Can Propolis Be a Useful Adjuvant in Brain and Neurological Disorders and Injuries? A Systematic Scoping Review of the Latest Experimental Evidence

Propolis has been used therapeutically for centuries. In recent years, research has demonstrated its efficacy as a potential raw material for pharmaceuticals and nutraceuticals. The aim of the present scoping review is to examine the latest experimental evidence regarding the potential use of propolis in protecting the brain and treating neurological disorders and injuries. A systematic scoping review methodology was implemented. Identification of the research themes and knowledge gap was performed. After applying the exclusion criteria, a total of 66 research publications were identified and retrieved from Scopus, Web of Science, Pubmed, and Google Scholar. Several key themes where propolis is potentially useful were subsequently identified, namely detoxification, neuroinflammation, ischemia/ischemia-reperfusion injury/traumatic brain injury, Alzheimer’s disease, Parkinson’s disease, and epilepsy models, depression, cytotoxicity, cognitive improvement, regenerative medicine, brain infection, and adverse effects. In conclusion, propolis is shown to have protective and therapeutic benefits in alleviating symptoms of brain and neurological disorders and injuries, demonstrated by various in vitro studies, animal models, and human clinical trials. Further clinical research into this area is needed.


Introduction
Propolis is a natural, non-toxic, and resinous substance collected by bees to maintain hive homeostasis and to provide physical and biochemical protection to the hive [1][2][3]. Propolis has been used therapeutically for centuries as it possesses various biological activities including antimicrobial, anti-inflammatory, anti-cancer, and antioxidant properties [4][5][6]. Several preliminary clinical studies have also demonstrated the efficacy of propolis as an adjuvant for treating Sars Cov-2 infections [7,8]. Therefore, propolis appears to be a promising raw material for the future development of new therapeutic compounds. The biological activities and therapeutic properties of propolis are shown to be due to its content of plant secondary metabolite compounds such as phenolics and terpenoids [9].
Another exciting area of research is the use of propolis in treating neurological and brain disorders. However, the efficacy of propolis in this particular area has not been thoroughly explored. The main objectives of the present scoping review are to investigate the landscape of propolis research, identify the knowledge and research gap, and provide guidance for future research investigating the potential therapeutic uses of propolis in treating brain and neurological injuries, either as pharmaceuticals or nutraceuticals.

Methods
The scoping review was performed in accordance to the guidelines provided by Peters et al. and Munn et al. [10,11]. The four-phase flow diagram of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) was followed [12].

Search Strategy and Study Selection
The guiding question for the present scoping review was as follows: Can propolis be used as adjuvant therapy and/or to protect brain and treat neurological disorders? Two independent reviewers (F.Z. and S.T.) performed the searches up to 5 August 2021. The databases searched were Scopus, Pubmed, Web of Science, and Google Scholar. Table S1 illustrates the search strategy and the terms included in the search. Limited keyword searches were used for Google Scholar as expansive keyword searches appeared to be redundant.
The objective of the present scoping review is to evaluate the latest experimental evidence in the potential use of propolis, and therefore the search was limited to the last ten years of research: 2012-2021. In addition, we also focus on the studies that evaluate propolis as a whole and not the individual bioactive components of propolis. Consequently, we did not use the terms that describe individual bioactive components of propolis such as caffeic acid phenethyl ester (CAPE), pinocembrin, apigenin, and so on. However, during the search process, if the articles that described the individual bioactive components of propolis appeared, we would include them in the screening process. Moreover, we excluded studies that use synthetic derivatives of propolis bioactive compounds.

Eligibility Criteria
Any article that describes the potential use of propolis in protecting the brain or treating neurological disorders was selected including in vitro studies, animal models, and human clinical trials. We included all articles from all fields of science and technology. The titles and abstracts were analyzed and selected according to the eligibility criteria. Review studies were excluded as they might impart biases to the present study. Only articles that were written in English were included.

Data Collection
Two reviewers (F.Z. and S.T.) assessed the search results independently. If any disagreement arose on the eligibility criteria of a particular article, the disagreement was resolved through discussion and consensus. The studies that were both included and excluded were recorded in Mendeley. The duplicates were then removed. The collected articles were then screened by analyzing the titles, keywords, and abstracts. The articles that did not fit in the guiding question were subsequently removed. For the remaining articles, further screening was performed by analyzing the full texts. For articles where we could not find the full text, we analyzed the abstracts and subsequently included the articles if the abstracts clearly stated the experimental methods, analyses, and detailed results.
The following data of the resulting articles were then collected and tabulated in Microsoft Excel; full reference, types of study, types of propolis extract and/or propolis bioactive compounds, geographic locations of the propolis source, and measured outcome. The reviewers subsequently analyzed the titles, abstracts, and full texts and categorized the included studies into the appropriate themes.

Results
The initial search resulted in 3624 scientific articles. Duplicates of 2683 were subsequently removed. Further screening based on the titles and abstracts excluded 799 articles. The full texts of the 142 articles were then analyzed and screened. The final screening resulted in 66 articles. Figure 1 illustrates the screening process. The qualitative analysis of the 66 included articles was performed and the articles were categorized into several themes. Table 1 illustrates the themes represented in the included studies; brain infection (2%), ischemia/ischemia-reperfusion/traumatic brain injuries/aneurysm (14%), detoxification (24%), Alzheimer's disease model (9%), Parkinson's disease model (8%), epilepsy model (3%), cognitive improvement (6%), regenerative medicine (3%), neuroinflammation/pain/oxidative stress (12%), depression & stress models (9%), cytotoxicity (8%), adverse effects (2%), and others (2%). Table 2 summarizes the themes, references, and the main findings alongside the types of propolis extracts and bioactive compounds used in the included studies. In addition, Figure 2 illustrates the types of studies in the included articles, namely animal models (67%), cell cultures (22%), in vitro (6%), randomized placebo-controlled human clinical trials (4%), and case reports (1%). The percentages are rounded to the nearest whole number.  Propolis induced the expression of the protective heat-shock protein (hsp)-70 and reduced the expression of inflammatory markers such as caspase-3 and apoptosis inducing factor in traumatic brain injury animal model.   Propolis inhibited the expression of NOS, NO, TNF-α and caspase-3 in the cerebral cortex (CC), cerebellum (CB) and brain stem (BS) of kainic acid-induced rats.  [53] Animal model (mice, n = not specified) Ethanolic extract up to 1000 mg/kg BW India High concentration of propolis extract up to 1000 mg/kg BW did not negatively affect the histological appearance of organs, including the brain. [54] Cell cultures (astroglia cell line/SVGp12) Ethanolic extract 10-100 µg/mL CAPE and Chrysin 5-50 µM
Adverse effects [77] Case report Not specified 50 g/day for 3 days

Turkey
Propolis appeared to induce psychotic episodes in a thirty four year old male.

Geographical Sources of Propolis and/or Bioactive Compounds Percentage (%)
Bioactive compounds 30

Discussion
The largest body of experimental evidence found in the present scoping review was in the detoxification theme. The therapeutic properties of propolis and its bioactive compounds appear to be due to their anti-inflammatory properties. In animals and cell cultures which were subjected to chemical and radiation toxicity, propolis was consistently demonstrated to reduce the expression of inflammatory and oxidative markers such as malonaldehyde (MDA), tumor necrosis factor-α (TNF-α), nitric oxide (NO), and inducible nitric oxide synthase (iNOS), while increasing and maintaining antioxidant parameters, namely superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione (GSH) [23,27,[29][30][31][32]34,36]. In addition, it inhibited apoptosis by reducing the expression of genes associated with apoptosis signaling pathways; protein-coding gene Bax, cytochrome-c, cas-3, cas-8, and p53 genes [24,25]. It was also evident that propolis protected cell membranes and prevented further deterioration of the tissue morphology associated with toxicity [23,26,28,33,35,37].
The neuroprotective effect of propolis was also demonstrated in terms of alleviating symptoms associated with aneurysm, ischemia, ischemia-reperfusion and traumatic brain injuries. The anti-inflammatory properties of propolis were shown to play a significant role in attenuating the negative effect of these injuries. Propolis reduced the expression of interleukin-6 (IL-6), TNF-α, matrix metalloproteinase-2 (MMP-2), MMP-9, monocyte chemotactic protein-1 (MCP-1), and iNOS, while increasing the expression of protective proteins such as heat shock protein-70 (hsp70) [14,[16][17][18]20]. It also inhibited the development of histopathology associated with these injuries and in some cases promoted the development of myelinated fibers [15,17,21]. More importantly, propolis was shown to significantly ameliorate the impairment of sensory-motor and other physical indices in animals subjected to these injuries [15,18,21,22].
Unsurprisingly, propolis was shown to be effective in attenuating symptoms of neuroinflammation, pain, and oxidative stress. Propolis was consistently shown to reduce inflammation markers such as vascular cell adhesion molecule-1 (VCAM-1), nuclear factor kappa B (NF-kB), mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK)-associated markers in artificially induced inflammation in both cell cultures and animal models. It also reduced the expression of reactive oxygen species (ROS) and pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α [63,64,[66][67][68]. In one study, propolis was shown to prevent the migration of leukocytes into the inflammation site [65]. Propolis also appeared to upregulate the expression of zinc-finger protein A20 during inflammation; a novel anti-inflammatory mode of action of propolis [61].
Moreover, the anti-depressant properties of propolis were demonstrated in various animal model studies. Propolis reduced the level of corticosterone and adenocorticotropic hormones in stressed and depressed animals [71,74]. Apoptosis of neurons in the brain regions such as hippocampus and prefrontal cortex was also inhibited by propolis [72,73]. In addition, it modulated the expression of inflammatory markers such as TNF-α, IL-1β, IL-6, kynurenine (KYN) levels, indoleamine-2,3-dioxygenase activity,5-hydroxytryptamine (5-HT), brain-derived neurotrophic factor (BDNF), and glucocorticoid receptors [73][74][75][76]. The modulation of the endocrines and biochemical markers resulted in the attenuation of depressive behavior and cognitive impairment in the animals.
In the neurological and neurodegenerative disease models, namely Alzheimer's disease, Parkinson's disease, and epilepsy, propolis also showed potential therapeutic benefits. Propolis was demonstrated to reduce amyloid fibrillation and reduce the impact of amyloid accumulation [39,42]. In addition, propolis inhibited the activity of both acetylcholinesterase and butyrylcholinesterase in a dose-dependent manner [40][41][42][43]. In the Parkinson's disease and epilepsy models, propolis reduced neuronal loss and improved the histopathology associated with these diseases [46,47,49,69,70]. In all of these disease models, propolis consistently reduced the expression of inflammatory markers, maintained the antioxidant status, and improved motor/cognitive scores of the animals [44,45,48,69].
One study showed that propolis could potentially be used as an adjuvant for treating brain infection [13]. Nosratiyan et al. (2021) and Shao et al. (2021) demonstrated that propolis can be used in regenerative medicine as it induced axon myelination and oligodendrocyte progenitor cells (OPC) differentiation. Propolis was also shown to be cytotoxic towards cancerous brain cells, i.e., glioblastoma cells, astrocytes, and astroglial cells [50][51][52]54]. However, Kalia et al. (2014) observed no cytotoxicity in organs, including the brain of normal mice fed up to 1000 mg propolis extract/ kg body weight.
Arguably, the most important studies were the translation of the therapeutic benefits of propolis into humans demonstrated in randomized, placebo-controlled clinical trials (RCTs). The present scoping review managed to identify three RCTs (n = 246 subjects in total). Propolis was shown in all of these studies to improve cognitive function of geriatric subjects measured by various standardized tests; Cognitrax, MMSE, and ADAScog. The cognitive improvement appeared to be correlated to the improvement of the serum level of inflammatory markers such as IL-6, TGFβ1, hs-CRP, and serum level of other biochemical markers namely total cholesterol, LDL, urea, creatinine, and uric acid. No adverse event was recorded in these studies [55,57,58]. However, we identified a case report where propolis appeared to induce psychotic episodes in a thirty-four-year-old male in Turkey [77]. Based on various human clinical trials in other areas of propolis research, propolis appears to be generally safe in humans [9,[80][81][82][83][84]. Figure 4 summarizes the potential use of propolis as an adjuvant therapy in brain and neurological disorders and injuries. In the present review, the reviewers adopted a comprehensive and systematic search strategy in order to objectively fulfill the aim of the study. A broad range of studies from all fields of science and technology was collected and analyzed. The reviewers limited the search to studies that were published in the last 10 years, to provide coverage of the latest experimental evidence in the field. However, the reviewers only assessed and included English language articles, which could potentially lead to missing studies from non-English databases, as it is apparent most studies originated from non-English speaking countries. The reviewers also did not assess the quality of the included studies in order to include as many studies and to provide as broad coverage as possible. In addition, the reviewers did not perform meta-analysis as it is not appropriate due to the heterogeneity of the included studies.

Future Directions and Concluding Remarks
One of the main criticisms often aimed at natural product research is the lack of characterization of the main bioactive compounds. This can be found in the included studies as 31% of the studies did not provide clear identification of the types of propolis extract used. In addition, 19% of the studies did not indicate the geographical location of the propolis source. We propose that all future studies investigating the biological activities of propolis should include at least two pieces of information, namely the types of extract and clear geographical location of the propolis source. Basic chemical analyses, where possible, of the propolis extract, such as total phenolics and/or flavonoids, should be performed. These steps would standardize propolis research, significantly assist in replication studies, and further solidify the potential therapeutic uses of propolis.
In addition, studies that investigated the biological activities of propolis may refrain from concluding certain phenolics, flavonoids, or terpenoids that impart its biological activities, unless it was clearly demonstrated. It appears the therapeutic benefit of propolis may lie in the synergistic effect of various compounds rather than individual compounds [65]. Furthermore, the majority of propolis extracts used were extracted using organic solvents such as ethanol and methanol. Concerns with regard to chemical toxicity, contamination, religious and cultural reasons often arise due to the use of organic solvents for extraction purposes. Another promising area of research is the use of safer and greener chemical alternatives such as natural deep eutectic solvents (NADES), glycerol, and propylene glycol [85][86][87]. Moreover, the majority of the studies used propolis from a single species of bees; the European honey bee (Apis mellifera). Future research could also explore the potential therapeutic properties of propolis harvested from the hives of other bee species such as Apis cerana and Meliponini bees (stingless bees). In conclusion, the present scoping review demonstrates that propolis is a promising therapeutic substance, either as pharmaceuticals or nutraceuticals, for protecting the brain and treating neurological disorders and injuries.