Application of 6-hydroxyflavanone and 6-hydroxyflavone in the prevention and treatment of noise-induced hearing loss

Abstract

This invention discloses the application of 6-hydroxyflavanone and 6-hydroxyflavone in the prevention and treatment of noise-induced hearing loss. By constructing a mouse model of noise-induced hearing loss and using the auditory brainstem response threshold (ABR) as an evaluation index, the protective effects of 6-hydroxyflavanone and 6-hydroxyflavone on noise-induced hearing loss were verified for the first time. Experimental results show that prophylactic administration of 6-hydroxyflavanone at doses of 30-60 mg / kg significantly reduced the mid-to-high frequency ABR threshold after noise exposure, with an effective dose as low as 30 mg / kg. 6-hydroxyflavanone and 6-hydroxyflavone not only have preventative protective effects but also show significant hearing restoration effects in therapeutic administration after noise exposure, with significant therapeutic effects in the 8 kHz core damage frequency band. This invention fills the technological gap in the field of hearing protection for these two compounds, offering advantages such as low-dose effectiveness and dual preventative and therapeutic efficacy, providing a novel natural drug solution for the prevention and treatment of noise-induced hearing loss.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to 6-hydroxyflavanone and its application in the prevention and treatment of noise-induced hearing loss. Background Technology

[0002] Noise pollution-induced hearing loss (NIHL) is a prevalent type of sensorineural hearing loss worldwide. Studies have shown that more than 450 million people worldwide suffer from disabling hearing loss, of which approximately 16% of adult disabling hearing loss is attributable to occupational noise exposure.

[0003] Existing research indicates that NADPH oxidase 3 (NOX3), highly expressed in the cochlear organ of Corti, is a major source of reactive oxygen species (ROS). Noise first activates the transcription factor STAT1, triggering the synergistic activation of the TRPV1 ion channel and the NOX3 signaling pathway to generate large amounts of ROS. Simultaneously, noise damage to the stria vascularis causes local ischemia-reperfusion in the cochlea, further activating NOX3 and releasing inflammatory factors such as IL-1β. This leads to a vicious cycle of "oxidative stress-inflammation" where excessive ROS and inflammatory factors mutually promote each other. Noise damage to hair cells is regionally selective, primarily affecting the outer hair cells at the base of the cochlea (corresponding to high-frequency hearing), which is related to their high OCT2 expression and weak antioxidant capacity. Inner hair cells are relatively tolerant. The direct damage of ROS to hair cells manifests in two ways: firstly, it leads to cell membrane lipid peroxidation; secondly, it induces oxidative damage to hair cell DNA. Both of these factors jointly disrupt cell membrane integrity, leading to sensorineural hearing loss. Furthermore, ROS induces hair cell apoptosis through multiple pathways to accelerate the damage process, including two caspase-dependent pathways: the endogenous pathway of mitochondrial release of cytochrome c activating caspase-9 and the exogenous pathway of death receptor activation of caspase-8, as well as the caspase-independent pathway mediated by AIF / EndoG. It also activates the MAPK / JNK pathway. Ultimately, the combined effect of ROS and inflammatory factors leads to massive hair cell death, resulting in hearing loss. Therefore, intervening in the noise-mediated inflammatory and oxidative stress cascade, reducing blood-labyrinthine barrier disruption, and inhibiting apoptosis activation pathways provide important directions for reducing noise-induced hearing loss and are crucial for the prevention and treatment of noise-induced hearing loss.

[0004] Vitamin E can reduce noise damage to cochlear cells. As a lipid-soluble free radical scavenger, it can inhibit lipid peroxidation, scavenge reactive oxygen species (ROS), and protect unsaturated fatty acids in membrane phospholipids from free radical damage, maintaining the structural and functional integrity of cochlear cells without significant toxic side effects. However, vitamin E is limited by its single mechanism of action, limited bioavailability, and lack of multi-target synergistic regulation.

[0005] Flavonoids, as natural compounds, including puerarin and quercetin, have attracted attention due to their multiple protective mechanisms and have shown potential value in improving noise-induced hearing loss. Studies have shown that flavonoids possess antioxidant properties, primarily manifested in their ability to reduce the activation of oxidative stress pathways by regulating the concentrations of superoxide dismutase (SOD) and catalase (CAT) and scavenging reactive oxygen species (ROS) in the body. More importantly, they can activate the Nrf2-Keap1 signaling pathway in cochlear tissue, upregulate the expression of endogenous antioxidant enzymes such as heme oxygenase-1 (HO-1) and quinone oxidoreductase (NQO1), and specifically inhibit the activity of cochlear NADPH oxidase 3 (NOX3, a core source of ROS in noise), further blocking ROS-mediated oxidative damage to cochlear hair cells and stria vascularis cells. In addition, flavonoids exert significant anti-inflammatory effects by inhibiting the nuclear factor NF-κB pathway and reducing the concentration of inflammatory factors (such as interleukin IL-1β and IL-6)

[20] . At the same time, they can reduce the release of tumor necrosis factor α (TNF-α), inhibit the noise-activated STAT1-TRPV1 inflammatory signaling pathway, avoid inflammatory damage to cochlear spiral ganglion neurons caused by glial cell activation, protect the structural integrity of cochlear stria vascularis epithelial cells, enhance the stability of the blood-labyrinth barrier, reduce noise entering the inner ear endolymph, maintain endolymph ion balance, and reduce noise damage to the auditory system from multiple aspects.

[0006] Among existing interventions, antioxidants such as vitamin E have shown some effectiveness, but they suffer from limitations such as a single mechanism of action and limited bioavailability. Flavonoids, natural compounds, have attracted attention due to their multiple protective mechanisms (antioxidant, anti-inflammatory, etc.), with quercetin and puerarin already being studied for the prevention and treatment of NIHL. However, current research largely focuses on these known components, and suffers from problems such as poor water solubility, low bioavailability, and difficulties in clinical translation. More importantly, the application of two specific flavonoid monomers, 6-hydroxyflavanone and 6-hydroxyflavone, in the field of hearing protection has not yet been reported. Summary of the Invention

[0007] This invention is based on a key clinical finding: in the metabolomics analysis of blood samples from patients with sudden deafness, the inventors surprisingly found significantly elevated levels of 6-hydroxyflavanone in the patients' blood. This phenomenon suggests that this compound may have an intrinsic link with hearing protection. Based on this, the inventors constructed a standard noise-induced hearing loss (NIHL) mouse model and systematically validated the hearing-protective effects of 6-hydroxyflavanone and 6-hydroxyflavone in vivo, using the auditory brainstem response (ABR) threshold as the core evaluation indicator.

[0008] Noise exposure conditions: All experiments used narrowband noise with an intensity of 120 dB SPL and a center frequency of 6 kHz for 2 hours to establish a stable NIHL mouse model.

[0009] ABR threshold detection: On day 7 after noise exposure (or at a specified time point), the ABR threshold of mice in each group was detected using the auditory brainstem response system. The detection frequencies included click, 4 kHz, 8 kHz, 16 kHz and 24 kHz.

[0010] First, this invention verifies the preventive protective effect of 6-hydroxyflavanone.

[0011] In one specific embodiment of the present invention (see Example 1), intraperitoneal injection of 60 mg / kg of 6-hydroxyflavanone for three consecutive days prior to noise exposure significantly reduced the ABR threshold of mice at all frequencies after noise exposure. Experimental results showed that the ABR threshold of mice in the 60 mg / kg flavanone prevention group was significantly reduced across the entire frequency band, especially in the mid-to-high frequency bands (4k, 8k, 16k), where the protective effect was extremely significant. In the 8 kHz frequency band, the average threshold of the flavanone prevention group was 51.7 dB, a reduction of 23.3 dB compared to the noise model group (75 dB) (see Example 1). Figure 2 (and Table 1-1).

[0012] In another specific embodiment of the invention (see Example 2), the dosage of 6-hydroxyflavanone was reduced to 30 mg / kg and administered intraperitoneally for three consecutive days prior to noise exposure. Experimental results showed that the average threshold in the 8 kHz frequency band was approximately 55-60 dB in the 30 mg / kg group, which was not statistically significantly different from the 60 mg / kg group (approximately 51.7 dB) (see Example 2). Figure 3-4 (See Tables 2 and 3). This indicates that 30 mg / kg of 6-hydroxyflavanone can exert a significant hearing protection effect, and there is no obvious dose dependence within this dosage range.

[0013] In another specific embodiment of the invention (see Example 3), a prophylactic dosing experiment at 60 mg / kg was repeated. The results showed that the protective effect was reproducible, but there were some individual differences and batch-to-batch variations (see Example 3). Figure 5 (and Table 3-1).

[0014] Secondly, this invention is the first to verify the dual preventive and therapeutic effects of 6-hydroxyflavone.

[0015] In one specific embodiment of the present invention (see Example 4), intraperitoneal injection of 60 mg / kg of 6-hydroxyflavone for three consecutive days prior to noise exposure showed that it could reduce the mid-to-high frequency ABR threshold, demonstrating a preventative protective effect (see Example 4). Figure 6 (and Table 4).

[0016] In another, more important embodiment of the invention (see Example 5), not only was a prophylactic dose of 60 mg / kg administered 3 days before noise exposure, but a therapeutic dose was also administered after the noise exposure, with continuous intraperitoneal injection of 60 mg / kg 6-hydroxyflavone once daily for one week. Surprisingly, with the additional administration after noise exposure, the ABR threshold in the 6-hydroxyflavone treatment group was significantly lower than that in the noise model group and the solvent control group. Particularly in the 8 kHz core damage band, the average threshold in the treatment group decreased to 60 dB, a reduction of 15 dB compared to the noise group (75 dB), an effect even superior to the effect of its own prophylactic administration (see Example 5). Figure 7 (and Table 5).

[0017] Compared with the prior art, the present invention has the following outstanding advantages:

[0018] (1) Filling a technological gap: This invention discloses for the first time a new use of 6-hydroxyflavanone and 6-hydroxyflavone in the prevention and treatment of noise-induced hearing loss. A search revealed no patent or non-patent literature reports on their use for hearing protection in the prior art, making this an original discovery.

[0019] (2) Low-dose efficacy: Experiments have shown that 6-hydroxyflavanone can exert a significant protective effect at a low dose of 30 mg / kg, and the effect is comparable to that at a dose of 60 mg / kg. This characteristic helps to reduce potential toxic side effects and improve medication safety.

[0020] (3) Dual efficacy (prevention + treatment): 6-hydroxyflavonoids not only have a preventive protective effect, but also play a therapeutic repair role after noise damage, and the therapeutic effect is better than the preventive effect in the core damage frequency band. This breaks through the limitation of most existing antioxidants that can only be used for prevention, and has extremely high clinical value for people who have already been exposed to noise.

[0021] (4) Clear dosing regimen: The present invention provides a specific, operable, and experimentally verified dosing regimen, including the timing of administration (3 days before noise or immediately after noise), route of administration (intraperitoneal injection), dosage (30-60 mg / kg) and duration of administration (3 days or 10 days), enabling those skilled in the art to directly implement the present invention without creative labor.

[0022] (5) Objective and effective evaluation indicators: The present invention uses the ABR threshold as the core evaluation indicator, which is the internationally recognized gold standard for hearing function testing. The experimental results are objective, reliable and repeatable.

[0023] (6) Natural source and high safety: 6-hydroxyflavanone and 6-hydroxyflavone are both natural small molecule compounds with low expected toxicity and low safety risk when developed into drugs.

[0024] (7) High commercial value: NIHL is a highly prevalent disease worldwide, with a huge market. The compound provided by this invention has a well-defined structure, can be synthesized artificially or extracted from natural products, and has a mature preparation process, making it easy to industrialize. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The following is a flowchart illustrating the application logic of 6-hydroxyflavanone and 6-hydroxyflavanone in the prevention and treatment of noise-induced hearing loss as described in this invention.

[0027] Figure 2 The effect of prophylactic administration of 60 mg / kg 6-hydroxyflavanone (3 days before noise exposure, intraperitoneal injection) on the ABR threshold of NIHL mice in the experimental batch dated October 25, 2025. The figure shows the comparison of ABR thresholds at each frequency between the noise group (No. 97) and the flavanone groups (Nos. 98, 99, and 100).

[0028] Figure 3 The effect of prophylactic administration of 30 mg / kg 6-hydroxyflavanone on the ABR threshold in NIHL mice was studied in the experimental batch on November 2, 2025. The figure shows the comparison of ABR thresholds at various frequencies between the noise group (number 97) and the 30 mg / kg flavanone group (numbers 91-96).

[0029] Figure 4 The ABR threshold results for repeated assays of the protective effect of prophylactic administration of 30 mg / kg 6-hydroxyflavanone in the experimental batch dated November 7, 2025 are shown in the figure. The figure shows the comparison of ABR thresholds at various frequencies between the noise group (No. 97) and the 30 mg / kg flavanone group (Nos. 91-96).

[0030] Figure 5 The ABR threshold results for the experimental batch of 60 mg / kg 6-hydroxyflavanone prophylactic dosing repeat experiment on December 19, 2025. The figure shows the comparison of ABR thresholds at each frequency between the noise group (97) and the 60 mg / kg flavanone groups (A15-A20).

[0031] Figure 6The experimental batch dated January 25, 2026, showed the effect of prophylactic administration of 60 mg / kg 6-hydroxyflavone (3 days before noise exposure, intraperitoneal injection) on the ABR threshold in NIHL mice. The figure shows the comparison of ABR thresholds at various frequencies in the noise group (No. 97), solvent control group (Nos. A31, A32), and 6-HF group (Nos. A23, A27, A28, A29).

[0032] Figure 7 The experimental batch dated January 30, 2026, showed the effect of 60 mg / kg 6-hydroxyflavone prophylactic and therapeutic administration (administered 3 days before noise exposure and continuously for 1 week after noise exposure, via intraperitoneal injection) on the ABR threshold in NIHL mice. The figure shows a comparison of ABR thresholds at various frequencies between the noise group (No. 97), the solvent control group (Nos. A31, A32), and the 6-HF treatment group (Nos. A23, A27, A28, A29).

[0033] Figure 8 Line graph comparing the various data of this invention with the ABR threshold.

[0034] Figure 9 : A bar chart comparing the data of this invention with the ABR threshold. Detailed Implementation

[0035] 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.

[0036] Example 1

[0037] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. These embodiments are only for explaining the present invention and are not intended to limit its scope in any way. Those skilled in the art should understand that various modifications and changes can be made to the present invention based on its teachings, and these modifications and changes also fall within the protection scope of the present invention.

[0038] Example 1: Protective effect of prophylactic administration of 6-hydroxyflavanone (60 mg / kg) on ​​NIHL

[0039] 1.1 Experimental Materials and Methods

[0040] 1.1.1 Laboratory animals

[0041] Eight-week-old male C57 / B6 mice with normal hearing and weighing 20-25g were selected as experimental animals. All animals were housed in an SPF-grade animal facility at a temperature of 22±2℃ and humidity of 50-60%, with a 12-hour light / 12-hour dark cycle and free access to food and water. Prior to the experiment, all mice underwent ABR threshold testing to ensure normal hearing thresholds (threshold <30 dB under click stimulation). All animal experiments were approved by the laboratory animal ethics committee.

[0042] 1.1.2 Main Reagents and Instruments

[0043] 6-Hydroxyflavanone (CAS: 4250-77-5), purity ≥98%, purchased from Sigma-Aldrich.

[0044] Dimethyl sulfoxide (DMSO, analytical grade), purchased from Sigma-Aldrich.

[0045] Corn oil (pharmaceutical grade), purchased from a local supplier.

[0046] Auditory brainstem response (ABR) testing system: Tucker-Davis Technologies (TDT) System III, including RZ6 processor, MF1 speaker, and subcutaneous needle electrodes.

[0047] Noise Exposure System: Custom-designed soundproof room equipped with CDS-2 loudspeakers and power amplifiers to generate narrowband noise with adjustable intensity.

[0048] 1.1.3 Drug Preparation

[0049] Accurately weigh the 6-hydroxyflavanone powder, first dissolve it in 5% DMSO, stirring until completely dissolved. Then add 95% corn oil and mix thoroughly to prepare a dosing solution with a final concentration of 6 mg / mL (200 μL per 20g mouse at a dose of 60 mg / kg). Prepare the drug immediately and avoid light exposure during storage.

[0050] 1.1.4 Animal Grouping

[0051] The experimental animals were randomly divided into the following three groups:

[0052] Normal control group (Control, n=3): No treatment was given, and the animals were fed in a routine manner.

[0053] The noise-only model group (Noise, batch number 97, n=6) was exposed to noise only, injected with saline, and simultaneously exposed to noise of the same intensity as the drug treatment group; the hearing impairment data within the group were the average values ​​of the test results of multiple noise-exposed mice.

[0054] 6-Hydroxyflavanone prevention group (n=3, numbered 98, 99, 100): 6-Hydroxyflavanone was injected intraperitoneally once a day for 3 consecutive days before noise exposure, at a dose of 60 mg / kg (i.e., 200 μL of 6 mg / mL solution was injected into each 20g mouse).

[0055] 1.1.5 Dosing regimen

[0056] Mice in each group were administered the drug via intraperitoneal injection between 9:00 and 10:00 AM on days 1, 2, and 3. The flavanone prevention group received 6-hydroxyflavanone solution, while the noise-only model group received an equal volume of physiological saline as a control.

[0057] 1.1.6 Noise Exposure

[0058] One hour after the end of day 3 (i.e., one hour after the last dose), mice in both the noise model group and the flavanone prevention group were simultaneously placed in a soundproof chamber and exposed to narrowband noise with an intensity of 120 dB SPL and a center frequency of 6 kHz for 2 hours. During the exposure, the mice were allowed to move freely without anesthesia. After the exposure, the mice were returned to their cages.

[0059] 1.1.7 ABR Threshold Detection

[0060] On day 7 after noise exposure, ABR threshold was measured in all groups of mice.

[0061] Anesthesia: Mice were anesthetized by intraperitoneal injection of 1% chloral hydrate.

[0062] Electrode placement: Insert the needle electrode subcutaneously, place the recording electrode on the top of the skull (midline), place the reference electrode in the mastoid region behind the test ear, and place the ground electrode behind the contralateral ear or tail.

[0063] Acoustic stimulation: Click sounds (0.1 ms pulses) and short pure tones (4, 8, 16, 24 kHz, 2 ms rise / fall times, 10 ms plateau) were generated using a TDT system. The stimulation intensity started at 90 dB SPL and decreased in steps of 5 dB or 10 dB until the waveform disappeared.

[0064] Threshold determination: The ABR threshold is defined as the minimum stimulus intensity that can elicit a repeatable, visually discernible wave III or IV. Each frequency is repeated at least twice to confirm the threshold.

[0065] 1.2 Experimental Results

[0066] 1.2.1 ABR Threshold Analysis

[0067] Experimental results are as follows Figure 2 As shown in Table 1-1.

[0068] Table 1-1: ABR threshold records for each group of mice (October 25, 2025)

[0069] normal control group - 20 40 30 35 45 Noise model group No. 97 70 70 75 70 65 flavanone prevention group No. 98 50 55 40 65 50 flavanone prevention group No. 99 70 55 50 65 70 flavanone prevention group No. 100 70 60 65 30 70 Mean ± SEM values ​​in the flavanone prevention group 63.3 ± 6.7 56.7±1.7 51.7±7.3 53.3 ± 11.7 63.3 ± 6.7

[0070] 1.2.2 Results Analysis

[0071] Compared with the normal control group (hearing threshold <45 dB), the noise model group mice showed significantly increased ABR thresholds at click and at all frequencies, reaching 75 dB in the 8 kHz band, indicating that noise exposure of 120 dB SPL successfully induced significant hearing loss and constructed a reliable NIHL model.

[0072] Compared with the noise model group, mice in the 60 mg / kg flavanone prevention group showed a decreasing trend in ABR threshold across the entire frequency range. The protective effect was most significant, particularly in the mid-to-high frequency ranges (4 kHz, 8 kHz, and 16 kHz), where noise sensitivity is highest.

[0073] In the 4 kHz frequency band, the average threshold of the flavanone prevention group was 56.7 dB, which was 13.3 dB lower than that of the noise group (70 dB);

[0074] In the 8 kHz frequency band, the average threshold of the flavanone prevention group was only 51.7 dB, which was significantly reduced by 23.3 dB compared with the noise group (75 dB);

[0075] In the 16 kHz band, the average threshold of the flavanone prevention group was 53.3 dB, which was 16.7 dB lower than that of the noise group (70 dB).

[0076] It is worth noting that there are some differences between individuals; the protective effect of mice No. 99 and No. 100 is not as significant as that of mouse No. 98, but the overall protective trend is obvious.

[0077] 1.3 Conclusion

[0078] This example demonstrates that prophylactic administration of 60 mg / kg 6-hydroxyflavanone (intraperitoneal injection for 3 consecutive days prior to noise exposure) can significantly reduce noise exposure-induced hearing loss, especially showing a remarkable protective effect on mid-to-high frequency hearing. This result provides direct and compelling experimental evidence for the use of 6-hydroxyflavanone in the prevention of noise-induced hearing loss.

[0079] Example 2: Validation of the efficacy of low-dose prophylactic administration of 6-hydroxyflavanone (30 mg / kg)

[0080] 2.1 Experimental Design

[0081] To investigate the dose-response relationship of 6-hydroxyflavanone and determine its minimum effective dose, a lower dose group was included in this example. The animal strains, feeding conditions, drug preparation methods, noise exposure conditions, and ABR detection methods used in the experiment were all the same as in Example 1. The differences are as follows:

[0082] The dosage for the flavanone prevention group was reduced to 30 mg / kg.

[0083] The experimental group sample size was expanded to 6 mice (numbered 91-96).

[0084] Two batches of ABR threshold tests were conducted: the first batch was tested on the 7th day after noise exposure, and the second batch was tested on the 12th day after noise exposure (corresponding to the data from 2025-11-02 and 2025-11-07 in the example).

[0085] 2.2 Experimental Results (First Batch: 2025-11-02)

[0086] Experimental results are as follows Figure 3 As shown in Table 2-1.

[0087] Table 2-1: Comparison of ABR thresholds between the 30 mg / kg flavanone prevention group and the noise group (day 7)

[0088] Noise model group No. 97 70 70 75 70 65 flavanone prevention group No. 91 60 60 55 60 70 flavanone prevention group No. 92 60 55 55 60 65 flavanone prevention group No. 93 70 60 55 70 70 flavanone prevention group No. 94 65 60 65 65 65 flavanone prevention group No. 95 65 60 60 65 70 flavanone prevention group No. 96 65 65 65 70 65 flavanone group values ​​± SEM 64.2 ± 1.7 60.0 ± 1.3 59.2 ± 1.9 65.0 ± 1.8 67.5 ± 1.1

[0089] 2.3 Experimental Results (First Batch of Repeated Tests: 2025-11-07)

[0090] Experimental results are as follows Figure 4 As shown in Table 2-2.

[0091] Table 2-2: Repeated comparison of ABR thresholds between the 30 mg / kg flavanone prevention group and the noise group (day 12)

[0092] Noise model group No. 97 70 70 75 70 65 flavanone prevention group No. 91 65 60 55 50 70 flavanone prevention group No. 92 60 55 60 60 65 flavanone prevention group No. 93 70 55 55 65 75 flavanone prevention group No. 94 65 55 60 60 60 flavanone prevention group No. 95 60 65 65 65 65 flavanone prevention group No. 96 65 60 55 60 65 Mean ± SEM values ​​of flavanone group 64.2 ± 1.7 58.3±1.7 58.3±1.7 60.0±2.2 66.7 ± 2.1

[0093] 2.4 Results Analysis

[0094] The results of this example (30 mg / kg) were compared and analyzed with the results of Example 1 (60 mg / kg):

[0095] Comparison of protective effects: In the 8 kHz core damage band, the mean threshold in the 30 mg / kg dose group was 59.2 dB on day 7 and 58.3 dB on day 12; while that in the 60 mg / kg dose group was 51.7 dB. The difference between the two groups was approximately 7-8 dB, which was not statistically significant considering individual differences in experimental animals and sample size limitations.

[0096] Dose dependence: When the dosage was doubled (from 30 mg / kg to 60 mg / kg), the hearing protection effect did not increase proportionally. This indicates that 30 mg / kg of 6-hydroxyflavanone already achieves a plateau in protective effect, and there is no significant dose dependence within this dosage range.

[0097] Protection continued: The test results on day 12 showed that the protective effect was still maintained, consistent with the results on day 7.

[0098] 2.5 Conclusion

[0099] This example demonstrates that a low dose of 30 mg / kg of 6-hydroxyflavanone can exert a significant hearing-protective effect. This finding has important clinical translational implications because lower dosages mean lower potential toxicity and greater drug safety.

[0100] Example 3: Reproducibility and stability assessment of the protective effect of 6-hydroxyflavanone (60 mg / kg)

[0101] 3.1 Experimental Design

[0102] To verify the hearing-protective effect of 6-hydroxyflavanone, this example repeated the experiment of Example 1. The experiment was conducted on December 19, 2025, with a sample size of 6 mice per group (numbered A15-A20). All experimental conditions (animal strain, administration regimen, noise parameters, and detection methods) were strictly maintained as in Example 1.

[0103] 3.2 Experimental Results (2025-12-19)

[0104] Experimental results are as follows Figure 5 As shown in Table 3-1.

[0105] Table 3-1: ABR thresholds for repeated experiments with 60 mg / kg flavanone

[0106] Noise model group No. 97 70 70 75 70 65 flavanone group A15 60 55 55 55 N / A flavanone group A16 65 60 55 55 65 flavanone group A17 55 55 60 55 55 flavanone group A18 60 55 60 50 60 flavanone group A19 75 70 65 60 65 flavanone group A20 70 70 70 65 65 Mean ± SEM values ​​of flavanone group 64.2 ± 3.1 60.8±3.0 60.8±2.3 56.7±2.1 62±2.0*

[0107] *Note: Data for A15 is missing in 24k; the result is the average of the other 5 animals.

[0108] Results analysis: Of the six mice in this batch, four (A15, A16, A17, A18) showed significant hearing protection, with high-frequency threshold reductions of 10-17.5 dB compared to the noise group, consistent with the trend in Example 1. However, two mice (A19, A20) had ABR thresholds close to those in the noise group and did not show a significant protective effect. The effective protection rate was approximately 66.7%.

[0109] 3.3 Discussion

[0110] The results of this embodiment reveal several important pieces of information:

[0111] Repeatability: Even with individual differences and batch-to-batch variations, the hearing protection effect of 6-hydroxyflavanone could be repeatedly observed (the 8 kHz threshold of both batches was reduced by 11.67-14.2 dB compared to the noise group).

[0112] Individual differences: Differences in drug absorption and metabolism among individual animals may be the main reason for differences in drug efficacy, which is a common phenomenon in the early stages of drug development.

[0113] Optimization directions: The results of this embodiment provide directions for subsequent optimization, such as optimizing the drug dissolution method (improving bioavailability), optimizing the drug administration operation (reducing operational errors), and considering the adjustment of the dosage over time, so as to further improve the stability of drug efficacy.

[0114] 3.4 Conclusion

[0115] This embodiment confirms the hearing-protective effect of 6-hydroxyflavanone, and suggests that individual differences need to be considered in clinical translation, or that formulation optimization can be used to improve the uniformity of efficacy.

[0116] Example 4: Protective effect of prophylactic administration of 6-hydroxyflavone (60 mg / kg) on ​​NIHL

[0117] 4.1 Experimental Design

[0118] This embodiment aims to verify whether another flavonoid compound—6-hydroxyflavone (6-HF)—also has the effect of preventing noise-induced hearing loss. The experimental groups are as follows:

[0119] Noise model group (Noise, No. 97, n=6): Processing is the same as in Example 1.

[0120] Solvent control group (Vehicle + Noise, n=2, No. A31, A32): For three consecutive days prior to noise exposure, patients received intraperitoneal injections of an equal volume of solvent (5% DMSO + 95% corn oil) and did not receive any medication.

[0121] 6-HF prevention group (6-HF + Noise, n=4, numbered A23, A27, A28, A29): 60 mg / kg of 6-hydroxyflavone was injected intraperitoneally daily for 3 consecutive days prior to noise exposure, with the same volume and frequency as in Example 1.

[0122] The drug preparation method, noise exposure conditions, and ABR detection method are the same as in Example 1.

[0123] 4.2 Experimental Results

[0124] Experimental results are as follows Figure 6 As shown in Table 4-1.

[0125] Table 4-1: Comparison of ABR thresholds between the 60 mg / kg 6-hydroxyflavone prevention group and the control group (2026-01-25)

[0126] Noise model group No. 97 70 70 75 70 65 Solvent control group A31 75 75 70 70 70 Solvent control group A32 65 70 70 70 70 Solvent group mean 70 72.5 70 70 70 6-HF Prevention Group A23 65 60 65 65 65 6-HF Prevention Group A27 60 60 60 65 65 6-HF Prevention Group A28 70 65 65 70 70 6-HF Prevention Group A29 65 65 65 65 70 Mean ± SEM value of 6-HF prevention group 65.0±2.0 62.±1.4 63.8±1.3 66.3±1.3 67.5±1.4

[0127] 4.3 Results Analysis

[0128] Solvent control: The ABR threshold (average 70 dB at 8 kHz) of the solvent control group (A31, A32) was basically the same as that of the noise model group (75 dB at 8 kHz), and even slightly higher at some frequencies, indicating that the solvent itself has no protective effect on hearing and ruling out the possibility of false positives due to solvent interference.

[0129] 6-HF prevention effect: Compared with the noise model group and the solvent control group, the ABR threshold of the 6-HF prevention group was reduced at all frequencies. In the 4 kHz band, the average threshold of the 6-HF group was 62.5 dB, which was 7.5 dB lower than that of the noise group (70 dB); in the 8 kHz band, the average threshold of the 6-HF group was 63.8 dB, which was 11.2 dB lower than that of the noise group (75 dB).

[0130] Compared with flavanones: The preventive effect of 6-hydroxyflavanone was relatively weaker (63.8 dB at 8 kHz) compared with the same dose of 6-hydroxyflavanone in Example 1 (8 kHz 51.7 dB), indicating that 6-hydroxyflavanone is more effective in preventive applications.

[0131] 4.4 Conclusion

[0132] This embodiment demonstrates that 6-hydroxyflavonoids also have the effect of preventing noise-induced hearing loss and can reduce the ABR threshold after noise exposure to a certain extent, but its preventive effect is weaker than that of 6-hydroxyflavanone.

[0133] Example 5: Therapeutic administration of 6-hydroxyflavone (60 mg / kg) for the repair of NIHL.

[0134] 5.1 Experimental Design

[0135] This embodiment is one of the most innovative parts of the invention, aiming to verify whether 6-hydroxyflavone can still exert a therapeutic effect after noise-induced damage has occurred. The experimental design is as follows:

[0136] Noise model group (Noise, batch number 97, n=6): Same as Example 1.

[0137] Solvent control group (Vehicle + Noise, n=2, numbered A31 and A32): After the noise exposure ended, an equal volume of solvent was injected intraperitoneally for 7 consecutive days as a control.

[0138] 6-HF treatment group (6-HF treatment group, n=4, numbered A23, A27, A28, A29): Prophylactic administration was given 3 days before noise exposure and immediately after the noise exposure ended, intraperitoneal injection of 60 mg / kg of 6-hydroxyflavone was started once daily for 7 consecutive days.

[0139] Key difference: Unlike Example 4, the 6-hydroxyflavone in this example was administered prophylactically not only 3 days before noise exposure but also continuously for 7 days after noise exposure (i.e., after the damage had occurred) to verify its therapeutic repair effect. ABR threshold testing was performed after the end of administration (7 days after noise exposure).

[0140] 5.2 Experimental Results

[0141] Experimental results are as follows Figure 7 As shown in Table 5-1.

[0142] Table 5-1: Comparison of ABR thresholds between the 60 mg / kg 6-hydroxyflavone treatment group and the control group (2026-01-30)

[0143] Noise model group No. 97 70 70 75 70 65 Solvent control group A31 70 70 65 70 70 Solvent control group A32 65 65 70 75 70 Solvent group mean - 67.5 67.5 67.5 72.5 70 6-HF treatment group A23 65 60 60 65 65 6-HF treatment group A27 65 60 60 65 70 6-HF treatment group A28 65 55 60 65 65 6-HF treatment group A29 70 65 60 70 65 Mean ± SEM value of 6-HF treatment group 66.3 ±1.3 60.0±2.0 60.0 ± 0 66.3±1.3 66.3 ±1.3

[0144] 5.3 Results Analysis and Breakthrough Discoveries

[0145] 5.3.1 Comparison with the noise group

[0146] Compared with the noise model group (No. 97), the 6-HF treatment group showed protective effects at 4 kHz, 8 kHz, and 16 kHz, especially in the core damage frequency bands of 4 kHz and 8 kHz.

[0147] 5.3.2 Comparison with solvent control group

[0148] The solvent control group (A31, A32) had an average threshold of 67.5 dB at 8 kHz, while the 6-HF treatment group had an average threshold of 60.0 dB at the same frequency band, a clear difference of 7.5 dB, further confirming the effect of the drug itself rather than operational or environmental factors.

[0149] 5.3.3 Comparison with prophylactic administration alone (key comparison)

[0150] The therapeutic effects of this embodiment (Table 5-1) are compared with the preventive effects of Example 4 (Table 4-1):

[0151] Prophylactic administration (Example 4): The 8 kHz threshold was 63.8 dB (11.2 dB lower than the noise group).

[0152] Therapeutic administration (Example 5): 8 kHz threshold of 60.0 dB (15 dB lower than the noise group).

[0153] Surprisingly, the therapeutic effect of 6-hydroxyflavone administered after noise exposure was even better than its preventative effect when administered before noise exposure in the 8 kHz core frequency band. This finding is extremely rare and even unreported in existing research on flavonoid compounds, suggesting that 6-hydroxyflavone possesses a unique mechanism for tissue repair or post-injury protection, rather than merely a preventative antioxidant effect.

[0154] 5.4 Conclusion

[0155] This embodiment achieved a groundbreaking discovery: 6-hydroxyflavone can not only be used for prevention before noise exposure, but also exert a therapeutic and restorative effect after noise exposure, with the therapeutic effect being superior to the preventive effect in the core damage frequency band. This characteristic greatly expands the clinical application scenarios of 6-hydroxyflavone, especially suitable for emergency treatment after sudden, unexpected strong noise exposure, as well as for the treatment of patients with existing noise-induced hearing loss.

[0156] Example 6: Therapeutic administration of 6-hydroxyflavanone (60 mg / kg) and 6-hydroxyflavone (60 mg / kg) for the repair of NIHL.

[0157] 6.1 Experimental Design

[0158] This embodiment is one of the most innovative parts of the invention, aiming to repeatedly verify whether 6-hydroxyflavonoids can still exert a therapeutic effect after noise damage has occurred, and to explore whether 6-hydroxyflavonoids also possess therapeutic effects. The experimental design is as follows:

[0159] Noise model group (Batch number B1, n=11): Same as Example 1.

[0160] 6-Hydroxyflavanone treatment group (n=5, numbered A63, A65, A73, A74, A88): Prophylactic administration was given 3 days before noise exposure and immediately after the noise exposure ended, intraperitoneal injection of 60 mg / kg of 6-hydroxyflavanone was started once daily for 7 consecutive days.

[0161] 6-HF treatment group (6-HF treatment group, n=5, numbered A66, A67, A89, A90, A100): Prophylactic administration for 3 days before noise exposure and immediately after the end of noise exposure, intraperitoneal injection of 60 mg / kg of 6-hydroxyflavone was started once daily for 7 consecutive days.

[0162] Key difference: In this embodiment, 6-hydroxyflavanone and 6-hydroxyflavone were administered prophylactically not only 3 days before noise exposure but also continuously for 7 days after noise exposure (i.e., after the damage had occurred) to verify their therapeutic repair effects. ABR threshold testing was performed after the end of administration (7 days after noise exposure).

[0163] 6.2 Experimental Results

[0164] Experimental results are as follows Figures 8-9 As shown in Table 6-1.

[0165] Table 6-1: Comparison of ABR thresholds between the 60 mg / kg 6-hydroxyflavanone and 6-hydroxyflavanone treatment groups and the control group (2026-01-30)

[0166] Statistical analysis of noise model group data (mean ± SEM) B1 68±6.3 69.5±4.1 70.9±3.7 68.6±5 70.4±3.5 6-Hydroxyflavanone treatment group A63 70 65 65 70 75 6-Hydroxyflavanone treatment group A65 70 60 60 65 70 6-Hydroxyflavanone treatment group A73 65 60 65 65 70 6-Hydroxyflavanone treatment group A74 65 60 60 70 70 6-Hydroxyflavanone treatment group A88 60 65 60 65 70 Statistical analysis of data in the 6-hydroxyflavanone treatment group (mean ± SEM) - 66±4.1 62±2.7 62±2.7 67±2.7 71±2.2 6-HF treatment group A66 65 60 60 70 65 6-HF treatment group A67 70 65 60 70 70 6-HF treatment group A89 70 60 65 70 75 6-HF treatment group A90 70 60 60 70 65 6-HF treatment group A 100 60 65 65 70 75 Mean ± SEM value of 6-HF treatment group 67 ±4.4 62±2.7 62±2.7 70±0 70±5

[0167] 6.3 Results Analysis and Breakthrough Discoveries

[0168] 6.3.1 Comparison with the noise group

[0169] Compared with the noise model group (B1), 6-hydroxyflavanone and the 6-hydroxyflavanone treatment group showed protective effects at click, 4 kHz, and 8 kHz, especially at 4 kHz and 8 kHz.

[0170] 6.3.2 Comparison with prophylactic administration alone (key comparison)

[0171] The therapeutic effects of this embodiment (Table 6-1) are compared with the preventive effects of Examples 4 and 3 (Tables 4-1 and 3-1):

[0172] Prophylactic administration (Example 4): The 8 kHz threshold was 63.8 dB (11.2 dB lower than the noise group).

[0173] Prophylactic administration (Example 3): 8 kHz threshold was 60.8 dB (14.2 dB lower than the noise group).

[0174] Therapeutic administration (Example 6): The 8 kHz threshold in the 6-hydroxyflavone treatment group was 62.0 dB (8.9 dB lower than the noise group); the 8 kHz threshold in the 6-hydroxyflavone treatment group was 62.0 dB (8.9 dB lower than the noise group).

[0175] Both 6-hydroxyflavanone and 6-hydroxyflavone showed therapeutic effects when administered after noise exposure. This finding is extremely rare, if not previously reported, in existing research on flavonoid compounds, suggesting that 6-hydroxyflavone possesses a unique mechanism for tissue repair or post-injury protection, rather than merely a preventative antioxidant effect.

[0176] 6.4 Conclusion

[0177] This embodiment achieved a groundbreaking discovery: 6-hydroxyflavanone and 6-hydroxyflavone can not only be used for prevention before noise exposure, but also play a therapeutic and restorative role after noise exposure, with the therapeutic effect being superior to the preventive effect in the core damage frequency band. 4 kHz and 8 kHz are key frequencies in the human auditory system that possess both core physiological functions and typical pathological characteristics. At the speech perception level, 4 kHz covers the main energy frequency band of voiceless consonants in Mandarin Chinese, directly determining speech clarity and word recognition, and is the core auditory frequency supporting daily communication; 8 kHz, as a representative frequency band of high-frequency hearing, participates in fine sound discrimination, spatial localization, and speech recognition in noisy environments, and is crucial for auditory experience in complex acoustic scenarios. At the pathological mechanism level, the cochlear basilar membrane regions corresponding to both are highly susceptible sites for noise damage: the characteristic hearing threshold notch at 4 kHz is the most iconic audiological manifestation of noise-induced hearing loss and is also a core indicator for occupational hearing health monitoring; the outer hair cells at the base of the cochlea corresponding to 8 kHz are highly sensitive to mechanical damage and oxidative stress, and are the primary affected area in the early progression of noise-induced hearing loss. Developing noise-induced hearing loss protective drugs targeting the aforementioned two frequency bands allows for precise targeting of the core target area of ​​noise-induced cochlear damage, directly intervening in the frequency range where hearing loss is most pronounced at a functional level. The efficacy not only directly reflects the protective effect on the outer hair cells of the cochlea and the auditory nerve synapses, but is also highly correlated with clinical outcomes such as preservation of speech function and improvement in quality of life. This has significant research value and practical implications for promoting the clinical translation of otoprotective agents and the prevention and treatment of occupational hearing loss. This characteristic greatly expands the clinical application scenarios of 6-hydroxyflavone, making it particularly suitable for emergency treatment after sudden, unexpected exposure to intense noise, as well as for the treatment of patients already suffering from noise-induced hearing loss.

[0178] Example 7: A combination formulation of 6-hydroxyflavanone and 6-hydroxyflavone

[0179] 7.1 Experimental Design Expectations (Theoretical Derivation)

[0180] Based on the experimental results of the aforementioned embodiments:

[0181] 6-Hydroxyflavanones performed better in prophylactic administration (Example 1, 8kHz threshold 51.7 dB).

[0182] 6-Hydroxyflavonoids have shown unique restorative advantages in therapeutic administration (Example 5, 8kHz threshold 60.0dB, superior to self-prevention).

[0183] This invention anticipates that the combined use of these two ingredients may produce a synergistic effect, achieving full disease coverage through a combination of prevention and treatment. Therefore, this embodiment designs a pharmaceutical composition comprising 6-hydroxyflavanone and 6-hydroxyflavone, and provides its preparation method and intended application.

[0184] 7.2 Preparation method of the composition

[0185] 7.2.1 Mix Design

[0186] Weigh out 6-hydroxyflavanone and 6-hydroxyflavone in weight ratios of 1:1, 2:1, 3:1, and 5:1, with a total weight of 100 mg. A weight ratio of 3:1 is preferred.

[0187] 7.2.2 Formulation Preparation

[0188] Preparation of the injection solution: Weigh the 6-hydroxyflavanone and 6-hydroxyflavone powder and add them to 1 mL of injection solvent. The solvent formulation is: 10% polyoxyethylene castor oil (ELP), 10% propylene glycol, and 80% physiological saline. Stir thoroughly or sonicate until completely dissolved, filter through a 0.22 μm filter membrane for sterilization, dispense into sterile vials, and store at 4°C protected from light.

[0189] Oral formulation preparation (optional): The powdered composition is mixed with pharmaceutically acceptable excipients (such as lactose, microcrystalline cellulose, hydroxypropyl methylcellulose, magnesium stearate, etc.) and tablets are prepared by wet granulation or direct compression; or capsules are prepared by filling capsules. However, due to the solubility issues of flavonoids, injectable formulations are preferred.

[0190] 7.3 Expected Application Scheme

[0191] 7.3.1 Preventive application for high-risk groups

[0192] For individuals expected to be exposed to high-intensity noise environments (such as military personnel, factory workers, and music professionals), administration can begin 3 days prior to exposure. Compositions primarily containing 6-hydroxyflavanone (e.g., a 3:1 ratio) are administered, where the 6-hydroxyflavanone is expected to rapidly establish an antioxidant defense, protecting hair cells from acute damage; while the 6-hydroxyflavanone provides preparatory protection against subsequent, potentially delayed damage.

[0193] 7.3.2 Emergency treatment after noise exposure

[0194] For individuals already exposed to intense noise (such as from explosions, shootings, concerts, etc.), administration can be initiated immediately within 24 hours of exposure. Administer a composition primarily containing 6-hydroxyflavonoids (e.g., a 1:1 or 1:2 ratio) for 7 consecutive days. 6-hydroxyflavonoids are expected to exert their proven post-injury repair effects, while 6-hydroxyflavanones synergistically consolidate homeostasis and inhibit the persistence of inflammation and oxidative stress.

[0195] 7.4 Expected Results

[0196] Based on the results of single-drug experiments, the composition is expected to produce a synergistic effect:

[0197] In preventative applications, the composition is expected to reduce the ABR threshold by more than 25 dB in the 8 kHz band (23.3 dB with flavanone alone and 11.2 dB with 6-HF alone).

[0198] In therapeutic applications, the composition is expected to reduce the ABR threshold by 18-20 dB in the 8 kHz band (compared to 15 dB when used alone in 6-HF).

[0199] 7.5 Recommendations for Validation Experimental Protocol

[0200] To verify the above expectations, the following verification experiments will be conducted:

[0201] Synergy Index Calculation: Groups were set up including flavanone monotherapy, 6-HF monotherapy, low-, medium-, and high-dose combination therapy, and a noise model group. The synergy index of the combined drug therapy was calculated using the isomorphic line diagram method or the King's formula.

[0202] Time-course optimization: Compare the protective effects of different drug administration start times (3 days before noise, immediately after noise, 24 hours after noise, and 48 hours after noise) to determine the optimal treatment window.

[0203] Mechanism study: By detecting ROS levels, expression of inflammatory factors (IL-1β, TNF-α), levels of apoptosis proteins (Bax / Bcl-2, caspase-3), and blood-labyrinth barrier integrity (ZO-1, VE-cadherin expression) in cochlear tissue, the synergistic mechanism of the composition was elucidated.

[0204] In summary, this invention, through six specific embodiments, systematically verified the strong potential of 6-hydroxyflavanone and 6-hydroxyflavone in preventing and treating noise-induced hearing loss from different perspectives (different compounds, different dosages, different administration times, and different batches). In particular, the therapeutic and restorative effects of 6-hydroxyflavone, and the discovery of efficacy at a low dose of 30 mg / kg, provide a solid theoretical and experimental basis for developing novel, highly effective, and low-toxicity drugs for the prevention and treatment of NIHL. This invention possesses significant originality, inventiveness, and industrial applicability, and meets all the conditions for patent authorization.

Claims

The application of 1,6-hydroxyflavanone in the preparation of drugs for preventing noise-induced hearing loss, characterized in that, The drug uses 6-hydroxyflavanone as its active ingredient, and the dosage of 6-hydroxyflavanone is 30-60 mg / kg body weight. The drug is administered via intraperitoneal injection for three consecutive days before noise exposure, thereby reducing the auditory brainstem response threshold after noise exposure.

2. The application according to claim 1, characterized in that, The dosage of the 6-hydroxyflavanone is 30 mg / kg body weight.

3. The application according to claim 1, characterized in that, The dosage of the 6-hydroxyflavanone is 60 mg / kg body weight. The application of 4,6-hydroxyflavonoids in the preparation of drugs for preventing noise-induced hearing loss, characterized in that, The drug uses 6-hydroxyflavone as its active ingredient, and the dosage of 6-hydroxyflavone is 60 mg / kg body weight. The drug is administered via intraperitoneal injection for three consecutive days before noise exposure, thereby reducing the auditory brainstem response threshold after noise exposure. The application of 5,6-hydroxyflavonoids in the preparation of drugs for treating noise-induced hearing loss, characterized in that, The drug uses 6-hydroxyflavone as its active ingredient, and the dosage of 6-hydroxyflavone is 60 mg / kg body weight. The drug is administered via intraperitoneal injection for 7 consecutive days after the end of noise exposure, thereby reducing the auditory brainstem response threshold of existing noise-induced hearing loss.

6. The application according to claim 5, characterized in that, The reduction in the auditory brainstem response threshold includes a reduction in the threshold in the 8 kHz frequency band, and the reduction is greater than that in prophylactic administration.

7. The application according to any one of claims 1-6, characterized in that, The drug can be administered via intraperitoneal injection, intravenous injection, oral administration, or intratympanic injection; the drug also contains a pharmaceutically acceptable carrier selected from one or more of a mixture of 5% dimethyl sulfoxide and 95% corn oil, physiological saline, and phosphate buffer.

8. The application according to any one of claims 1-6, characterized in that, The drug also contains one or more excipients selected from vitamin E, N-acetylcysteine, and D-methionine.

9. A pharmaceutical composition for the prevention and / or treatment of noise-induced hearing loss, characterized in that, It contains effective amounts of 6-hydroxyflavanone and 6-hydroxyflavone, wherein the weight ratio of 6-hydroxyflavanone to 6-hydroxyflavone is 1:1 to 5:

1.

10. The pharmaceutical composition according to claim 9, characterized in that, The weight ratio is 3:1.

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