Potential Role of Propolis in the Prevention and Treatment of Metabolic Diseases

Propolis is a resinous mixture with a complex chemical composition, produced by honeybees and stingless bees from a variety of vegetal sources. In the last decades, propolis was extensively researched, multiple studies confirming its anti-inflammatory, antioxidant, antimicrobial, and wound-healing properties. More recently, due to an exponential increase in the number of patients with metabolic diseases, there is also a growing interest in the study of antidiabetic, antihyperlipidemic, and anti-obesity effects of propolis. The aim of this review was to evaluate the potential role of propolis in the prevention and treatment of metabolic diseases like diabetes mellitus, dyslipidemia, and obesity. The preclinical in vivo and in vitro pharmacological models investigating antidiabetic, antihyperlipidemic, and anti-obesity effects of propolis were reviewed with a focus on the putative mechanisms of actions of several chemical constituents. Additionally, the available clinical studies and an evaluation of the safety profile of propolis were also presented.


Introduction
Propolis, commonly known as the "bee glue", is a natural resinous mixture produced by honeybees (mostly Apis mellifera) and some other bees, such as stingless bees from resinous and gummy substances gathered from leaves, buds, sap flows, trichomes, and other actively exuding plant structures. Honeybees take the vegetable materials with their mandibles and mix them with some salivary enzymes like alpha-amylase, betaamylase, maltase, or some esterases. Other bees, such as stingless bees species produce propolis by collecting resinous material from plants and mixing it with beeswax and soil to form the so called geopropolis. Bees are using propolis to protect hives by blocking the cracks, sealing the spaces, and smoothing out the internal walls to maintain a constant inner temperature and to attain an internal aseptic environment [1][2][3][4][5].
Bee products have been used since ancient times as important bioresources because of their widely beneficial properties. Egyptians, Greeks, and Romans reported the biological properties of propolis for lesion healing. Aristotle, Dioscorides, Pliny, and Galen described some of the medicinal properties of propolis and they used propolis as antiseptic and mouth disinfectant as well as for healing wounds. In the medieval period, these applications of propolis were spread by Arabian physicians. The Incas used propolis as an antipyretic. Since the 18th century, propolis was first included in the London Pharmacopeia as an official drug. Between, the 17th and 20th centuries, propolis became popular in Europe, are being analyzed, the number of constituents is constantly increasing, new compounds being reported in studies published between 2018 and 2021, such as new flavanones and phenantrendiol derivatives in African samples, and new prenylflavonoids in Asian samples [19][20][21].
Another tropical propolis type, Clusia propolis or Cuban red propolis is the one originating from resin exuded by the flowers of different Clusia species found in Cuba and Venezuela [4,52]. It is rich in isoflavones, isoflavanes, flavonoids, and isoprenylated benzophenones [31,47].
The specificity of Mediterranean propolis, that seems to originate from cypress, is the high concentration of terpenoids (mainly totarol and diterpenic acids: isocupressic, communic, pimaric, imbricatoloic, abietic acids). This type is found in Greece, Malta, Sicily, Turkey, and Algeria [4,28,37,42,47,52]. If this propolis is obtained solely of cypress trees, the extract does not contain flavonoids, nor phenolic acids, but only diterpenes totarol and totarolon at high concentrations [50].
In the last decades, propolis has gained extensive popularity as a functional food and dietary supplement. In order to extract propolis for commercial purposes, ethanol, glycerol, and water are the main solvents employed, other solvents being also available. Ethanol is currently the most used solvent to obtain low wax propolis extracts rich in biologically active compounds. Recently, new methods of extracting propolis have been studied in order to replace the conventional ethanolic extraction method. One of the most promising methods is supercritical fluid extraction, which has the capacity to retain the antioxidant properties of the obtained propolis extracts through its use of low temperatures, which is an important characteristic for the pharmaceutical and food industries [54].

Preclinical Studies Investigating the Effects of Propolis in Metabolic Diseases
Our review identified 22 preclinical in vivo and in vitro studies, which were focused on the investigation of the effects of propolis in metabolic diseases like diabetes mellitus, dyslipidemia, or obesity ( Table 2). The in vivo animal models used to evaluate antidiabetic effect of propolis used streptozotocin (STZ), alloxan, D-glucose, or fructose to induce specific pathological modifications in glucose metabolism leading to chronic hyperglycemia, a key factor associated with cardiovascular complications. In these studies, the administration of propolis reduced the rise of blood glucose and ameliorated insulinemia with protective effects on pancreatic beta cells in chemically induced diabetes mellitus [55][56][57][58]. Additionally, several in vitro studies demonstrated inhibitory effects of propolis on several enzymes involved in glucose metabolism (alpha-glucosidase, maltase, or alpha-amylase), with the reduction of digestive absorption of glucose, which may also contribute to the overall antidiabetic effect [59,60,66].
For the evaluation of the effects of propolis in dyslipidemia, the majority of the in vivo experimental models used a high-fat diet in order to induce an increase in the serum concentration of cholesterol and triglycerides and only one model used sodium nitrate to induce hypercholesterolemia [68]. Another experimental approach was to use genetically engineered animals like APOE2 or LDL r-/-transgenic mice, which develop severe dyslipidemia due to alterations of enzymes and receptors involved in cholesterol metabolism [70][71][72]. In all experiments, the administration of propolis decreased the concentration of total cholesterol, LDL, and triglycerides. Additionally, propolis proved to be protective against the development of aortic lesions and arterial atherogenesis in transgenic animals [71].
The experimental models used to study the anti-obesity effect of propolis used C57BL/6J mice, in which the weight gain was diet induced. The treatment with propolis caused a reduction of body weight gain and an increased thermogenesis in adipose tissue, also reducing the accumulation of visceral adipose tissue [75,76].

Clinical Studies Investigating the Effects of Propolis in Metabolic Diseases
The effects of propolis in metabolic diseases were investigated in eight clinical studies focused on diabetes, obesity, or diabetic complications like diabetic foot ulcer (Table 3). In diabetic patients there is a significant risk of macrovascular or microvascular complications with a high mortality rate and impaired quality of life.  [83,84].
The presented clinical studies have some limitations, being represented by small scale randomized placebo controlled trials (RCTs) using a reduced number of enrolled patients. Therefore, larger clinical trials with a superior statistical significance are needed in order to warrant a possible clinical use of propolis in diabetes mellitus, its complications, or other metabolic diseases.

Inhibition of Alpha-Amylase and Alpha-Glucosidase in Diabetes Mellitus
Alpha-amylase and alpha-glucosidase are digestive enzymes necessary for the breakdown of complex molecules like starch or maltose to glucose, which can be absorbed into the bloodstream and subsequently used as energy source. The most important of the two enzymes is alpha-glucosidase, situated on the brush border of the small intestine which is capable of hydrolyzing disaccharides to alpha-glucose. The inhibition of alpha-glucosidase can decrease the glucose absorption and finally the amount of glucose in the bloodstream [85].
Several drugs with inhibitory effects on alpha glucosidase can mitigate postprandial hyperglycemic peaks, being useful in the treatment of type 2 diabetes. However, multiple adverse effects like abdominal cramps or diarrhea could reduce patient adherence to treatment [85], therefore natural products with alpha glucosidase inhibitory effect may become successful drug candidates for the management of diabetes mellitus. The study of Pujirahayu et al., 2019 tested the inhibitory effect of several triterpenes from propolis (cycloartenol, ambonic acid, mangiferonic acid, and ambolic acid) on alpha-glucosidase. The results showed that mangiferonic acid from propolis had the strongest inhibitory effect on alpha-glucosidase with an IC50 of 3.46 µM/mL (Figure 1) [86].

Modulation of Insulin Receptor Signaling in Diabetes Mellitus
Insulin receptor signaling leading to the translocation of glucose transporters on the membrane of hepatocytes, adipocytes, and skeletal muscle cells is a key process involved in the regulation of glucose, lipid, and energy metabolism [87]. The modulation of insulin receptor signaling in different steps of the intracellular pathway can augment the response to insulin in several types of tissues and subsequently reduce insulin resistance [87].
Several insulin receptor signaling modulators of natural origin have been tested with promising results. The research of Liu et al., 2018 proved that two important chemical constituents from propolis, galangin, and pinocembrin modulated insulin receptor signaling acting via Akt/mTOR pathway. The two compounds reduced insulin resistance by upregulating IR, Akt, and GSK3β and downregulating the phosphorylation of IRS. It is known that in diabetes the serine/threonine phosphorylation of IRS may cause a reduction of insulin signal transduction, therefore the intracellular effect of galangin and pinocembrin from propolis can restore insulin receptor sensitivity and alleviate insulin resistance, as shown in Figure 1 [88]. Additionally, the study of Nie et al., 2017 showed that caffeic acid phenethyl ester (CAPE) present in the chemical composition of propolis was able to enhance p-Akt, inhibiting simultaneously p-JNK, amplifying insulin effects at receptor level with a reduction of insulin resistance in diabetic mice [89].

Anti-Inflammatory Mechanisms in Dyslipidemia and Atherosclerosis
Recently, atherosclerosis is increasingly regarded as an inflammatory condition at vascular level, an inhibition of inflammatory processes being considered a valuable strategy to reduce the progression of endothelial lesions. Hence, IL-6, a pro-inflammatory cytokine produced mainly by macrophages can favor the development and progression of atherosclerosis. In humans, a clinical trial (Bacchiega et al., 2017) proved that IL-6 is a major player in the inflammatory events leading to atherosclerosis and the blockade of this cytokine with specific inhibitors like tocilizumab may reduce cardiovascular risk, unfortunately with significant adverse reactions [90]. Other cytokines like IL-17 have inhibitory roles, the study of Simon et al. proving that elevated levels of IL-17 are associated with better outcomes in patients with myocardial infarction, due to atherosclerosis [91].
Propolis proved to be a significant inhibitor of IL-6 in experimental models of inflammation and, in addition, the study of Fang et al., 2013 proved that propolis decreased the level of IL-6 while increasing IL-17 in a rodent model of dyslipidemia and atherosclerosis [71]. A previously published study of Bachiega et al., 2012 showed that cinnamic and coumaric acids from propolis significantly inhibited IL-6 production in macrophages from BALB/c mice [92].

Antioxidant Mechanisms in Dyslipidemia
A series of in vitro and in vivo experimental models have shown that oxidative stress is directly involved in the pathogenesis of atherosclerosis, the life-threatening consequence of dyslipidemia. In the vascular wall, oxidized low density lipoproteins are internalized in macrophages with the formation of foam cells which promote cell proliferation and endothelial dysfunction. In hypercholesterolemic animals, atherosclerotic processes Additionally, the study of Nie et al., 2017 showed that caffeic acid phenethyl ester (CAPE) present in the chemical composition of propolis was able to enhance p-Akt, inhibiting simultaneously p-JNK, amplifying insulin effects at receptor level with a reduction of insulin resistance in diabetic mice [89].

Anti-Inflammatory Mechanisms in Dyslipidemia and Atherosclerosis
Recently, atherosclerosis is increasingly regarded as an inflammatory condition at vascular level, an inhibition of inflammatory processes being considered a valuable strategy to reduce the progression of endothelial lesions. Hence, IL-6, a pro-inflammatory cytokine produced mainly by macrophages can favor the development and progression of atherosclerosis. In humans, a clinical trial (Bacchiega et al., 2017) proved that IL-6 is a major player in the inflammatory events leading to atherosclerosis and the blockade of this cytokine with specific inhibitors like tocilizumab may reduce cardiovascular risk, unfortunately with significant adverse reactions [90]. Other cytokines like IL-17 have inhibitory roles, the study of Simon et al. proving that elevated levels of IL-17 are associated with better outcomes in patients with myocardial infarction, due to atherosclerosis [91].
Propolis proved to be a significant inhibitor of IL-6 in experimental models of inflammation and, in addition, the study of Fang et al., 2013 proved that propolis decreased the level of IL-6 while increasing IL-17 in a rodent model of dyslipidemia and atherosclerosis [71]. A previously published study of Bachiega et al., 2012 showed that cinnamic and coumaric acids from propolis significantly inhibited IL-6 production in macrophages from BALB/c mice [92].

Antioxidant Mechanisms in Dyslipidemia
A series of in vitro and in vivo experimental models have shown that oxidative stress is directly involved in the pathogenesis of atherosclerosis, the life-threatening consequence of dyslipidemia. In the vascular wall, oxidized low density lipoproteins are internalized in macrophages with the formation of foam cells which promote cell proliferation and endothelial dysfunction. In hypercholesterolemic animals, atherosclerotic processes were favored by the generation of reactive oxygen species which induced an increased oxidation of LDL [72].
Propolis has a high content of antioxidant molecules, being able to decrease lipid peroxidation and the generation of reactive oxygen species with positive effects on the cardiovascular system.  demonstrated that propolis was able to prevent left ventricular hypertrophy (LVH) and atherogenesis in hypercholesterolemic mice, due to its ability to eliminate superoxide and hydroxyl radicals and the reduction of CD40L expression [70].
The chemical constituents from propolis responsible for the antioxidant effect are considered to be polyphenols and flavonoids, present in all types of propolis in different concentrations, influenced by plant origin, bee species, temperature, or geographic factors. The study of Kocot et al., 2018 identified specific propolis compounds like 3,5dicaffeoylquinic acid, artepillin C or 3,4,5-tricaffeoylquinic acid as being responsible for the antioxidant effect [93].

Activation of FFA4 Receptor with Positive Effects in Obesity
Obesity is a complex and multifactorial disease, which can lead to an inflammatory condition triggered by the toll-like receptor 4 (TLR4), with a role in the etiology of cardiovascular diseases [94]. The inflammatory response from obesity can be mitigated by some unsaturated fatty acids like eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), which are agonists on the free fatty acid receptor 4 (FFA4). This G-protein coupled receptor is present in the enteric nervous system but also in adipocytes, pancreas, or brain, being involved also in the regulation of energy homeostasis, appetite control, or adipocyte differentiation. The activation of FFA4 receptor leads to a reduction of the kinase activated by the growth factor beta (TAK1) activity and consequently an inhibition of IKK-β/NF-kβ and JNK/AP-1 pathway with anti-inflammatory consequences [94].
The study of Cho et al., 2020 found that several phenolic constituents from propolis like pinocembrin, chrysin, and galangin were able to activate the FFA4 receptor in vitro. Pinocembrin, a flavanonic compound from propolis, was the most potent activator of FFA4 receptor with potential applications in the pharmacological management of obesity and its complications [95].

Safety Profile of Propolis
Propolis is a natural product that is widely accepted by patients at a time when natural products are increasingly popular, to the detriment of chemically produced drugs. Although several in vivo studies in animals and humans aimed at demonstrating certain therapeutic effects of propolis were performed, they were not focused on the determination of adverse effects and toxicity, as propolis is generally recognized as safe (GRAS) [96]. Most of the chemical constituents in propolis are harmless and well tolerated if the doses are not too high. It is estimated that ingestion of 70 mg propolis/day is potentially nontoxic for the organism, however, exceeding the dose of 15 g/day may cause adverse effects [15,97]. The major compounds in propolis belong to the class of polyphenols (flavonoids, phenolic acids, and their esters). It is considered that except for caffeic acid phenethyl ester (CAPE), all other polyphenols have a low order of acute oral toxicity, but the toxicity of individual compounds was rarely tested. A study showed that galangin, an important chemical constituent from propolis, had no toxicity in doses up to 320 mg/kg in Wistar rats [98]. Pinocembrin, another active constituent from propolis was found to be non-toxic and non-mutagenic in doses up to 100 mg/kg in rats [99].
In humans, the occurrence of adverse effects following the administration of propolis has been observed both in oral and in local administration to the skin or throat. As a direct result of topical application of cosmetic and pharmaceutical formulations, adverse reactions included dermatitis, urticaria, swelling, and ulcerative gingivitis, especially in atopic patients. The study of Walgrave et al., 2005, reported that 1.2-6.6% of dermatitis patients were sensitive to propolis [100]. In general, adverse reactions were moderate, but literature also mentioned cases of patients with anaphylactic shock with laryngeal edema induced by the local administration of propolis [101]. The studies aimed to assess the allergenic potential of propolis have revealed that the major allergen in propolis is LB-1, which is a mixture of three isomeric pentenyl caffeates [15,101].
Despite the numerous studies focused on its chemical composition and beneficial effects, a chemical standardization of propolis is necessary, in order to be officially accepted into the mainstream of health care systems. However, due to its complex and variable chemical composition, it is difficult to find universally valid criteria. Literature mentions Bankova's approach, which considers that a quantification by group of structurally related compounds is more appropriate [102]. Future studies will continue the efforts to standardize propolis, for a safer and more effective administration.

Conclusions
Our review identified 22 preclinical and 8 clinical studies, which proved a series of favorable effects of propolis in diabetes mellitus, dyslipidemia, and obesity. Inhibition of alpha-glucosidase, modulation of insulin receptor signaling, reduction of IL-6, and activation of FFA4 receptors were the most important mechanisms of action identified for several chemical constituents from propolis: galangin, pinocembrin, mangiferonic acid, and CAPE. Additional studies are needed to ascertain the importance of propolis as a useful functional food for the prevention and treatment of metabolic diseases.

Conflicts of Interest:
The authors declare no conflict of interest.