Recent Update on the Anti-Inflammatory Activities of Propolis

In recent years, research has demonstrated the efficacy propolis as a potential raw material for pharmaceuticals and nutraceuticals. There is limited report detailing the mechanisms of action of propolis and its bioactive compounds in relation to their anti-inflammatory properties. Thus, the aim of the present review is to examine the latest experimental evidence (2017–2022) regarding the anti-inflammatory properties of propolis. A systematic scoping review methodology was implemented. After applying the exclusion criteria, a total of 166 research publications were identified and retrieved from Scopus, Web of Science, and Pubmed. Several key themes related to the anti-inflammatory properties of propolis were subsequently identified, namely in relation to cancers, oral health, metabolic syndrome, organ toxicity and inflammation, immune system, wound healing, and pathogenic infections. Based on the latest experimental evidence, propolis is demonstrated to possess various mechanisms of action in modulating inflammation towards the regulatory balance and anti-inflammatory environment. In general, we summarize that propolis acts as an anti-inflammatory substance by inhibiting and downregulating TLR4, MyD88, IRAK4, TRIF, NLRP inflammasomes, NF-κB, and their associated pro-inflammatory cytokines such as IL-1β, IL-6, IFN-γ, and TNF-α. Propolis also reduces the migration of immune cells such as macrophages and neutrophils, possibly by downregulating the chemokines CXCL9 and CXCL10.


Introduction
Propolis is a resinous substance collected by bees to provide physical and biochemical protection to the nests. The biological activities of propolis include antibacterial, antiviral, antifungal, anti-parasitic, antioxidant, and anti-inflammatory. Due to its wide-ranging therapeutic properties for humans, propolis has been used to treat ailments for centuries. Historical records show that ancient Egyptians, Greeks, Persians, Romans, Indians, Mayans, and Australian Aborigines independently developed the therapeutic use of propolis. The more recent scientific research demonstrates that the therapeutic properties of propolis are due to its content of plant secondary metabolites such as phenolics and terpenoids [1][2][3].
The present review article attempts to collate and investigate the recent development of propolis as an anti-inflammatory substance. The term 'propolis' in the present study includes propolis from all propolis-producing bees, such as European honeybees (Apis mellifera) and stingless bees of the genera such as Trigona, Melipona, Geniotrigona, Heterotrigona, and Tetragonula.

Immune System Modulation
Inflammation is a coordinated process triggered by the immune system usually in response to microbial infections and/or tissue damage. Inflammatory response is considered successful if the triggers are terminated by an attenuation and resolution of inflammation processes, regulated by anti-inflammatory cytokines and other mediators. However, injurious inflammation occurs if the deleterious inflammatory triggers could not be eliminated and therefore the homeostatic baseline is shifted towards persistent inflammatory environment marked by the constant infiltration of leukocytes and elevated level of proinflammatory cytokines [4][5][6]. Propolis with its inflammation-modulating properties could play a role in regulating the components of the immune system (Table 1). Table 1. Anti-inflammatory properties of propolis in terms ofimmune system modulation.
Doxorubicin and geopropolis alone significantly increased TNF-α and IL-10 production in human monocytes in the presence of LPS. In contrast to the stimuli alone, the combination of doxorubicin and geopropolis increased the production of TNF-α and IL-10.
Geopropolis reduced IL-1β production in the absence of LPS; however, IL-1β production increased in treated monocytes when treated with doxorubicin while remaining at baseline levels when combining with geopropolis.
In the presence or absence of LPS, all stimuli reduced the synthesis of IL-6. In comparison to the control, cells treated with doxorubicin, geopropolis, or both raised the expression of the NF-κB gene, and geopropolis reduced the phosphorylation of IκBα.
[ 15] Cameroon, Ghana, Indonesia, United Kingdom Ethanolic Extract Not determined Propolis extract reduced the pro-inflammatory cytokines production of TNF-α, IL-1β, and IL-6 in LPS-activated THP-1-derived macrophage cells TNF-α production was considerably reduced with P-UK1, P-C, and P-Ind2, while propolis extracts P-UK4 and P-C had the best anti-IL-1β effects.
All propolis extracts strongly reduced the production of the pro-inflammatory IL-6.
[17] For both forms of propolis microencapsulation (SPM and RPM), a substantial reduction in paw volume was seen, indicating a potential anti-inflammatory activity of the SPM and RPM.
Due to the significance of inflammatory inhibition, SPM produced a stronger anti-inflammatory activity than RPM. [18] Indonesia (Tetragonula sapiens) Ethanolic extract Not determined In LPS-induced RAW 264.7 macrophages cells, propolis reduced the production of tumor necrosis factor-alpha (TNF-α), inducible nitric oxide synthase (iNOS), and nitric oxide (NO) [19] Iran Ethanolic extract Caffeic acid, galangin, quercetin, chrysin The expression of Cox2 was significantly inhibited by co-treatment with PEEP on RAW 264.7 cells in all tested dosages (p < 0.001). In considerable doses of PEEP (0.15 and 1.5 µg/mL), treatment with PEEP resulted in an inhibitory effect on the production of IL-1β. PEEP therapy at a dosage of 15 µg/mL substantially reduced the expression of IL-6 mRNA. [20] Italy Hydroethanolic extract Galangin, pinocembrin, CAPE IL-1β and IL-6 levels in human peripheral blood mononuclear cells (PMBC) were slightly increased by PP at a concentration of 25 µg/mL.

Geographical Sources of Propolis Types of Extract Bioactive Compounds Measured Outcome References
In vitro and ex vivo

Malaysia (Heterotrigonaitama) Ethanolic extracts Terpenoids
In RAW 264.7 macraphoages, the H. Itama propolis terpenoid-rich extract showed relative low antioxidant effect but inhibited inflammatory response by decreasing the inflammatory mediators iNOS, IL-1β, IL-10, and increasing the antioxidant mediators HO-1. The study also revealed strong anti-inflammatory activity of terpenoids extracted from the propolis sample. [23] Morocco Hydroethanolic extracts The propolis extracts suppressed the secretion of IL-1β and IL-6 with less effect on TNFα. Propolis also reduced the levels of nitric oxide formed by LPS-stimulated macrophages. Propolis extracts exert an anti-inflammatory effect by lowering the production of IL-10, inhibition of pro-inflammatory cytokines and by the metabolic reprogramming of LPS activity in macrophages. [28] In vivo The parameters evaluated were relative percent survival (RPS), the specific IgM antibody level and the expression profiles of several immune-related genes. The results showed propolis could effectively protect the experimental fish against A. salmonicida infection with an RPS of 89.47%. The gene expression data revealed induction of differential expression of the pro-inflammatory cytokines, MHC class I, T-cell markers and Ig markers in immunized fish, with higher levels in liver and spleen than in head kidney and gill.
Phosphorylation of FcεRI signaling molecules Lyn, Akt and ERK was downregulated in basophils treated with propolis. Propolis also inhibited IgE-CAI and attenuated intestinal anaphylaxis, which involves basophils and basophil-derived IL-4.
Several in vivo studies demonstrated the effect of propolis in modulating the immune system towards regulatory profile and anti-inflammatory environment. Propolis stimulated the trans-differentiation of M1 macrophages to D11b + , Gr-1 + myeloid-derived suppressor cells (MDSCs) in visceral adipose tissue and the peritoneal cavity of lean and obese mice. This trans-differentiation had an anti-inflammatory outcome [29]. In newborn Egyptian-Nubian goat kids, propolis supplementation significantly increased the serum IgG and IgA immunoglobulin levels and reduced the serum pro-inflammatory cytokine levels (IFNγ, TNF-α, IL-1β, and IL-6) [32]. Hsieh [26] demonstrated that propolis attenuated the increase in neutrophil infiltration caused by intraperitoneal injection of monosodium urate (MSU). Propolis also inhibited the MSU-induced IL-1β, active caspase-1, IL-6, and MCP-1 expression in peritoneal lavage fluid [26].
Moreover, propolis and its bioactive compounds especially artepillin C, increased the TNFR2 expression through the IRF4/cMyc axis in Tregs in mice, suggesting the potential of propolis in up-modulating Tregs for the purpose of attenuating inflammatory diseases induced by auto-immune conditions [30]. In an allergic inflammation mouse model, propolis suppressed the IgE/antigen-induced expression of IL-4, IL-6, and IL-13 in basophils. Phosphorylation of FcεRI signaling molecules Lyn, Akt, and ERK was inhibited in basophils treated with propolis. Propolis also down-regulated IgE mediatedchronic allergic inflammation (IgE-CAI) and attenuated basophils-and-basophil-derived IL-4-induced intestinal anaphylaxis [35].
Several studies have demonstrated that propolis exhibits several aspects of proinflammatory activity in immunosuppressive environment, suggesting the ability of propolis to act as an immunomodulator and/or immune-restoring substance. Oliveira [11] showed that propolis could potentially combat the immunosuppressive effect of chemotherapy drug doxorubicin. Propolis increased the expression of TLR-4, TNF-α, NF-κB, and IL-10 in the monocytes exposed to doxorubicin. Interestingly, the expression of IL-1β and the phosphorylation of IκBα in doxorubicin-exposed monocytes were reduced in the presence of propolis [11]. Further, propolis restored the expression of HLA-DR, TNF-α, and IL-6 in Docetaxel-induced immunosuppression of monocytes [15]. Moreover, propolis was also able to induce the proliferation of lymphocyte, the expression of IL-4 and IFN-γ, and promote antibody response with the dominant IgG1 pattern, comparable to Freund's and Alum adjuvants in HIV vaccine candidates [34].

Cancers
The involvement of inflammatory processes has been well studied and documented in tumorigenesis and carcinogenesis. Inflammation plays a significant role in all stages of cancer development [36,37]. Therefore, the use of anti-inflammatory compounds has been linked to lower the risk of cancers, namely esophageal, stomach, liver, and pancreatic cancer [38]. Propolis, as a natural product, with its anti-inflammatory properties could potentially play a role in alleviating cancer progression and its associated symptoms [3].
In the present review, there are five included studies that are related to role of propolis as an anti-inflammatory substance and cancers. Most are in vitro and in vivo studies. In MDA-MB-231 breast cancer cell lines, propolis has been shown to reduce the expression of Toll-like Receptor-4 (TLR4) signaling pathway molecules, such as TLR4, Myeloid differentiation primary response 88 (MyD88), interleukin-1 receptor-associated kinase 4 (IRAK4), TIR-domain-containing adapter-inducing interferon-β(TRIF), and Nuclear factor kappa B (NF-κB) p65 [39]. Uncontrolled TLR4 activation is linked to the progression in many cancers [40]. Propolis also reduced the expression of pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6, and NLR family pyrin domain containing 3 (NLRP3) inflammasome in MDA-MB-231 breast cancer cell lines. In breast cancer animal models, propolis consumption appeared to reduce the population of IL-10 and TGF-β Expressing-CD4+CD25+ Regulatory T cells [41]. The reduction in the expression of IL-10 and TGF-β is thought to be beneficial in cancers as they are both immunosuppressive cytokines that contribute to the development of cancers [42,43].
In addition, propolis down-regulated the expression of NLR family pyrin domain containing-1 (NLRP1) protein and the mRNA of pro-inflammatory cytokines IL-1α,IL-βandIL-18 in human melanoma cell A375 [44]. Moreover, the tumor volume and the multiplicity of colorectal carcinomas in colorectal cancers in rats were reduced by ethanolic extract of propolis. The reduction in tumor volume and multiplicity correlated with the reduction in the expression of the inflammation-associated proteins: inducible nitric oxide synthase (iNOS), TNF-α, NF-κB, and glutathione peroxidase-2 [45].
Moreover, Darvishi [46] found that the breast cancer patients that were supplemented with propolis, prior to chemotherapy, had a significant improvement in terms of oxidant/antioxidant balance compared to baseline, based on the measurement of serum IL-2, protein carbonyl, and TNF-α. Conversely, the placebo group patients had significant increase in terms of those pro-inflammatory markers following chemotherapy [46]. Table 2 summarizes the included studies describing the anti-inflammatory activities of propolis in relation to cancers.  Propolis treatments reduced the population of IL-10 and TGF-β Expressing-CD4+CD25+ Regulatory T cells. [41]

Human clinical trials
Iran Not mentioned Not determined Significant decrease in the antioxidant-oxidant balance in the Propolis group. There was increased serum level of the anti-inflammatory factor TGF-β. The serum levels of MMPs were decreased in the Propolis group. [46]

Dental/Oral-Related Disorders and Diseases
Propolis with its antimicrobial, antioxidant, and anti-inflammatory properties has been shown to be efficacious in treating and alleviating dental/oral-related disorders and diseases [48,49]. Peycheva [50] demonstrated that the expression of pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α, IL-17A, IL-18, and INF-γ was significantly reduced in the gingival crevicular fluid isolated from adolescents with moderate gingivitis after receiving toothpaste containing propolis [50]. Similar observations on the effect of propolis in reducing the expression of pro-inflammatory cytokines were also made in studies investigating the effect of propolis on human periodontal ligament cells and human dental pulp cells and tissues [51][52][53]. In addition, propolis appeared to inhibit high glucose-induced NLRP3 inflammasome activation by downregulating the expression of NLRP3, caspase-1 and IL-1β mRNA and their associated protein levels [54]. Moreover, the anti-inflammatory effect of propolis on the expression of pro-inflammatory cytokines such as IL-1β and TNFαwas also demonstrated in various animal models [55,56]. Table 3 summarizes the use of propolis in dental/oral-related disorders based on its anti-inflammatory properties.  [54]

Indonesia
Ethanolic extract incorporate into gel

Not determined
Propolis increased the expression of TGF-β in the tension side of alveolar bone in the orthodontic tooth movement animal models. [57] Iraq Ethanolic extract Not determined Irrigation with propolis sample as an adjunct to SRP, significantly decreased the serum IL-1β, TNF-α, and MDA levels in experimental periodontitis rats when compared with rats that were treated by SRP with vehicle irrigation. [58] Not determined Ethanolic extract 8.0% artepillin C and 0.14% culifolin In gingival irritation model mice, topical application of propolis ointment promoted wound healing and reduced the infiltration of inflammatory the expression of IL-1β and TNF-α. In human gingival fibroblasts, propolis suppressed an increase in IL-1β and TNF-α upon stimulation with H 2 O 2 [55] Not determined Not determined Not determined In irradiated rats with tongue damage and oral mucositis, propolis reduced the expression of myeloperoxidase and TNF-α. [56]

Human clinical trials
Brazil Aqueous extract Not determined Patients subjected to gingival curettage treated with propolis had better improvement in terms of plaque index (PI), probing pocket depth (PPD), bleeding on probing (BOP), and reduction in interleukin-1β (IL-1β), compared to patients that were prescribed 1% tetracycline. [59] China 95% of Propolis powder (poplar type) mixed with 0.3 mL of normal saline (1:1.5 wt./vol) to form a paste Quercetin, galanga, and chrysin (indentified by the manufacturer) Only at 4 h did the patients in the propolis group have lesser pain scoresthan the calcium hydroxide group. Acute increase in pain scores (flare-up) was slightly higher in the propolis group.
[60] In primary teeth from children aged 5-10 years following pulpotomy, propolis treatment appeared to be more efficacious compared to standard treatment Formocresol based on histological examination showing thick and continuous dentin bridge formation with minimal inflammation (leukocyte infiltration). [62] Not determined Not determined Not determined Propolis-containing toothpaste significantly reduced the plaque accumulation and inflammatory response, especially the IL-1β and IL-6 levels in patients with gingivitis. [63] Not determined Hydroethanolic extract Not determined In patients with leukemia treated with chemotherapy, patients in the propolis treatment had lower incidence of oral mucositis compared to the control group that were given traditional Chinese medicine.
The propolis group patients had significantly shorter oral mucositis recovery time. The mRNA expression of inflammatory cytokines such as IL-22 and TNF-α, and chemokines CXCL9 and CXCL10 were also significantly less in the propolis group. [64] Not determined Not determined Not determined Propolis mouthwash significantly reduced papillary bleeding index. [65] Importantly, human clinical trials were carried out to investigate and demonstrate the use of propolis, especially with regards to its anti-inflammatory properties in dental and oral-related issues.Sukmawati [59] found that in patients subjected to propolis treatment following gingival curettage had better recovery parameters such as improvement in terms of plaque index (PI), probing pocket depth (PPD), bleeding on probing (BOP), and reduction in inflammatory interleukin-1β (IL-1β), when compared to patients treated with the conventional treatment tetracycline [59]. Additionally, in the primary teeth of children aged 5-10 years following pulpotomy, propolis treatment was more efficacious compared to standard treatment Formocresol based on histological examination which showed thick and continuous dentin bridge formation with minimal inflammation induced by leukocyte infiltration [62].
Propolis-containing toothpaste significantly reduced the plaque accumulation and the salivary expression of inflammatory cytokines, especially the IL-1β and IL-6 levels in patients with gingivitis [63]. Propolis was as effective as 0.1% triamcinolone in alleviating pain in erythema in symptomatic oral lichen planus [61]. Kiani [65] demonstrated that propolis mouthwash can effectively decrease gingival inflammation and bleeding, without causing tooth discoloration or staining [65].
Moreover, Lv and Qu (2021) compared the efficacy of propolis and traditional Chinese medicine in alleviating the symptoms of chemotherapy-induced oral mucositis in leukemia patients. They observed that the patients in the propolis treatment had lower incidence of oral mucositis compared to the control group that were given traditional Chinese medicine. In addition, the propolis group patients had significantly shorter oral mucositis recovery time. The mRNA expression of inflammatory cytokines such as IL-22 and TNF-α, and chemokines CXCL9 and CXCL10 were also significantly lower in the propolis group [64].
Shabbir [60] investigated the efficacy of propolis paste as an intracanal medicament on postoperative endodontic pain intensities in comparison to calcium hydroxide in patients with single-rooted necrotic teeth with visible periapical. There was no statistically significant difference in terms of the mean pain scores. However, the incidence of flare-up was slightly higher in the propolis group (17% of the patients) compared to calcium hydroxide group (12%) [60].

Metabolic Syndrome-Related Disorders and Diseases
Metabolic syndrome has a set of well-defined criteria as follows: abdominal obesity (population specific), increased triglycerides (≥150 mg/dL or ≥1.7 mmol/L), reduced HDL (<40 mg/dL for men and <50 mg/dL for women), increased blood pressure (systolic ≥ 130 mm Hg and/or diastolic ≥ 85 mm Hg), and increased fasting glucose (>100 mg/dL or 5.5 mmol/L) [66][67][68]. Metabolic syndrome is a precursor of chronic diseases such as type 2 diabetes mellitus, chronic kidney disease, non-alcoholic fatty liver disease, dementia and Alzheimer's disease, and cardiovascular diseases [69]. The 2017-2018 data showed that metabolic syndrome affects 34% of US population and up to 25% of the population in other developed countries, showing no signs of abating [70]. One of the hallmarks of metabolic syndrome is the chronic inflammation where propolis could potentially play a role in alleviating [69].
The majority of the studies identified and included in the present review that are related to metabolic syndrome were animal models (in vivo) ( Table 4). Sakai [71] demonstrated the infiltration of epididymal fat by macrophages and T cells was reduced significantly by propolis consumption in the mice fed with high-fat diet [71]. Propolis was also shown to reduce insulin resistance, body weight gain, and fat accumulation induced by high fat diet in mice. Additionally, propolis significantly reduced the mRNA expression level of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in a dose-dependent manner, while increasing the anti-inflammatory IL-10 expression in hepatic and adipose tissues [72]. Similar result was also observed in the hypercholesterolemia rabbits. Propolis consumption reduced the level of C-reactive protein, IL-6, and TNF-α, and inhibited the expression of CD68, TLR4, NF-κB p65, MMP-9, and TNF-α in the carotid arteries [73]. Further, Cardinault [74] found that propolis induced the expression of Nrf2-target genes in adipocytes, transactivated Nrf2 response element and the Nrf2 DNA-binding in mice [74]. In high-fat-fed mice, poplar propolis reduced the expression of mRNAs coding for inflammation, namely TNF-α and chemokine C-C motif ligand 5 (Ccl5) and Ccl2. However, Baccharis and Dalbergia propolis appeared to increasemRNA expression. [76] China Ethanolic extract Protocatechuic acid, caffeic acid, p-coumaric acid, ferulic acid, daidzein, cinnamic acid, quercetin, naringenin, genistein, keampferol, hesperetin, and biochanin A EEP treatment reduced insulin resistance induced by high fat diet (HFD)-fed. EEP supplementation improved impaired glucose tolerance and reduced the gain in body weight and fat accumulation in HFD-fed mice.
EEP treatment significantly reduced the mRNA expression levels of TNF-α, IL-1β, and IL-6 in a dose-dependent manner, but increased that of IL-10 in hepatic and adipose tissues. [72] China Ethanolic extract 3-O-acetyl pinobanksin, chrysin, pinocembrin, pinobanksin, and CAPE In hypercholesterolemia rabbits, propolis ameliorated restenosis by reducing the plasma levels of C-reactive protein, interleukin-6, and tumor necrosis factor-α (TNF-α), and inhibiting the expression of CD68, TLR4, NF-κB p65, MMP-9, and TNF-α in the carotid arteries [73]  In streptozotocin-induced diabetic rats, propolis attenuated the increase in the expression of nuclear factor kappa B (NF-κB), tumor necrosis factor-α, interleukin(IL)-1β and caspase-3. In addition, propolis increased the expression of IL-10 and inhibited the proliferation cell nuclear antigen in the liver of diabetic rats.
Propolis also ameliorated the inflammation of the liver by reducing the number of the hepatocytes with pyknotic nuclei and inflammatory infiltration.

Geographical Sources of Propolis Types of Extract Bioactive Compounds Measured Outcome References
In vitro

Taiwan
Ethanolic extract Propolins C, D, F, G, and H In Streptozotocin/High-Fat Diet-induced rats, propolis delayed the progression of type 2 diabetes mellitus by reducing the severity of β-cell damage indicated by the HOMA-β and β-cell mass.
Propolis also significantly reduced the pro-inflammatory cytokines especially TNF-α and IL-1β. [81] Turkey Ethanolic extract  When compared to the L-NAME group, NOS-inhibited (hypertensive) rats treated with propolis had significantly lower levels of NF-κB in testis tissue. [83] Turkey Hydroethanolic extract Not determined In cardiovascular rat models, propolis reduced the expression of d NF-κB levels. [84] Human clinical trials
The cognitive improvement correlated with the decrease in serum pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. Propolis-treated group also had an increase in serum TGF-β1. [86] China Ethanolic extract of propolis Not determined In diabetic patients, propolis increased serum GSH, flavonoids, and polyphenols. In addition, propolis decreased serum lactate dehydrogenase.
In addition, serum IL-6 was significantly increased in the Chinese propolis group. [87] Iran Not determined Total flavones and flavonols: 8.4%, total flavanones and dihydroflavonols: 4.6% and total phenolic compounds: 28% Patients with Type 2 Diabetes Mellitus.
In comparison to the placebo group, blood levels of high-sensitivity CRP and TNF-α significantly reduced after the administration of Iranian propolis.
On day 90, there was a decrease in serum IL1-β and TNF-α in the propolis group. [88] Iran Ethanolic extract Not determined Propolis attenuated hepatic steatosis and fibrosis in patients with nonalcoholic fatty liver disease (NAFLD). Propolis significantly reduced the inflammatory marker hs-CRP. [89] Many other animal studies had confirmed the ability of propolis in reducing the level and expression of pro-inflammatory cytokines in metabolic syndrome-related disorders/diseases. However, Cardinault [76] compared three different types of propolis namely poplar, Baccharis, and Dalbergia and found that poplar propolis reduced the expression of mRNAs coding for TNF-α, chemokine C-C motif ligand 5 (Ccl5), and Ccl2, while Baccharis and Dalbergia propolis appeared to increase these mRNA expression in high fat-fed mice. Interestingly, the poplar and Baccharis propolis had similar polyphenol profile, suggesting it could be other types of bioactive compounds that contribute to this difference in effect [76].
More importantly, several human clinical trials confirm the efficacy of propolis in attenuating inflammation in patients with metabolic syndrome-related chronic diseases. The marker of inflammation MCP-1 (monocyte chemoattractant protein-1) level in urine gradually decreased in the propolis-treated chronic kidney disease patients [85]. Zhu [86] demonstrated that, in a 24-month trial, propolis-treated elderly subjects had significant improvement in Mini-Mental State Exam (MMSE) scores compared to placebo group. The cognitive improvement correlated with the decrease in serum pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. Propolis-treated patients also had an increase in serum TGF-β1 [86]. Zakerkish [88] demonstrated that patients with type 2 diabetes mellitus treated with propolis had significant reduction in the serum level of high-sensitivity CRP (hs-CRP), IL1-β, and TNF-α [88]. Further, Soleimani [89] demonstrated that propolis attenuated hepatic steatosis and fibrosis in non-alcoholic fatty liver disease (NAFLD) patients. Propolis consumption also significantly reduced the serum level hs-CRP in NAFLD patients [89]. Furthermore, Gao [87] found that propolis increased serum glutathione (GSH), flavonoids, and polyphenols, and reduced serum lactate dehydrogenase in diabetic patients. However, serum IL-6 appeared to increase in the propolis group [87].

Organ Toxicity and Inflammation
Inflammatory responses triggered by several factors, namely toxic compounds, pathogens, and damaged cells, can promote acute and/or chronic inflammation in various organs, such as the liver, kidney, lung, brain, cardiovascular, gastrointestinal tract, and reproductive organs, leading to persistent cellular and tissue damage [90,91]. Due to its anti-inflammatory properties, propolis has a potential therapeutic use in this regards (Table 5). Medjeber [92] found that in peripheral blood mononuclear cells isolated from Celiac disease patients, propolis significantly inhibited the expression of nitric oxide and interferon-γ while significantly increasing IL-10 expression. Propolis also downregulated the iNOS expression and the activity of NF-κB and pSTAT-3 transcription factors [92]. In the animal models of ulceritis colitis, propolis had a protective effect in the form of the reduction in inflammatory infiltrates, histological damage, the expression of inflammatory mediators such as TNF-α and NO, and the activity of inflammatory enzymes COX-1 and COX-2 [93,94]. In addition, the inflammatory markers in the colonic inflammation: IL-1β, IL-6, and MCP-1 were reduced by propolis. Propolis also increased the expression of TGF-β. Further, propolis increased the diversity and richness of gut microbiota populations while reducing the population of the unfavorable Bacteroides spp. [95]. Propolis also appears to be gastric-protective through similar modes of action [96,97].  In epirubicin-induced cardiotoxicity and nephrotoxicity rat models, propolis attenuated necrosis, edema, and inflammatory infiltrates in the heart and kidney cells. [102] Algeria Ethyl acetate phase of ethanolic extract

Not determined
Propolis reduced the expression of TNF-α and prostaglandin E2 in associated with the inflammationpaw edema.
[104] The histological assessment showed the protective effect of propolis compared to control. P10 treated animals had lymphocyte-rich inflammatory infiltration, whereas polymorphonuclear-rich infiltration shown in the P100. P10 had lower inflammatory scores and histological damage compared to P100 and control.
Propolis downregulated myeloperoxidase (MPO) activity. [93] Brazil Not determined Not determined Mice were exposed to chronic cigarette smokes for 60 days to develop a condition mimicking the chronic obstructive pulmonary disease (COPD) and treated with propolis orally for another 60 days.
Histological analysis showed that propolis recovered alveolar spaces, alveolar septa, and elastic fibers. Propolis also increased and decreased the expression of MMP-2 and MMP-12, respectively-promoting the process of tissue repair.
Propolis appeared to promote lung repair via via macrophage polarization from M1 to M2 and the downregulation of IGF1 expression in a Nrf2-independent manner.
[105] [107] Brazil Ethanolic extract Not determined L-arginine was administered in groups two and three to cause Acute Pancreatitis. Two 250 mg/100 g of body weight (BW) intraperitoneal (IP) injections of L-arginine produced in isotonic saline (20 percent 0.15 M sodium chloride) were given one hour apart. After two hours after L-arginine injection, the rats in group three received daily treatments of Brazilian green propolis alcohol extract at a dose of 100 mg/kg BW for seven days.
Serum analysis of proinflammatory (IL-1β, IL-6, and TNF-α) was lower in group three (propolis-treated acute pancreatitis rat group) than in group two (acute pancreatitis rat group).
HEGP (100 and 300 mg/kg) showed reduction in histological changes inthe gastric mucosa. HEGP (100 and 300 mg/kg) showed reduction in histological changes in the gastric mucosa. Treatment with HEGP accelerated the healing process promoting a notable recovery of the ulcer base. The pre-treatment with HEGP (300 mg/kg, p.o) increased the mucin-like glycoproteins staining by 67%. HEGP treatment showed ducts of glands with less damage, the ducts with higher amounts of mucus and a basal region with intense PCNA staining, suggesting the enhancement in cellular proliferation. HEGP had a direct DPPH scavenging radical ability in a concentration-dependent manner. Oral pre-treatment with HEGP (300 mg/kg) reduced the ROS production as well as lipoperoxidation levels.HEGP at doses of 100 or 300 mg/kg reduced the SOD activity by 11% and 26%, and increased the CAT activityby 77% and 82%, respectively. Moreover, the GST activity was increased by 20% in animals that received HEGP at 100 or 300 mg/kg. HEGP prevented the increase in MPO activity in ethanol-induced acute gastric ulcer, reducing the activity by 93%. [96] Brazil Hydroethanolic extract Phenolics and flavonoids In rats exposed to excessive UVB irradiation, propolis was shown a photoprotective properties: UV-B exposed rats had comparable appearance to control rats where mast cell infiltration was comparable to controls.

In vivo
Brazil Ethanolic extract CAPE, p-coumaric acid, trans-cinnamic acid, aromadendrin, artepillin C In septic rat model, propolis attenuated the increase in the expression of Toll-like receptor 4 and nuclear factor-kappa B proteins in lung and renal tissues and macrophage infiltration.

China Hydroethanolic extract
In mice with alcohol-induced depressive symptoms, propolis reduced the expression of serum levels of LPS and fatty-acid-binding protein 2 (FABP2). Propolis also reduced the inflammatory cytokines: TNF-α, IL-6, and IL-18 in the spleen of the mice. [116] Denmark Aqueous extract of propolis (PWE) Caffeic acid, ferulic acid, isoferulic acid, vanillin, and cinnamic acid) PWE was successful in protecting against the elevation in the intestinal marker of inflammation TNF-α and MPO activity in radiation-induced intestinal mucosal injury. [117] Egypt Methanolic extract of propolis Not determined In ulcerative colitis, propolis methanol extract administration resulted in a dose-dependently significant reduction in both inflammatory mediators, TNF-α and NO The inflammatory enzymes COX-1 and COX-2 could be inhibited by propolis methanol extractin vivo. [94]

Geographical Sources of Propolis Types of Extract Bioactive Compounds Measured Outcome References
In vivo [120] In the indomethacin-induced gastric ulcer animal models, propolis exhibited gastroprotective properties by increasing PGE 2 level and reducing the expression of inflammatory cytokines such as TNF-α, IL-1β, and IL-6.

Geographical Sources of Propolis Types of Extract Bioactive Compounds Measured Outcome References
In vivo
Propolis did not affect immune cell infiltration and lung function. Propolis attenuated carrageenan-induced hind paw edema and plasma TNF-α level. [127] Turkey Water-soluble propolis extract Not determined Propolis pretreatment significantly reduced I/R-induced IL-6 level elevation in the ovary.
Caspase-3 and TNF-α immunoreactivity in the ovary was minimal in the I/R (ischemia-reperfusion injury) + Propolis group. [128] Not determined Not determined Not determined Propolis alleviated the severity of concanavalin A-induced hepatitis. Propolis attenuated the increase in serum level of the inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) as well as the profibrotic cytokine TGF-β. [129] Not determined Not determined Not determined Propolis alleviated the inflammation in nitrosamine-induced liver injury in animals.

In vivo
Not determined Ethanolic extract Not determined When compared to control mice, the CCl 4 -treated mice group showed a noticeably higher expression of HSP70. propolis caused a significant decrease in the expression level of HSP70 when given orally to CCl 4 -treated mice.
When compared to the liver lysates from control animals, CCl 4 significantly increased the amounts of these proinflammatory cytokines (IL-1β, IL-6, and TNF-α).
The levels of proinflammatory cytokines in CCl 4 -treatedmice were markedly reduced in mice treated with propolis supplementation. [131] Not determined Not determined Not determined When compared to healthy control hamsters, propolis was effective in reducing the neurotoxic effects of propionic acid (PPA), showing minimal changes in IL-6 and IL-10 levels. [132] Not determined Not determined Not determined In rats exposed to CCl 4 , propolis attenuated the increase in TNF-α. [133] Human clinical trials

Indonesia Propolis compress Not determines
The propolis compress treatment group had the highest percentage with a score of 0 or no phlebitis (60%) and the lowest with early signs of phlebitis (40%). [134] Iran Propolis tablet Not determined Propolis significantly attenuated the symptoms in moderate persistent asthma patients, compared patients given placebo. Propolis was also shown to reduce eosinophilic inflammation associated with asthma.
[135] In elderly women suffering from rheumatoid arthritis, propolis consumption (24 weeks) did not improve disease severity in terms of Disease Activity Score in 28 joints using erythrocyte sedimentation rate (DAS28-ESR).
Propolis also did not affect secondary endpoints, other disease activity assessment (DAS28 using C-reactive protein, simplified disease activity index, clinical disease activity index), ultrasonographic evaluation of synovitis, activities of daily living, quality of life, changes in cytokine levels, and adverse events. [136] Brazil

Green propolis Not determined
Brazilian green propolis demonstrated a tendency to reduce inflammation based on the reduction in serum hs-CRP in the hemodialysis patients. [137] Moreover, propolis was shown to reduce the level of prostaglandin E2 (PGE2)-a potent inflammatory mediator in the peritoneal exudates and TNF-α in the liver of epirubicininduced hepatotoxicity in rats [101]. Tsuchiya [106] demonstrated that in acetaminopheninduced hepatocellular necrosis in animal models, propolis significantly reduced inflammatory cell infiltration and down-regulated the mRNA expression of IL-1β and IL-10 [106]. Other groups have also confirmed the hepatoprotective capacity of propolis against hepatotoxic compounds through its anti-inflammatory activities by down-regulating and/or inhibiting the expression of NF-κB, COX-2, transforming growth factor β (TGF-β), Bcl-2, and IL-6 [120,121,125,129,131]. Similar protective mechanisms and anti-inflammatory activities of propolis were also observed in the retina, skin, pancreas, spleen, kidneys, ovary, and heart [100,102,104,108,110,112,118,120,128].
The protective effect of propolis is also observed in lung inflammation caused by environmental toxins. Propolis significantly down-regulated and up-regulated Nrf2 and HO-1 expression, respectively, in a dose-dependent manner in nicotine-induced pulmonary damage [115]. In addition, Barroso [105] investigated the effect of propolis in the chronic obstructive pulmonary disease (COPD) animal models. The mice were exposed to chronic cigarette smokes for 60 days to develop a condition mimicking the chronic obstructive pulmonary disease (COPD) and treated with propolis orally for another 60 days. Histological analyses showed that propolis recovered alveolar spaces, alveolar septa, and elastic fibers. Propolis also increased the expression of MMP-2 while simultaneously decreased the expression MMP-12-inducing tissue repair. Interestingly, propolis promoted the recruitment of leukocytes, including macrophages, withoutROSrelease. Furthermore, the lung repair induced by propolis was demonstrated to be through macrophage polarization (from M1 to M2) and the downregulation of IGF1 expression in a Nrf2-independent manner [105].
The protective effect has been observed in several human clinical trials. Mirsadraee [135] demonstrated propolis consumption significantly attenuated the clinical and physiological symptoms of asthma-often considered as an inflammatory disease. Asthma control test (ACT) score, Forced expiratory volume in 1 s (FEV1), FV1/Forced vital capacity and expiratory flows, and fractional exhaled nitric oxide (FENO) were all significantly improved in the propolis group compared to the placebo. Further, eosinophilia was significantly decreased in the propolis patients whereas the placebo group recorded a significant increase during the trial period [135].
Further, Silveira [137] showed that propolis supplementation significantly reduced inflammatory marker hs-CRP level in hemodialysis patients. In addition, safety parameters such as amylase, aspartate aminotransferase (AST) and creatine phosphokinase (CPK) were not affected [137]. However, Matsumoto [136] found that propolis supplementation in the elderly women suffering from rheumatoid arthritis did not improve disease severity in terms of Disease Activity Score in 28 joints using erythrocyte sedimentation rate (DAS28-ESR). Moreover, propolis also did not improve any of the secondary endpoints such as DAS28 using C-reactive protein, simplified disease activity index, clinical disease activity index, ultrasonographic evaluation of synovitis, activities of daily living, quality of life, changes in cytokine levels, and adverse events [136].

Pathogenic Infections
In the light of evolution, inflammation is a conserved process and an important first line of defense in invertebrates as well as vertebrates against pathogenic invaders. Therefore, inflammation affects both the host and the pathogens, and an uncontrolled inflammation has been shown to be one of the primary drivers of many diseases [138,139]. Reboucas-Silva [140] demonstrated that propolis reduced the viability of Leishmania (Viannia) braziliensis promastigotes and parasite burden inside the infected macrophages. Dry propolis extract significantly modified the inflammatory profile of the macrophages by downregulating the TGF-β and upregulating TNF-α levels, whereas the alcoholic and glycolic extracts upregulated the IL-10 level [140]. This suggests different types of propolis extracts modulate inflammatory signaling pathway differently (Table 6). Propolis reduced, in a dose-dependent effect, the viability of Leishmania (Viannia) braziliensis promastigotes and helped controlled the parasite burden inside the infected macrophages. Dry propolis extract significantly modified the inflammatory profile of murine macrophages by reducing the TGF-β and IL-10 production, while upregulating TNF-α. All three types of propolis extract reduced nitric oxide (NO) and superoxide levels in activated L. braziliensis-infected macrophages.
[140] Propolis did not reduce the expression of IL-2, TNF-α, IFN-γ and IL-17 in the adherent cells of the healthy donors and ATL patients.
When adherent cells from healthy donors were infected with Leishmania braziliensis, propolis pre-treatment increased the expression of IL-6, IL-17, and reduced the expression of IL-10. [141]
krusei and C. parapsisolis, and it reduced in vitro yeast cell adhesion to human keratinocytes at sub-inhibitory concentrations. BOP demonstrated significantly low toxicity in Galleria melonella larvae at antifungal doses. [142] Iraq Hydroethanolic extraction followed dissolution in 8% L-lysine

Not determined
Propolis reduced the expression of IL-1β and NLRC4 inflammasome through activation of autophagy following P. aeruginosa infection.

Korea Ethanolic extract
Korean propolis decreased the levels of these pro-inflammatory cytokines-IL-8, IL-1β, IL-12, and TNF-α, in a dose-dependent manner.It dose-dependently inhibited the expression of COX-2 and iNOS mRNA and protein expression induced by H. pylori infection. It also attenuated the phosphorylation of ERK, JNK, and p38 MAPKs in H. pylori-infected AGS cells in a dose-dependent manner. It significantly inhibited the NF-κBsignaling pathway in H.
pylori-infected AGS cells by suppressing IκBα phosphorylation. It not only had radical scavenging activity, which significantly elevated in a dose-dependent manner but also upregulated the protein and mRNA expression of anti-oxidant enzymes, such as HO-1, NQO1, GCLM, and SOD1 dose-dependently. Korean propolis increased the promoter binding activity of Nrf2 to the anti-oxidant relative. [144]

In vivo
Algeria Ethanolic extract of propolis (EEP) Not determined The concentration of serum TNF-α in the cystic echinococcosis (CE) mice group was significantly reduced after receiving EEP orally, and levels were remarkably similar to those in the Control group.
In the spleen of CE mice, there was a significantly elevated iNOS, TNF-α, and NF-κB/p50 immunoreactivity; however, following propolis therapy, this immunoreactivity significantly decreased. [145] Egypt Ethanol Propolis Extract Not determined All BHV-1-infected groups had higher blood and cellular levels of TNF-α, IL-2 and IFN-γ after receiving propolis extracts at 50 µg/mL for seven days following inoculation.

In vivo
Egypt Used in powder form as a dietary supplement Not determined Dietary PR supplementation was able to (p < 0.05) reduce the negative impact of E. coli challenge on egg number, egg weight and feed intake by 20%, 7% and 18%, respectively, compared to the laying hens fed on basal diet and challenged with E. coli. A significant reduction (p < 0.05) was noticed in TNF-α by 13% and in both IL-1β and plasma corticosterone by 37%. Dietary PR minimized (p < 0.05) the elevation of MDA concentration induced by E. coli challenge by 45% in PR + EC group and retuned back to the normal level compared to control.
Data also showed a significant (p < 0.05) improvement in the antioxidant status by increasing the concentration of TAC and SOD, by 84% and 20%, respectively, compared to C group. Total white blood cells (TWBCs) were reduced (p < 0.05) by 15.8%, while heterophile/lymphocyte (H/L) ratio was increased (p < 0.05) by 1.8 folds in PR + EC groups. The lymphocyte proliferation of T-cells was suppressed (p < 0.05) by 29.5% and B-cell proliferation was inhibited (p < 0.05) by 15.8%, in PR+EC group. PR supplementation caused a significant increase (p < 0.05) of villi height. PR significantly (p < 0.05) increased call viability (CV)% by 40% compared to C group. PR also normalized the Foxo3 expression to similar levels of the C group [147] Saudi Arabia Hydromethanolic extract Not determined Plasmodium chabaudi infection model in mice. Propolis reduced oxidative damage by downregulating the malondialdehyde (MDA) and increasing the catalase (CAT) activity and the glutathione (GSH) levels. Propolis appeared to increase the level of pro-inflammatory cytokines such as IFN-γ, TNF-α, GM-CSF and G-CSF. [148] Not determined Ethanolic extract Not determined In anthrax animal models, propolis reduced the TNF-α expression. [149] In addition, Dos Santos Thomazelli [141] investigated the effect of propolis on Humanderived peripheral blood mononuclear cells from American Tegumentar Leishmaniasis (ATL) patients and healthy donors. They found that propolis pre-treatment increased the expression of IL-4 and IL-17 and downregulated IL-10, either in the presence or absence of the L. braziliensis infection [141]. Moreover, propolis reduced the expression of IL-1β and NLRC4 inflammasome in the bone marrow-derived macrophages through autophagy following Pseudomonas aeruginosa [143]. Propolis was also demonstrated to reduce Helicobacter pylori infection-induced increase in pro-inflammatory interleukins and mediators, namely IL-8, IL-12, IL-1β, TNF-α, COX-2, and iNOS. Propolis also inhibited the phosphorylation of ERK, JNK, and p38 MAPKs in H. pylori-infected cells in a dose-dependent manner [144].

Wound Healing
Propolis with its anti-inflammatory properties could play a significant role in wound healing process [49]. Most of the studies that were identified and included in this particular theme are in vivo studies (Table 7). Corrêa [150] found that wounded animals that were given propolis had significant improvement in wound healing. The animals in the propolis group had less neutrophil and macrophage in the wounded tissue. In addition, they also had reduced expression of inflammatory transcription factor pNF-κB protein, and inflammatory cytokines, such as TGF-β, TNF-α and IL-6 [150]. Additionally, Sahib [151] showed that propolis-treated wound had significantly increased epithelial closure rate accompanied by decreased level of neutrophils and macrophages and increased level of fibroblasts and blood vessels, compared to the non-treated group [151].
Conversely, there were studies demonstrating that propolis induced inflammation during the wound healing process followed by the substantial decrease in inflammation. It appears that the up-regulation of inflammation results in speedier wound healing progression. Marquele-Oliveira [152] found that propolis-enriched cellulose membrane induced higher level of inflammation (neutrophil infiltration) in the wound compared to control group animals in the second day. However, the propolis group had substantially less inflammation in the seventh day and had faster rate of wound healing [152]. In addition, Picolotto [153] observed increased level of inflammation in the form of the upregulation of TNF-α and TGF-β and the recruitment of leukocytes at day 7 in the propolis-treated animal model of diabetic wounds. These inflammatory markers became statistically insignificant compared to control group at day 14. More importantly, the wound healing and closure were significantly better in the propolis-enriched cellulose membrane group [153].
Additionally, Eyarefe [154] found that propolis treatment appeared to induce higher level of inflammatory response in the wound. Propolis induced higher level of inflammatory infiltrates such as monocytes, macrophages, eosinophils, mast cells, and platelets. However, this inflammatory response regressed at day 8 and induced significantly better wound healing compared to the untreated control group. The control group had significantly worse wound healing response and persistent elevation of inflammation through to day 16 [154]. Moreover, the effect of solvents and/or carriers of the propolis extract should also be considered. Saritaş [155] found that the effect of propolis on bowel wound healing animal models with regards to the pro-inflammatory cytokines was less clear due to the solvent effect [155].
In terms of human clinical trials, Mujica [156] demonstrated the efficacy of propoliscontaining spray in treating human diabetic foot wounds. Propolis-treated patients had significantly higher reduction in wound area:~4 cm 2 , compared to control group:~3 cm 2 ; approximately 33.3% higher reduction in wound area in propolis treated patients over the course of the trial (8 weeks). In addition, propolis also improved the inflammatory profile of the tissues. Propolis treatment significantly increased the glutathione (GSH) and GSH/glutathione disulfide (GSSG) ratio, reduced the pro-inflammatory TNF-α, and upregulated the anti-inflammatory IL-10 levels of the treated tissue [156]. Table 7. Anti-inflammatory properties of propolis in wound healing. Wound healing properties. The animals were wounded and fed 100 mg propolis extract/kg body weight of propolis daily for 9 days.

Sources of Propolis
Significant improvement in wound healing following propolis administration, measured in terms of skin area. The animals in the propolis group also had less neutrophil and macrophage infiltration in the wounded tissues. The propolis group also had reduced expression of inflammatory transcription factor pNF-κB protein, and inflammatory cytokines, such as TGF-β, TNF-α and IL-6. [150] Brazil Ethanolic extract p-coumaric acid and artepillin C In wound healing models, there was a severe inflammatory infiltration in all groups, with the BC/PP membrane group showing the most.
When compared to the other groups, the BC/PP samples revealed higher levels of neutrophil involvement on the second day. The greater level in the BC/PP group exhibited a substantial decrease in neutrophil infiltration by the seventh day.
Propolis-enriched membrane induced an intense pro-inflammatory action when compared to BC membrane and SHAM in the beginning of the treatment, followed by a significant reduction in the inflammatory process during the follow-up of the wounds. Propolis induced a faster wound healing rate. [152] Brazil Ethyl acetate and butane extract Benzoic acid, anisaldehyde, daidzein, liquiritigenin, isoliquiritigenin, formononetin, medicarpin, vestitol, isovestitol, neovestitol, biochanin A, vesticarpan, retusapurpurin TNF-α levels in red propolis-stimulated wounds were considerably greater at day 7. Expression of pro-inflammatory cytokines and TGF-β (anti-inflammatory) peaked 7 days after injury, and pro-inflammatory cytokine and TGF-β expression were greater in the groups treated with bacterial cellulose membrane combined with red propolis. Propolis also promoted leukocyte recruitment. These inflammatory responses induced faster wound healing.

In vivo
Brazil Ethanolic extract Not determined Tissues exposed to cements containing EERP had a tissue reaction similar to that of the tissues exposed to cements without propolis, demonstrating that they were biocompatible in rat subcutaneous tissue. The addition of propolis resulted in healing, and propolis did not interfere in the repair process. [157] Brazil Not determined Not determined Propolis did not affect the wound healing process in terms of the presence of inflammatory cells, fibroblasts, collagen, neovascularization, granulation tissue and re-epithelialization. [158] Indonesia Propolis gel Not determined The topical application of propolis gel upregulated the expression of FGF-2 and fibroblasts in promoting ulcer healing in streptozotocin-induced diabetic rats. [159] Iran Ethanolic extract Not determined Oral propolis alone and in combination with 30 ppm SNPs provided anti-inflammatory effects and increase fibroblast proliferation and collagen deposition in experimental wounds thus increasing the rate of healing. Propolis + 60 ppm SNPs had a cytotoxic effect. [160] Iran Ethanolic extract Flavonoids, phenols, terpenoids, steroids, alkaloids, resins, saponins, coumarins A significant decrease was observed in the numbers of neutrophils and macrophages while a significant increase was observed in number of fibroblasts and blood vessels. Epithelial closure rate was significantly increased in the EEP-treated wounds. Powerful effect of propolis on accelerating wound healing was observed as the healing process of excision wound in albino rats treated with the EEP ointment started earlier and had a faster course than the comparative group. [151] Iran Oral propolis (100mg/kg) Not determined The rate of wound healing decreased significantly in the propolis group by day 12. By day 12, there was a significant decrease in number of neutrophil and eosinophil. By day 12, there was a significant increase in fibroblast count and collagen fiber density.
[160] After the rats were anesthetized, each rat's dorsum was cleaned with alcohol and chlorhexidine before being prepared for aseptic surgery. For each treatment, three identical circular full-thickness skin incisions measuring 6.5 ± 0.5 mm in diameter were made on the dorsum of each rat. According to the experimental plan, two drops (0.1 mL) of each agent (propolis, propylene glycol, silver sulfadiazine) were administered to the wounds twice a day.
The inflammatory cellular response was much greater (p = 0.002) in the propolis extract group. Monocytes and macrophages were significantly higher in the PE group (p = 0.003).
Other inflammatory cells such as eosinophils, mast cells, and platelets were much more prevalent in the PE group (p = 0.001).
However, the inflammatory infiltration regressed at day 8 in the propolis treated group, whereas the untreated group had signs of inflammation through to day 16.
The propolis treated group had the highest rate of wound healing. [154] Nigeria Hydroethanolic extract Not determined In MRSA-infected wound model rats, propolis promoted wound healing and bacterial clearance with regression of neutrophil infiltration and attenuated platelet reduction. [161] Portugal Ethanolic extract Not determined Wound scratch assay using NHDF cells presented better results with Propolis Extract 2 at 0.5%. The samples also promoted cell migration, demonstrating the wound-healing potential. A significant reduction in the margins of the scratch was observed, maximum with Propolis Extract 2 at 0.5%. [162] Turkey Ethanolic extract Not determined In bowel wound healing animal models, propolis appeared to have beneficial effect on wound healing. However, the effect of propolis on inflammatory cytokines such as TNF-α, IL-1, and IL-6 was less certain, perhaps due to the presence of ethanol solvent in the propolis preparation.
[155] In rabbits, the treatment of Amniotic membrane transplantation + Topical water-based propolis extract shows significant improvement in the defect area of the corneal alkali burn eyes.
The wound healing process of corneal alkali burn eyes was shown to be sped up by the combination of propolis and amniotic membrane.
There was no difference in the severity of the inflammatory alterations between the Amnion membrane transplantation + Propolis group, the Propolis only therapy group, and the control group. [163]

Human clinical trial
Chile Not determined (propolis spray)

Not determined
From the examination of diabetic wound tissues applied with propolis spray, TNF-α was significantly decreased and IL-10 was increased.
Propolis promoted a reduction in the wound's area by an average of 4 cm 2 compared to control patients. The healing was related to an increase in the connective tissue deposit. [156]

Others
The anti-inflammatory properties of propolis have also been demonstrated in wideranging studies (Table 8). In vitro studies demonstrated the inhibition of propolis extract from various geographical sources in terms of the hyaluronidase activity [164]. In addition, propolis extracts also downregulated the expression of pro-inflammatory cytokines, such as TNF-α, IL-6, IL-1β, MCP-1, ICAM-1, and VCAM-1, and their associated gene expression, including miR-19a-3p, miR-203a-3p, and miR-27a-3p [165]. Further, an in vivo study found that propolis reduced the expression of H1R and IL-9 genes, protein kinase Cδ (PKCδ), and nuclear factor of activated T-cells (NFAT) signaling pathways in rhinitis animal model [166].
In another allergic animal model, propolis reduced IL-13 production and eosinophilic infiltration and induced the frequency and number ofpolymorphonucler myeloid-derived suppressor cells (PMN-MDSC) [167].
In terms of human clinical trials, Fudhali [134] demonstrated that propolis compress significantly reduced phlebitis incidence associated withintravenous therapy [134]. Susan [168] reported the potential use of propolis as an anti-nociceptive therapy postsurgery [168]. Moreover, Soleimani [169] found that propolis supplementation reduced the pro-inflammatory cytokine IL-6 level in male military cadets subjected to Cooper 12-min run test and running-based anaerobic sprint test [169]. Measuring the effect of brown and green propolis on the expression of miRNAs associated with inflammatory responses. 1. miR-19a-3p and miR-203a-3p, which target mRNA coding for TNF-α, were upregulated by propolis. The increase in both miRs reduces the expression of TNF-α.
[165]   In both the low protein diet + propolis group and the standard protein diet + propolis group, green propolis was able to sustain a low inflammatory infiltrate within 7 days, but it was unable to do so within 15 days in the organisms with protein deficiencies.
[179] Propolis treatment also induced a decrease in IL-13 production on lymph node cell culture re-stimulated with OVA compared to non-treated allergic mice. A significant decrease in the mucus production as well as in the cellular infiltration were found in propolis-treated allergic mice.
Together these show anti-inflammatory role of propolis in the OVA-induced Th2 inflammation.This reduction was followed by a decrease in the frequency and number of eosinophils. propolis treatment induced a significant increase in frequency and number of PMN-MDSC. [167] Brazil Hydroethanolic extract Not determined In intra-abdominal adhesion rat models, propolis, delivered as intraperitoneal meshes, did not have any effect on adhesions and histological characteristics including inflammatory characteristics.

Method
The search strategy for the present review was carried out systematically. Three independent authors (F.Z., A.C., and K.C.) performed the searches for peer-reviewed manuscripts published from 2017 up to May 2022. The guiding statement was as follows: Propolis as an anti-inflammatory substance. The databases searched were Scopus, Pubmed, and Web of Science.The keywords used were: (Propolis or bee glue) AND (inflammat* or inflammas* or cytokine or chemokine). We excluded the terms that describe the individual bioactive compounds of propolis such ascaffeic acid phenethyl ester (CAPE), pinocembrin, and quercetin, as we opted to focus on the experimental evidence using propolis as a whole. Moreover, the exclusion criteria were review articles, in silico articles, non-English language documents, research papers that describe the combination of propolis with other compounds and/or ingredients.
All articles describing the potential use of propolis as an anti-inflammatory substance; in vitro, ex vivo, in vivo, and human clinical trials were selected. The studies were recorded in Mendeley and the duplicates subsequently removed. The selected articles were then screened by analyzing the titles, keywords, abstracts, and full texts. The articles that did not fit in the guiding statement and the set criteria were then removed. If any disagreement arose on the eligibility of a particular article, the disagreement was resolved through the discussion with the other authors. The following data were then collected and tabulated in Microsoft Excel: types of propolis extract, bioactive compounds, geographic locations of the propolis source, types of study, outcome of the study, and references. The authors (F.Z., A.C., and K.C.) then categorized the included studies into the appropriate themes: cancers, dental/oral-related disorders and diseases, immune system modulation, metabolic syndrome-related disorders and diseases, organ toxicity and inflammation, pathogenic infections, wound healing, and others. These categories were formed based on the apparent general themes illustrated by the included studies. Figure 1 illustrates the search and selection processes.

General Discussion and Conclusions
The inflammatory signaling cascades and processes are initiated by the disruption of tissue homeostasis, usually by pathogenic infection or tissue damage. The damage signals such as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) are then recognized by TLRs. TLRs subsequently activates MyD88-dependent signaling pathway by inducing the phosphorylation of the inhibitory IκB protein by IKK. Consequently, NF-κB is activated by its release from IκB protein and translocated into the nucleus. NF-κB then upregulates the expression of the inflammationrelated genes. As a result, various cytokines and chemokines, that are intimately involved in inflammation modulation, such as IL-1β, IL-6, IL-8, IL-10, IL-12, IL-15, IFN-γ, and TNF-α are produced. These cytokines then promote the migration of neutrophils and monocytes to the site of injury/infection. In addition, mast cells and macrophages induce further migration by releasing leukotrienes, histamines, and prostaglandins. Moreover, neutrophils also release non-specific toxic compounds such as reactive oxygen and nitrogen species (ROS/RNS) and proteases that are detrimental towards the pathogens and the host cells and tissues. Macrophages and dendritic cells then phagocytose the antigens. These cells subsequently migrate to lymphoid tissues and induce the differentiation of naïve T cells (Th0) into various effector and regulatory cells, such as Th1, Th2, Tregs, and Th17 cells, which in turn induce the production of other sets of cytokines. Finally, the resolution of inflammation is initiated when the neutrophil-induced switch of leukotrienes (produced by macrophages and other immune cells) to lipoxins. In addition, the expression of Fas ligand, protectins, and resolvins promotes the apoptosis and neutrophils. Apoptotic neturophils and other cellular debris are then phagocytosed by macrophages [186][187][188].
Inflammation involves many complex signaling pathways that are tightly regulated and therefore an uncontrolled, unresolved, and chronic inflammation could be significantly detrimental to the host. Figure 2 (summarized from Tables 1-8) demonstrates various mechanisms of action of propolis in modulating inflammation towards the regulatory balance, anti-inflammatory environment, and consequently, speedier resolution. In general, propolis acts as an anti-inflammatory substance by inhibiting and downregulating TLR4, MyD88, IRAK4, TRIF, NLRP inflammasomes, and their associated pro-inflammatory cytokines, such as IL-1β, IL-6, IFN-γ, and TNF-α. Propolis also reduces the migration of immune cells, such as macrophages and neutrophils, possibly by downregulating chemokines CXCL9 and CXCL10.
An opposite response is observed in some of the studies related to wound healing. Propolis appears to promote an intense inflammatory response in the initial wound healing process. However, the inflammation is then markedly reduced shortly after the initial burst. As a result, propolis-treated wounds are generally healed significantly faster compared to untreated wounds. More importantly, the anti-inflammatory (and immune-modulating) properties of propolis have been shown, not only in in vitro, ex vivo, and in vivo studies, but also in various human clinical trials with consistent results such as the reduction in serum and tissue inflammatory markers: IL-1β, IL-6, TNF-α, and hs-CRP, and the reduction in immune cell infiltration in the inflammation sites. Moreover, the anti-inflammatory effect was demonstrated in the studies using propolis extracts sourced from a wide-ranging of geographical sources and types of bees, solidifying the consistency of the anti-inflammatory properties of propolis.
In the present review, the authors adopted a comprehensive and systematic search strategy in order to objectively fulfill the aim of the study. A broad range of studies from all relevant fields of science and technology was collected and analyzed. The authors limited the search to studies that were published from 2017-2022, to provide coverage of the latest experimental studies in the field. However, the authors only assessed and included English language articles, which could potentially lead to missing studies from non-English databases, as it is clear that 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. Moreover, the reviewers did not perform any meta-analysis due to the heterogeneity of the included studies. Figure 2. Various mechanisms of action of propolis in modulating inflammation towards the regulatory balance. This figure is constructed based on the biochemical and molecular markers investigated in the 166 included papers. Propolis appears to inhibit and downregulate TLR4, MyD88, IRAK4, TRIF, and NLRP inflammasomes, and their associated pro-inflammatory cytokines such as IL-1β, IL-6, IFN-γ, and TNF-α. Propolis also decreases the migration of immune cells such as macrophages and neutrophils, possibly by downregulating chemokines CXCL9 and CXCL10.
The authors also noted that a proportion of the included studies did not identify the types of extract, geographical locations, and/or bioactive compounds of the propolis extracts used in the studies (Tables 1-8). We consistently come across this trend in the field of propolis research. In order to solidify the development and application of propolis, including reproducibility, as nutraceutical and/or pharmaceutical products, we suggest that all propolis research should include at least three types of identification, namely bee species, geographical sources, and types of extract. If possible, chemical analyses such as total phenolics and/or flavonoids should be included.