthol 2g, eucalyptus oil 3mL, refined sesame oil 3mL, soybean lecithin 2g, Fritillaria cirrhosa extract 2g, loquat leaf extract 1g, Tween 0.1g, ascorbic acid 0.2g, sodium sorbate 0.2g, and distilled water 20mL.

[0098] Preparation Method: The menthol and eucalyptus oil are dissolved in an oil phase, and then the emulsifier, co-emulsifier, Chinese herbal extract, antioxidant, preservative, and distilled water are added. The mixture is fully emulsified using an emulsifier to produce an oil-in-water submicron emulsion. Finally, the emulsion is mixed with physiological saline to produce a spray containing 2% menthol (g / v) and 3% eucalyptus oil (v / v) or other concentrations.

[0099] Example 20 Novel Menthol Eucalyptus Oil Antitussive Spray

[0100] [Formula] The novel menthol eucalyptus oil antitussive spray formula described in this embodiment includes the following components: menthol 5g, eucalyptus oil 8mL, corn oil 4mL, soybean lecithin 6g, pinellia extract 2g, platycodon extract 2g, Tween 0.4g, ascorbic acid 0.5g, sodium benzoate 0.2g, and distilled water 60mL.

[0101] Preparation Method: The menthol and eucalyptus oil are dissolved in an oil phase, and then the emulsifier, co-emulsifier, Chinese herbal extract, antioxidant, preservative, and distilled water are added. The mixture is fully emulsified using an emulsifier to produce an oil-in-water submicron emulsion. Finally, the emulsion is mixed with physiological saline to produce a spray containing 5% menthol (g / v) and 8% eucalyptus oil (v / v) or other concentrations.

[0102] Animal Experiment 1: Study on the Antitussive Efficacy of Menthol and Eucalyptus Oil

[0103] Experimental instruments

[0104] (1) Water Purifier: Labconco, USA

[0105] (2) Low temperature refrigerator: Forma Scientific, USA

[0106] (3) Precision balance: AG245, Mettler Toledo, Sweden

[0107] (4) TruboBoy nebulizer inhaler, Bühler GmbH, Germany

[0108] (5) Video recording equipment

[0109] Experimental reagents

[0110] (1) Menthol: Jiangxi Xinsen Natural Plant Oil Co., Ltd. (Batch No.: 2022-02-10)

[0111] (2) Eucalyptus oil: Jiangxi Xinsen Natural Plant Oil Co., Ltd. (Batch No.: 2022-02-28)

[0112] (3) Soybean lecithin: provided by the sample delivery unit (Batch No.: SY-SO-211001)

[0113] (4) Olive oil: Jiangxi Xinsen Natural Vegetable Oil Co., Ltd. (Batch No.: 2022-01-08)

[0114] (5) Codeine: Sinopharm Group Industrial Co., Ltd., batch number: 200311

[0115] (6) Sodium citrate: Sinopharm Group Industrial Co., Ltd., batch number: 20210108

[0116] (7) Other reagents were of domestic analytical grade.

[0117] experimental animals

[0118] Guinea pigs (100, 250–300 g, half male and half female) were purchased from Beijing Weitonglihua Animal Science Company, with certificate numbers 110011220104932418 and 110011220104932542. Humidity was maintained at 40–60% and the light cycle was 12 h dark and 12 h light. Animals had free access to water and food. Animals were housed in a standard animal room. All procedures and protocols for this experiment adhered to the Regulations for Animal Experimentation.

[0119] Experimental process

[0120] The basic experimental process is shown in Figure 1. The guinea pigs were placed in a glass bell jar and stabilized for 1 minute. Then, they were inhaled with compressed aerosol of 0.4M citric acid solution (the blank group was replaced with normal saline) for 10 minutes, and the number of coughs from the start of the aerosol to the end was recorded. Guinea pigs with more than 10 coughs and less than 30 coughs in the initial screening were retained for formal experiments. Guinea pigs that passed the screening were randomly divided into groups (no less than 10 in each group) and subjected to aerosol inhalation of solvent, menthol, eucalyptus oil, or a combination of menthol and eucalyptus oil for 10 minutes. After inhaling the test substance for 10 minutes, they were then aerosolized and inhaled with 0.4M citric acid solution. The number of coughs from the start of the aerosol to the end was recorded again by video. Among them, the time from the aerosol inhalation of citric acid to the first cough was the latent period. The cough induction period was recorded by video recording.

[0121] Experimental animal groups:

[0122] Table 1 Grouping and dosing regimen Note: Eucalyptus oil % (V / V), menthol % (g / V)

[0123] Drug preparation

[0124] (1) 0.4M sodium citrate: Accurately weigh sodium citrate, add it to an appropriate volume of pure water, sonicate until completely dissolved, and dilute to 10 -4 M standby.

[0125] (2) Codeine: Accurately weigh codeine and grind it repeatedly in a mortar. Add appropriate amount of physiological saline and dissolve it by ultrasonication.

[0126] (3) Preparation of test compounds: First, prepare the oil phase. Dissolve 2g of menthol in 10mL of olive oil (menthol group); or evenly mix 3.6mL of eucalyptus oil with 6.4mL of olive oil (eucalyptus oil group); or 2g of menthol + 3.6mL of eucalyptus oil / 0.6g of menthol + 3.6mL of eucalyptus oil / 2g of menthol + 1.2mL of eucalyptus oil, and prepare 10mL of solution with the corresponding volume of olive oil (menthol and eucalyptus oil combination group). Add 5g of soybean lecithin (previously cut into pieces) to 10mL of olive oil (control group) or the above drug groups. Then, slowly stir in a 42℃ water bath to dissolve the soybean lecithin and form a uniform oil phase. Place the above prepared oil phase on a magnetic stirrer and slowly add double distilled water while stirring to fully emulsify the oil phase and water phase; the final volume is 100mL, and the emulsion with the corresponding drug concentration is obtained.

[0127] Statistical analysis

[0128] All experimental data are expressed as mean ± SEM. SSPS16.0 data processing software was used for analysis of variance and t-test between groups. The data were integrated and the conclusions were evaluated. P < 0.05 was considered statistically significant.

[0129] Experimental results

[0130] The results are shown in Figure 2 and Table 2. Compared with the control group, which received only nebulized saline, 0.4 M sodium citrate significantly induced cough in guinea pigs (P < 0.01). Nebulization of the active drug codeine phosphate at a dose of 30 mg / mL for 10 minutes before citric acid-induced cough significantly increased cough latency and reduced cough frequency (P < 0.01), demonstrating that the citric acid-induced cough system is established and can be used to evaluate antitussive drug efficacy. Nebulization of 2% menthol, 3.6% eucalyptus oil, or a combination of menthol and eucalyptus oil at varying concentrations significantly increased the latency of sodium citrate-induced cough and reduced cough frequency within 10 minutes (P < 0.01). The combination of 2% menthol and 3.6% eucalyptus oil demonstrated the greatest antitussive effect, with significantly fewer coughs than either menthol or eucalyptus oil alone, and even outperforming the 30 mg / mL codeine phosphate group, suggesting further exploration and development potential.

[0131] Table 2 Effects of menthol and eucalyptus oil on the latency time and frequency of cough induced by sodium citrate ##, P < 0.01 vs control group, **, P < 0.01 vs solvent group.

[0132] in conclusion

[0133] 2% menthol, 3.6% eucalyptus oil, and their combination at different concentrations significantly increased cough latency and reduced cough frequency in guinea pigs compared to the control group given only solvent by nebulization. Among them, the combination of 2% menthol and 3.6% eucalyptus oil had the strongest antitussive effect, and the number of coughs was significantly less than that of 2% menthol or 3.6% eucalyptus oil alone, which shows value for further exploration and development.

[0134] Animal Experiment 2: Study on the antitussive effects of menthol and eucalyptus oil on LPS-induced cough in guinea pigs

[0135] Experimental instruments

[0136] (1) Water Purifier: Labconco, USA;

[0137] (2) Low temperature refrigerator: Forma Scientific, USA;

[0138] (3) Precision balance: AG245, Mettler Toledo, Sweden;

[0139] (4) TruboBoy nebulizer inhaler, Bühler GmbH, Germany;

[0140] (5) Video recording equipment: Logitech camera (C302)

[0141] Experimental reagents

[0142] (1) Menthol: Jiangxi Xinsen Natural Plant Oil Co., Ltd. (Batch No.: 2022-02-10)

[0143] (2) Eucalyptus oil: Jiangxi Xinsen Natural Plant Oil Co., Ltd. (Batch No.: 2022-02-28)

[0144] (3) Soybean lecithin: Shenyang Tianfeng Biopharmaceutical Co., Ltd. (Batch No.: SY-S0-211001)

[0145] (4) Olive oil: Jiangxi Xinsen Natural Vegetable Oil Co., Ltd. (Batch No.: 2022-01-08)

[0146] (5) Codeine: Sinopharm Group Co., Ltd., batch number: 20210901;

[0147] (6) Sodium citrate: Sinopharm Group Co., Ltd., batch number: 20210108;

[0148] (7) Lipopolysaccharide (LPS): Sigma, catalog number: L3129, batch number: 0000135946;

[0149] (7) Other reagents were of domestic analytical grade.

[0150] experimental animals

[0151] Guinea pigs (160 animals, 250–300 g, half male and half female) were purchased from Xinjian Rabbit Farm in Dashiju Town, Xinchang County, with certificate number 20220908Cdzz0412999409. The animals were housed in a standard animal room. All procedures and protocols were in compliance with the Regulations on Animal Experimentation. The animals were housed in a standard animal room.

[0152] Production license: SCXK (Zhejiang) 2020-0005

[0153] License: SCXK(Zhejiang)2021-0019

[0154] Experimental methods

[0155] Exploring new models for antitussive

[0156] At present, there are few reports on inflammatory cough models at home and abroad. In order to be closer to the actual occurrence of clinical cough, this application explored an inflammatory cough model in the early stage. This application uses LPS combined with citric acid to establish a cough model. This application found that after the use of LPS for early inflammation induction, a lower concentration of citric acid (0.2M) can induce significant cough in guinea pigs, which is significantly better than using 0.2M citric acid alone, and the cough induction effect is comparable to the conventional cough-inducing dose of 0.4M.

[0157] Overall test process

[0158] After acclimating for 5 days, the guinea pigs were placed in glass bell jars on the sixth day. After stabilization for 1 minute, they were subjected to compressed inhalation of a 0.4 M citric acid solution (normal saline was used instead of the control group) for 10 minutes. The number of coughs from the start of the inhalation to the end of the inhalation was recorded. Guinea pigs with more than 10 but less than 30 coughs during the initial screening were retained for the final experiment. Guinea pigs were randomly divided into groups (no fewer than 8 per group) and inhaled a nebulized solution of solvent, menthol, eucalyptus oil, or a combination of menthol and eucalyptus oil for 10 minutes. 15 minutes after inhalation of the test substance, 1 mg / mL LPS was then inhaled. 4 hours after LPS inhalation, the nebulized solution of solvent, menthol, eucalyptus oil, or a combination of menthol and eucalyptus oil was inhaled for 10 minutes. 15 minutes later, 0.2 M citric acid solution was inhaled. The number of coughs from the start of the inhalation to the end of the inhalation was recorded using video. The latency period is the time from aerosol inhalation of citric acid to the first cough. The control group used solvent for aerosolization and cough induction. The experimental flow chart is shown in Figure 3.

[0159] Experimental animal grouping

[0160] Table 3 Grouping and dosing regimen

[0161] Drug preparation

[0162] (1) 0.4 M citric acid: Accurately weigh citric acid and add it to an appropriate volume of pure water to a final concentration of 0.4 mol / L. Ultrasonicate until completely dissolved.

[0163] (2) 0.2 M citric acid: Accurately weigh citric acid and add it to an appropriate volume of pure water to a final concentration of 0.2 mol / L. Ultrasonicate until completely dissolved.

[0164] (3) Codeine: Accurately weigh codeine and grind it repeatedly in a mortar. Add appropriate amount of physiological saline and dissolve it by ultrasonication.

[0165] (4) LPS preparation: Accurately weigh LPS, add an appropriate amount of physiological saline to make the final concentration of 1 mg / mL, and vortex until completely dissolved.

[0166] (5) Preparation of test compound: 4 g of menthol was dissolved in 10 mL of olive oil (menthol group), or 0.45 mL of eucalyptus oil was uniformly mixed with 9.55 mL of olive oil (eucalyptus oil group), and then 5 g of soybean lecithin (pre-cut into pieces) was added to 10 mL of olive oil (control group) or the above drug group. Then, in a 42°C water bath, the soybean lecithin was slowly stirred to dissolve and form a uniform oil phase. The prepared oil phase was placed on a magnetic stirrer and double distilled water was slowly added while stirring to fully emulsify the oil phase and the water phase. The final volume was 100 mL, and an emulsion with the corresponding drug concentration was obtained. Different amounts of menthol and eucalyptus oil were dissolved in olive oil to prepare menthol and eucalyptus oil combination groups.

[0167] Statistical analysis

[0168] All experimental data are expressed as mean ± SEM and analyzed using univariate analysis with SPSS 16.0 data processing software. The data were then integrated and evaluated. Synergistic effects were analyzed using SPSS factorial interaction and main effects analysis. P < 0.05 was considered statistically significant.

[0169] Experimental Results (I) Inhibitory Effects of Menthol Combined with Eucalyptus Oil on LPS Combined with Citric Acid-Induced Cough

[0170] Guinea pigs were induced to cough by nebulizing 1 mg / mL LPS for 4 hours, followed by nebulizing 0.2 M citric acid for 10 minutes. The frequency of coughs and cough latency within 10 minutes were observed. The results showed that 0.2 M citric acid significantly increased the frequency of coughs in guinea pigs compared with the control group (P < 0.01). Furthermore, nebulizing 30 mg / mL codeine before nebulizing LPS and 0.2 M citric acid significantly reduced the frequency of coughs and increased the cough latency in guinea pigs (P < 0.01). This indicates that the LPS combined with citric acid cough-inducing system is established and can be used to evaluate the efficacy of antitussive drugs.

[0171] Menthol, eucalyptus oil, or a combination of menthol and eucalyptus oil were nebulized before nebulization of LPS and 0.2 M citric acid. The results showed that nebulized 0.06%, 0.25%, 1.0% and 4% menthol could reduce the number of coughs in guinea pigs in a dose-dependent manner, among which the 1.0% and 4% doses had a significant inhibitory effect on the number of coughs (P<0.01-0.05); nebulized 0.05%, 0.15% and 0.45% eucalyptus oil could reduce the number of coughs in guinea pigs in a dose-dependent manner, among which the 0.15% and 0.45% doses had a significant improvement effect (P<0.01-0.05); menthol doses of 0.06%, 0.25% and 1.0% and eucalyptus oil doses of 0.05% and 0.15% were combined to prepare mixed preparations. The results showed that each combination preparation could significantly improve the number of coughs induced by LPS combined with 0.2M citric acid (P<0.01-0.05) (Figure 4A). Although each drug-treated group was able to increase the latency of guinea pig cough induced by LPS combined with 0.2M in a dose-dependent manner, the latency time within each group varied greatly. Statistically, only 0.45% eucalyptus oil, 0.25% menthol and 0.15% eucalyptus oil combined, and 1.0% menthol and 0.15% eucalyptus oil combined had significant improvement effects compared with the model group (P<0.01-0.05) (Figure 4B).

[0172] Table 4 Effects of menthol and eucalyptus oil on cough induced by LPS combined with citric acid (Mean ± SEM) ☆The sum of cough suppression rates of menthol and eucalyptus oil alone.

[0173] (II) Synergistic analysis of menthol combined with eucalyptus oil

[0174] Based on the above analysis, the present application found that menthol, eucalyptus oil, and their combination were all effective in improving LPS- and citrate-induced cough in guinea pigs. To further analyze whether the combination of menthol and eucalyptus oil has a synergistic effect, the present application compared the menthol and eucalyptus oil combination groups with the respective monotherapy groups and performed a factorial analysis with interaction.

[0175] The results showed that the combined use of 0.06% menthol and 0.15% eucalyptus oil significantly improved cough frequency compared to 0.06% menthol alone (Figure 5A, Table 5). Menthol at doses of 0.25% and 1.0%, when combined with 0.05% and 0.15% eucalyptus oil, respectively, significantly improved cough frequency compared to either menthol or eucalyptus oil alone (Figure 5C, Table 5). This suggests that combined therapy has significant advantages over menthol or eucalyptus oil alone. However, in a factorial analysis with an interaction effect, no significant difference was found between the combination of menthol and eucalyptus oil, indicating no synergistic effect (P>0.05). This is despite the fact that the combined effects of 0.25% and 1.0% menthol with 0.05% and 0.15% eucalyptus oil, respectively, were significantly better than, or at least comparable to, the combined effects of the two drugs alone (Table 4). The above results show that the combination of menthol and eucalyptus oil shows good antitussive effect under this model condition and has long-term development prospects.

[0176] Table 5 Statistical table of the inhibitory effect of menthol and eucalyptus oil on cough induced by LPS combined with citric acid *, P < 0.05, **, P < 0.01 vs. model group; +, P < 0.05, ++, P < 0.01 vs. group using only the same dose of eucalyptus oil; $, P < 0.05, $$, P < 0.01 vs. group using only the same dose of menthol.

[0177] (III) Comparison of the antitussive effects of drugs in the original model and the new inflammatory cough model

[0178] To better compare and interpret the experimental results from the traditional and new models, selected results from the two experiments are summarized in Table 6. In the original model, 2% menthol suppressed cough by 37.39%, while 3.6% eucalyptus oil suppressed cough by 33.88%. The concentration ratio at which the two drugs achieved similar antitussive effects was approximately 1:2. The cough suppression rate of 2% menthol plus 3.6% eucalyptus oil was 68.11%, demonstrating a significant enhancement of the antitussive effect of the combined drug combination. In the new model, 4% menthol suppressed cough by 39.78%, while 0.15% eucalyptus oil suppressed cough by 36.47%. The concentration ratio at which the two drugs achieved similar antitussive effects when used alone was approximately 20:1. These results indicate that the antitussive effect of menthol on inflammatory coughs is reduced, while that of eucalyptus oil is significantly enhanced. When the two drugs are used in combination in the new model, 1% menthol + 0.15% eucalyptus oil can achieve a cough suppression rate of 67.57%. It can be seen that the dosage of both drugs can be significantly reduced in inflammatory coughs, especially the dosage of eucalyptus oil.

[0179] Comparing drug concentrations required to achieve similar cough suppression rates, menthol achieved a 37.39% cough suppression rate at a 2% concentration in the original model, while achieving a 39.78% cough suppression rate at a 4% concentration in the new model. Therefore, menthol's antitussive effect on inflammatory coughs decreased approximately twofold. Because the sodium citrate concentration used to induce coughs in the new model was 0.2M, half the concentration used in the original model, menthol's actual antitussive effect on inflammatory coughs decreased approximately fourfold. Eucalyptus oil, on the other hand, showed the exact opposite effect. While the cough suppression rate reached 33.88% at a 3.6% concentration in the original model, it reached 36.47% at a 0.15% concentration in the new model, representing a 24-fold increase in antitussive efficacy. Also, considering that the sodium citrate concentration used to induce coughs in the new model was only half that used in the original model, eucalyptus oil's actual antitussive efficacy against inflammatory coughs increased at least 12-fold.

[0180] Two animal studies have shown that menthol and eucalyptus oil have different antitussive mechanisms: menthol is superior to eucalyptus oil for non-inflammatory coughs, while eucalyptus oil is significantly superior to menthol for inflammatory coughs. Combining these two drugs can complement each other's strengths and achieve a synergistic effect.

[0181] Table 6 Comparison of experimental results of traditional citrate guinea pig antitussive model and new inflammatory cough antitussive model

[0182] in conclusion

[0183] Currently, there are no dedicated models for inflammatory cough, both domestically and internationally. This application establishes a new inflammatory cough model using LPS combined with 0.2M citric acid, a novel approach. Previous model exploration also found that LPS combined with 0.2M citric acid induced significantly more coughs in guinea pigs than 0.2M citric acid alone. Simultaneous nebulization of 30mg / mL codeine significantly reduced the number of coughs and increased cough latency in guinea pigs, demonstrating that the LPS combined with citric acid cough-inducing system is established and can be used to explore and evaluate the efficacy of inflammatory antitussive drugs.

[0184] In the above model, the present application further found that 0.06%, 0.25%, 1.0% and 4% menthol can reduce the number of guinea pig coughs in a dose-dependent manner, among which the 1.0% and 4% doses have a significant improvement effect on the number of coughs; 0.05%, 0.15% and 0.45% eucalyptus oil can reduce the number of guinea pig coughs in a dose-dependent manner, among which the 0.15% and 0.45% doses have a significant improvement effect; menthol doses of 0.25% and 1.0% combined with 0.05% and 0.15% eucalyptus oil can significantly improve LPS combined with 0.2M citric acid-induced coughs, and are significantly better than the use of each drug alone. In particular, by comparing the results with the original model, it was found that the efficiency of menthol on inflammatory coughs was reduced, while the sensitivity of eucalyptus oil to inflammatory coughs was significantly enhanced, indicating that the combination of menthol and eucalyptus oil can achieve a synergistic antitussive effect in terms of the mechanism of action. In conclusion, the antitussive preparation composed of menthol and eucalyptus oil has the value of further exploration and development.

[0185] References

[0186] 1. Banner, KH, F. Igney, and C. Poll, TRP channels: emerging targets for respiratory disease. Pharmacol Ther, 2011.130(3):p.371-84.

[0187] 2.Zhou,Y.,et al.,Sensitivity of bronchopulmonary receptors to cold and heat mediated by transient receptor potential cation channel subtypes in an ex vivo rat lung preparation.Respir Physiol Neurobiol, 2011.177(3):p.327-32.

[0188] 3.Laude, EA, AHMorice, and TJGrattan, The antitussive effects of menthol, camphor and cineole in conscious guinea-pigs. Pulm Pharmacol, 1994.7(3):p.179-84.

[0189] 4.Plevkova,J.,et al.,The role of trigeminal nasal TRPM8-expressing afferent neurons in the antitussive effects of menthol.J Appl Physiol(1985),2013.115(2):p.268-74.

[0190] 5.Millqvist,E.,E.Ternesten-Hasseus,and M.Bende,Inhalation of menthol reduces capsaicin cough sensitivity and influences inspiratory flows in chronic cough.Respir Med,2013.107(3):p.433-8.

[0191] 6.Buday,T.,et al.,Modulation of cough response by sensory inputs from the nose-role of trigeminal TRPA1 versus TRPM8 channels.Cough,2012.8(1):p.11.

[0192] 7.Wise,P.M.,P.A.Breslin,and P.Dalton,Sweet taste and menthol increase cough reflex thresholds.Pulm Pharmacol Ther,2012.25(3):p.236-41.

[0193] 8.Paul,I.M.,Therapeutic Options for Acute Cough Due to Upper Respiratory Infections in Children.Lung,2012.190(1):p.41-44.

[0194] 9.Paul,I.M.,et al.,Vapor Rub,Petrolatum,and No Treatment for Children With Nocturnal Cough and Cold Symptoms.Pediatrics,2010.126(6):p.1092-1099.

[0195] 10.Her,L.,et al.,Efficacy and Safety of Eucalyptus for Relieving Cough:A Systematic Review and Meta-Analysis of Randomized Controlled Trials.Journal of Integrative and Complementary Medicine,2022.

[0196] 11.Fischer,J.and U.Dethlefsen,Efficacy of cineole in patients suffering from acute bronchitis:a placebo-controlled double-blind trial.Cough,2013.9(1):p.25.

[0197] 12.Kardos,P.,Khaletskaya,O.& Kropova,O.,Efficacy and safety of Cineole in the treatment of patients with acute bronchitis:results of an open-label randomized clinical phase III study.Clinical Phytoscience,2021.Volume 7,Article number:83.

[0198] 13.Juergens,U.R.,et al.,Anti-inflammatory activity of 1.8-cineol(eucalyptol)in bronchial asthma:a double-blind placebo-controlled trial.Respir Med,2003.97(3):p.250-6.

[0199] 14.Juergens,U.R.,Anti-inflammatory properties of the monoterpene 1.8-cineole:current evidence for co-medication in inflammatory airway diseases.Drug Res(Stuttg),2014.64(12):p.638-46.

[0200] 15.Bachir,R.G.and M.Benali,Antibacterial activity of the essential oils from the leaves of Eucalyptus globulus against Escherichia coli and Staphylococcus aureus.Asian Pac J Trop Biomed,2012.2(9):p.739-42.

[0201] 16.Mulyaningsih,S.,et al.,Antibacterial activity of essential oils from Eucalyptus and of selected components against multidrug-resistant bacterial pathogens.Pharm Biol,2011.49(9):p.893-9.

Claims

1. A novel menthol eucalyptus oil antitussive spray, characterized in that: The novel menthol eucalyptus oil antitussive spray contains an active ingredient, the active ingredient is menthol and a second active ingredient, and the second active ingredient is eucalyptus oil or eucalyptol; The w / v concentration of menthol in the novel menthol eucalyptus oil antitussive spray is 0.01-20%, and the v / v concentration of the second active ingredient is 0.01-20%; The novel menthol eucalyptus oil antitussive spray is an oil-in-water emulsion, and further comprises: An oil phase, wherein the oil phase is selected from at least one of olive oil, soybean oil, corn oil, avocado oil, tea seed oil, sunflower oil, peanut oil, sesame oil and refined rapeseed oil; An emulsifier, wherein the emulsifier is selected from at least one of lecithin, phospholipids, gelatin, gum arabic, gum tragacanth, sorbitan fatty acid, polysorbate, Pluronic F-68, polyoxyethylene fatty acid esters, polyoxyethylene fatty acid alcohol ethers and polyoxyethylene polyoxypropylene copolymers.

2. The novel menthol eucalyptus oil antitussive spray according to claim 1, characterized in that: The w / v concentration of menthol in the novel menthol eucalyptus oil antitussive spray is 0.25-1%, and the v / v concentration of the second active ingredient is 0.05-0.15%; Alternatively, the w / v concentration of menthol in the novel menthol eucalyptus oil antitussive spray is 1-3%, and the v / v concentration of the second active ingredient is 2.5-5%.

3. The novel menthol eucalyptus oil antitussive spray according to claim 1 or 2, characterized in that: The novel menthol eucalyptus oil antitussive spray further comprises one or more of the following ingredients: an emulsifier, wherein the emulsifier is selected from at least one of Tween, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl cellulose, sodium alginate, gum tragacanth, gum arabic, cetyl alcohol, glyceryl monostearate, stearic acid, stearyl alcohol, bile acid, bile salt, oleic acid and oleate; An antioxidant, wherein the antioxidant is selected from at least one of vitamin E, sulfites, pyrosulfites, sodium thiosulfate, methionine, glutathione, ascorbic acid, ascorbyl palmitate, thiourea, propyl gallate, butylated hydroxyanisole and di-tert-butylated cresol; A preservative, wherein the preservative is selected from at least one of sorbic acid and its salts, benzoic acid and its salts; The flavoring agent is selected from at least one of sugar, maltitol, non-sugar sweeteners and fruit flavoring agents.

4. The novel menthol eucalyptus oil antitussive spray according to claim 3, characterized in that: The sugar includes but is not limited to one or more of sucrose, glucose, fructose and isomerized sugar; The non-sugar sweeteners include but are not limited to natural sweeteners and / or artificial sweeteners; The natural sweeteners include but are not limited to one or more of licorice, stevia and monk fruit; The artificial sweeteners include, but are not limited to, one or more of saccharin sodium, cyclamate and acesulfame potassium.

5. The novel menthol eucalyptus oil antitussive spray according to claim 3, characterized in that: The sugars include, but are not limited to, sucrose, glucose, fructose or isomerized sugars; The non-sugar sweetener includes natural sweetener or artificial sweetener; The natural sweeteners include liquorice, stevia or monk fruit; The artificial sweeteners include saccharin sodium, cyclamate or acesulfame potassium.

6. The novel menthol eucalyptus oil antitussive spray according to claim 3, characterized in that: The sugar is selected from one or more of sucrose, glucose, fructose and isomerized sugar; The non-sugar sweetener is selected from natural sweeteners and / or artificial sweeteners; The natural sweetener is selected from one or more of liquorice, stevia and monk fruit; The artificial sweetener is selected from one or more of saccharin sodium, cyclamate and acesulfame potassium.

7. The novel menthol eucalyptus oil antitussive spray according to claim 3, characterized in that: The fruit flavoring agent includes natural fruit flavoring agent and / or artificial fruit flavoring agent; The natural fruit flavoring agent includes but is not limited to one or more of strawberry, blueberry, blackberry, sweet orange, tangerine, grapefruit, lemon, grape, apple, pineapple, passion fruit, mango, banana, lychee, longan, pear, plum, watermelon, chestnut, jujube and hawthorn.

8. The novel menthol eucalyptus oil antitussive spray according to claim 3, characterized in that: The novel menthol eucalyptus oil antitussive spray also contains other antitussive and expectorant Chinese and Western medicinal ingredients, which include but are not limited to one or more of dextromethorphan, carbocisteine, ambroxol, bromhexine, loquat, pinellia, fritillaria, platycodon, snow pear, ophiopogon, lily, almond, trichosanthes, mulberry bark, anemarrhena and radix stemonae.

9. Use of the novel menthol eucalyptus oil antitussive spray according to any one of claims 1 to 8 in the preparation of medicines or preparations for treating various types of coughs, asthma, upper respiratory tract infections, pharyngitis or rhinitis.

10. Use of the novel menthol eucalyptus oil antitussive spray according to any one of claims 1 to 8 in the preparation of oral cleaning preparations.

11. The use according to claim 9, wherein the cough comprises an inflammation-related cough caused by pathogen infection.

12. The use according to claim 9, 10 or 11, characterized in that: The novel menthol eucalyptus oil antitussive spray is topically administered through the respiratory tract.

13. Use of any one of menthol, eucalyptus oil and eucalyptol as a main active ingredient in the preparation of medicines or preparations for treating various types of coughs, asthma, upper respiratory tract infections, pharyngitis and rhinitis.

14. Use of any one of menthol, eucalyptus oil and eucalyptol as a main active ingredient in the preparation of a preparation for oral cleaning.

15. Use of eucalyptus oil or eucalyptol in combination with menthol in the preparation of medicines or preparations for treating various types of coughs, asthma, upper respiratory tract infections, pharyngitis and rhinitis, wherein the preparation does not contain any active ingredients other than eucalyptol and menthol.

16. Use of eucalyptus oil or eucalyptol in combination with menthol in the preparation of a preparation for oral cleaning, wherein the preparation does not contain any active ingredients other than eucalyptol and menthol.

Images