Discussion
Otomycosis refers to fungal infections of the external ear, including the auricle, auditory canal, eardrum, and middle ear. The symptoms include otorrhea, otalgia, itchiness, and it is usually a recurrent disease [12, 13]. Fortunately, this entity is not life-threatening, and complications are uncommon except in immunocompromised patients as it is an opportunistic infection. However, this disease presents difficulties for the otolaryngologist and the patient, as it has a high recurrence rate and residual disease. Additionally, it necessitates long-term treatment and monitoring [13]. The mainstay of treatment for otomycosis is thorough removal and cleaning of the infected area with topical antifungal ear drops [14]. Topical medications are preferable to systemic medications because they have a low risk of systemic side effects and a high concentration in the targeted infected area.
Aspergillus and Candida are the most prevalent fungal species isolated in otomycosis [13]. There has been a report of growing resistance to classical antifungal agents that contain azoles, polyenes, and echinocandin, which are usually effective againstAspergillus and Candida [15]. This is described as clinical resistance, which is the inability to eliminate a fungal infection despite the antifungal agents having in vitro action against the pathogen [15]. Currently, there is no FDA-approved antifungal otic solution for otomycosis treatment [16]. Physicians have struggled to discover the most efficient antimycotic medication to treat this ailment since there are numerous medicines available with varying antimycotic properties. This opens up the research of using herbal plants as an alternative drug to treat otomycosis.
According to the World Health Organization, herbal plants are one of the ideal sources for obtaining a range of medicines [17]. Hence, these plants should be studied further to evaluate their benefits, safety, and effectiveness. Henna leaf (Lawsonia inermis) has long been valued for its healing and medicinal properties since ancient times.  The therapeutic effects of various components of Henna are due to its biologically active compound of 2-hydroxynapthoquinone (Lawsone) [3]. Studies have shown that henna leaf extracts in Malaysia inhibit the development of pathogenic fungi [4]. Although commonly used as a natural remedy to treat skin, outer ear, and hair lesions, the safety status and possible ototoxic effects of henna leaves are still to be reported. Ototoxicity is identified as functional disability and cellular degradation of the inner hair cells by therapeutic drugs and chemical compounds and is categorized into cochleotoxicity and vestibulotoxicity [18].
Two Malaysian henna extracts were used, which are aqueous extract and ethanol extract, which, their fungitoxic activity has been established [4]. Yaroko et al. tested the antifungal activity of Malaysian henna leaf extract against two prevalent otomycotic pathogenic fungi, which were Aspergillus niger and Candida albicans . He discovered that the aqueous extract of Malaysian Henna is superior in inhibiting the growth of A. niger . In contrast, ethanol extract is more effective in suppressing the growth of C. Albicans [4]. Three concentrations of aqueous and ethanol henna extracts were used in this study; 25mg/ml, 50mg/ml, and 75mg/ml. Statistical analysis revealed significant inhibition of A. niger with aqueous extract concentrations of 75mg/ml and 50mg/ml, while ethanol extract was significant with a concentration of 75mg/ml only. Meanwhile, henna ethanol extract demonstrated near-significant inhibition againstC. Albicans with a 75mg/ml concentration. The least inhibition was recorded with a 25mg/ml concentration for both extracts [4]. However, to ensure that the substance is administered into the ear canal and absorbed by the inner ear and middle ear mucosa, aqueous and ethanol henna at a concentration of 25% were used and instilled transtympanically [4]. Nadjib et al. also studied using the same concentration of 25% by mixing 25g of powdered henna plant with 100 ml of different solvents to test against C.albicans and various microbes [9]. Meanwhile, Zakaria et al. recommended 20mg/ml as the best concentration to inhibit many pathogenic fungi in aqueous or ethanol extract [19]. This concentration has been shown to have a sufficient antifungal effect in a thin diluted solution. It is suitable for physicians and patients to administer in the form of an ear drop.
Wistar rats are widely used in otological studies and were chosen in our study [20, 21]. The middle and inner ear anatomy is nearly identical to humans. The rat cochlear comprises two and a half turns, making it a good model for cochleotoxicity and vestibulotoxicity research [22]. The rats were all three months old when used in the experiment, equivalent to a 20-26-year-old human [23]. This is due to the widespread recognition of the age connection between mice and humans, particularly regarding neurological development.
The four established vestibular parameters observed in rats after transtympanic instillation of ototoxic agents were dyskinetic head movements and circling, tail hanging, air righting reflex, and contact inhibition of righting reflex [11]. A normal baseline vestibular parameters were ensured in all rats following the inclusion criteria. The henna extract was injected into the right middle ear of each rat for constant observational behaviour. Vestibular parameters were recorded at four hours, twenty-four hours, seventy-two hours, one week, two weeks, and three weeks after transtympanic instillation. Acute vestibular changes can be observed in rats as early as 4 hours and as late as three weeks after transtympanic instillation. This is because rats can reestablish normal postural locomotor function following a temporary vestibular insult on one side [23]. The findings recorded by blinded observers were zero or normal responses in all rats at every vestibular interval tested. Hence, statistical analysis could not be implemented in this study.
To conclude, henna extract in aqueous and ethanol extract can safely be used in otomycosis without vestibulotoxic effect in an animal model, even with a tympanic membrane perforation. Malaysian Henna showed potential to be used as an alternative topical antifungal eardrop in treating otomycosis. We recommend conducting a histological study of the cochlear in an animal model to assess the effect on inner hair cells to fully establish the safety efficacy of Henna before human clinical trials.