Propolis and Their Active Constituents for Chronic Diseases

Propolis is a mass of chemically diverse phytoconstituents with gummy textures that are naturally produced by honeybees upon collection of plant resins for utilization in various life processes in beehives. Since ancient times, propolis has been a unique traditional remedy globally utilized for several purposes, and it has secured value in pharmaceutical and nutraceutical areas in recent years. The chemical composition of propolis comprises diverse constituents and deviations in the precise composition of the honeybee species, plant source used for propolis production by bees, climate conditions and harvesting season. Over 300 molecular structures have been discovered from propolis, and important classes include phenolic acids, flavonoids, terpenoids, benzofurans, benzopyrene and chalcones. Propolis has also been reported to have diverse pharmacological activities, such as antidiabetic, anti-inflammatory, antioxidant, anticancer, immunomodulatory, antibacterial, antiviral, antifungal, and anticaries. As chronic diseases have risen as a global health threat, abundant research has been conducted to track propolis and its constituents as alternative therapies for chronic diseases. Several clinical trials have also revealed the potency of propolis and its constituents for preventing and curing some chronic diseases. This review explores the beneficial effect of propolis and its active constituents with credible mechanisms and computational studies on chronic diseases.


Introduction
Propolis emerged as a unique naturally derived resinous remedy possessing diverse medical importance that has gained popularity worldwide over the centuries [1]. Currently, tremendous clinical trials have begun to reveal the medicinal importance of propolis and push this remedy in pharmaceutical and nutraceutical areas. Honey bees contribute as pollinators in natural ecosystems and provide beeswax, propolis, royal jelly, venom and apitherapy as medicinal and nutritional resources for human health [2]. Propolis is loaded with diverse constituents, and this diversity varies depending on the type of honeybee species, plant source used for propolis production by bee, climate conditions and harvesting season [3,4]. The bioactive profile of propolis shows a correlation with ecosystems, especially the diverse variety of floral resources that play a key role in the survival and biodiversity of bees [5]. Over three hundred chemical components, including flavonoids, terpenoids, phenolic acids, benzofuranes, benzopyranes and chalcones, are major constituents of propolis which have biological activity [3]. Massive experimental trials have reported that propolis, especially that derived from Brazil, China, Taiwan and Iran, and its constituents have biomedical beneficiary activities, such as antidiabetic, anti-inflammatory, antioxidant, anticancer, immunomodulatory, antibacterial, antiviral, antifungal and anticaries activities [3,4,6]. Chronic diseases remain a global threat, with the most frequent being diabetes, chronic obstructive pulmonary disease (COPD), tuberculosis, downregulates the formation of growth factors, vascular endothelial growth factors and signaling pathways such as IL and NF-κB, and also reduces the transcription of the cell cycle along with inducing apoptosis [24]. For the proliferation of cells, balance among cyclic-dependent kinases (CDKs), CDK inhibitors and cyclins is essential and is altered by propolis components. Kabala-Dzik et al. compared the effectiveness of CAPE along with caffeic acid against a human breast cell line and concluded that higher cytolysis and cell death were observed in CAPE than in CA [25]. Flavonoids of propolis extracts from Asian traditional medicine decrease the cellular division of the estrogen receptor (ER), which is essential in cancers such as breast, ovaries, endothelium (ER-α) and bone, vessels of blood, bone and lungs (ER-β) [26]. It also inhibits telomerase reverse transcriptase, which leads to distortion of telomeres and eventually cell death [27].
Propolis possesses antiviral properties against various DNA and RNA viruses, especially poliovirus (PV), herpes simplex virus [28], influenza virus [29], rhinovirus [30], SARS-CoV-2 [31,32] and many more ( Figure 1). Propolis possesses potential inhibitory efficiency on protein kinase (PAK1), angiotensin-converting enzyme (ACE-2), STAT-3, RNA-dependent RNA polymerase (RdRp), transmembrane serine protease 2 (TMPRSS2) and spike glycoprotein (SGp), especially in the management of SARS-CoV-2, which shows potent antiviral properties [33][34][35]. ACE-2 and TMPRSS2 are responsible for cellular entry, PAK1 induces lung fibrosis, STAT-3 activates the proinflammatory response and RdRp and SGp mediate viral endocytosis [34,36]. It targets the entire viral cycle, ensuring effectiveness, and is widely used as a therapeutic molecule, along with being adjuvant to vaccination approaches. Globally, diabetes is a major concern to the community, and various approaches are being developed to control the disease. Propolis works on various targets to treat diabetes mellitus (DM). Contributing modifications in the body, such as an exaggerated hexosamine pathway activated PF-C, increased glycation end products and oxidative stress, can elevate DM [37]. Propolis derived from the vicinity of Kayseri Province provides a strong antioxidant effect and reduces oxidative stress by inhibiting malonaldehyde levels, Globally, diabetes is a major concern to the community, and various approaches are being developed to control the disease. Propolis works on various targets to treat diabetes mellitus (DM). Contributing modifications in the body, such as an exaggerated hexosamine pathway activated PF-C, increased glycation end products and oxidative stress, can elevate DM [37]. Propolis derived from the vicinity of Kayseri Province provides a strong antioxidant effect and reduces oxidative stress by inhibiting malonaldehyde levels, which leads to improved glucose metabolism [37][38][39]. Miranda et al. stated that Brazilian green propolis significantly reduced TNF-α, but did not interfere with other interleukins [40]. Algerian propolis is a miraculous bundle of bioactive constituent molecules that shows effectiveness against various diseases with few to no side effects. It has acceptable pharmacokinetic properties that facilitate its utilization [41].
All the crystal structures of protein were prepared using the Protein Preparation Workflow tool of Maestro, where they were minimized by optimization of hydrogen bonds. The phytoconstituents of propolis were sketched using ChemDraw and prepared using the LigPrep tool. The grid was generated using the Receptor Grid Generation module by tackling the centroid of co-crystallized ligands of all the protein structures. The GLIDE tool was used for extra precision (XP) docking. The docking protocol was validated by calculating all atom RMSD between the docked structure and co-crystallized ligand, which ranged between 0.37 and 0.09 Å for all protein structures.
Hesperidin, myricetin, quercetin and kaempferol were found to have good docking scores against the selected protein structures of SARS-CoV-2. Table 1 describes the docking scores of the constituents. The receptor-ligand interaction diagrams of the best docked compound hesperidin against the promising targets of COVID-19 (docking score against Mpro: −9.59 kcal/mol, spike-ACE2 RBD: −9.25 kcal/mol and RdRp: −8.91 kcal/mol) are shown in Figure 2. Hesperidin interacts with Mpro by forming hydrogen bonds with His41 (key amino acid of the catalytic dyad), Phe140 and Glu166. It was also found to interact with the spike ACE2 RBD by forming hydrogen bonds with Glu406, Gln409, Gly496 and Gln498 amino acid residues. Hydrophilic interactions were also observed with RdRp by forming hydrogen bonds with Tyr546, Arg836, Ala840, Asp858 and Arg865. The other phytoconstituents were also observed to have similar interactions with SARS-CoV-2 proteins. Only CAPE showed very poor interaction with RdRp, possessing a docking score of −2.07 kcal/mol.  Mycobacterium tuberculosis is the causative agent for tuberculosis. Mycolic acid is the active component of the bacterial cell wall and has become the most promising target to combat the disease. InhA and DprE1 are the key enzymes involved in the synthesis of mycolic acid [43]. Hesperidin, myricetin, quercetin and kaempferol showed good potential to inhibit these enzymes when investigated using molecular docking. Among all the constituents depicted in Table 2, hesperidin showed a good docking score against InhA (−11.95 kcal/mol) and DprE1 (−10.79 kcal/mol). It was found to interact with Met98 and Gln100 of InhA by forming hydrogen bonds ( Figure 3A), as well as with Ala244 and Asp318 of DprE1 ( Figure 3B). Limonin showed comparatively poor interactions with InhA and poorer interactions with DprE1. CAPE also poorly interacts with DprE1.  Propolis phytoconstituents have been associated with cell proliferation and tumor growth modulation pathways such as pI3k/Akt, EGFR and caspase [17]. In this case, one of the important constituents of propolis, hesperidin, was discovered to interact with the Mycobacterium tuberculosis is the causative agent for tuberculosis. Mycolic acid is the active component of the bacterial cell wall and has become the most promising target to combat the disease. InhA and DprE1 are the key enzymes involved in the synthesis of mycolic acid [43]. Hesperidin, myricetin, quercetin and kaempferol showed good potential to inhibit these enzymes when investigated using molecular docking. Among all the constituents depicted in Table 2, hesperidin showed a good docking score against InhA (−11.95 kcal/mol) and DprE1 (−10.79 kcal/mol). It was found to interact with Met98 and Gln100 of InhA by forming hydrogen bonds ( Figure 3A), as well as with Ala244 and Asp318 of DprE1 ( Figure 3B). Limonin showed comparatively poor interactions with InhA and poorer interactions with DprE1. CAPE also poorly interacts with DprE1.  Mycobacterium tuberculosis is the causative agent for tuberculosis. Mycolic acid is the active component of the bacterial cell wall and has become the most promising target to combat the disease. InhA and DprE1 are the key enzymes involved in the synthesis of mycolic acid [43]. Hesperidin, myricetin, quercetin and kaempferol showed good potential to inhibit these enzymes when investigated using molecular docking. Among all the constituents depicted in Table 2, hesperidin showed a good docking score against InhA (−11.95 kcal/mol) and DprE1 (−10.79 kcal/mol). It was found to interact with Met98 and Gln100 of InhA by forming hydrogen bonds ( Figure 3A), as well as with Ala244 and Asp318 of DprE1 ( Figure 3B). Limonin showed comparatively poor interactions with InhA and poorer interactions with DprE1. CAPE also poorly interacts with DprE1.  Propolis phytoconstituents have been associated with cell proliferation and tumor growth modulation pathways such as pI3k/Akt, EGFR and caspase [17]. In this case, one of the important constituents of propolis, hesperidin, was discovered to interact with the Propolis phytoconstituents have been associated with cell proliferation and tumor growth modulation pathways such as pI3k/Akt, EGFR and caspase [17]. In this case, one of the important constituents of propolis, hesperidin, was discovered to interact with the aforementioned receptors, as evidenced by its high binding affinity with these receptors.
It exhibited docking scores of −13.43 kcal/mol against EGFR, −13.93 kcal/mol against pI3K and −7.84 kcal/mol against caspase-3 ( Table 3). The hydroxyl and keto groups of hesperidin were found to interact with the Ala722, Gly724, Met793, Asp800 and Asp855 amino acid residues of EGFR by forming hydrogen bonds ( Figure 4A). Hesperidin was observed to exhibit two π-π stacking interactions with the Trp812 and Tyr867 residues of pI3K. It was also found to form hydrogen bonds with Ala805, Val882, Lys890 and Asp964 of pI3K ( Figure 4B). It was also observed that hesperidin forms hydrogen bonds with the Gly122, Glu123, Glu124 and Glu167 residues of caspase-3 ( Figure 4C). Limonin was found to have less binding affinity for these three receptors than hesperidin. aforementioned receptors, as evidenced by its high binding affinity with these receptors. It exhibited docking scores of −13.43 kcal/mol against EGFR, −13.93 kcal/mol against pI3K and −7.84 kcal/mol against caspase-3 ( Table 3). The hydroxyl and keto groups of hesperidin were found to interact with the Ala722, Gly724, Met793, Asp800 and Asp855 amino acid residues of EGFR by forming hydrogen bonds ( Figure 4A). Hesperidin was observed to exhibit two π-π stacking interactions with the Trp812 and Tyr867 residues of pI3K. It was also found to form hydrogen bonds with Ala805, Val882, Lys890 and Asp964 of pI3K ( Figure 4B). It was also observed that hesperidin forms hydrogen bonds with the Gly122, Glu123, Glu124 and Glu167 residues of caspase-3 ( Figure 4C). Limonin was found to have less binding affinity for these three receptors than hesperidin.  Gray sticks indicate ligands, atom-type color sticks indicate amino acid residues of the receptor and yellow and red dotted lines represent hydrogen bonds. π-π stacking formed between the ligands and the receptor, respectively.
As per the above molecular docking study, hesperidin was the best phytoconstituent of propolis involved in COVID-19, TB and cancer, and can be evaluated further for better therapeutic development.

Chronic Diseases with Reported Therapeutic Properties of Propolis and its Active Constituents
Propolis has been effectively evaluated with medicinal importance in various chronic diseases. Brazilian propolis was found to reduce renal dysfunction by reducing proteinuria and oxidation stress levels [44,45]. Various varieties of propolis have been attributed to benefits in heart dysfunction, including the influence of inflammatory factors [46]. The anticancer activity of propolis and its constituents is commonly associated with blocking the localization of NF-кB, inducing ROS and regulating gene expression [47]. Propolis has potent antioxidant properties and beneficial effects on the inflammatory state as well as on oxidative stress conditions in RA patients [48]. The potential effects of propolis and its constituents against well-known chronic diseases are summarized below. Gray sticks indicate ligands, atom-type color sticks indicate amino acid residues of the receptor and yellow and red dotted lines represent hydrogen bonds. π-π stacking formed between the ligands and the receptor, respectively. As per the above molecular docking study, hesperidin was the best phytoconstituent of propolis involved in COVID-19, TB and cancer, and can be evaluated further for better therapeutic development.

Chronic Diseases with Reported Therapeutic Properties of Propolis and Its Active Constituents
Propolis has been effectively evaluated with medicinal importance in various chronic diseases. Brazilian propolis was found to reduce renal dysfunction by reducing proteinuria and oxidation stress levels [44,45]. Various varieties of propolis have been attributed to benefits in heart dysfunction, including the influence of inflammatory factors [46]. The anticancer activity of propolis and its constituents is commonly associated with blocking the localization of NF-κB, inducing ROS and regulating gene expression [47]. Propolis has potent antioxidant properties and beneficial effects on the inflammatory state as well as on oxidative stress conditions in RA patients [48]. The potential effects of propolis and its constituents against well-known chronic diseases are summarized below.
A summary of some well-known chronic diseases as well as propolis and its constituents against them.

Chronic Kidney Disease
CKD has been a well-known cause of suffering and mortality in recent decades [49]. CKD is observed with the majority of pathogenic complications involving inflammatory and oxidative stress conditions. Oxidative stress is said to be the main causative pathway for the progression of CKD [50]. While various medicaments are available for reducing and controlling complications arising in CKD, new remedies with enhanced medicinal benefits and few side effects are still in demand. In recent years, propolis has been investigated for its potential action regarding repairing kidney tissue injuries and returning normal kidney function in acute kidney injury and CKD [51]. Some of the important clinical trials examining the activity of propolis and its constituents against CKD have been summarized in Table 4.
Silveira et al. studied a double-blinded, randomized, placebo-controlled model in CKD patients and concluded that Brazilian green propolis extract was capable of significantly reducing proteinuria in diabetic and non-diabetic CKD patients. A dose of 500 mg/day for 12 months of treatment resulted in a reduction in proteinuria and a significant decrease in urinary monocyte chemoattractant protein-1, but no change in estimated GFR or blood pressure [45]. Nada Oršolić et al. examined the effect of Croatian propolis extract in a diabetic mouse model and reported the healing action of propolis preparation on diabetic hepatorenal damage. This effect is proposed to occur through the antioxidant activity of propolis [52]. Ahad et al. examined the nephroprotective effects of chrysin (a bioactive constituent of propolis) in a STZ/HFD-induced T2DM Wistar albino rat model and revealed a reduction in oxidative stress and renal inflammation by chrysin by inhibiting the TNFα pathway, restoring renal function [53]. Jessica Granados-Pineda et al. explored the preventive effect of pinocembrin (a bioactive constituent of Mexican brown propolis) using a diabetic nephropathy in vivo model. Pinocembrin shows a preventive effect on the progression of dyslipidemia and kidney damage. However, at advanced stages, acceleration of disease progression by pinocembrin was observed [54]. Wei Zhu et al. proposed the preventive property of propolis on hepatorenal injury through blocking peroxidation of lipids, and induction of antioxidant enzymatic activity upon observing the beneficiary effect of Brazilian propolis and Chinese propolis against diabetic Sprague Dawley rats induced with hepatorenal injuries [55]. Iranian propolis was shown to reduce blood glucose levels (BGLs), improve antioxidant levels and diminish histopathological changes in the T1DM rat model [56]. Teles et al. demonstrated renal protective effects in a severe CKD model with a reduction in kidney injury-related inflammation and oxidative stress conditions. Brazilian red propolis diminished the progression of proteinuria in 5/6 renal ablation model Wistar rats, partially prevented heart hypertrophy, and resulted in a 24% higher rate of survival compared to untreated rats [44]. Ulusoy et al. examined the healing property of propolis on methotrexate-induced renal damage by noting the amount of heat shock protein-70 expression returning to baseline levels with the improvement of nephrotoxicity [57]. The neuroprotective action of propolis in a rodent model was studied by Chang et al. who mentioned the inhibitory activity of propolis on SMAD 2/3-dependent pathways and further suppressed the activation of SMAD-independent JNK/ERK [58].

Rheumatoid Arthritis
Rheumatoid arthritis (RA) falls into the class of autoimmune diseases comprising oxidative stress and chronic inflammatory events. Inflammation progresses in arthritis due to NF-κB activation mediated through ROS [59,60]. Several factors have been reported to facilitate the release of IL-1, IL-6 and TNF-α-like proinflammatory cytokines [59]. Depressing inflammatory conditions may be beneficial in RA [61]. The suppressive effect of an ethanolic extract of propolis was estimated by Park et al. against an arthritis rat model, and they proposed an anti-inflammatory effect resulting from inhibition of prostaglandin production [62]. Orsi et al. demonstrated the non-specific immunological response of propolis associated with macrophage activation and nitric oxide inhibition [63]. Propolis can also prevent inflammation by hindering the NF-κB pathway and decreasing ROS levels [48]. The chemical composition of propolis has powerful anti-inflammatory properties capable of regulating immune cell functions and reducing the immune response mediated through cytokines [64]. CAPE is a flavonoid-like bioactive compound of propolis that specifically inhibits NF-κB activation [65]. Polyphenolic constituents found in propolis possess free radical scavenging activity against ROS [66], and the polyphenolic profile of propolis has a direct correlation with its antioxidant properties [67]. Tanaka et al. reported that the suppressive effects of Brazilian propolis possess beneficial effects in a mouse model of RA and proposed that the suppressive effect results from a reduction in IL-17 production by blocking the differentiation of IL-17-secreting cells, also denoted as Th17 cells [68]. However, a double-blinded, randomized, controlled trial performed by Matsumoto et al. mentioned the lack of beneficial activity of propolis in patients with RA [69].

Cancer
Various preclinical studies have reported the chemopreventive and anticancer properties of different types of propolis and its major constituents, such as flavonoids and polyphenols [70]. Table 5 summarizes such important clinical trials. Portuguese propolis has effective antiproliferative activity in human renal cell carcinoma (RCC), and free radical-induced erythrocyte damage is also protected by propolis [71]. CAPE (a bioactive constituent of propolis) was reported to have anticancer properties by inhibiting NF-κB, caspase and Fas signaling activation in MCF-7 cells [72]. Kamiya et al. revealed potential inhibition of MCF-7 breast cancer cells by Brazilian red propolis extract and CAPE. They concluded that the anticancer activity was due to the induction of DNA fragmentation, CCAAT/enhancer binding protein homologous protein expression and caspase-3 activity [73]. Xuan et al. suggested that the antitumor activity of the ethanol extract of Chinese propolis (EECP) and its bioactive constituents mainly persist due to regulation of the annexin A7 and p53 proteins, mitochondrial membrane potential and ROSs, as well as that inhibition of NF-κB causes apoptosis in cancer cells [74]. Chang et al. found that Chinese propolis and CAPE exert inhibitory effects on the proliferation and migration of the TLR4-positive MDA-MB-231 cell line. Action may be increased by TLR4 signaling pathway blockades, apoptosis and autophagy. Cell proliferation was significantly reduced by 25 µg/mL and 50-100 µg/mL CAPE and EECP [75]. The cytotoxic and antiproliferative potency of Cuban red propolis was determined by Herrera et al. and reported on the MDA-MB 231 tumor cell line, and the inhibitory effect of propolis was proposed to occur through the induction of mitochondrial dysfunction, resulting in ROS-associated necrosis [76]. A concentration of 100 µg/mL was able to attain 71% cytotoxicity [76]. The anticancer potency of Chinese poplar propolis was examined by Li et al. in a MDA-MB-231 cancer cell line stimulated with LPS in an inflammatory microenvironment [77]. They illustrated a propolis-mediated reduction in proinflammatory cytokine production and a negative effect on angiogenesis, proliferation and migration of tumor cells. A concentration of 25-200 µg/mL noticeably inhibited the metastasis of breast cancer [77]. Propolis collected from Poland was evaluated by Milena et al., and promising antiproliferative activity was found against colon, lung, breast and glioma cancer cell lines [78].

Diabetes
Diabetes mellitus (DM) is a chronic disease that arises from either a decline in insulin synthesis (type 1 diabetes mellitus, T1DM) or a decrease in insulin sensitivity (type 2 diabetes mellitus, T2DM) that leads to hyperglycemia. Recent studies ( Table 6) have proven that elevated glycemic levels and other diabetic complications are well controlled by natural bioactive compounds and dietary supplements [79]. Yajing Li et al. observed improved insulin sensitivity and lipid levels in a T2DM rat model after treatment with encapsulated propolis, which was obtained from the A. mellifera L. species of honeybees found in northern China. A total of 100 male Sprague-Dawley rats were given a high-fat diet and streptozotocin to induce diabetes. Low (50 mg/kg), medium (100 mg/kg) and high (200 mg/kg) doses of encapsulated propolis were administered to T2DM rats, and 10 mg/kg pioglitazone was used as a positive control. The fasting blood glucose, fasting serum insulin and triglyceride levels were found to be decreased, which was comparable with the control, pioglitazone, although there was no change in total cholesterol or body weight [80]. Chihuahua propolis was evaluated for its hypoglycemic effect on a T2DM mouse model, and it was revealed to have antioxidant activities [81]. The beneficial effect of Brazilian propolis was examined by Aoi et al. on Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Eight weeks of treatment resulted in the control of BGL and a decrease in insulin levels in blood. Furthermore, propolis also improved cardiovascular and metabolic complications [82]. Chen et al. showed that the ethanolic extract of Taiwanese green propolis plays a preventive role on damaged pancreatic β-cells in STZ-and HFD-induced T2DM rat models, as well as that disease progression can be retarded upon propolis treatment [83]. Brazilian propolis was found to protect mesenteric adipose tissues by regulating immune cell density and reducing the amplitude of diabetes [84].   [77] Investigations of the potency of propolis and its beneficial effects on diabetes complications were also performed in a human diabetic model. Iranian propolis treatment for 8 weeks at 3 times the dose of 500 mg can control glucose levels and inflammatory conditions in T2DM patients [85]. A similar clinical study showed a significant decrease in fructosamine and oxidized LDL as well as an increase in catalase activity [86]. Fukuda et al. revealed an improvement in glomerular filtrating dysfunction by Brazilian green propolis, probably by resisting the worsening of uric acid levels, inflammation and GFR, which generally occur in T2DM patients [87]. Zhao et al. applied Brazilian green propolis treatment to T2DM patients and interpreted the elevation of patients' antioxidant statuses [88]. Chinese propolis was reported to enhance the serum antioxidant framework and thereby improve antioxidant defensive activity in T2DM patients [89]. Samadi et al. examined the effects of 12 weeks of 300 mg doses of propolis administered three times daily, which could control lipid content and FBG by improving diabetic symptoms in patients [90]. Zakerkish et al. found improved glucose metabolism in T2DM patients; furthermore, their study also supported beneficial effects on renal and hepatic impairments [91].

Tuberculosis
Tuberculosis (TB) is a chronic contagious respiratory infection in which the causative pathogen Mycobacterium tuberculosis (Mtb) infects the lung. The treatment of drug-resistant TB is often incurable, and new strategies are now a worldwide concern. Constituents of propolis showing antitubercular activity are promising new therapies to treat tuberculosis [92,93]. Scheller et al. correlated the infectious effects of various mycobacterial strains and the antitubercular potency of the ethanolic extract of propolis (EEP), where seven strains were found to be susceptible to propolis treatment [94]. EEP was established to have a synergistic effect upon enhancing the effectiveness of anti-tuberculosis drugs [95]. Essential oil obtained from green propolis in Brazil is composed of important chemical constituents [96]. This essential oil was evaluated by Quintino et al. and showed inhibitory potency against Mtb, with MIC= 64 µg/mL and MIC = 62.5 µg/mL found for Mycobacterium avium [97]. Volatile oil from brown propolis possesses promising antimycobacterial activity against Mtb and M. avium, with MIC = 50 µg/mL and 62.5 µg/mL, respectively [98]. Sawicki et al. observed metabolic and transcriptomic changes in Mtb by conducting an in vitro study of an ethanolic extract of Nepalese propolis, with MIC = 8 µg/mL for the Trigona species and MIC = 32 µg/mL for the mellifera species [99].

COPD
Chronic obstructive pulmonary disease emerges with inflammatory symptoms associated with the respiratory pathway. These are characterized by resistance to airflow due to abnormalities in the respiratory path upon exposure to toxic gases or particles [100]. Machado et al. revealed a protective effect of Brazilian green propolis on respiratory inflammation in a mouse model [101]. Investigations of herbal products in treating COPD have increased [102]. Khayyal et al. examined two months of treatment with silver sachets (propolis extract) in asthma patients and showed improvement in immunological parameters, severe attacks and pulmonary dysfunction in patients [103]. Kolarov et al. studied the beneficial effect of propolis in combination with N-acetylcysteine, which was able to suppress cough in COPD patients [104]. This combination was proposed to suppress the development of coughing and, thereby, reduce the incidence of severe attacks in COPD patients [105].

Cardiovascular Disease
CVD is related to dysfunction of the heart and blood circulation, and is a rising threat with major morbidity as well as health and economic burdens globally. It is elevated in conditions such as angina, stroke, rhythm and heart failure [106]. Reduction in diameter and resisting flow of blood in blood vessels are general conditions of CVD [107]. Table 7 illustrates the beneficial effects of propolis and its constituents in CVD. Balion et al. explored the inhibitory property of propolis on mitochondrial respiration in a rat model. Aqueous (AqEP), polyethylene glycol-aqueous (Pg-AqEP), and ethanolic propolis extracts (EEP) have been shown to influence mitochondrial and ROS production in astrocytic C6 cells. PC EEPs (2 to 4 g/mL) provided less protracted protection against ischemia-induced superoxide generation and mortality. Pg-AqEP and Ag-EP (but not EEP) both effectively protected cultures from hypoxia-induced increases in TNF, IL-1, and IL-6. Only Pg-AqEP (not AqEP or EEP) reversed the hypoxia-induced reduction in mitochondrial basal and ATP-coupled respiration rates while also dramatically increasing mitochondrial respiratory capacity. The extent of the inhibitory effect is dose-dependent and affected by substrates utilized for respiration [108]. Zhang et al. examined the in vitro anti-platelet aggregation action of a water extract of propolis in a dose-dependent manner [109]. Majiene et al. explored the inhibitory property of propolis on mitochondrial respiration in a rat model. The extent of the inhibitory effect is dose-dependent and affected by substrates utilized for respiration [108]. Wang et al. proposed a beneficial effect of the total flavonoid constituents of propolis by regulating the expression of connexin 43 on CHF in a rat model [110]. Chao et al. examined propolis treatment against carotid restenosis with improvement in disturbed cardiac functions [111]. They looked at how propolis (125 and 250 mg kg −1 day −1 ) affected carotid restenosis in hypercholesterolemic rabbits. In hypercholesterolemic rabbits, propolis dramatically decreased the degree of carotid restenosis, prevented neointima hyperplasia, lowered blood lipid profiles, and boosted antioxidative activity. Furthermore, propolis decreased the expression of CD68, TLR4, NF-B p65, MMP-9 and TNF-in the carotid arteries, and also lowered plasma levels of C-reactive protein, interleukin-6 and TNF-. Propolis has a protective impact on carotid restenosis in rabbits, which is connected with controlling blood lipids and decreasing oxidative stress and inflammation, and its anti-inflammatory action may be related to suppressing the TLR4-mediated NF-B signaling pathway. In ApoE-/-mice, a 14-week combined therapy of simvastatin (a cholesterol-lowering medicine) and pinocembrin dramatically lowered blood lipid levels, enhanced endothelial function and reduced atherosclerotic symptoms [112]. Platelet aggregation has been linked to atherosclerosis and atherothrombosis, two complicated multifactorial chronic inflammatory illnesses caused by fat and other macromolecule buildup in artery walls. CAPE (15 and 25 M) substantially reduced collagen-stimulated platelet aggregation. It was reported that CAPE exhibited cardioprotective effects in short-term myocardial ischemia in rats through a reduction in xanthine oxidase (XO) and adenosine deaminase (ADA) activities, as well as antioxidant effects [106,113]. Table 7. Summary of clinical trials which experimented with the activity of propolis and its constituents against CVD.

Other Chronic Diseases
Propolis has many active constituents and is utilized for medicinal value in many diseases [115]. Chen et al. reported that a small molecule extracted from green propolis improved metabolic syndrome in a glycemic mouse model by influencing hepatic glucose metabolism and lowering the levels of glucose and lipids [116]. The antiviral potency of propolis against SARS-CoV-2 in vitro was determined by Refaat et al., who reported a potency comparable to that of remdesivir [117]. A molecular docking study showed that constituents of ethanolic propolis extracts are capable of having of inhibitory activity against ACE-2 receptors for SARS-CoV-2, especially rutin, which exhibits the best inhibition potential of −8 kcal/mol binding energy [118]. Khayrani et al. performed a similar molecular study with Sulawesi propolis compounds, where five constituents were found to have high potential affinity for ACE-2 receptors [119]. Egyptian propolis has also been reported as a promising candidate in combatting against SARS-CoV-2 using computational modeling [120]. Several other computational studies reported those supporting the affinity of propolis constituents toward SARS-CoV-2 [121][122][123]. The bioactive constituent of propolis CAPE has been proven to have a neuroprotective effect in in vivo models of Parkinson's disease [124]. Zulhendri et al. summarized the therapeutic value of propolis on the brain regarding neurological disorders and injuries, suggesting that its suppressive effect on oxidative stress and inflammatory mediators also influences protein-coding gene expression and reduces apoptosis in various disorders [125].

Current Scenario of Preclinical and Clinical Development
Propolis is highly accepted for the treatment of various chronic diseases (Table 8). A preclinical study conducted in 36 mice regarding the management of high diet-based hypertension concluded that when propolis was administered for a week, it showed significant therapeutic efficacy. Propolis promotes wound healing in just 3 days and increases collagen and fibroblasts at higher concentrations when compared to the control group [109]. Moura et al. concluded that propolis attains characteristics such as angiogenesis and fibroproliferative processes by studying a sponge implanted animal model [126]. Mesenteric adipose tissue plays a crucial role in the evolution of T2DM and is significantly targeted by propolis and its derivative. Ichi et al. suggested a noticeable reduction in weight by decreasing the adipose tissue reservoir in obese mice [127]. The remarkable anti-inflammatory activity of artepellin c, found in Brazilian green propolis, was cited by Paulino et al. as one of the main causes of damage to the lungs and other organs. They outlined their preclinical findings on rat paw edema. The average reduction in inflammation stimulated by carrageenan was reduced by 38% within 6 h [128]. With the support of much preclinical data, clinical trials have become crucial to ensuring therapeutic effects similar to those observed in other animals. The following demonstrates the clinical trials intended to prove the efficacy of propolis in numerous diseases.

Conclusions and Future Directions
Propolis is a wondrous molecule with a broad spectrum of action. It consists of over 300 active phytochemicals with a wide variety of effects on various chronic diseases. CAPE, rutin, quercetin, CA and many important chemical moieties are present in propolis, which helps in the management of various diseases, such as DM, CVD, allergy to SARS-CoV-2 and inflammation. Additionally, it is also beneficial for gastrointestinal disorders (giardiasis anti-H. pylori), gynecological care (recurrent vulvovaginal candidiasis) and oral health (halitosis gingivitis). Numerous clinical trials are ongoing on propolis. As a known molecule, it has a wide spectrum of exploration in the treatment of many diseases. It has certain limitations, such as allergic reaction (respiratory or epidermal symptoms), mouth ulceration and irritation, but they are not very common. In the future, a more detailed study on propolis and its incorporation in the treatment of various diseases will be required, from the smallest inflammation to life-threatening diseases such as cancer. It is crucial to attain critical knowledge related to its active constituents, its interaction with numerous receptors and its subsequent pharmacological actions. The long-term effect of propolis administration is of keen interest, and must be determined before developing it into a potential therapeutic moiety.