Introduction

Meniere's Disease (MD) is an inner ear disorder characterized by symptoms such as vertigo, hearing loss, tinnitus, and ear fullness [1-3]. The current medical interventions primarily aim to alleviate symptoms rather than address the root cause [4-6]. Despite various available treatments, there is no universally acknowledged cure for MD. The condition's underlying mechanisms are associated with endolymphatic hydrops in the inner ear [3,7-9]. Endolymphatic hydrops refers to a pathological anatomical condition where the structures surrounding the endolymphatic space undergo distension due to an increase in the volume of endolymph [10-12]. While some treatments like the Meniett device and endolymphatic sac surgery exist, their efficacy remains controversial [13]. Although Traditional Chinese Medicine (TCM) is reported by some individuals to have positive outcomes for Meniere's syndrome, scientific evidence supporting its effectiveness is limited [14,15].

Our comprehensive clinical and animal studies address this gap. Dingxuan, a traditional Chinese medicine composed of coptis, ginger pinellia, perilla, magnolia officinalis, ginger zuru, Jiaoshan gardenia, light tempeh, poria, dried ginger, and raw oysters, was the focus of this study. The primary objective was to assess through a clinical trial the effectiveness of Dingxuan in relieving acute vertigo episodes. The study also aimed to evaluate Dingxuan's safety, particularly concerning liver and kidney toxicity, even at seven times the standard dosage. Betahistine was initially approved in Europe in 1970 for addressing Ménière's disease, serving as an anti-vertigo medication [16,17]. Frequently prescribed to manage balance disorders and alleviate symptoms of vertigo, Betahistine functions as a histamine analog [18,19]. It enhances microcirculation in the inner ear by inducing vasodilation and mitigating the accumulation of fluid within the inner ear [20,21]. Additionally, a comparison between Dingxuan and betahistine in a guinea pig model explored the potential of Dingxuan in modulating the water channels AQ2 and AQ5, regulating AVP-mediated signaling via cAMP, and improving inner ear labyrinth hydrops. The promising findings suggest Dingxuan's potential in targeting water channel activity, laying the groundwork for further research and clinical trials to delve into its broader implications and efficacy in human subjects.

Materials and Methods

Plant Names

Dingxuan comprises a blend of medicinal plants, featuring key components such as Chinese goldthread, Pinellia ternata, Perilla frutescens, Officinal magnolia, bamboo shavings, gardenia, fermented soybeans, and Poria cocos. Additionally, it includes adjuvants like Zingiber officinale and raw oyster shell.

Clinical Study

The clinical efficacy of Dingxuan was substantiated through a pre-post trial, involving a significant cohort of 30 patients diagnosed with Meniere's Disease following the Clinical Practice Guideline for Meniere's Disease [22,23]. Diagnosis entailed a thorough patient history, characterized by discrete episodes of vertigo lasting 20 minutes or more, accompanied by initially low frequency sensorineural hearing loss, aural fullness, and tinnitus [24,25]. Diagnostic workup included audiometry, contrast-enhanced MRI of the internal auditory canals, and the exclusion of other conditions presenting similar symptoms, such as syphilis, autoimmune inner ear disease, perilymphatic fistula, superior semicircular canal syndrome, lyme disease, multiple sclerosis, vestibular paroxysms, and temporal bone tumors [26,27]. Additionally, a history of migraine was considered due to its high cooccurrence rate [24].

The assessment of Dingxuan's efficacy encompassed various parameters, including the frequency, severity, and duration of vertigo attacks. The Grading of Inner Ear Symptoms and Function in Vestibular Disorders (GISFaV) self-rating scale and the Dizziness Handicap Inventory (DHI) were also employed [28-30]. DHI gauges self-perceived handicapping effects related to vestibular system disease, with its final version consisting of 25 items, including 7 physical questions, 9 functional questions, and 9 emotional questions, resulting in a total score of 100 points (4 points for each item) [31,32]. Higher scores indicate more severe handicaps, with a maximum score of 100 and a minimum score of 0 [30,33]. Supplementary (Table 1) provides comprehensive information on the DHI scales. The GISFaV self-assessment scale is primarily used to evaluate subjective symptoms related to vestibular disorders. It relies on self-reported information from individuals experiencing vestibular symptoms. Supplementary (Table 2) offers ample detail on the GISFaV scales.

Table 1: The Dizziness Handicap Inventory (DHI)

Table 2: Medication duration

This pre-post trial was carried out at the Otorhinolaryngology outpatient department of Wuhu Traditional Chinese Medicine Hospital in Wuhu, China, spanning the period from October 15, 2023, to January 15, 2024. The research included and managed a group of 30 patients using Dingxuan. To be eligible for a Meniere's Disease diagnosis in this study, patients had to exhibit key symptoms like distinct episodes of vertigo lasting 20 minutes or longer, coupled with initial low-frequency sensorineural hearing loss, aural fullness, and tinnitus [2,9,34]. Exclusion criteria required the exclusion of other possible causes for similar symptoms, such as vestibular migraine, labyrinthitis, perilymphatic fistula, acoustic neuroma, benign paroxysmal positional vertigoor, endolymphatic sac tumor, autoimmune inner ear disease, and certain central nervous system disorders [35-39]. The study (KY2022-005) was approved by the ethics committee of Wuhu Traditional Chinese Medicine Hospital, and written informed consent was secured from all eligible participants before enrollment.

Animal Study

All animal procedures were conducted with prior approval from the Wannan Medical College Committee on Animal Care and aligned with ethical standards of the National Research Council Guide for the Care and Use of Laboratory Animals. Guinea pigs and mice resided in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility at Wannan Medical College, maintained at a controlled environment (21-23°C, 12-hour light-dark cycle). Guinea pigs received standard guinea pig chow, and nutritional guidelines for mice in laboratory settings were strictly followed [40]. Guinea pigs basic diet is unlimited amounts of commercial high-fiber guinea pig pellets, supplemented with smaller timothy or other low-calcium hay as well as Vitamin C each day [41].

Modeling Process

The guinea pig experiment encompasses six groups, including a control group, a Betahistine group, a model group, and a drug administration group (comprising low, medium, and high doses). The control group serves as the untreated control, while the other five groups undergo a literature-based method involving intraperitoneal injection of desmopressin acetate to induce membranous labyrinth hydrops. An initial intraperitoneal injection of 4 μg/kg desmopressin acetate(dDAVP) (Hybio, Pharmaceutical Co., Ltd) is administered, followed by a 7-day continuous injection [42,43]. Subsequently, the dose is increased to 6 μg/kg for an additional 3 days, aiming to induce cochleosaccular hydrops, a sensitive finding in Meniere's Disease [44,45]. Ten days after modeling, the intragastric administration of betahistine and Dingxuan (low, medium, and high dosage groups) is carried out based on body weight. This administration occurs once a day for seven consecutive days. Dingxuan is orally administered three times a day, with each dose consisting of one bag (10g/bag). The guinea pig dosage for intragastric administration is determined using a conversion coefficient of 5.42, based on the comparison between humans and guinea pigs, considering an adult standard weight of 60kg [46,47]. Guinea Pig Dosage of 2.71g/kg/d is recorded as the medium dose of the Dingxuan group administered by gavage to guinea pigs. The high dose is calculated as 5.42 g/kg/d, and the low dose is 1.36g/kg/d. The Betahistine group(1.084g/kg) receives treatment according to established procedures [19,48].

Guinea Pig Vertigo Scoring Criteria

We established a standardized method to assess nystagmus in guinea pigs, defining vertigo scoring criteria as follows: Score 0: No symptoms. Indication: Absence of observable vertigo symptoms in guinea pigs. Description: The animal maintains typical behavior, movement, and posture without any signs of imbalance or abnormal eye movements (nystagmus). Score 1: Mild nystagmus. Indication: Guinea pigs exhibit mild nystagmus, characterized by involuntary rhythmic eye movements. Description: Subtle, noticeable eye movement occurs without significantly affecting overall behavior or posture. Score 2: Mild nystagmus with curling up and lying down. Indication: Guinea pigs display both mild nystagmus and additional behaviors such as curling up and lying down. Description: Alongside mild nystagmus, the guinea pig may intermittently exhibit a tendency to curl up or lie down, indicating a mild to moderate level of vertigo. Score 3: Severe nystagmus with curling up and lying down. Indication: Guinea pigs show severe nystagmus, characterized by intense, sustained involuntary eye movements. Description: The animal demonstrates pronounced vertigo symptoms, including severe nystagmus, and frequently curls up or lies down, suggesting a substantial impairment of balance and coordination.

These defined scoring criteria offer a systematic approach for evaluating the severity of vertigo symptoms in guinea pigs, encompassing both nystagmus observations and associated behaviors.

Hepatotoxicity and Renal Toxicity Assays

For toxicity assessment, Dingxuan was administered at an elevated dosage of 34.8 g/kg/d, which is 70-fold over the mouse standard dose. The primary focus was scrutinizing potential hepatotoxicity and renal toxicity. The animals were humanely euthanized following approved ethical guidelines [49,50]. The liver and kidneys were thoroughly rinsed in ice-cold physiological saline to eliminate excess blood. Subsequently, the tissue slices were fixed in formalin, embedded in paraffin, sectioned, and subjected to Hematoxylin and Eosin (H&E) staining [51,52]. Microscopic examination was conducted to identify any structural changes. The outcomes derived from histological analyses were quantified. Statistical analyses were performed to identify significant distinctions between the control and treatment groups.

Serum samples were collected and Assay kits were employed, following manufacturer instructions(BUN, AST, ALT, and CREA assay kit, Jiancheng Biotechnology Co., Ltd). A BUN assay kit (diacetyl oxime colorimetric method) and AST/ALT assay kits (microplate methods) were used to measure hepatotoxicity, while a creatinine assay kit (sarcosine oxidase method) assessed renal toxicity [53].

Hematoxylin and Eosin (H&E) Staining

Cochlear tissues were isolated, processed, and stained to assess differences in membranous labyrinth hydrops among guinea pig groups. The tissue was then refrigerated at 4°C for 48 hours. Subsequently, the specimen was transferred to a 10% EDTA decalcifying solution(Feijing Technology Co.Ltd) (pH 7.2) for about 21 days, with the solution changed daily. After decalcification, the excess softened bone was carefully removed, trimmed as needed, and rinsed with running water. The tissue was then dehydrated in a graded series of ethanol, made transparent in xylene, and embedded in paraffin. Paraffin blocks were cut in 4 μm thick slices parallel to the modiolus. The images with staining were subjected to analysis using Image J software to quantify the cross-sectional area of both the scala media and scala vestibule [54-56].

Enzyme-Linked Immunosorbent Assay (ELISA)

Following an 8-day recovery period, the guinea pigs were randomly allocated into five groups: three receiving varying doses of Dingxuan, another receiving betahistine, and a control group. The animals were subjected to a seven-day treatment regimen. Guinea pigs were administered general anesthesia through intraperitoneal injection with 10% chloral hydrate (3.5 ml/kg). Subsequently, blood samples were collected for the detection of AVP (Jianglai Technology Co. Ltd) and cAMP (Jianglai Technology Co. Ltd) activities using Enzyme-Linked Immunosorbent Assay (ELISA). For the enzyme-linked immunosorbent assay, 50 μL of the sample was incubated for 60 minutes in designated wells. AVP antibody incubation, secondary antibody addition, enzyme reaction initiation, color development, and absorbance measurement followed using a microplate reader at the appropriate wavelength [15,57,58].

Western Blot Analysis

For protein quantification, cochlear tissue from the right ear of guinea pigs was swiftly frozen at -80°C. The finely sliced tissue received 100 μl of RIPA protein lysis buffer (for every 10 mg) and 10 μl of PMSF (for every 1 ml of RIPA lysis buffer) [59,60]. Subsequent centrifugation at 12,000 rpm for 30 minutes at 4°C yielded the protein extract supernatant [61,62]. After 12% SDS-PAGE gel electrophoresis, proteins transferred to a membrane were blocked in 5% skimmed milk powder solution. ECL luminescent solution incubation, conducted in the dark, was followed by exposure, image collection, and band gray value analysis using Image J image processing software [13,63-65].

Statistical Analysis

The results are presented as means ± Standard Error of the Mean (SEM), and statistical analyses were performed using GraphPad Prism 9 software. For Western blot and Hematoxylin and Eosin (HE) staining experiments, unpaired two-tailed Student's t-tests were employed for data analysis. Significance was considered at P < 0.05.

Results

Effective reliefs for Meniere’s symptoms reduce both vertigo frequency and intensity.

An acute episode of vertigo and other associated symptoms such as tinnitus, nausea, and vomiting manifest often frighten the patient. For this study, inclusion criteria encompassed patients experiencing a period of remission and newly diagnosed individuals over 30 years of age and under 65 years. Following 18 days of Dingxuan treatment, there was a notable decrease in the mean frequency of vertigo attacks among patients with Ménière's disease. Specifically, the frequency decreased from an onset time of 55.56 hours to 0.06 hours. Relief of symptoms with Dingxuan treatment can occur as early as 4 days and is typically achieved within a maximum period of 10 days. The difference between pre-treatment and post-treatment became significant from the end of the fourth day of treatment onward. The duration of treatment varies depending on whether the patient's vertigo symptoms resolve Supplementary (Table 3). 19 individuals among the 30 patients treated had GISFaV scores exclusively at 0, representing 63.3% of the overall population. This implies that, on the 18th day, the post-treatment group showed a 63.3% of patients with GISFaV scores of zero, while the pre-treatment group maintained a 0% rate Supplementary (Table 4). While 11 patients continue to experience symptoms such as vertigo, tinnitus, or visual vertigo, a comparison of the cumulative duration and frequency of vertigo attacks before and after treatment revealed that Dingxuan still significantly reduces Ménière syndrome's impact (Figure 1A). The vertigo intensity score showed more frequent improvement in post-treatment compared to pre-treatment (Figure 1B). Associated symptoms such as tinnitus, aural fullness, nausea, and vomiting were also more frequently improved in post-treatment than in pre-treatment. Symptoms absent from the post-treatment records in (Table 3) suggest their disappearance Supplementary (Table 1). Similarly, six out of the seven items in the Dizziness Assessment Rating Scale showed more improvement in the post-treatment group than in the pre-treatment group. Dingxuan post-treatment significantly reduced Dizziness Handicap Inventory(DHI) scores more frequently than pre-treatment (Figure 1C) and Supplementary (Table 2).

Table 3: The Grading of Inner Ear Symptoms and Function in Vestibular Disorders (GISFaV) self-rating scale

Table 4: The Dizziness Handicap Inventory (DHI) score

Figure 1A: Clinical trail of Dingxuan (A) Study protocol and participant exclusion.

Figure 1B: Clinical trail of Dingxuan (B) Assessment of dizziness disorder scale scores in Meniere’s disease patients. The Dizziness Handicap Inventory (DHI) after treatment significantly decreases compared to pre-treatment (p < 0.05) (n=30).

Figure 1C: Clinical trail of Dingxuan (C) Evaluation of the improvement in the frequency of vertigo attacks in Meniere’s disease, showing a significant improvement (p < 0.05)(n=30).

Both Dingxuan and Betahistine alleviate vertigo, impacting inner ear fluid dynamics.

Betahistine is commonly used to treat vertigo and balance disorders, particularly associated with Ménière's disease [66]. Betahistine is believed to have a vasodilatory effect on the blood vessels in the inner ear, improving blood flow. Additionally, it may influence the release of histamine, acting as a histamine agonist, and affect the sensitivity of the vestibular system [67]. In our study, we conducted a comparative analysis involving two groups of guinea pigs, with one group receiving Betahistine and the other Dingxuan.

Several clinical and experimental investigations have proposed the potential involvement of Arginine Vasopressin (AVP) in the progression of endolymphatic hydrops48. Findings from studies indicate that the administration of a significant quantity of AVP can induce endolymphatic hydrops in guinea pigs [68]. Histological sections from these studies demonstrated the dilation of the scala media, coupled with the extension of Reissner's membrane toward the scala vestibuli. On the tenth day following the administration of arginine vasopressin to guinea pigs, we compared the vertigo index between the Dingxuan and Betahistine-treated groups. Both Dingxuan and Betahistine demonstrated the ability to improve the cochlear duct area of the inner ear in guinea pigs and reduce membranous labyrinth hydrops (Figure 2A). Both Dingxuan and Betahistine are postulated to influence fluid dynamics within the inner ear. The results indicated a significant improvement in the vertigo index in guinea pigs treated with both Dingxuan and Betahistine, with a statistically significant difference observed (Figure 2B). The Hypothalamic-Pituitary-Adrenal (HPA) axis is a complex neuroendocrine system that plays a crucial role in the body's response to stress and the regulation of various physiological processes [69]. Research suggests that stress-related hormonal changes and increased cortisol levels may contribute to vascular changes, inflammation, and fluid retention in various parts of the body, including the inner ear [70]. This therapeutic effect is attributed to the positive modulation of the Hypothalamic-Pituitary-Adrenal (HPA) axis4. Dingxuan exhibits the ability to decrease AVP and cAMP levels, suggesting its role in regulating plasma AVP levels through the HPA axis (Figure 2C). Additionally, Dingxuan contributes to maintaining fluid homeostasis within the inner ear by influencing AQP2 and AQP5 to regulate the volume and pressure of endolymph (Figure 2D). Dingxuan reduces the activation of vasopressin receptor 2, thereby inhibiting the AVP-AQP2 system, crucial for antidiuresis and maintaining plasma osmotic stability. This leads to a benign modulation of the AVP-AQP2 system, effectively mitigating membranous hydrops.

Figure 2A: Comparative Study of Dingxuan and Betahistine in the Treatment of Ménière Syndrome Induced by Membranous Hydrops in Guinea Pigs. (A) Micrographs of sectioned guinea pig cochleae show mid modiolar sections through normal and hydropic guinea pig cochlea. The endolymphatic compartment has been highlighted, revealing marked enlargement of the endolymphatic space and distension of Reissner’s membrane. Various membranous structures in the ear, including those bounding the saccule, utricle, and ampullae of the semi-circular canals, may be displaced to varying degrees. The bar graph indicates that Dingxuan can reduce membranous labyrinth hydrops in guinea pigs, with the high dose exhibiting a similar effect to betahistine(n=10)

Figure 2B: Comparative Study of Dingxuan and Betahistine in the Treatment of Ménière Syndrome Induced by Membranous Hydrops in Guinea Pigs. (B) Mean dizziness curves illustrate the maximal performance of guinea pigs on the rotating beam. Results are expressed in dizziness scores as a function of posttreatment time in days (on the abscissa)(n=10).

Figure 2C: Comparative Study of Dingxuan and Betahistine in the Treatment of Ménière Syndrome Induced by Membranous Hydrops in Guinea Pigs. (C) Dingxuan demonstrates the ability to decrease AVP and cAMP levels, suggesting its role in regulating plasma AVP levels through the HPA axis(n=10).

Figure 2D: Comparative Study of Dingxuan and Betahistine in the Treatment of Ménière Syndrome Induced by Membranous Hydrops in Guinea Pigs. (D) Dingxuan contributes to maintaining fluid homeostasis within the inner ear by influencing AQP2 and AQP5 to regulate the volume and pressure of endolymph. Compared with betahistine, Dingxuan rescues fluid homeostasis(n=10).

Elevated dosage in mice reveals no adverse effects on body weight, organ indices, or liver/kidney function.

In conducting in vivo animal acute toxicity experiments in mice using Dingxuan, we administered the herbal remedy to mice at a dose 69.6 times higher than the normal dosage. Subsequently, we closely monitored the mice for potential side effects at this elevated dose. The change in body weight of mice serves as an indicator of the effects or impact of a particular intervention, treatment, or condition. A comparison of the body weight gain between the experimental group and the blank group revealed no statistically significant difference (p>0.05) (Figure 3A). The body weight of mice in both groups exhibited an increase following administration. An upward trend in body weight typically indicates growth and overall well-being in mice. The mouse organ index, which reflects the relative size of specific organs concerning the overall body weight, can also suggest various physiological conditions. We assessed the heart, lung, spleen, liver, and kidney organ indices, comparing them between the administration group and the placebo group. Monitoring these indices provides valuable information about the impact of interventions or experimental conditions on organ health and physiology in mice. No changes in organ indices suggested that there were no alterations in organ development, function, or response to treatments in administration group (p>0.05)(Figure 3B).

Figure 3A: Study of safety and tolerance of Dingxuan (A) Body Weight Analysis, The body mass index of mice, with an equal distribution of male and female subjects in each group (n=10), revealed no significant changes in body weight between the Dingxuan and control groups.

Figure 3B: Study of safety and tolerance of Dingxuan (B) Organ Mass Index, Evaluation of the main organ mass index, including the heart, liver, lungs, spleen, and kidneys, showed no remarkable differences between the Dingxuan and control groups. This comprehensive assessment considered variations in body weight (n=10).

Monitoring for hepatotoxicity and nephrotoxicity is crucial in assessing the safety of drugs or interventions, as damage to the liver or kidneys can have serious health implications. Regular assessments of liver and kidney function, including laboratory tests and imaging studies, are typically conducted to detect and manage potential toxic effects on these organs. We evaluated the levels of Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), Urea Nitrogen (BUN), and Creatinine (CRE) in the serum of two mouse groups. No significant differences were observed between the two groups (Figure 3C). Moreover, the examination of mouse liver and kidney tissue sections offers valuable insights into the respective organs' conditions, revealing structural and functional characteristics, identifying potential abnormalities, and assessing responses to treatments or conditions. The findings indicated that Dingxuan had no adverse effects on mouse liver and kidney (Figure 3D).

Figure 3C: Study of safety and tolerance of Dingxuan (C) Serum Biochemical Analysis, Analysis of serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen (BUN), and creatinine (CRE) displayed no significant differences between the two mouse groups(n=10).

Figure 3D: Study of safety and tolerance of Dingxuan (D) Tissue Section Examination, Microscopic examination of liver and kidney tissue sections provided valuable insights into the structural and functional characteristics of these organs(n=10). The assessment aimed to identify potential abnormalities and evaluate organ responses to Dingxuan treatments or conditions.

Discussion

Meniere's Disease (MD) is a complex inner ear disorder marked by troubling symptoms, including vertigo, hearing loss, tinnitus, and aural fullness [2]. Since its original diagnosis in the 1860s, the precise mechanisms underlying MD development remain elusive [9]. Researchers have identified Endolymphatic Hydrops (ELH), an abnormal accumulation of endolymph fluid in the inner ear's membranous labyrinth, as a prominent histopathological feature [5]. Current treatments primarily target management of MD symptoms rather than addressing the root cause, lacking definitive cures. Meniere's syndrome complexity presents clinical challenges, and existing medications have inherent pitfalls. Traditional Chinese herbal medicines, known for their intricate formulations, can restore body homeostasis and address specific symptoms [71,72]. The synergistic effects of multiple herbs, particularly those with anti-inflammatory properties [52,73], are considered beneficial in reducing inner ear inflammation linked to Meniere's syndrome.

In this study, patients meeting inclusion criteria, including those in remission and newly diagnosed individuals aged 30 to 65 years, exhibited significant improvements after 18 days of Dingxuan treatment. The mean frequency of vertigo attacks markedly decreased, indicating the potential efficacy of the treatment. Betahistine, an anti-vertigo medication, is widely prescribed for balance disorders and vertigo relief, and gained initial registration in Europe in 1970 for Meniere's disease [17]. Our study explored the effects of Betahistine and Dingxuan, an herbal prescription widely used for Meniere's disease management. AVP, a hormone regulating water reabsorption in the kidneys, could contribute to abnormal fluid retention in elevated levels, potentially causing Meniere's disease symptoms [74,75]. cAMP, a second messenger in cellular processes, influences protein expression [62,76,77]. AQP2, vital for water reabsorption, and AQP5, regulating water movement across cell membranes, play roles in osmotic balance [78-81]. In comparison to betahistine, Dingxuan showed promising outcomes in a guinea pig model. Dingxuan modulated water channels AQ2 and AQ5, regulated AVP-mediated signaling through cAMP, and alleviated inner ear labyrinth hydrops. Importantly, no signs of liver and kidney toxicity were observed even at seventy times the standard dosage. This Chinese patent medicine holds potential clinical applications in targeting water channel activity, introducing a novel avenue for Meniere's syndrome treatment. Further research and clinical trials are necessary to explore its broader implications and efficacy in human subjects.

Conclusion

While grappling with the complexities of Meniere's disease management, this study illuminates the strides taken in comprehending its pathophysiology and advancing tailored treatments. Dingxuan emerged as a promising contender, showcasing efficacy in modulating water channels and regulating AVP-mediated signaling. Its capacity to ameliorate inner ear hydrops is noteworthy, accompanied by an absence of discernible liver or kidney toxicity even at elevated dosages. The prospects for Dingxuan as a Chinese patent medicine targeting water channel activity in clinical applications appear auspicious.

Declaration of competing interest

No conflicts of interest exist; the research adheres to ethical standards and transparently reports funding sources and affiliations.