Atopic Dermatitis (AD) is a chronic skin inflammatory disorder affecting 20% of children and up to 10% of adults worldwide (Laughter et al., 2021). Clinical manifestations of AD include dryness, erythema, itchy skin, and histological findings illustrate spongiosis and infiltration of inflammatory cells around the upper dermal layer (Han et al., 2016). Immune dysregulation as well as a plethora of genetic and environmental factors significantly contribute to the multifactorial etiology and complexed pathophysiology of a debilitating skin disorder like AD. With regards to immune dysregulation, AD has long been understood to predominantly express skewness towards a systemic T-helper type 2 (Th2)-dominant immune response (Renert-Yuval et al., 2021). With the expression of a Th2-skewed immunity, AD patients tend to exhibit an increase in cytokine expression, with interleukins 4 and 13 (IL-4 and IL-13) being the two pivotal cytokines that orchestrate the pathogenesis of this disease as well as elevated levels of high-affinity IgE receptor known as Fc epsilon receptor I (FcεRI). Studies have reported that the initiation of AD is closely associated with Th2 cytokines whereas the disease progression and chronicity are correlated with a delayed surge of Th1 cytokines mainly interferon- γ (IFN- γ) in the chronic phase, thus presenting itself as a biphasic inflammation (Grewe et al., 1998). Amongst the many prevalent hallmarks of AD, it is often correlated with a high level of circulating IgE and an elevated mast cells (MC) count in AD skin lesions (An et al., 2020).

Originally deriving from the bone marrow, MCs are the only terminally differentiated hematopoietic cells that express the c-Kit tyrosine kinase receptor and its ligand stem cell factor (SCF), to synergistically aid in mast cell proliferation and maturation in peripheral tissues (Mekori et al., 1995). Extensive studies in the literature have established the role of MCs as key contributors in the development and propagation of IgE-mediated hypersensitivity reactions as well as in innate and adaptive immune responses. Additionally, MCs are eminently located around the blood and lymphatic vessels, hair follicles and glandular structures of the skin. Mast cells found in the skin could be easily distinguished from mucosal MCs by their protease content as the former contains both chymase and tryptase in their granules whereas only tryptase is present in the latter (Stone et al., 2010). Upon exposure and sensitization to an allergen, activated B cells undergo differentiation into plasma cells to produce IgE, which readily cross-links with an antigen via high-affinity IgE receptors (FcεRI) on the MCs surface. This in turn stimulates IgE-mediated activation of MCs whereby subsequent degranulation induces the release of cytokines (i.e., IL-4, IL-5 and IL-13), preformed lipid mediators namely leukotrienes and platelet activating factor (PAF) which could further intensify the progression of AD (Kulka and Befus, 2003).

To date, the management of AD mainly focuses on the restoration of a normal functional skin barrier as well as to elicit anti-inflammatory and immunosuppressing effects, in a stepwise manner according to disease severity and symptoms (Lebwohl et al., 2013). Currently, standard treatment regimens for AD involve the use of moisturizing emollients, topical corticosteroids (TCs) and topical calcineurin inhibitors (TCIs) for mild-to-moderate AD; and the reliance on systemic immunosuppressant drugs such as azathioprine and mycophenolate mofetil or phototherapy, especially for more severe AD cases (Akhavan and Rudikoff, 2008; Lee HJ et al., 2016). However, studies have emphasized an increased risk of unwanted side effects such as skin atrophy, telangiectasia, acneiform eruptions and many more due to long-term usage of TCs and TCIs. Consequently, to address an unmet need in establishing a “gold standard” treatment for AD, profound interest lies in unravelling the development of a safe, efficacious as well as cost-effective anti-atopic agents derived from traditional herbs or natural sources (Choi JH et al., 2017; Nygaard et al., 2017).

Recent studies are actively demonstrating the potential pharmacological actions of traditional herbs and their active constituents as they might possess anti-inflammatory, antibacterial, antifungal, immunosuppressive activities and many more. Aloe vera (L.) Burm.f. is a plant widely prized in the medicinal, skincare and beauty industry owing to its wound healing and immunomodulatory properties. Based on a review carried out by Sánchez et al. (2020), the Chinese, Ayurvedic and Arabian medicine highly utilize Aloe vera (L.) Burm.f. as a treatment for constipation, helminth infections, and eczema respectively. In studies performed by Kim et al. (2010) and Finberg et al. (2015), the efficacy of a medicinal plant like Aloe vera (L.) Burm.f. in treating AD could be proven by its ability to modulate immunological responses in AD when administered via oral and topical routes. Once known as folk medicine, this stemless plant has gained a well-deserved reputation in ameliorating AD due to its profound anti-inflammatory properties and effectiveness in wound healing (Zari and Zari, 2015). Moreover, local inhabitants have been using herbs as one of the methods in managing diseases including skin related problems. As evidently seen in Nigeria, residents in Keffi are known to utilize the leaves of Senna alata (L.) Roxb. as a treatment for eczema by applying the herb extract to the affected areas (Mowobi et al., 2016) and this herb was proven to elicit dermatophytic activities due to its bioactive compounds (Oladeji et al., 2020). As reported by Hennebelle et al. (2009), Senna alata (L.) Roxb. was used not only as a topical treatment but also as an oral medicament against skin diseases including eczema. In consolidation, Senna alata (L.) Roxb. and Aloe vera (L.) Burm.f. were administered via the two most common routes of drugs administration, which further elucidates the primary focus of this review on highlighting both oral and topical routes of administration. A systematic review can therefore assist in analyzing the recent and relevant data from studies investigating the potential therapeutic effects of herbs and their active constituents on AD via two common routes of drug administration namely oral and topical. On top of that, an astonishing array of animal models have been developed in the field of skin diseases for drug discovery particularly in mice whereby they develop spontaneous skin lesions after prolonged exposure to various allergens and closely mimic the features of human AD. Therefore, this systematic review consists of a total of 103 in vivo studies of AD models that were being divided into two categories with the first section studying the oral administration of herbs and their active constituents (n = 48) followed by the topical route (n = 55). This review paper was commissioned:

i. to screen recent 6 years of publications (2014–2020) on oral and topical administration of herbs and their active constituents in murine models of AD

Over the past few decades, there has been a tremendous surge in the public’s shifting interest towards the reliance on natural herbal remedies as alternative therapeutic options to conventional Western allopathic medicine to treat AD. Essentially, plant-based or herbal medicine are natural products consisting portions of plants or unpurified plant extracts that possess potential bioactive compounds offering a myriad of medicinal and nutritive values possibly working synergistically, to affect multiple targets that contribute to the pathogenesis of AD. To date, the cornerstone of AD management commonly involves therapeutic intervention with emollients, TCs, TCIs and systemic immunosuppressants, especially for more severe AD cases (Carr, 2013; Nygaard et al., 2017). Nonetheless, inter-individual drug response variability and the potential risk of adverse effects from long-term use such as skin atrophy, tachyphylaxis and telangiectasia, are the key rationale behind a shifting interest towards herbal drug development with lesser side effects (Thomsen 2014; Montes-Torres et al., 2015). With that goal in mind, this systematic review was drawn from 103 studies evaluating the efficacy of novel herbs and their active constituents, synthesized from various preparations including crude extracts, compounds isolated from the herbs or mixtures of different herbs applied via oral and topical administration in in vivo murine AD models.

The use of murine models is needed to aid in drug discovery and testing of novel treatments as they are capable of mimicking different aspects of the pathophysiology of human AD. Mice models of AD are generally classified into three categories: 1) mice induced by epicutaneous sensitization with allergens, 2) spontaneous mice models and 3) genetically modified mice that either over-express a transgene or lack of selective endogenous genes. In this review paper, the majority of the studies used allergen-induced AD models, which are relatively more convenient as the usage of exogenous allergens permits dose- and time-controlled induction (Kim et al., 2019). Also, out of 48 oral and 55 topical herbs administration studies involving AD in murine models, the majority used BALB/c mice strain (oral: n = 31, topical: n = 25), followed by NC/Nga (oral: n = 14, topical: n = 20). BALB/c mice have been widely known to trigger a Th2-dominant immune response, therefore, propagating the development of AD (Lee et al., 2010). On the other hand, NC/Nga mice were investigated to develop approximately 50% of spontaneous AD-like skin lesions when they are kept in conventional housing with uncontrolled air but remain normal and healthy under specific pathogen-free (SPF) conditions. However, this particular mouse model requires repeated applications of a hapten to fully develop AD (Shiohara et al., 2004).

Gender considerations remain an important facet in in vivo pharmacology studies as a substantial body of literature supports the notion that male mice are highly preferred in comparison to female due to the ease of handling, less variation in results and cost-effectiveness. Studies on orally administered herbs in this review correspond to this notion as male mice (n = 28) and female mice (n = 14) were used. Contrastingly, this was not constant with studies involving topical herb administration as female mice were found to lead the number with n = 30 and male (n = 25). However, this gap was not of significant difference and male mice are indeed more favourable in AD research. According to Wald and Wu (2010), female murine models confer a 4-day oestrous cycle which therefore accounts for the use of a larger number of animals as compared to males in order to keep their cycle in synchrony, and this in turn makes it less economical. Additionally, the fluctuation of hormones across their oestrus cycle results in behavioural variability in female mice as seen in the production of a female sex hormone called oestrogen which was reported to negatively regulate the wound healing process of AD-like skin lesions (Mukai et al., 2016). Taken together, researchers more typically eliminate the use of female murine models on the presupposition that they elicit intrinsic variation as compared to males, which could possibly interfere with the efficacy of herbs of interest in ameliorating AD. It was also brought to attention that a range between 6 and 8 weeks were the most commonly used murine age groups across the studies in this review, involving oral (n = 24) and topical (n = 43) herb administration. In murine, the maturation of T cells was at 8 weeks of age, which is crucial for the production of T-cell-dependent antibodies (Holladay and Smialowicz, 2000; Jackson et al., 2017).

A vast array of inducer agents was utilized across the studies on orally and topically administered herbs with the majority of them involving the use of DNCB (n = 52) either individually or in combination with DfE or Biostir-AD ointment (Choi and Kim, 2014a; Choi et al., 2014b; Bae et al., 2016; Jeong et al., 2018) and oxazolone (Jegal et al., 2017; Lee et al., 2018; Jo et al., 2019). DNCB exposure can create covalent conjugates with skin proteins to undergo further processing and presented to T cells for activation. Besides, repeated exposure to DNCB permits the accumulation of macrophages at the sensitization site, hence exemplifying an allergic inflammatory response as seen in AD (Yan et al., 2019). In fact, Ku et al. (2018) mentioned that morphological changes in the skin lesions of DNCB-sensitized mice revealed some of the major histopathological findings associated with human AD such as thickened epidermis, massive infiltration of CD4⁺ T-cells producing pro-inflammatory cytokines as well as increased infiltration of inflammatory cells i.e., mast cells and eosinophils. Apart from DNCB, several other chemical compounds were also used to induce AD-like lesions in topical application of herb section of this review, including DNFB (n = 3), oxazolone (n = 6), repetitive cutaneous sensitization with DfE ointment (n = 12), MC903 (n = 1), and phthalic anhydride (n = 1). As we dived into the induction period, it was noticeable that the average duration of exposure to an inducer lasted between 3 and 4 weeks, with the shortest being a week. However, in a study conducted by Jung et al. (2015), DNCB exposure lasted for approximately 8 weeks (∼55 days). DNFB (n = 8), DfE (n = 6) and FITC (n = 4) are among other inducers that were mentioned in this orally administered herb section of this review. In terms of duration, FITC marked the shortest period of AD induction with only 3 days then the mice were sacrificed. This is because the induction was only for the initial stage of AD and the articles from Tao et al. (2017) and Wang et al. (2017) did not mention any data on dermatitis score, serum IgE level and mast cell count which are the hallmarks of AD hence it was difficult to assess the AD severity. On the other hand, DfE recorded the longest period of AD induction which is 8 weeks.

Interestingly, nine out of 14 studies on topically administered herbs and their active constituents and five out of six studies on orally administered herbs and their active constituents were subjected to repeated application of DfE, subsequently after barrier disruption with a chemical substance mainly known as sodium dodecyl sulfate (SDS). SDS functions to facilitate the development of AD-like skin lesions by eliminating the lipid lamella of the stratum corneum. With reference to Supplementary Tables S1, S3, the oral and topical application of herbs and their active constituents was found to ameliorate AD symptoms via restoration of the skin barrier function (Cha et al., 2016; Yang JH et al., 2016; Cha HY et al., 2017; Cha KJ et al., 2017; Kang et al., 2018; Lee et al., 2018; Zhanxue et al., 2018; Cho et al., 2019; Jo et al., 2019; Park J et al., 2019; Park JH et al., 2019). Breakdown in the architecture of the stratum corneum in response to barrier disruption methods with either SDS or tape stripping, further permits the entry of allergens across the skin to interact with local antigen-presenting cells and immune effector cells. Previous research in the literature illustrated that repetitive application of DfE is capable of contributing to the onset and development of AD via the activation of inflammasomes in the epidermal keratinocytes (Jin et al., 2016). Restoration of an intact skin barrier in AD serves as a valuable therapeutic target since it acts as the first line of defence from various microbes and environmental allergens, as well as decreases in trans-epidermal water loss (TEWL). Subsequently, we found out that two studies of oral herbs and their active constituents' administration and 13 studies using topical herbs and their active constituents' administration were shown to decrease TEWL, whereas five studies on orally treated herbs and their active constituents and seven studies on topically treated herbs and their active constituents showed increased levels of epidermal barrier proteins such as filaggrin, involucrin and loricrin.

Until recently, the etiology of AD could possibly be accounted by two opposing hypotheses brought up by Silverberg and Silverberg (2015). The “outside-in” hypothesis supports the notion impairments in the epidermal barrier are solely responsible for the onset of inflammatory lesions in AD and an essential structural protein filaggrin, possesses a fundamental role in maintaining skin barrier integrity, skin hydration and pH by minimizing water loss (Li, 2014; Malik et al., 2017). In contrast, there are some who propose the “inside-out” hypothesis in which a dysregulated immune response is the key event that propagates the inflammatory cascade in AD and skin barrier disruption is an epiphenomenon (Boguniewicz and Leung, 2011; Silverberg and Silverberg, 2015). In this review, 102 studies could be accounted by the outside-in theory as murine models were introduced to the experimental allergens repeatedly in order to disrupt epidermal barrier integrity. Additionally, 12 studies in this review reported that oral and topical herbs and their active constituents' administration elicited an increase in levels of epidermal barrier proteins such as filaggrin, involucrin and loricrin. Hence, it allows us to justify that orally and topically administered herbs and their active constituents confers the potential to accelerate barrier recovery by up-regulating the expression of such vital structural proteins of the skin.

To fully comprehend the origins of AD, a molecular link has been well-documented between the elevated serum IgE levels and marked infiltration of mast cells. During AD inflammation, an allergen penetrates through the skin and gets readily bound to MCs by cross-linking to the high-affinity IgE receptor, FcɛRI, expressed on their cell surface (Brown et al., 2008; Amin, 2012). Upon the activation of MCs, degranulation occurs within seconds, leading to the subsequent release of preformed secretory granules containing biologically active inflammatory mediators and proteins including histamine (cutaneous inductor of itch), PGs and leukotrienes, as well as serine proteases i.e. mast-cell chymase and tryptase (Kawakami et al., 2009; Siebenhaar et al., 2018). Among the 48 studies on orally administered herbs and their active constituents, 37 of them were reported to measure serum IgE levels and 22 studies measured the number of MCs and reported congruent decrease. Decreased levels in both of these crucial parameters suggest that the oral route of herbs and their active constituents’ administration is capable of alleviating the allergic responses. On the other hand, across the 45 studies investigating IgE levels as an outcome parameter for topically administered herbs and their active constituents, it was found that 39 of these studies measured serum IgE and six studies investigated plasma IgE levels. Fortunately, serum or blood IgE levels successfully decreased after treatment with topical herbs and their active constituents in all 45 studies. However, it is important to note that total serum IgE may not reflect accurate levels of systemic IgE since raised concentrations are present in many patients who have no evidence of allergy. As a result, accurate measures of allergen-specific IgE could assist in defining the bona fide mechanism of novel topically applied herbal drugs in immunity (Qiu et al., 2020). In addition, from the studies involving topical route of administration, 39 herbs and their active constituents were found to significantly decrease the extent of mast cell infiltration into the skin. Moreover, it seems that the reduction of MCs infiltration can be associated with a decrease in MCs degranulation as well as down-regulation of IL-4 and IL-13 cytokines secretion. Therefore, this implies that MCs may serve as a key contributing factor to the suppression of Th2 cytokines in AD-like skin lesions via topical herbs and their active constituents' administration.

The extensive array of mediators secreted from FcεRI-mediated mast cell degranulation can ultimately promote inflammation, resulting in the appearance of macroscopic and microscopic skin lesions which are associated with AD. The severity of AD-like skin lesions could be investigated macroscopically according to the acronym widely known as SCORAD (SCORing Atopic Dermatitis) index. This scoring system serves as a clinical tool to assess the extent and severity of eczema by evaluating the common characteristic skin symptoms of AD including 1) erythema, 2), erosion, 3) dryness and 4) lichenification. In our review, dermatitis severity was measured in 26 studies using oral administration of herbs in which all 26 herbs and their active constituents were able to reduce the dermatitis severity score. The same results were obtained for treatment with topical herbs and their active constituents as the dermatitis severity score was found to decrease in 19 studies. On top of that, there was a study performed by Aslam et al. (2018), investigating on a single compound named thymoquinone, which used both routes of drug administration. Results from this study illustrated that both routes were able to alleviate the dermatitis severity score in murine models of AD. However, more in-depth studies need to be performed to thoroughly compare the efficacy of thymoquinone via different routes of administration before definitive conclusions can be drawn.

The immunological hallmark of AD is characterized by a predominant expression of Th2-inflammatory cytokines (i.e., IL-4, IL-5 and IL-13) in the acute phase followed by a prolonged activation of Th1-type cytokines (i.e., IFN-γ and TNF-α) in the chronic phase of AD (Brunner et al., 2017; Choi et al., 2014b). As demonstrated in Supplementary Table S1, 19 studies showed a simultaneous decrease in both Th1 and Th2 cytokines after treatment with orally administered herbs and their active constituents. Apart from that, 11 articles were found to study the Th2 cytokines independently and results showed a reduction in the level of cytokines. Without much anticipation, four studies measured the cytokines released from both types of helper T cells reported an unpredictable finding as orally administered herbs and their active constituents decreased the level of Th2 cytokines but contrastingly, induced an increase in the level of Th1 cytokines (Wu et al., 2014; Kong et al., 2015; Wan et al., 2016; Han et al., 2018). A substantial body of literature have suggested that an aberrant Th2-immune response plays a key proximal role in the pathogenesis of AD, hence the two vital Th2 cytokines, IL-4 and IL-13 were investigated in 38 and 32 studies of topical administration of herb and their active constituents, respectively. Besides, another Th2-mediated cytokine, IL-5 was measured in 11 studies. In all the studies mentioned above, topical application of herbs and their active constituents resulted in a marked reduction of IL-4, IL-5 and IL-13 expression. To address the classical Th1/Th2 paradigm in AD, TNF-α and IFN-γ levels were investigated as representative Th1 cytokines in 24 and 16 studies, respectively. Across all these studies, topical herbal application induced a decrease in TNF-α and IFN-γ levels, however, this was not achieved in a study by Yang HJ et al. (2017) whereby an unexpected asynchronism in both Th1 cytokines was reported after treatment. This finding could be accounted by the “Th1/Th2 imbalance” in the immunological hallmark of AD, in which Th1 responses are capable of antagonizing the development of Th2 cells, hence producing a contrasting effect in the levels of both types of cytokines (Herberth et al., 2010). On the other hand, extremely low and undetected levels of IFN-γ were reported by Yang J et al. (2017), possibly due to a Th2-driven inflammation whereby IL-4 was able to truncate the key Th1 cytokines namely IFN-γ (Brunner et al., 2017).

Recent progress in molecular biology has provided insights into the potential role of nuclear factor kappa B (NF-κB) transcription factor family in the progression and maintenance of AD (Tanaka et al., 2007). Studies have illustrated that activation of the NF-κB pathway is accountable for the regulation and chronicity of inflammatory skin diseases like AD. NF-κB participates in the differentiation of T cells into various subsets comprising of Th1, Th2 and Th17. Moreover, NF-κB functions to increase the expression and transcription of numerous cytokines, growth factors and chemokines such as IL-1β, IL-6 and IL-8, which are highly linked to the inflammatory cascade in AD (Tak and Firestein, 2001; Liu et al., 2017). In this review, 10 articles on orally administered herbs and their active constituents managed to inhibit the NF-κB pathway and similar results were recorded from 10 articles on topically administered herbs and their active constituents. Apart from NF-κB pathway, the phosphorylation of mitogen-activated protein kinases (MAPKs) signalling pathway is closely associated with the production of pro-inflammatory mediators which could trigger and exacerbate AD. Owing to the importance of MAPK signalling molecules i.e. ERK, JNK and p38, in the activation, proliferation, migration and degranulation of immune cells involved in AD, therefore pharmacological inhibition of this pathway may serve as an attractive strategy for the treatment of allergic disorders (Huang et al., 2019). Inhibition of MAPK activation were seen in eight articles after administration of herbs and their active constituents via oral route whereas 11 studies illustrated an inhibition of MAPK phosphorylation after administration of herbs and their active constituents topically, as illustrated by a decrease in ERK, JNK and p38 protein kinases. As such, these findings propose that both NF-κB and MAPK signalling pathway serve as the potential avenues of investigative research in the development of a new therapeutic agent to treat AD.

Throughout this systematic review, we found that serum IgE, infiltration of mast cells and the levels of Th1 and Th2 cytokines were evaluated the most in 82, 62 and 53 studies respectively. These three biomarkers are interrelated in the pathogenesis of AD as depicted in Figure 3. In brief, allergen penetration stimulates T cells to secrete cytokines (IL-4, IL-5, IL-13, IFN-γ, etc.). The released cytokines in turn trigger nearby B cells to produce IgE which then bind to the high affinity receptor IgE, FcɛRI on MCs. The synthesis and release of inflammatory mediators and cytokines can be induced by IgE-dependent mechanism (Williams and Galli, 2000: Kulka and Befus, 2003). The mechanism of action of these herbs and their active constituents may involve the inhibition of 1) binding of IgE to the FcɛRI, 2) the release of mediators and cytokines from MCs, or 3) synthesis of IgE from B cells. Although the exact mechanism was not mentioned in each of these studies, the biomarkers of AD related to these three parameters were found to be alleviated. However, it may be difficult to compare the data from different studies as various investigators used different experimental approaches to assess the outcome of AD such as ELISA, histological analysis and dermatitis severity. Reports may also vary considerably depending on the methodological details provided that could potentially lead to risk of bias. Generally, most studies included in this systematic review did not describe their methodologies precisely which could lead to an underestimation or overestimation of the findings. Thus, it is suggested that animal experiments should be properly designed and carried out well with transparency in every step especially blinding in performance and assessment to avoid any bias. Data should be reported entirely and reasons for any dropout data should also be provided. With all the data gathered from 103 studies evaluating the efficacies of oral and topical administered herbs along with their active constituents in alleviating AD and the underlying mechanism of actions using various animal models and inducers, it is hoped that this systematic review will aid in the evidence-based selection of animal models for AD in the future.

This systematic review has summarized 103 articles into several aspects based on PICO; in which the most common population (P) or choice of animal model used was BALB/C mice (n = 56), due to the fact that they lean towards a Th2-dominated immune response which, therefore, suit the disease of interest, AD. As for the intervention (I), this review consisted of both oral and topical administration routes of herbs and their active constituents in AD-like murine models and the most common positive control (C) drug chosen among these studies was the topical corticosteroid known as dexamethasone (n = 34). A substantially large number of these studies (n = 75) were conducted in Korea and none of the studies analysed the same herb and only two articles by Aslam et al. (2018) and Tsang et al. (2016) were understood to compare the different routes of administrations of the same herb mixture and compound. Findings from these two studies showed that both oral and topical routes of herb and their active constituents’ administration were able to alleviate AD, however, the authors did not make a distinct comparison as to which route was more effective than the other. The common outcome (O) parameters of AD assessed amongst these studies include the histological findings of AD-like skin lesions, serum IgE levels, mast cell count and Th1/Th2 cytokines. Generally, both oral and topical administration of herbs and their active constituents elicited the potential to decrease the AD parameters mentioned above. Besides, large attention was also drawn towards the production and expression of Th2 cytokines in these studies. Upon tabulation of the vast array of findings, a myriad of results obtained from majority of the oral and topical studies demonstrated that herb extracts, mixtures and compounds isolated from herbs could markedly suppress IgE levels and MCs infiltration or degranulation. Theoretically, upon successful inhibition of IgE-mediated activation of MCs, they therefore hinder the subsequent degranulation and secretion of pro-inflammatory cytokines and preformed mediators. Taken together, these findings allow us to propose that the anti-AD effects could be attained by down-regulation of IgE-mediated MCs activation in in vivo models. Therewithal, MAPK and NF-κB pathways are worth to dive into for future research endeavours as the inhibition of these signalling pathways have been recognized as a potential therapeutic candidate against AD. It is also important to note that majority of the studies included in this systematic review mentioned about compliance with animal welfare as well as conflict of interest statement among the authors. However, most studies did not distinctly elaborate on examiner blinding, sample size calculation and animal allocation. As a result, there is ample room for methodological improvement in these studies as the authors could declare these aspects in order to circumvent an unclear risk of bias. Therefore, future research should implement these crucial details to enhance the quality of studies.