1. IntroductionNatural products have been widely used as a rich source for antimicrobials with structural as well as chemical diversity for the purpose of drug discovery. Since antimicrobial resistance increases for market antibiotics once they are used in clinical practice, the need for nature-based alternatives has increased [1,2].The scabies itchy mite, Sarcoptes scabiei, is a medically important pathogenic ectoparasite of human as well as other mammals worldwide [3]. The mites burrow deep inside the stratum corneum layer resulting in inflammation, immunity reactions, and progressive skin hyper-keratinization, usually followed by bacterial infections or renal/cardiac ailments [4]. Scabies was added to the World Health Organization (WHO) list of neglected tropical diseases in 2017, which highlights the substantial global health and economic burden, especially in low income populations [5]. Similar to human infection, rabbits are the most affected animals by Sarcoptes infection, known as ‘mange’ which results in economic losses of rabbit colonies, reduction in their productivity, and lethality in the case of absence of treatment [3]. Mange in rabbits is usually treated with synthetic acaricides such as ivermectin, doramectin, organophosphates, and pyrethroids, which have serious side effects and environmental hazardous impacts [6,7,8,9].Mites exert a long co-evolution with their hosts and stimulate the host’s keratinocytes with their eggs, feces, saliva, and enzymes resulting in inflammation [10]. Later, they stimulate fibroblasts, endothelial cells, and immune effector cells including mast cells, macrophages, and lymphocytes which carry antigens from the mites to the lymphatic tissue to start the adaptive immune responses [11]. As a result, a cross talk occurs between inflammatory and anti-inflammatory cytokines as well as chemokines, which results in the appearance of scabies symptoms [11].The synthetic acaricides mainly work on mange mites nerve axons by modifying the kinetics of ligand-gated ion channels of mange mites and breaking their life cycles [6,7,12,13]. Disadvantages include drug resistance, treatment delay due to the absence of a significant effect on tissue healing, environmental toxicity, and high cost especially for resource-poor farmers who extensively deal with mammals in the developing world [14]. Natural products are an excellent alternative to synthetic drugs due to the richness of bio-actives that have been widely used for fighting against parasitic infections [11]. Such phytoconstituents attain broad spectrum efficacies, multiple mechanisms of action, as well as tolerable toxicity [15,16].Coconut palm (Cocos nucifera, Arecaceae) is native to Southeast Asia and India, and is considered one of the essential fruit palms in the world [17]. Besides edible applications due to high nutritional value, they can produce biodegradable detergents, cleaning products, cosmetics, toiletries, and plasticizers [18]. The coconut fruit ethanolic extract reveals the presence of phenols, tannins, anthocyanidins, flavonoids, triterpenes, steroids, and alkaloids, while the oil has been reported to contain medium-chain fatty acids. The edible oil attains antioxidant, anti-inflammatory, antiviral, anti-inflammatory, antimicrobial, and immunomodulatory efficacies and has been reported to have emollient as well as wound healing efficacies [3].In the present study, the phytochemical composition as well as GC-MS profiling of coconut seed extract (CSE) was employed. Additionally, the scabicidal potential of CSE on sarcoptic mange in rabbits has been investigated via in vitro, in vivo, histopathology, mRNA expression, and docking analysis for the first time to open the gate for incorporating natural candidates towards proper and safe management of sarcoptic mange in rabbits. Figure 1 depicts the framework of the present investigation. 3. DiscussionThe pathogenesis of scabies is quite complicated and various mechanisms are implicated such as the persistence of the parasite, microvascular/endothelial dysfunction, aggravated immune response (via the imbalance between pro-inflammatory and anti-inflammatory cytokines), as well as the induction of permanent oxidative stress caused by the mites burrowing deep into the skin, which directly affects both structure and function of the skin [23]. All these factors combine to hinder proper treatment, especially with the fact that most synthetic agents act on mite killing rather than immunomodulation or tissue healing [4]. Accordingly, plant-derived phytochemicals with wide therapeutic potential with negligible side effects can safely act as alternatives for synthetic candidates for the eradication of infectious diseases [16,24]. In this sense, the coconut palm has been reported to possess antioxidant, anti-inflammatory, antimicrobial, and immunomodulatory properties with an emphasis on the presence of a high content of lauric, palmitic, stearic, and oleic acids which have proved efficacy against scabies in a number of studies [25,26,27,28]. It showed antibacterial activity against Staphylococcus aureus and Candida skin infections such as oral and vaginal candidiasis with significant results [29]. Interestingly, studies have demonstrated that coconut seed oil may alter gene expression in inflammatory responses, with suppression of pro-inflammatory cytokines and enhancement of protective barriers in the skin [30]. To the best of our knowledge, the acaricidal effect of CSE against Sarcoptes scabeiei has never been investigated.Hence, the present study investigated the GC/MS composition as well as the phytochemical composition of crude seed extract and evaluated the scabicidal potential of the CSE against scabies mites in both in vitro and in vivo assays. The GC-MS analysis revealed the presence of a prevailing percentage of oleic, palmitic, and stearic acids, known as medium-chain fatty acids, which is a further encouraging step as they are reported to demonstrate repellent activities against many arthropods [31]. The application of CSE did not reveal any signs of skin irritation, inflammation, or restlessness during and after the application, coincident with the reported safety of topical coconut extract [32]. The safety of the oil was ascertained by studies that reported the efficacy of coconut palm as antifungal, antibacterial, anti-inflammatory, and anti-eczema without allergic outputs [18]. Our results revealed that CSE could exert a cidal effect on Sarcoptes scabiei mites 24 h post application, which shows a strong acaricidal effect, as reported [6,31]. The mites’ death positively affected the inflammatory reactions in the animals’ skin, which started to show healthy signs such as stoppage of inflammation and hyperkeratosis, appearance of new skin layers, and growth of new hair, which was coincident with reports of successful treatment of rabbit mange [14]. This complete recovery was observed after 3 weeks, while the healing of the ivermectin group extended to the end of the experiment (4 weeks) without complete healing. The histopathological results showed improvement in both the dermis and the epidermis, expressed in a decrease in inflammatory cells as well as an absence of remnants of mites in the skin layers of the treated animals [6]. The improvement is mainly due to the death of mites and cessation of inflammation, pruritis, skin destruction, and scale formation [21]. On the other hand, slow changes occurred to the skin layers in the ivermectin group during treatment, where some inflammatory cells as well as remnants of dead mites were still observed at the end of the study. This may be attributed to the concentrated effects of ivermectin towards mites, as well as the normal itching and allergic sensations caused by ivermectin topical application, which prolongs inflammation and further delays the appearance of healthy signs [33].To gain insight into the modulative effects of CSE on the pathophysiology of scabies, epidermal keratinocytes as the first line of defense against harmful external invaders should be implemented [34]. Keratinocytes attain recognition receptors on their surfaces, allowing them to recognize various pathogens and initiate immune responses, via secretion of cytokines, chemokines, and anti-microbial peptides, which contribute to the recruitment of inflammatory cells [34,35]. Keratinocytes play a significant role in the regulation of skin immune homeostasis and any imbalance in their activities can develop a disease [36,37,38]. Our results showed that a large number of genes in both skin keratinocytes and fibroblasts are differentially expressed in response to live burrowing scabies mites, or their products (as saliva or eggs), which further activate other cell types [39,40]. Thus, there is a complex interplay (cross talk) among many other cell types in the skin (including lymphocytes, endothelial cells, LCs, and dendritic cells) in response to scabies, leading to evolution of inflammation and oxidative stress states [40]. This might enhance reactive oxygen species such as hydrogen peroxide (H2O2), which induces lipid peroxidation and causes harmful alterations to both structure and permeability of the skin [41,42,43]. In our study, amelioration of the altered oxidant/antioxidant balance towards normal in treated animals indicated the potential antioxidant action of coconut, supported by faster clinical and parasitological recovery. Antioxidants are thought to help manage wound oxidative stress and hence speed up wound healing [44]. They play a critical role in controlling the damage that biological components such as DNA, protein, lipids, and body tissue may sustain in the presence of reactive species [44]. The ROS scavenging potential of the extract might be the cornerstone in the management protocol since high levels of reactive oxygen species (ROS) at the wound site are the main promotor of collagen breakdown, destruction of the extracellular matrix (ECM), reduction in angiogenesis and re-epithelialization, and increasing pro-inflammatory cytokines, all which prolong inflammation [44,45].Reports state that penetrating mites sensitize the skin keratinocytes and tend to suppress the body’s immunity by downregulating the gene expression of i-CAM-1 (an intracellular adhesion molecule on the endothelial cell surface), which reduces the supply of blood and immunity cells to the area of penetration and diminishes both lymphocytes’ and neutrophils’ protective mechanisms [46]. On the contrary, the infection upregulates the chemokine MCP-1, which activates immune cells to promote inflammation [47]. Likewise, the regulatory T cells (type 1) are elicited to secret IL-10, which is an anti-inflammatory cytokine that plays a crucial role in preventing inflammatory and autoimmune pathologies in humans so clinical symptoms are unobserved until 4–6 wks after a person becomes infested with scabies mites [11,46]. Additionally, the keratinocytes sensitized by mite products tend to increase expression of VEGF, which is also increased by the mites themselves to increase angiogenesis, through which the mites enhance the surrounding blood supply to obtain requirements from the food, a process that further exaggerates inflammation [11]. As a result, KGF receptor signaling is diminished, which further reduces the proliferation rate of epidermal keratinocytes at the wound edge, resulting in delayed re-epithelialization of the wound. The inflammation also causes a significant elevation of matrix metalloproteinase (MMP-9) which belongs to MMPs—a group of hydrolase enzymes expressed in many overwhelming cases such as wounds, osteoarthritis, ischemia, and infectious diseases [46,48]. MMP-9 is implemented in nearly all parasitic infections for purposes of tissue remodeling and generally results in the downregulation of ECM syntheses such as collagen II and aggrecan [49]. The biological activities of MMPs are strictly controlled by TIMP-1 (tissue inhibitor of metalloproteinase) and upon the imbalance in the MMPs/TIMPs ratio, proteolysis occurs [50].Accordingly, reversing the activity of this series of interconnected genes may act as a valuable therapeutic strategy in scabies progression [51]. The topical application of CSE led to a significant decrease in the expression of IL-1β, IL-6, IL-10, MMP-9, VEGF, and MCP-1, and an increased gene expression level of i-CAM-1, KGF as well as TIMP-1, all of which are expressed in Figure 9 [51]. As a result, this led to a decrease in pro-inflammatory cytokines and an increase anti-inflammatory ones as well as an improvement in host immunity towards invading mites, which could reverse the unhealthy signs and result in decreasing inflammation, improving re-epithelialization and fast recovery [50,51].The in silico docking was performed on the individual phytoconstituents of CSE to present a computational explanation to the previous findings and also explain whether the extract could affect the life cycle of the mites themselves causing their death [52]. The results clearly showed that most compounds acquired sufficient affinity towards the screened proteins, especially gondoic acid (11-eicosenoic acid) which possessed higher affinities towards IL-1β, IL-6, and VEGF with higher binding energy scores [53]. Interestingly, among many compounds which showed good affinities towards the GST protein, the sugar compound 3″(1‴-O-β-D-glucopyranosyl)-sucrose showed the highest affinity score. GST is a chief enzyme for mites which might contribute to insecticidal resistance by playing a central role in the detoxification of xenobiotic and endogenous compounds in mites [22]. It should be taken into consideration that studies reported the acaricidal effect of sugar derivatives such as erythritol, which was reported as a safe insecticide [52].Collectively, it is clear that the fatty acids content of coconut seed extract may share a great role in the fast recovery of animals from scabies, which explains why coconut seed extract was used in a previous study side by side with oils as a cream base for aloe vera gel to treat skin damages occurred with scabies infection [54,55]. Previous studies reported the anti-inflammatory, skin permeation, and enhancement of the angiogenesis of FAs, where they moisturize the dry desquamated skin by traversing deeper into the different layers of the skin, diminish the fibroblasts and other inflammatory cells in the scarring tissues, and promote collagen formation which enhances wound healing [56,57]. This was coincident with a previous study which stated that FAs contained in coconut seed have soothing and curative effects on the skin, as well as hair-growth-promoting effects which greatly aid in clinical recovery via the enhancement of skin smoothness and fur growth in rabbits [58].