Uses of Acorus tatarinowii essential oil in the preparation of drugs for the prevention and treatment of sensorineural hearing loss

Abstract

This invention belongs to the field of biomedical technology and discloses the application of Acorus tatarinowii essential oil in the preparation of drugs for the prevention and treatment of sensorineural hearing loss. Using Acorus tatarinowii essential oil as the core active ingredient, the key active substances of Acorus tatarinowii essential oil are identified as β-asarone and isomethyleugenol, which can be prepared into oral formulations and / or ear drops. Animal experiments and human safety tests have confirmed that Acorus tatarinowii essential oil can significantly reduce noise-induced hearing threshold shift, protect the integrity of outer hair cells and the band synaptic function of inner hair cells in the cochlea, inhibit the activation of local inflammatory pathways in the cochlea, and has no toxicity to major organs or skin and tympanic membrane irritation. This invention solves the technical pain points of existing drugs for the prevention and treatment of sensorineural hearing loss, such as poor compliance, significant systemic side effects, and unclear active ingredients, and provides a safe, effective, and scenario-adaptable prevention and treatment solution.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of Acorus tatarinowii essential oil in the preparation of drugs for the prevention and treatment of sensorineural hearing loss. Background Technology

[0002] Sensorineural hearing loss is a group of hearing impairment diseases centered on dysfunction of the cochlear hair cells, auditory nerve, or auditory center. It encompasses various subtypes, including noise-induced hearing loss, sudden deafness, drug-induced hearing loss, and age-related hearing loss, and its pathogenesis is complex and diverse. These diseases are often characterized by insidious or sudden onset, rapid progression, and irreversible pathological damage, severely impacting patients' auditory function and quality of life, and have become a pressing public health issue worldwide.

[0003] Currently, there are still significant limitations in the prevention and treatment of sensorineural hearing loss in clinical practice: the efficacy of systemically administered antioxidant, anti-inflammatory, and neuroprotective drugs is greatly affected by the timing of administration, individual differences, and the severity of injury, and they are prone to causing systemic adverse reactions, leading to poor patient compliance; although local ear administration has the potential advantages of strong targeting and fewer systemic side effects, existing ear drop preparations mostly focus on infectious inflammation or postoperative management, and there is a lack of targeted preparations for the core pathological aspects of sensorineural hearing loss. At the same time, it faces technical bottlenecks such as low tympanic membrane barrier permeability, insufficient drug transmembrane transport efficiency, and poor stability and quantitative controllability of active ingredients.

[0004] In addition, natural products have shown potential in anti-inflammatory, antioxidant and neuroprotective effects due to their multi-component synergistic effects. However, existing research is mostly at the basic level. In the application of sensorineural hearing loss, there are still problems such as unclear effective components and insufficient compatibility of administration methods with ear physiology. There is a lack of systematic and standardized prevention and treatment programs that can meet clinical needs.

[0005] The following common technical challenges exist in the current field of sensorineural hearing loss prevention and treatment: First, there is a lack of portable, highly compliant, and readily applicable topical ear medications suitable for rapid application before and after exposure; second, the targeting and effective exposure of candidate small molecules to key pathological processes in the inner ear have not yet been resolved; and third, there are technical gaps in the identification of active ingredients, reproducible quality control, and stable drug delivery for complex components derived from natural products.

[0006] Therefore, there is an urgent need to provide a prevention and treatment solution based on clearly defined small molecule active ingredients, adapted to the local ear delivery pathway, and with controllable quality, in order to protect the structure and function of the inner ear in noise exposure-related multi-pathway damage. Summary of the Invention

[0007] The purpose of this invention is to provide a solution that addresses the technical pain points of existing drugs for the prevention and treatment of sensorineural hearing loss, such as unclear effective ingredients, significant systemic side effects, insufficient suitability for local ear administration, and difficulty in quality control. The solution uses Acorus tatarinowii essential oil, which has a clearly defined key active ingredient, as the core, and can be administered via both oral and ear drops, thus achieving dual-pathway administration, which is safe, effective, suitable for different scenarios, and has controllable quality.

[0008] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0009] The use of Acorus tatarinowii essential oil in the preparation of drugs for the prevention and treatment of sensorineural hearing loss. The effective active ingredients of the drugs include Acorus tatarinowii essential oil. The key active substances in Acorus tatarinowii essential oil are β-asarone and isomethyleugenol. The drugs include oral preparations and / or ear drops.

[0010] Preferably, sensorineural hearing loss includes noise-induced hearing loss.

[0011] Preferably, the dosage form of the oral formulation includes one of tablets, capsules, soft capsules, oral solutions, self-microemulsion drug delivery systems, or nanoemulsions.

[0012] Preferably, the daily dose of Acorus tatarinowii essential oil in oral preparations is 0.5-200 mg / kg.

[0013] Preferably, the ear drop formulation includes one of ear drops, microemulsion, nanoemulsion, heat-sensitive gel, or ion-sensitive gel.

[0014] Preferably, the content of Acorus tatarinowii essential oil in the ear drops preparation is 0.01%-10%, and the volume of a single dose per ear is 0.02-0.2 mL.

[0015] Preferably, the timing of drug administration includes one of the following: pretreatment 0.5-24 hours before noise exposure, protection during exposure, or early intervention 0-24 hours after exposure.

[0016] Preferably, the oral formulation is administered 1-2 times daily for 1-14 consecutive days.

[0017] Preferably, the ear drops are administered 1-2 times daily for 1-14 consecutive days.

[0018] Preferably, the quality control of the drug adopts a gas chromatography-mass spectrometry fingerprinting and liquid chromatography-mass spectrometry quantitative method, and the similarity of the gas chromatography-mass spectrometry fingerprints of β-asarone and isomethyleugenol in the drug is greater than 0.90.

[0019] This invention clarifies the oral and / or ear drop administration modes of Acorus tatarinowii essential oil, forming a stratified and synergistic precision intervention system targeting the multi-stage pathological mechanisms of sensorineural hearing loss. The oral preparation of Acorus tatarinowii essential oil can address the problem of systemic inflammatory signals being transmitted to the cochlear target organ after noise exposure; the ear drop preparation, leveraging the physicochemical advantages of Acorus tatarinowii essential oil's high oil solubility and small molecular weight, acts directly on the external auditory canal, compensating for the limitations of potentially insufficient local drug concentration and slower onset of action in the cochlea when administered systemically. This method is non-toxic to major organs and causes no skin or tympanic membrane irritation, ensuring systemic safety and covering the entire window of pre-exposure treatment, protection during exposure, and early post-exposure intervention, ultimately significantly improving the overall prevention and treatment effect.

[0020] Preferably, the combination of drugs includes the combination of anti-inflammatory adjuvant drugs or the combination of physical protection.

[0021] Preferably, the anti-inflammatory adjuvant includes one of N-acetylcysteine ​​or a magnesium salt.

[0022] Preferably, physical protection includes either earplugs or earmuffs.

[0023] Preferably, the preparation method of Acorus calamus essential oil includes removing impurities from dried Acorus calamus rhizomes, pulverizing them, soaking them in deionized water for 1-3 hours, distilling them at 95-100℃ for 5-7 hours, allowing the condensate to stand and separate into layers for 3-5 hours, taking the upper oil phase, drying and filtering to obtain Acorus calamus essential oil.

[0024] Preferably, the mass-to-volume ratio of Acorus calamus granules to deionized water is 1 kg: 1-10 L.

[0025] Preferably, the preparation method of Acorus calamus essential oil includes removing impurities from dried Acorus calamus rhizomes, pulverizing them, loading them into a supercritical CO2 extraction vessel, extracting for 2-4 hours, analyzing them through a vacuum separation vessel, purifying them with anhydrous ethanol in a gradient, and rotary evaporating them to obtain Acorus calamus essential oil.

[0026] Preferably, the extraction pressure is 28-32 MPa, the temperature is 38-42 °C, and the CO2 fluid flow rate is 18-22 L / h.

[0027] Preferably, the preparation method of Acorus calamus essential oil includes: removing impurities from dried Acorus calamus rhizomes and pulverizing them; adding 92-96% ethanol solution; reflux extraction at 45-55℃ for 1-3 hours; vacuum distillation at 55-65℃ and 0.06-0.1MPa to recover ethanol and obtain concentrated extract; adding the concentrated extract to deionized water, stirring evenly, extracting with petroleum ether 2-4 times to obtain petroleum ether phase, and vacuum distillation to obtain refined Acorus calamus essential oil.

[0028] Preferably, the mass-to-volume ratio of Acorus tatarinowii granules to ethanol solution is 1 kg: 1-10 L.

[0029] This invention also provides a method for preparing refined Acorus calamus essential oil, comprising:

[0030] After removing impurities from the dried rhizome of Acorus gramineus, it is pulverized into 15-25 mesh particles to obtain Acorus gramineus granules. Deionized water is added to the Acorus gramineus granules and soaked for 1-3 hours. The mixture is then distilled at 95-100℃ for 5-7 hours, and the condensate produced during distillation is collected. The condensate is allowed to stand for 3-5 hours to separate the layers, and the upper oil phase is separated. Anhydrous sodium sulfate is added and the mixture is dried for 10-15 hours. The oil is then filtered through a 0.2-0.25μm organic phase filter membrane to obtain refined Acorus gramineus essential oil.

[0031] Preferably, the mass-to-volume ratio of Acorus calamus granules to deionized water is 1 kg: 1-10 L.

[0032] This invention clarifies that the key active substances in Acorus tatarinowii essential oil are β-asarone and isomethyleugenol. The synergistic effect of the two not only enhances the combined anti-inflammatory and antioxidant effects, but also solves the problem of uncertain mechanism of action caused by the ambiguity of the effective components of natural products. This gives the drug a clear molecular target and a traceable mechanism for protecting against sensorineural hearing loss. When β-asarone and isomethyleugenol are used in combination, their effect on improving noise-induced hearing threshold shift is significantly better than that of using a single component. The hearing threshold protection effect is more prominent in the noise-sensitive mid-to-high frequency range such as 12-32kHz. This also shows that Acorus tatarinowii essential oil has extremely high application value in the preparation of drugs for the prevention and treatment of sensorineural hearing loss.

[0033] This invention also provides a method for preparing refined Acorus calamus essential oil, comprising:

[0034] After removing impurities from the dried rhizome of Acorus gramineus, it was pulverized into 35-45 mesh particles, placed in a supercritical CO2 extraction vessel, and extracted for 2-4 hours. The extracted oil was then separated by a vacuum separation vessel, purified by gradient with anhydrous ethanol, and obtained by rotary evaporation to obtain refined Acorus gramineus essential oil.

[0035] Preferably, the extraction pressure is 28-32 MPa.

[0036] Preferably, the extraction temperature is 38-42℃.

[0037] Preferably, the CO2 fluid flow rate is 18-22 L / h.

[0038] Preferably, the temperature of the separation vessel is 48-52℃ and the pressure is 4.8-5.2MPa.

[0039] This invention also provides a method for preparing refined Acorus calamus essential oil, comprising:

[0040] After removing impurities, the dried rhizomes of Acorus gramineus are pulverized into 15-25 mesh particles to obtain Acorus gramineus granules. A 92-96% ethanol solution is added to the Acorus gramineus granules, and the mixture is refluxed at 45-55℃ for 1-3 hours to obtain an extract. The extract is then subjected to vacuum distillation at 55-65℃ and 0.06-0.1 MPa to recover the ethanol, yielding a concentrated extract. The concentrated extract is added to deionized water, stirred evenly, and extracted 2-4 times with petroleum ether to obtain a petroleum ether phase. This phase is dried over anhydrous magnesium sulfate, and the petroleum ether is removed by vacuum distillation to obtain refined Acorus gramineus essential oil.

[0041] Preferably, the mass-to-volume ratio of Acorus tatarinowii granules to ethanol solution is 1 kg: 1-10 L.

[0042] This invention also provides a method for preparing an oral preparation of Acorus tatarinowii essential oil, comprising:

[0043] Refined Acorus calamus essential oil is mixed with starch, and deionized water is added and stirred to form a uniform soft material. The material is then extruded through a 15-25 mesh sieve to obtain wet granules. The wet granules are dried at 55-65℃ until the moisture content is less than 3%. After being granulated through a 15-20 mesh sieve, they are placed together with microcrystalline cellulose and magnesium stearate into a mixer and mixed at 25-35 rpm for 10-20 minutes. The mixture is then pressed to obtain an oral preparation of Acorus calamus essential oil.

[0044] Preferably, the mass ratio of refined calamus essential oil to starch is 1:10-50.

[0045] Preferably, the mass ratio of refined calamus essential oil to microcrystalline cellulose is 1:10-50.

[0046] Preferably, the mass ratio of refined calamus essential oil to magnesium stearate is 1:0.1-2.

[0047] This invention also provides a method for preparing an ear drop formulation of Acorus tatarinowii essential oil, comprising:

[0048] Polysorbate 80 and propylene glycol were added to deionized water and stirred at 30-40℃ until completely dissolved to form a homogeneous mixed solution. Refined Acorus tatarinowii essential oil was added to the mixed solution and emulsified at 900-1100 rpm for 15-25 min to form a homogeneous emulsion. The pH was adjusted to 6.9-7.1 and the mixture was sterilized by filtration through a 0.2-0.25 μm filter membrane to obtain the Acorus tatarinowii essential oil ear drops preparation.

[0049] Preferably, the mass ratio of refined calamus essential oil to deionized water is 1:10-200.

[0050] Preferably, the mass ratio of refined calamus essential oil to polysorbate 80 is 1:1-10.

[0051] Preferably, the mass ratio of refined calamus essential oil to propylene glycol is 1:10-50.

[0052] This invention utilizes Acorus tatarinowii essential oil, with β-asarone and isomethyleugenol as the key active ingredients, to prepare a drug for preventing noise-induced hearing loss. Therefore, it offers the following beneficial effects: it achieves precise multi-stage intervention in noise-induced inner ear hair cell damage, zoster synaptic lesions, and inflammatory responses. This ensures both accessibility and compliance for systemic medication and rapid onset and targeted enrichment for local application. Furthermore, animal experiments and human safety tests have confirmed that the drug is non-toxic to major organs, non-irritating to the skin and tympanic membrane, and exhibits strong quality control and high batch-to-batch consistency. Therefore, this invention provides a novel prevention and treatment solution for sensorineural hearing loss with a clear mechanism, safety, effectiveness, broad applicability, and ease of industrialization, offering an innovative and feasible technical path for hearing protection in various scenarios, including occupational exposure and recreational activities. Attached Figure Description

[0053] Figure 1 This is a schematic diagram of the overall experimental timeline for Experiment Example 1.

[0054] Figure 2 This is a schematic diagram showing the changes in ABR hearing thresholds at different frequencies in mice of different treatment groups in Experiment Example 1.

[0055] Figure 3 This is a schematic diagram showing the changes in ABRI wave latency in mice of different treatment groups after noise exposure in Experiment Example 1.

[0056] Figure 4 This is a schematic diagram showing the changes in ABRI wave amplitude after noise exposure for different treatment groups in Experiment Example 1.

[0057] Figure 5 This is a schematic diagram showing the morphological changes in different parts of the cochlea in mice of different treatment groups in Experiment Example 1.

[0058] Figure 6 This is a schematic diagram showing the survival rate of outer hair cells in different parts of the cochlea in mice of different treatment groups in Experiment Example 1.

[0059] Figure 7 This is a schematic diagram showing the number of functional banded synapses in the inner hair cells of mice in different parts of the cochlea in different treatment groups of Experiment Example 1.

[0060] Figure 8 This is a schematic diagram showing the changes in body weight of mice in different treatment groups during gavage in Experiment Example 2.

[0061] Figure 9 This is a schematic diagram showing the changes in ABR hearing thresholds at different frequencies in mice of different treatment groups in Experiment Example 2.

[0062] Figure 10This is a schematic diagram showing the histological comparison of major organs in mice of different treatment groups in Experiment Example 2.

[0063] Figure 11 This is a schematic diagram showing the serum alanine aminotransferase levels in mice under different treatment groups in Experiment Example 2.

[0064] Figure 12 This is a schematic diagram showing the serum aspartate aminotransferase levels of mice in different treatment groups in Experiment Example 2.

[0065] Figure 13 This is a schematic diagram of serum urea nitrogen levels in mice under different treatment groups in Experiment Example 2.

[0066] Figure 14 This is a schematic diagram showing the serum creatinine concentrations of mice in different treatment groups in Experiment Example 2.

[0067] Figure 15 This is a schematic diagram showing the changes in blood routine indicators of mice in different treatment groups in Experiment Example 2.

[0068] Figure 16 This is a schematic diagram of the overall experimental timeline for Experiment Example 3.

[0069] Figure 17 This is a schematic diagram showing the changes in ABR hearing thresholds at different frequencies in mice of different treatment groups in Experiment 3.

[0070] Figure 18 This is a schematic diagram showing the changes in ABRI wave latency in mice of different treatment groups after noise exposure in Experiment Example 3.

[0071] Figure 19 This is a schematic diagram showing the changes in ABRI wave amplitude in mice of different treatment groups after noise exposure in Experiment 3.

[0072] Figure 20 This is a schematic diagram showing the morphological changes in different parts of the cochlea in mice of different treatment groups in Experiment 3.

[0073] Figure 21 This is a schematic diagram showing the survival rate of outer hair cells in different parts of the cochlea in mice of different treatment groups in Experiment Example 3.

[0074] Figure 22 This is a schematic diagram showing the number of functional banded synapses in the inner hair cells of mice in different parts of the cochlea in different treatment groups of Experiment Example 3.

[0075] Figure 23 This is a schematic diagram showing the results of gene ontology enrichment analysis of differentially expressed genes in mice of different treatment groups in Experiment Example 3.

[0076] Figure 24 This is a schematic diagram showing the results of pathway enrichment analysis of differentially expressed genes in the cochlea of ​​mice in different treatment groups in Experiment Example 3.

[0077] Figure 25 This is a schematic diagram showing the results of Toll-like receptor signaling pathway enrichment analysis of differentially expressed genes in the cochlea of ​​mice in different treatment groups in Experiment Example 3.

[0078] Figure 26 This is a schematic diagram showing the results of the enrichment analysis of differentially expressed genes in the cochlea of ​​mice in different treatment groups in Experiment Example 3, specifically the TNF signaling pathway.

[0079] Figure 27 This is a schematic diagram of the gas chromatography of Acorus calamus essential oil in Experiment Example 4.

[0080] Figure 28 This is a schematic diagram of the high-performance liquid chromatography (HPLC) of Acorus calamus essential oil in Experiment Example 4.

[0081] Figure 29 This is a schematic diagram of the chemical structure of the two in-ear monomer components detected in Experiment Example 4.

[0082] Figure 30 This is a schematic diagram of the overall experimental timeline for Experiment Example 5.

[0083] Figure 31 This is a schematic diagram showing the auditory brainstem reflex threshold results of mice in the isomethyleugenol group and the β-asarone group of Experiment Example 5.

[0084] Figure 32 This is a schematic diagram showing the auditory brainstem reflex threshold results of mice in the group treated with isomethyleugenol + β-asarone in Experiment Example 5.

[0085] Figure 33 This is a schematic diagram of the latency of wave I of the auditory brainstem reflex in mice of different experimental groups in Experiment 5.

[0086] Figure 34 This is a schematic diagram of the amplitude of wave I of the auditory brainstem reflex in mice of different experimental groups in Experiment 5.

[0087] Figure 35 This is a schematic diagram showing the relative expression level of IL-1β mRNA in BV2 cells after treatment with isomethyleugenol in Experiment Example 6.

[0088] Figure 36 This is a schematic diagram showing the relative expression level of IL-6 mRNA in BV2 cells after treatment with isomethyleugenol in Experiment Example 6.

[0089] Figure 37 This is a schematic diagram showing the relative expression level of TNF-α mRNA in BV2 cells after treatment with isomethyleugenol in Experiment Example 6.

[0090] Figure 38 This is a schematic diagram showing the relative expression level of IL-1β mRNA in BV2 cells after treatment with β-asarone in Experiment Example 6.

[0091] Figure 39 This is a schematic diagram showing the relative expression level of IL-6 mRNA in BV2 cells after treatment with β-asarone in Experiment Example 6.

[0092] Figure 40 This is a schematic diagram showing the relative expression level of TNF-α mRNA in BV2 cells after treatment with β-asarone, as shown in Experiment Example 6.

[0093] Figure 41 This is a schematic diagram of the human local safety evaluation test results of Acorus calamus essential oil in Experiment Example 7.

[0094] Figure 42 This is a schematic diagram of the test results for the safety evaluation of the external auditory canal and tympanic membrane of Acorus tatarinowii essential oil in Experiment Example 8.

[0095] Figure 43 This is a schematic diagram showing the clinical efficacy test results of Acorus tatarinowii essential oil in Experiment Example 9. Detailed Implementation

[0096] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0097] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0098] Example 1:

[0099] Preparation of refined Acorus calamus essential oil: Dried Acorus calamus rhizomes were pulverized to 20 mesh after removing impurities, yielding Acorus calamus granules. Deionized water was added to the granules, and the mixture was soaked for 2 hours. The mixture was then distilled at 100℃ for 6 hours, and the resulting condensate was collected. The condensate was allowed to stand for 4 hours to separate into layers. The upper oil phase was separated, dried with anhydrous sodium sulfate for 12 hours, and then filtered through a 0.22 μm organic phase filter membrane to obtain refined Acorus calamus essential oil. The mass-to-volume ratio of Acorus calamus granules to deionized water was 1 kg: 5 L.

[0100] Preparation of oral preparations of Acorus calamus essential oil: Refined Acorus calamus essential oil was mixed with starch, and deionized water was added and stirred to form a uniform soft material. The mixture was then extruded through a 20-mesh sieve to obtain wet granules. The wet granules were dried at 60℃ until the moisture content was 2%, and then granulated through an 18-mesh sieve. These granules were then mixed with microcrystalline cellulose and magnesium stearate in a mixer at 30 rpm for 15 minutes, and then pressed to obtain the oral preparation of Acorus calamus essential oil. The mass ratio of refined Acorus calamus essential oil to starch was 1:20, the mass ratio of refined Acorus calamus essential oil to microcrystalline cellulose was 1:30, and the mass ratio of refined Acorus calamus essential oil to magnesium stearate was 1:1.

[0101] Preparation of Acorus calamus essential oil ear drops: Polysorbate 80 and propylene glycol were added to deionized water and stirred at 35°C until completely dissolved to form a homogeneous mixture. Refined Acorus calamus essential oil was then added, and the mixture was emulsified at 1000 rpm for 20 minutes to form a homogeneous emulsion. The pH was adjusted to 7.0, and the solution was filtered through a 0.22 μm filter and sterilized to obtain the Acorus calamus essential oil ear drops preparation. The mass ratio of refined Acorus calamus essential oil to deionized water was 1:100, the mass ratio of refined Acorus calamus essential oil to polysorbate 80 was 1:5, and the mass ratio of refined Acorus calamus essential oil to propylene glycol was 1:20.

[0102] Example 2: The only difference between this example and Example 1 is the preparation of refined Acorus calamus essential oil.

[0103] Preparation of refined Acorus calamus essential oil: After removing impurities from dried Acorus calamus rhizomes, they were pulverized to 40 mesh particles and placed in a supercritical CO2 extraction vessel. The extraction pressure was set at 30 MPa, the extraction temperature at 40℃, the CO2 fluid flow rate at 20 L / h, and the extraction time at 3 h. The mixture was then separated by a vacuum separation vessel at 50℃ and 5 MPa. Gradient purification was performed with anhydrous ethanol, followed by rotary evaporation to obtain refined Acorus calamus essential oil.

[0104] Example 3: The only difference between this example and Example 1 is the preparation of refined Acorus calamus essential oil.

[0105] Preparation of refined Acorus calamus essential oil: Dried Acorus calamus rhizomes were pulverized to 20 mesh after removing impurities, yielding Acorus calamus granules. 95% ethanol solution was added to the granules, and the mixture was refluxed at 50℃ for 2 hours to obtain the extract. The ethanol was then recovered by vacuum distillation at 60℃ and 0.08 MPa, yielding a concentrated extract. The concentrated extract was added to deionized water, stirred thoroughly, and extracted three times with petroleum ether to obtain the petroleum ether phase. This phase was dried over anhydrous magnesium sulfate, and the petroleum ether was removed by vacuum distillation to obtain refined Acorus calamus essential oil. The mass-to-volume ratio of Acorus calamus granules to ethanol solution was 1 kg: 5 L.

[0106] Experimental Example 1: Test on the preventive and therapeutic effects of oral administration of Acorus tatarinowii essential oil on noise-induced hearing loss.

[0107] Test sample: The oral preparation of Acorus tatarinowii essential oil prepared in Example 1.

[0108] Test Methods: Six-week-old male C57BL / 6J mice were randomly divided into four groups: a control group, a noise group, a group receiving 50 mg / kg of Acorus tatarinowii essential oil, and a group receiving 100 mg / kg of Acorus tatarinowii essential oil. The 50 mg / kg Acorus tatarinowii essential oil was administered using the oral preparation of Acorus tatarinowii prepared in Example 1, with the essential oil content as the dosage, at a dose of 50 mg / kg. The 100 mg / kg Acorus tatarinowii essential oil was also administered using the oral preparation of Acorus tatarinowii prepared in Example 1, with the essential oil content as the dosage, at a dose of 100 mg / kg. A standard mouse noise-induced hearing loss model was used: bandpass noise (20-20 kHz, approximately 105 dBSPL) was provided by a loudspeaker in a closed sound chamber for 2 consecutive hours; sound pressure was calibrated near the animal's ear using a precision sound level meter. Following baseline ABR testing on Day 1, mice were administered medication daily via gavage according to their group starting from Day 1. Noise exposure was implemented on Day 8. ABR testing was repeated on Day 15 and Day 22. On Day 22, tissue samples were collected for morphological and synaptic assessment, and a short-term prevention and treatment protocol was adopted, continuing until the day of exposure. The ABR test included the presentation of pure tones and click sounds at 4, 8, 12, 16, 24, and 32 kHz using an externally amplified sound wave device under light anesthesia. Repeatable identifiable waveforms were used as threshold criteria, and the thresholds were recorded. Wave I latency and amplitude were recorded at a fixed sound pressure level of 70 dBSPL for auxiliary analysis. Electrodes were placed on the parietal bone, mastoid process behind the ear, and back of the mouse. Morphological assessment included the whole cochlear organ being fluorescently stained, Phalloidin labeling of the stylociliary tracts, and Myosin 7a labeling of hair cell bodies. Imaging was performed in the apical, middle, and basal circles, and the number of surviving OHCs was counted within a preset length and converted into OHC survival rate (%). Synaptic assessment included synaptic co-labeling at the same site, CtBP2 band-shaped presynaptic membrane labeling, and GluR2 postsynaptic membrane AMPA receptor labeling. The number of CtBP2-positive spots and their co-localization with GluR2 were counted per IHC (inner hair cell), expressed as the number of band-shaped synapses per IHC. Each band was counted and statistically analyzed independently. Data were analyzed using one-way or two-way ANOVA with concurrent post-hoc tests. The significance threshold was P < 0.05. * and ** in the figures indicate significant differences.

[0109] The overall experimental timeline of Experiment Example 1 is as follows: Figure 1 The diagram illustrates the sequential administration of Acorus tatarinowii essential oil, noise exposure, auditory brainstem response testing, and sample collection according to time points. The test results of the preventive effect of oral administration of Acorus tatarinowii essential oil on noise-induced hearing loss are shown below. Figure 2-7As shown, the changes in ABR hearing thresholds at different frequencies in mice of different treatment groups are as follows: Figure 2 As shown, the changes in ABRI wave latency after noise exposure in mice of different treatment groups are as follows: Figure 3 As shown, the changes in ABRI wave amplitude in mice of different treatment groups after noise exposure are as follows: Figure 4 As shown; morphological changes in different parts of the cochlea in mice from different treatment groups are as follows. Figure 5 As shown, the survival rate of outer hair cells in different parts of the cochlea of ​​mice in different treatment groups is as follows: Figure 6 As shown, the number of functional band synapses in the inner hair cells of mice in different parts of the cochlea in different treatment groups is as follows: Figure 7 As shown in the figure, the noise group exhibited significant threshold shifts in the 12-24kHz frequency band, which was significantly impaired. In both groups, oral administration of Acorus tatarinowii essential oil significantly reduced the thresholds in all frequency bands, with the most pronounced improvement in the mid-to-high frequencies. Wave I amplitude recovered, and latency approached that of the control, consistent with threshold protection. Furthermore, the noise group showed disordered and missing OHC columns in the mid / base loops. Oral administration of Acorus tatarinowii essential oil significantly improved OHC survival, with more pronounced improvement in the mid and base loops, suggesting structural protection of the cochlear mechanical amplification function. CtBP2-GluR2 colocalization was significantly reduced in the noise group. Oral administration of Acorus tatarinowii essential oil significantly restored the number of banded synapses per IHC and increased the colocalization ratio, most significantly in the mid / base loops, indicating a stabilizing effect on excitotoxicity-related synaptic detachment.

[0110] Experimental Example 2: Safety assessment test of oral preparations of Acorus tatarinowii essential oil.

[0111] Test sample: The oral preparation of Acorus tatarinowii essential oil prepared in Example 1.

[0112] Test Methods: Six-week-old male C57BL / 6J mice were randomly divided into four groups: a control group, a noise group, a group receiving 50 mg / kg of Acorus tatarinowii essential oil, and a group receiving 100 mg / kg of Acorus tatarinowii essential oil. The 50 mg / kg Acorus tatarinowii essential oil was administered using the oral preparation of Acorus tatarinowii prepared in Example 1, with the essential oil content as the dosage (50 mg / kg). The 100 mg / kg Acorus tatarinowii essential oil was also administered using the oral preparation of Acorus tatarinowii prepared in Example 1, with the essential oil content as the dosage (100 mg / kg). All groups received the medication by gavage daily for seven consecutive days starting from day 1, without noise exposure. Daily changes in mouse body weight were recorded. On day 7, an ABR test and organ histological evaluation were performed. The ABR test included the use of an externally amplified acoustic device to present pure tones and click sounds at 4, 8, 12, 16, 24, and 32 kHz under light anesthesia; threshold values ​​were recorded based on repeatable waveforms; the latency and amplitude of wave I were recorded at a fixed sound pressure level of 70 dBSPL for auxiliary analysis; electrodes were placed on the parietal bone, mastoid process behind the ear, and back of the mouse. Organ histological evaluation included the sacrifice of mice, followed by H&E staining of the heart, liver, spleen, lungs, and kidneys; detection of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (CREA) levels; and the measurement of white blood cells, red blood cells, hemoglobin, and platelets as complete blood count indicators. All data were analyzed using univariate or two-way ANOVA with concordant post-hoc tests. The significance threshold was P < 0.05. * and ** in the figures indicate significant differences.

[0113] Safety evaluation tests of oral preparations of Acorus tatarinowii essential oil, such as Figure 8-15 As shown, the changes in body weight of mice in different treatment groups during gavage are as follows: Figure 8 As shown, the changes in ABR hearing thresholds at different frequencies in mice of different treatment groups are as follows: Figure 9 As shown, the histological comparisons of major organs in mice from different treatment groups are as follows: Figure 10 As shown, the serum alanine aminotransferase levels in mice of different treatment groups are as follows: Figure 11 As shown, the serum aspartate aminotransferase levels in mice under different treatment groups are as follows: Figure 12 As shown, the serum urea nitrogen levels of mice in different treatment groups are as follows: Figure 13 As shown, the serum creatinine concentrations of mice in different treatment groups are as follows: Figure 14 As shown, the changes in blood routine indicators of mice in different treatment groups are as follows: Figure 15As shown in the figure. Throughout the entire administration period, different doses of Acorus tatarinowii essential oil had no significant effect on the body weight of mice, indicating that oral administration was well tolerated. ABR analysis showed no significant difference in the hearing threshold at baseline among the control group, noise group, and essential oil group, indicating that the essential oil itself does not affect normal hearing function and has good safety. HE staining results showed no obvious inflammatory infiltration, necrosis, or structural damage in major organs such as the heart, liver, spleen, lungs, and kidneys in the control group, noise group, and essential oil group, suggesting that oral administration of 100 mg / kg of Acorus tatarinowii essential oil does not cause histological abnormalities in major organs. Further blood biochemistry tests showed no significant differences in ALT, AST, blood glucose, and serum creatinine among the groups, suggesting that Acorus tatarinowii essential oil has no adverse effects on liver function, glucose metabolism, and kidney function. Blood routine analysis showed that indicators such as white blood cells, red blood cells, hemoglobin, and platelets were consistent between the essential oil group and the control group, with no statistically significant differences, further demonstrating that oral administration of Acorus tatarinowii essential oil has high systemic safety within the experimental dosage range.

[0114] Experimental Example 3: Test on the preventive and therapeutic effects of Acorus tatarinowii essential oil ear drops on noise-induced hearing loss.

[0115] Test sample: Acorus tatarinowii essential oil ear drops prepared in Example 1.

[0116] Test Methods: Six-week-old male C57BL / 6J mice were randomly divided into four groups: a control group, a noise group, a group receiving 25 mg / kg of Acorus tatarinowii essential oil, and a group receiving 50 mg / kg of Acorus tatarinowii essential oil. The 25 mg / kg Acorus tatarinowii essential oil was administered using the Acorus tatarinowii ear drops preparation prepared in Example 1, with the Acorus tatarinowii essential oil as the dosage, at a dose of 25 mg / kg. The 50 mg / kg Acorus tatarinowii essential oil was also administered using the same preparation, with the Acorus tatarinowii essential oil as the dosage, at a dose of 50 mg / kg. A standard mouse noise-induced hearing loss model was used: bandpass noise (20-20 kHz, approximately 105 dBSPL) was provided by a loudspeaker in a closed sound chamber for 2 consecutive hours; sound pressure was calibrated near the animal's ear using a precision sound level meter. Following baseline ABR testing on Day 1, ear drops were administered daily according to group assignments, with 2 μL instilled bilaterally into each external auditory canal each time. Noise exposure was implemented on Day 8. ABR testing was repeated on Day 15 and Day 22. On Day 22, tissue samples were collected for morphological and synaptic assessment, and a short-term prevention and treatment regimen was adopted, continuing until the day of exposure. Tympanic membrane integrity was confirmed by microscopic examination at the endpoint, with no perforation or significant effusion. The methods for noise exposure, ABR testing, external hair cell assessment, and banded synapse assessment were the same as in Case 1. After drug administration and noise exposure in each experimental group, mice were sacrificed at the endpoint time point. Bilateral cochleas were rapidly isolated, and the cochlea was peeled off and flash-frozen in liquid nitrogen. Total RNA was extracted from the cochlea using TRIzol reagent, and its concentration and purity were determined. Qualified samples were sent to a professional sequencing company to construct strand-specific mRNA libraries, and paired-end sequencing was performed on a high-throughput platform. After removing adapters and low-quality reads, the data were aligned to the mouse reference genome mm10 and gene quantification was performed. Differential expression analysis was conducted using DESeq2, and GO, KEGG, and GSEA pathway enrichment analyses were performed on differentially expressed genes. All data were analyzed using one-way or two-way ANOVA with concurrent post-hoc tests. The significance threshold was P < 0.05. "*" and "**" in the figures indicate significant differences.

[0117] The overall experimental timeline of Experiment Example 3 is as follows: Figure 16 The diagram shows the sequence of events, including ear drops of Acorus calamus essential oil, noise exposure, auditory brainstem response testing, and sample collection. The test results for the preventative effect of oral ear drops of Acorus calamus essential oil on noise-induced hearing loss are as follows: Figure 17-26 As shown, the changes in ABR hearing thresholds at different frequencies in mice of different treatment groups are as follows: Figure 17 As shown, the changes in ABRI wave latency after noise exposure in mice of different treatment groups are as follows: Figure 18 As shown, the changes in ABRI wave amplitude in mice of different treatment groups after noise exposure are as follows: Figure 19 As shown, the morphological changes in different parts of the cochlea in mice from different treatment groups are as follows: Figure 20As shown, the survival rate of outer hair cells in different parts of the cochlea of ​​mice in different treatment groups is as follows: Figure 21 As shown, the number of functional band synapses in the inner hair cells of mice in different parts of the cochlea in different treatment groups is as follows: Figure 22 As shown, the gene ontology enrichment analysis results of differentially expressed genes in mice from different treatment groups are as follows: Figure 23 As shown, the Kyoto Gene and Genome Encyclopedia pathway enrichment analysis results of differentially expressed genes in the cochlea of ​​mice in different treatment groups are as follows: Figure 24 As shown, the Kyoto Gene and Genome Encyclopedia pathway enrichment analysis results of differentially expressed genes in the cochlea of ​​mice in different treatment groups are as follows: Figure 25 As shown in the figure, the results of the enrichment analysis of differentially expressed genes in the cochlea of ​​mice in different treatment groups using the TNF signaling pathway are as follows: Figure 26 As shown in the figure, the noise group exhibited a significant threshold shift in the 12-32kHz range; both doses of Acorus tatarinowii essential oil ear drops significantly reduced the threshold, with P < 0.05 at multiple frequencies, showing the most significant improvement in the mid-to-high frequency range; simultaneously, wave I latency shortened and amplitude rebounded, consistent with the threshold improvement. Disordered and missing OHC arrangement was observed in the Middle / Base loops of the noise group; Acorus tatarinowii essential oil ear drops significantly improved OHC survival, with more pronounced improvement in the Middle and Base loops, suggesting structural protection of the cochlear mechanical amplification unit. CtBP2-GluR2 colocalization per IHC was significantly reduced in the noise group; Acorus tatarinowii essential oil ear drops dose-dependently restored synapse count, with increases observed in Apex, Middle, and Base loops, indicating a stabilizing effect on excitotoxicity-related synaptic detachment. GO enrichment analysis showed that noise exposure significantly activated typical inflammation-related biological processes such as "response to exogenous stimuli," "immune response," "response to cytokine stimulation," and "defense response." Ear drops of Acorus tatarinowii essential oil significantly downregulated these inflammation-related genes, suggesting its inhibitory effect on local cochlear inflammation. KEGG pathway analysis further revealed that noise-induced activation of inflammatory cascade pathways, including the TNF signaling pathway, IL-17 signaling pathway, NOD-like receptor pathway, and cytokine-receptor interaction; ear drops of Acorus tatarinowii essential oil downregulated the genes involved in these key pathways overall, indicating a broad-spectrum inhibitory effect on noise-induced immune-inflammatory responses. GSEA enrichment analysis showed that the NES values ​​of the Toll-like receptor signaling pathway mmu04620 and the TNF signaling pathway mmu04668 were also negatively enriched, both suggesting that Acorus tatarinowii essential oil can overall inhibit the activation trend of noise-induced inflammatory genes.

[0118] Experimental Example 4: Confirmation Test of the Composition of Acorus calamus Essential Oil and its Inhalation Components.

[0119] Test sample: Acorus tatarinowii essential oil ear drops prepared in Example 1.

[0120] Test Methods: Six-week-old male C57BL / 6J mice were randomly divided into two groups of equal numbers, including a control group and a group receiving 50 mg / kg of Acorus tatarinowii essential oil. The 50 mg / kg Acorus tatarinowii essential oil was administered using the Acorus tatarinowii ear drop preparation prepared in Example 1, with the essential oil content as the dosage, at a dose of 50 mg / kg. Administered once daily, 2 μL was instilled into each ear canal. After several days of continuous ear drops, the animals were sacrificed and the cochlea was collected. During the ear drops, the tympanic membrane integrity was confirmed using an otoscope, with no perforation or exudation. The components of the Acorus tatarinowii essential oil were identified using gas chromatography-mass spectrometry and high-performance liquid chromatography-mass spectrometry. The cochlear components after ear drops were verified using headspace gas genomics. 2 μL of the Acorus tatarinowii ear drop preparation prepared in Example 1 was instilled into both external auditory canals of the mice. At the endpoint, the animals were sacrificed and the cochleas were rapidly separated. The local components of the cochlea were detected using headspace gas genomics.

[0121] The results of confirmatory tests on the components of Acorus calamus essential oil and its components that can be inhaled are as follows: Figure 27-29 As shown, the gas chromatogram of Acorus calamus essential oil is as follows: Figure 27 As shown in the figure, the high performance liquid chromatography of Acorus calamus essential oil is as follows: Figure 28 As shown, the chemical structural diagrams of the two detected in-ear monomer components are as follows: Figure 29 As shown in the figure. GC-MS and HPLC-MS confirmed that Acorus tatarinowii essential oil contains major volatile aromatic components such as β-asarone and isomethyleugenol. The characteristic responses of β-asarone and isomethyleugenol were also observed in the cochlear tissue separated after the drug was applied.

[0122] Experimental Example 5: Test on the preventive and therapeutic effects of ear drops containing the ear-ingesting component of Acorus tatarinowii essential oil on noise-induced hearing loss.

[0123] Test sample: Refined Acorus calamus essential oil prepared in Example 1.

[0124] Test Methods: Six-week-old male C57BL / 6J mice were randomly divided into four groups: a control group, a noise group, an isomethyleugenol group, a β-asarone group, and an isomethyleugenol + β-asarone combination group. A standard mouse noise-induced hearing loss model was used: bandpass noise (20-20 kHz, approximately 105 dBSPL) was provided in a closed acoustic chamber via loudspeaker for 2 hours; sound pressure was calibrated near the animal's ear using a precision sound level meter. After baseline ABR testing on Day 1, ear drops (2 μL per ear) were administered daily according to the group, with each ear receiving bilateral external auditory canal drops. In the isomethyleugenol + β-asarone combination group, the volume ratio of isomethyleugenol to β-asarone was 1:1. Noise exposure was implemented on Day 8. ABR testing was repeated on Day 15 and Day 22. On Day 22, tissue samples were collected for morphological and synaptic assessment, and a short-term prevention and treatment regimen was implemented until the day of exposure. The integrity of the tympanic membrane was confirmed by end-point microscopic examination, with no perforation or significant effusion. The ABR test method was the same as in Case 3. All data were analyzed using one-way or two-way ANOVA with concurrent post-hoc tests. The significance threshold was P < 0.05. "*" and "**" in the figure indicate significant differences.

[0125] The overall experimental timeline for Example 3 is shown in Figure 30; the test results of the ear drops containing the ear-derived component of Acorus tatarinowii essential oil on the prevention and treatment of noise-induced hearing loss are shown in Figure 30. Figures 31-34 As shown, the auditory brainstem reflex thresholds of mice in the isomethyleugenol group and the β-asarone group are as follows: Figure 31 As shown, the auditory brainstem reflex thresholds of mice in the isomethyleugenol + β-asarone combination group are as follows: Figure 32 As shown, the latency of auditory brainstem reflex wave I in mice of different experimental groups is as follows: Figure 33 As shown, the amplitude of auditory brainstem reflex wave I in mice from different experimental groups is as follows: Figure 34 As shown, ABR pure tone threshold protection showed a significant threshold shift in the noise group from 12 to 32 kHz; both isomethyleugenol and β-asarone significantly reduced the threshold with a single drop, and the threshold shift was better when isomethyleugenol and β-asarone were used in combination than when used alone, indicating a synergistic effect; at the same time, the latency of wave I was shortened and the amplitude was restored, consistent with the improvement in the threshold.

[0126] Experimental Example 6: Test on the inhibitory effect of active ingredients of Acorus tatarinowii essential oil on the inflammatory response of BV2 cells.

[0127] Test sample: Refined Acorus calamus essential oil prepared in Example 1.

[0128] Test Methods: BV2 microglia were cultured in DMEM high-glucose medium containing 10% FBS and 1% antibiotics at 37℃ in a 5% CO2 incubator. Cells in the exponential growth phase were seeded into 6-well plates. A 0 μM drug concentration was used as the blank control group, while 50 μM and 100 μM were used as drug groups. The drugs used in the drug groups included isomethyleugenol and β-asarone. After 2 h of drug pretreatment, 1 μg / mL LPS was added to co-stimulate the cells with the corresponding drug concentrations for 6 h. The blank control group did not receive LPS. Total RNA was extracted from the cells, and the mRNA expression levels of three inflammatory factors (IL-1β, IL-6, and TNF-α) were detected by qPCR. GAPDH was used as an internal control for normalization. One-way ANOVA was used, with Tukey post-hoc test. The significance threshold was set at P < 0.05. "*" and "**" in the figure indicate statistical differences.

[0129] The test results of the inhibitory effect of the active ingredients of Acorus tatarinowii essential oil on the inflammatory response of BV2 cells are as follows: Figure 35-40 As shown, the relative expression levels of IL-1β mRNA in BV2 cells after treatment with isomethyleugenol are as follows: Figure 35 As shown, the relative expression levels of IL-6 mRNA in BV2 cells after treatment with isomethyleugenol are as follows: Figure 36 As shown, the relative expression levels of TNF-α mRNA in BV2 cells after treatment with isomethyleugenol are as follows: Figure 37 As shown, the relative expression levels of IL-1β mRNA in BV2 cells after treatment with β-asarone are as follows: Figure 38 As shown, the relative expression levels of IL-6 mRNA in BV2 cells after treatment with β-asarone are as follows: Figure 39 As shown, the relative expression levels of TNF-α mRNA in BV2 cells after treatment with β-asarone are as follows: Figure 40 As shown, after LPS stimulation, the expression levels of IL-1β, IL-6, and TNF-α mRNA in the blank control group were significantly upregulated; isomethyleugenol alone significantly inhibited the expression of the above inflammatory factors, showing a dose-dependent inhibition trend; isomethyleugenol had a strong inhibitory effect on the three inflammatory factors, and β-asarone had a more significant inhibitory effect on IL-1β and IL-6, and neither of them affected the basic cellular inflammation level; the inflammatory response is a key pathological link in sensorineural hearing loss, and most existing anti-inflammatory drugs are administered systemically, while the active ingredient of this invention can exert its anti-inflammatory effect through local administration, and the mechanism is clear, providing molecular-level evidence for the anti-inflammatory effect of the formulation.

[0130] Experimental Example 7: Evaluation Test of Local Safety of Acorus calamus Essential Oil in Humans.

[0131] Test sample: Refined Acorus calamus essential oil prepared in Example 1.

[0132] Test Method: Twenty healthy adult volunteers aged 18-60 years with no history of skin diseases or plant essential oil allergies were selected. Informed consent was obtained before the experiment. A skin area with a diameter of 1.5-2 cm behind the ear was selected as the test point, and the skin condition before application was photographed. The refined Acorus calamus essential oil prepared in Example 1 was applied evenly to the test area with a cotton swab. After natural drying, skin changes were observed and photographed at 30 minutes and 24 hours after application. The focus was on assessing whether there were any irritation reactions such as erythema, swelling, papules, itching, exudation, and pigmentation.

[0133] The results of the local safety evaluation test of Acorus calamus essential oil on the human body are as follows: Figure 41 As shown, after applying Acorus calamus essential oil, the skin appearance of the test area remained consistent with that before application: no erythema, swelling, or damage; no subjective discomfort such as itching or pain; no exudation or irritating skin lesions; and the skin color returned to normal after 24 hours with no pigmentation. Acorus calamus essential oil exhibits good biocompatibility with human skin and did not cause irritation or allergic reactions. After contact with the essential oil, the external auditory canal and tympanic membrane structures remained intact, and no tympanic membrane congestion, opacity, or irritating patches were observed; no edema, secretions, or erosion of the external auditory canal skin was observed; and no subjective discomfort such as burning, pain, or a feeling of muffled hearing was observed; the tympanic membrane surface remained consistent with the baseline state after administration. The results indicate that Acorus calamus essential oil has good tolerance and biocompatibility with human skin, and its local application in the external auditory canal and tympanic membrane demonstrates good safety, without causing irritation or damage, meeting the skin safety requirements for topical medication.

[0134] Experimental Example 8: Safety Evaluation Test of Acorus calamus essential oil in the external auditory canal and tympanic membrane.

[0135] Test sample: Acorus tatarinowii essential oil ear drops prepared in Example 1.

[0136] Test method: Twenty healthy adult volunteers with the same conditions as in Experiment 7 were selected. The Acorus tatarinowii essential oil ear drops prepared in Example 1 were applied to the ear canal entrance of the subjects using a gelatin sponge, allowing the essential oil to naturally adhere to the skin of the ear canal. The condition of the ear canal and tympanic membrane before administration was photographed with an otoscope. The essential oil was kept in contact for 10-15 minutes, and the local condition was recorded during administration. After the essential oil was removed, the condition of the ear canal and tympanic membrane surface was photographed again to assess whether adverse changes such as erythema, congestion, edema, exudation, erosion or perforation occurred.

[0137] The safety evaluation test results of Acorus calamus essential oil in the external auditory canal and tympanic membrane are as follows: Figure 42As shown, after contact with Acorus tatarinowii essential oil, the external auditory canal and tympanic membrane structures remained intact in all subjects. No tympanic membrane congestion, opacity, or irritation patches were observed. There was no edema, secretions, or erosion of the skin in the external auditory canal, and subjects experienced no subjective discomfort such as burning, pain, or a feeling of fullness in the ears. The tympanic membrane surface remained consistent with the baseline condition after administration. These results confirm that Acorus tatarinowii essential oil has good safety when applied topically to the external auditory canal and tympanic membrane, and does not cause irritation or damage. The safety of the tympanic membrane barrier is a core requirement for ear drops. Some existing ear drops may cause tympanic membrane irritation or damage. This invention addresses concerns about the local safety of ear drops through targeted human trials.

[0138] Experimental Example 9: Clinical efficacy test of Acorus calamus essential oil.

[0139] Test sample: Acorus tatarinowii essential oil ear drops prepared in Example 1.

[0140] Methods: Ninety patients with sudden deafness who were hospitalized in the ENT department of a hospital in the past year were selected. All patients met the Western medicine diagnostic criteria in the "Guidelines for the Diagnosis and Treatment of Sudden Deafness (2015)" and the TCM syndrome differentiation criteria in "Traditional Chinese Medicine Otorhinolaryngology". They were aged 18-70 years, regardless of gender, with normal function of important organs such as liver and kidneys, and signed informed consent forms and voluntarily participated in this study. Patients who were pregnant or lactating, had difficulty communicating or were unwilling to cooperate with the examination, had unclear syndrome types or complex syndromes, had other sensorineural hearing loss with clear etiologies, had organic diseases such as middle ear lesions or acoustic neuroma, had relevant treatment contraindications, or did not agree to sign informed consent forms were excluded. Patients were randomly divided into three groups: the experimental group and the control group, with 30 patients in each group. The control group received standard Western medicine treatment: 10 μg of alprostadil (a circulation-improving drug) was administered intravenously once daily, along with 40 mg of monosialotetrahexosylganglioside (a neurotrophic drug) administered intravenously once daily. Systemic corticosteroids, intratympanic corticosteroids, and batroxobin were added, either alone or in combination, depending on the patient's condition. The experimental group received standard Western medicine treatment plus ear drops of refined Acorus tatarinowii essential oil prepared in Example 1, applied to the affected ear. 0.6 ml of the Acorus tatarinowii essential oil ear drops were soaked in a 2×2 mm gelatin sponge and placed in the cartilaginous part of the external auditory canal, changing the drops daily. Both groups received the same nursing care, and the treatment course was 2 weeks. If the patient's hearing recovered to pre-illness levels during treatment, the treatment was terminated, and the patient was counted as cured. The pure-tone audiometry thresholds and average thresholds of damaged frequencies at each frequency were recorded before and after treatment for each group. "*" in the figure indicates statistical differences.

[0141] Clinical efficacy test results of Acorus calamus essential oil are as follows: Figure 43As shown, after 2 weeks of treatment, the control group showed that the pure tone audiometry threshold changes were concentrated in the low improvement range; the experimental group showed that the threshold changes were significantly higher than those in the control group, with most patients showing improvement in the range of 20 to 40 dB. The difference in pure tone audiometry threshold changes between the two groups was statistically significant, and the average threshold improvement in the experimental group was significantly better than that in the control group, confirming that the hearing improvement effect of Acorus tatarinowii essential oil combined with standard Western medicine treatment was more prominent.

[0142] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0143] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. The use of Acorus tatarinowii essential oil in the preparation of medicines for the prevention and treatment of sensorineural hearing loss, characterized in that: The effective active ingredient of the drug is Acorus tatarinowii essential oil, and the key active substances of Acorus tatarinowii essential oil are β-asarone and isomethyleugenol. The drug is an oral preparation and / or an ear drop preparation, and the sensorineural hearing loss is noise-induced hearing loss. The preparation method of the ear drops is as follows: Polysorbate 80 and propylene glycol are added to deionized water and stirred at 30-40℃ until completely dissolved to form a homogeneous mixed solution; refined Acorus tatarinowii essential oil is added to the mixed solution and emulsified at 900-1100 rpm for 15-25 min to form a homogeneous emulsion; the pH is adjusted to 6.9-7.1, and the mixture is filtered and sterilized through a 0.2-0.25μm filter membrane to obtain the Acorus tatarinowii essential oil ear drops preparation. The oral preparation is prepared by mixing refined Acorus calamus essential oil with starch, adding deionized water and stirring to form a uniform soft material, extruding and granulating through a 15-25 mesh sieve to obtain wet granules, drying the wet granules at 55-65℃ until the moisture content is less than 3%, granulating through a 15-20 mesh sieve, and then mixing them together with microcrystalline cellulose and magnesium stearate in a mixer at 25-35 rpm for 10-20 minutes, and pressing to obtain the Acorus calamus essential oil oral preparation.

2. The use of Acorus tatarinowii essential oil according to claim 1 in the preparation of a medicine for preventing and treating sensorineural hearing loss, characterized in that: The oral preparation is in the form of tablets or capsules, and the daily dose of Acorus tatarinowii essential oil in the oral preparation is 0.5-200 mg / kg.

3. The use of Acorus tatarinowii essential oil according to claim 1 in the preparation of a medicine for preventing and treating sensorineural hearing loss, characterized in that: The dosage form of the ear drops is one of ear drops, microemulsion and nanoemulsion, and the content of Acorus tatarinowii essential oil in the ear drops is 0.01%-10%, with a single dose volume of 0.02-0.2 mL per ear.

4. The use of Acorus tatarinowii essential oil according to claim 1 in the preparation of a medicine for preventing and treating sensorineural hearing loss, characterized in that: The timing of drug administration includes one of the following: pretreatment 0.5-24 hours before noise exposure, protection during exposure, or early intervention 0-24 hours after exposure.

5. The use of Acorus tatarinowii essential oil according to claim 1 in the preparation of a medicine for preventing and treating sensorineural hearing loss, characterized in that: The oral preparation is administered 1-2 times daily for 1-14 consecutive days; the ear drops are administered 1-2 times daily for 1-14 consecutive days.

6. The use of Acorus tatarinowii essential oil according to claim 1 in the preparation of a medicine for preventing and treating sensorineural hearing loss, characterized in that: The quality control of the drug was performed using a combination of gas chromatography-mass spectrometry fingerprinting and liquid chromatography-mass spectrometry quantitative analysis. The similarity of the gas chromatography-mass spectrometry fingerprints of β-asarone and isomethyleugenol in the drug was greater than 0.

90.

7. The use of Acorus tatarinowii essential oil according to claim 1 in the preparation of a medicine for preventing and treating sensorineural hearing loss, characterized in that: The combination of the drugs includes the combination of anti-inflammatory adjuvants or the combination of physical protection. The anti-inflammatory adjuvants include one of N-acetylcysteine ​​or magnesium salt, and the physical protection includes one of earplugs or earmuffs.

8. The use of Acorus tatarinowii essential oil according to claim 1 in the preparation of a medicine for preventing and treating sensorineural hearing loss, characterized in that: The method for preparing Acorus calamus essential oil includes removing impurities from dried Acorus calamus rhizomes, pulverizing them, soaking them in deionized water for 1-3 hours, distilling them at 95-100℃ for 5-7 hours, allowing the condensate to stand and separate into layers for 3-5 hours, taking the upper oil phase, drying and filtering to obtain Acorus calamus essential oil. The mass-to-volume ratio of Acorus calamus granules to deionized water is 1 kg: 1-10 L.

9. The use of Acorus tatarinowii essential oil according to claim 1 in the preparation of a medicine for preventing and treating sensorineural hearing loss, characterized in that: The method for preparing Acorus calamus essential oil includes removing impurities from dried Acorus calamus rhizomes, pulverizing them, placing them in a supercritical CO2 extraction vessel, extracting for 2-4 hours, analyzing the extract through a vacuum separation vessel, purifying them with anhydrous ethanol using a gradient, and rotary evaporating to obtain Acorus calamus essential oil. The extraction pressure is 28-32 MPa, the temperature is 38-42℃, and the CO2 fluid flow rate is 18-22 L / h.

10. The use of Acorus tatarinowii essential oil according to claim 1 in the preparation of a medicine for preventing and treating sensorineural hearing loss, characterized in that: The method for preparing Acorus calamus essential oil includes: removing impurities from dried Acorus calamus rhizomes and pulverizing them; adding a 92-96% ethanol solution; extracting under constant temperature reflux at 45-55℃ for 1-3 hours; and recovering the ethanol by vacuum distillation at 55-65℃ and 0.06-0.1MPa to obtain a concentrated extract; adding the concentrated extract to deionized water, stirring evenly, and extracting with petroleum ether 2-4 times to obtain the petroleum ether phase; and distilling under vacuum to obtain refined Acorus calamus essential oil, wherein the mass-to-volume ratio of Acorus calamus granules to ethanol solution is 1 kg: 1-10 L.

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