Propolis Protects Endotoxin Induced Acute Lung and Liver Inflammation Through Attenuating Inflammatory Responses and Oxidative Stress
Abstract
Propolis is a natural bee product, and it has many effects, including antioxidant, anti-inflammatory, antihepatotoxic, and anticancer activity. In this study, we aimed to explore the potential in vivo anti-inflammatory, antioxidant, and antiapoptotic properties of propolis extract on lipopolysaccharide (LPS)-induced inflammation in rats. Forty-two, 3- to 4-month-old male Sprague Dawley rats were used in six groups. LPS (1 mg/kg) was administered intraperitoneally to rats in inflammation, inflammation + propolis30, and inflammation+propolis90 groups. Thirty milligram/kilogram and 90 mg/kg of propolis were given orally 24 h after LPS injection. After the determination of the inflammation in lung and liver tissues by 18F-fluoro-deoxy-d-glucose–positron emission tomography (18FDG-PET), samples were collected. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), nitric oxide (NO), and DNA fragmentation were determined. The decrease of MDA levels in inflammation + propolis30 and inflammation + propolis90 groups was determined compared to the inflammation group in lung and liver tissues. The increase of SOD% inhibition in inflammation + propolis90 group was determined in liver, lung, and hemolysate compared to the inflammation group. Increased CAT activities in inflammation + propolis30 and inflammation + propolis90 groups were observed in liver tissue and hemolysate compared to inflammation group. In lung tissue, NO levels were lower in inflammation group compared to the control group, but DNA fragmentation levels were higher. 18F-FDG uptake of tissues in inflammation + propolis30 and inflammation + propolis90 groups was decreased compared to the inflammation group. In conclusion, the data of this study indicate that the propolis application may serve as a potential approach for treating inflammatory diseases through the effect of reducing inflammation and free oxygen radical production.
Evidence on the Health Benefits of Supplemental Propolis
A more common adverse effect of propolis administration is hypersensitivity, particularly in regard to topical application, resulting in allergic reactions, swelling, dermatitis and urticaria. In specific cases, an individual had severe swelling of the throat and anaphylactic shock upon topical application [ 44 ]. Severe adverse effects, such as laryngeal edema and anaphylactic shock, seldom have been reported [ 75 ], which are rarely attributed to a “natural” supplement like propolis. It is reported that 1.2–6.6% of individuals with dermatitis are sensitive to propolis [ 76 ]. As such, despite the positive safety profile, it is recommended people seek medical advice before supplementing or applying propolis products.
Based on previous animal studies and applying a safety margin when extrapolating to generally healthy humans, a safe dose of propolis has been reported to be 70 mg/day [ 27 ]. Interestingly, studies on pinocembrin, a component of propolis, have been conducted using 150 mg as a single dose [ 30 ]. When provided to conscious mice, the median lethal dose of propolis extract is more than 7.34 g/kg (LD50), confirming the product is generally safe. [ 42 , 43 ]. The difficulty with prescribing an accurate dose of propolis based on the investigated population, dosing regimens, compliance and product purity, is pronounced. As the phenolic compounds present within propolis vary based on geographical origin, the bioactivities will also vary significantly making it difficult to define a correct dosage [ 69 ].
A case report of a 59-year-old man with cancer of the bile duct was supplementing with propolis. The patient developed acute renal failure and was undergoing concurrent haemodialysis therapy. Fortunately, his renal function improved following the removal of propolis. However, based on the uncertainty of propolis as the cause of renal failure at that time, the patient continued ingesting propolis for the underlying cancer. Renal function deteriorated again after re-challenge and recovered following secondary withdrawal of propolis [ 75 ]. While renal biopsies where not taken in this particular case, concerns over the adverse effects of propolis on a vulnerable population are potentially warranted. It has been suggested that CAPE, a constituent of propolis, inhibits inducible nitric oxide synthase (iNOS) pathways which may decrease kidney perfusion and thus induce acute renal failure in at-risk patients [ 75 ].
Clinical investigation in mice and humans report that propolis and its constituents are generally well tolerated and non-toxic unless administered in very large quantities [ 1 , 28 , 30 ]. However, it should be noted that the adverse events and toxicity of propolis are rarely included as an outcome measure in human trials. Considered a “mere” herbal product, consumers rarely consider the potential adverse side effects of nutritional supplements that are perceived to be naturally derived.
The rate at which polyphenols are excreted in the urine appears to vary considerably between individuals. Those placed on a three-day high phenolic diet demonstrated this variable excretion with one participant excreting 8 of the 17 phenolic acids measured, another all 17 [ 39 ]. The reasons for individual variability in excretion are generally unknown, but may be related to ageing, renal function or inherent propolis properties. Ongoing research and overall consideration to the general health of consumers of propolis and the effect on renal function is warranted.
Once the propolis phenols reach the blood stream, their selective permeability across the blood–brain barrier and systemic elimination limit the therapeutic efficacy with regards to optimal brain function [ 73 ]. However, increasing evidence suggests certain propolis derivatives are capable of crossing the blood–brain barrier. Caffeic acid phenethyl ester (CAPE) is hydrolysed to caffeic acid within six hours of reaching the plasma [ 23 , 40 ] and CAPE has recently been shown to cross the blood brain barrier at least in rats [ 23 , 40 ]. The degree to which propolis polyphenols and metabolised derivatives are capable of crossing the blood–brain barrier depend on their lipophilicity with less polar polyphenols or metabolites O-methylated derivatives) capable of greater brain uptake than the more polar ones sulphated and glucuronidated derivatives) [ 74 ].
Propolis comprises of lipids, waxes and resins in a complex matrix with a large molecular weight, contributing to a poor bioavailability and absorption. The form of polyphenol administered (natural fruit, juice or extract) or the presence of multiple polyphenolics support synergistic effects and are important determinants of bioavailability [ 73 ]. Factors believed to contribute to poor bioavailability of polyphenols include digestive instability, poor transcellular efflux in intestinal cells, and rapid metabolism and excretion [ 13 ]. Since dietary polyphenols exist as esters, polymers or in glycosylated forms, they cannot be absorbed and must be hydrolysed by the intestinal enzymes or by the colonic microflora before absorption [ 73 ]. When in the intestinal system, poorly absorbed polyphenolic compounds are converted to smaller phenolic acids with improved bioavailability, aided by enzyme activity of the colonic microbiota [ 39 ]. As microbiota vary between people the inter-individuality in absorption and metabolism is being increasingly considered. Despite poor absorption percentages of bio-accessible phenolic compounds in propolis, the recovered amounts detected in plasma were still high due to their high initial contents compared to other food materials such as fruits and vegetables [ 66 ].
According to the HPLC analysis, the phenolic content of propolis is mainly composed of crysin, galangin, pinostrobin, pinobanksin and pinocembrin, the last being the most abundant flavonoid in propolis [ 12 , 66 ]. European and Asian propolis contain simple phenolic acids while lignans are the main compounds in propolis from warmer climates (Brazil) [ 1 ]. A study of New Zealand propolis found 70% of the flavonoid content was pinobanksin and pinocembrin [ 71 ], much higher than samples from Brazil, Uruguay and China [ 72 ]. The differences in structure and function of the various propolis products does cloud the ability to recommend clinical and research direction. In general health claims are likely to be isolated to a product generated from a particular geographical location.
Of note, propolis contains a wide variety of natural phenolic compounds, mainly flavonoids and phenolic acids which have been attributed to its successful use as an anti-inflammatory and healing agent [ 28 , 70 ]. The pharmacological activity of flavonoids is mainly due to their structural characteristics as tricyclic compound, and to the presence of radicals attached to their rings [ 6 ]. The polyphenolic content of various propolis samples varies significantly, ranging from 143 to 324 mg gallic acid equivalents/g and from 206 to 705 mg quercetin equivalents/g of ethanolic extracts of propolis, respectively [ 66 ]. With the great diversity of propolis sub-groups, classified according to their physico-chemical composition, however there is also a large variance in the content of flavonoids [ 25 ].
Propolis is derived from animals but is contains bioactives derived from plant-based diet of the bees. Multiple compounds have been identified in propolis from different locations, including phenolic acids, flavonoids, esters, diterpenes, sesquiterpenes, lignans, aromatic aldehydes, alcohols, amino acids, fatty acids, vitamins and minerals [ 6 ]. The botanical source of origin is highly varied, which is consistent with the native flora by which the bees have access. The number of constituents identified in any individual propolis samples is in the hundreds, depending on geographic origin [ 10 , 28 , 68 ]. Whether the propolis extract was prepared with ethanol or water also changes the constituent nutrient profile [ 10 ]. The variety of compounds and phytonutrients found in propolis somewhat explains the range of biological properties reported, from antimicrobial, antioxidant, anti-inflammatory, immune modulating, skin healing, skin aesthetic, cancer protection and anti-cavity among others [ 69 ].
Propolis is a plant-derived substance collected by honeybees from various sources and contains multiple polyphenolic constituents, mainly flavonoids and phenolic acids [ 64 ]. Propolis originates from a variety of countries including Argentina, Brazil, China, New Zealand, South Africa, Taiwan, Ukraine, United States and Uzbekistan with different geographical characteristics, and is categorised into three major classes depending on its predominant colour, either green, red or brown [ 28 , 65 , 66 ]. The different classifications and source of origins make it difficult to extrapolate health-related claims on any one propolis source [ 67 ].
4.2. Oxidative and Inflammatory Effects of Propolis
4.2.1. Free Radicals and Oxidative Stress The antioxidant activity of propolis and its constituents has been well documented [47], with the vast majority of outcomes demonstrating a reduction in oxidative stress markers [36]. In order to reduce the oxidative stress-induced tissue damage, endogenous antioxidant systems have developed protective mechanisms including enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), and antioxidant nutrients ascorbic acid, glutathione and flavonoids [28]. Malondialdehyde concentrations are commonly being used as potential oxidative stress biomarkers and indicators of oxidative lipid damage. The chemical structure of the constituent polyphenols enable propolis to effectively eliminate free radicals [31]. The flavonoids in propolis are powerful antioxidants, capable of scavenging free radicals and thereby protecting the cell membrane against lipid peroxidation [13]. The most important organ of the central nervous system, the brain, is more sensitive to free radical-induced damage because of its high use of oxygen, its high concentration of polyunsaturated fatty acids, and its low concentration of antioxidant molecules compared to other tissues [27]. In the brain, oxidative stress results in acute and chronic injury and plays an important role in the pathogenesis of neuronal damage. Propolis and its derivatives appear to prevent oxidative stress in radiation-injured brain tissue by decreasing the formation of lipid peroxidation and increasing the antioxidant enzyme activities, and also by inhibiting free radical generation [27]. These results suggest an important role of propolis and its constituents as an antioxidant and free radical scavenger on the oxidative stress in the radiation-injured brain tissue. Preclinical studies have also suggested that pinocembrin, a component of propolis, protects rat brain against oxidation and apoptosis induced by ischemia-reperfusion both in vivo and in vitro [30]. In transgenic mice at risk of Alzheimer’s, those given pinocembrin maintained adequate glutathione content and SOD activity [18]. Administration of brown propolis from Iran ameliorated neuronal damage of permanent cerebral artery occlusion in mice because it restored the antioxidant SOD and GPx activity, and enzyme ratio, as well as reduced lipid peroxidation [28]. Oxidative stress is a well-known cause of persistent chronic inflammation due to its ability to activate transcription factors such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein 1, tumour protein, hypoxia-inducible factor 1-alpha, peroxisome proliferator-activated receptor gamma and nuclear factor erythroid 2-related factor 2 (Nrf2) [3]. NF-κB is a protein complex that controls the transcription of DNA and is involved in cellular responses to stimuli such as stress, cytokines, free radicals, ultraviolet irradiation, oxidised low density lipoproteins (LDL) and bacterial or viral antigens. Propolis is able to activate the Nrf2 transcription factor which is a major regulator of antioxidant proteins. The binding of Nrf2 to the antioxidant response element leads to transcription of several antioxidant enzymes, including heme oxygenase-1, the regulatory and catalytic subunits of γ-glutamate-cysteine ligase, GPx, glutathione reductase, CAT, SOD and glutathione-S-transferase [33]. The clinical implications for the antioxidant activity of propolis are difficult to ascertain, however below we discuss the anti-inflammatory and neuroprotective properties of propolis, both which have an antioxidant basis.
4.2.2. Anti-Inflammatory Inflammation is defined as an interaction between the immune system and injured tissues designed to restore homeostasis via complex signalling pathways [77]. The anti-inflammatory activity of propolis appear related to its associated constituents: flavonoids, phenolic acids and their esters, terpenoids, steroids and amino acids, with CAPE being the most studied compound. The main mechanisms underlying the anti-inflammatory activity of propolis include: (1) the inhibition of cyclooxygenase (COX) and consequent inhibition of prostaglandin biosynthesis, (2) free radical scavenging as discussed below; (3) inhibition of nitric oxide synthesis; (4) reduction in the concentration of inflammatory cytokines; and (5) immunosuppressive activity [2]. COX is an enzyme that is responsible for the formation of prostaglandins, prostacyclin and thromboxane. Pharmacological inhibition of COX can provide relief from the symptoms of inflammation and pain [2]. CAPE inhibits the activity and expression of COX-2 in human oral epithelial cells and in rat models of inflammation [78]. In transgenic mice at risk of Alzheimer’s, three months of pinocembrin treatment confirms significant reductions in neuronal inflammatory markers (tumour necrosis factor alpha (TNF-α), interleukin-1 (IL-1) and interleukin-6 (IL-6) [17]. Pinocembrin reduces the level of proinflammatory cytokines (TNF-α, interleukin-1beta (IL-1β)), chemokines (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), inducible nitric oxide synthase (iNOS), and aquaporin-4 [4]. In a study on rats with non-alcoholic fatty liver disease, 200 mg/day propolis reduced TNF-α and IL-6, with effects attributed to the anti-inflammatory activity of propolis [16]. Pinocembrin appears to suppress the nuclear translocation of NF-κB and decrease TNF-α expression in the cerebral cortex and the hippocampus of diabetic mice, suggesting propolis alleviates cognition deficits by protecting neurons from inflammation injury [11]. Pinocembrin may reduce damage induced radiation by the reduction of oxidative stress, inflammatory and apoptotic markers [5]. Of significance, pinocembrin normalised the infarct size elevated by radiation. The anti-inflammatory actions of pinocembrin has been evidenced by the reduced expression of IL-1b, TNF-a and iNOS [5]. Not all studies report a reduction in inflammatory markers. In type-2 diabetic patients, TNF-α was significantly decreased after the treatment with Brazilian green propolis, however IL-1β and IL-6 were significantly increased [22]. Propolis, as well as some of its components, actively stimulated the secretion of several cytokines, including IL-1β and the pro-inflammatory effects resulted from increasing IL-1β production were likely counteracted by the anti-inflammatory effects of IL-6 [22]. Therefore, the combined action of Brazilian green propolis on chronic inflammation was favourable in those with type-2 diabetes.
4.2.3. Atherosclerosis and Cardio-Protective Activity The cardiovascular effects of propolis have been widely reported, although the underlying mechanisms have been poorly characterised. Results from research conducted tend to indicate that cardio-protective effects are the result of the antioxidant activity of propolis and its constituent compounds [79]. Epidemiological studies revealed that a diet rich in flavonoids is positively correlated with increased longevity and decreased incidence of cardiovascular disease [70]. Oxidative stress appears to play a role in the initiation of cardiovascular disease. Low density lipoprotein oxidation triggers the process of atherogenesis, which is the developmental process of plaques on artery walls, resulting in atherosclerosis (a thickening of artery walls and restriction of blood flow), and finally cardiovascular disease [40]. Beyond the well-recognised anti-oxidant effects, polyphenols interact with the generation of nitric oxide (NO) from vascular endothelium, which results not only in vasodilatation, but also to the expression of genes which protect the cardiovascular system [79]. Mice on a cholesterol-rich diet revealed that all propolis types (red, green or brown) diminished total cholesterol and elevated high density lipoprotein-cholesterol concentrations, and sclerotic legions in mice, suggesting trivial bioactive differences in regional product sourcing [15]. Polyphenols are known to inhibit numerous cellular enzymes like lipoxygenase, xanthine oxidase and phospholipase A2 and reduce LDL-peroxidation. In addition, flavonoids found in propolis are iron chelators and can therefore decrease the iron-dependent production of free radicals [80]. The good scavenging activity of propolis flavonoids requires the presence of a catechol moiety in the B-ring along with 3-OH moiety in combination with a 2–3 carbon double bond in the C-ring of flavonoids, resulting in increased iron chelation and inhibiting the rate of lipid peroxidation [80]. Thus, chelation raises the level of scavenging activity of flavonoids [80]. Pinocembrin, in particular, is thought to influence cardiovascular diseases based on its ability to regulate ApoE and reduce rho kinase [4]. Propolis also showed to be protective against cerebral occlusion in mice with decreased infarct volume and improved behavioural function [28]. With regards to a particular cardiovascular-related disease, stroke, dietary polyphenols may provide benefits for different phases of the disease. It has been suggested that a nutrient timing approach could be applied to the use of propolis and other polyphenols in the reduction of cardiovascular disease. During stages of disease prevention, polyphenols improve cerebral blood flow, prevent platelet aggregation and thrombosis and inhibit oxidative stress. For the early disease stage, polyphenols reduce inflammation and protect endothelial phase, thus interfering with ischemia death mechanisms such as apoptosis and necrosis [79]. The utilisation of polyphenols like propolis in targeted disease states may be worthy of follow up in future research to provide clinical insight around optimal use.
Propolis extract: health benefits
Propolis extract has become a key natural ingredient for pharmaceutical and food supplement products. The main reason is that it contains many bioactive compounds that are very beneficial for health.
Propolis has regained attention recently as consumers have an interest in a healthier lifestyle. Also, in products that strengthen their immune system. Let’s think about its broad applications in prehistoric times. Throughout history, propolis extract has been used to fight infection, heal wounds, or embalm corpses. However, if you ever underestimated its potential, learn that it is the reason why thousands of honeybees have been able to survive 50 days to the Canary Island’s volcano eruption.
By using the stocking of this resinous material to seal up the holes in their hive, they were able to protect themselves from toxic ash and deadly gas.
In this article, we will focus on what characteristics make propolis extract a fundamental beehive product for pharmaceutical and food supplement companies.
How is bee propolis extract produced?
Honey bees make propolis out of the resins and sap they collect from deciduous trees, sap flows and other botanical sources. Back in the hive, resins are mixed with wax, enzymes and honey; and the mixture is stocked for a whole lot of uses including the sterilizing and disinfection of the bee colony.
While propolis has many uses for bees, it is also highly beneficial for humans. In order to exploit its countless possibilities, propolis is extracted commercially with a suitable solvent; usually a clear grain type of alcohol.
Its extraction consists of dissolving the propolis collected into the solvent and afterwards straining out the solution. Doing so removes the inert material from the source of propolis while preserving the desired, bactericidal compounds.
Bioactive Compounds of Propolis
Propolis is a natural source of biologically active substances: essential oils, vitamins and other organic compounds that occur naturally as part of food chains and confer useful health benefits. Some of the most important are outlined below:
Phenolic compounds
Propolis is high in phenolic acids and polyphenols, which act as some of the oldest antiseptic agents. These compounds grant propolis extract a natural antibiotic property that protects the immune system and acts against microorganisms.
Terpenoids
Terpenoids are highly aromatic compounds with strong antifungal qualities that empower antimicrobial activity. Its presence in natural extracts help reduce the expression of inflammatory mediators, enhance skin penetration and boost energy levels.
Flavonoids
Flavonoids present in propolis are responsible for protecting body cells from oxidative damage that can potentially lead to disease.
Enzymes
Propolis has a mixture of salivary and enzymatic secretions that accelerate different enzymatic reactions, fostering the metabolism of cells, blood circulation and also the formation of collagen fibers.
European propolis market: claims and trends
During the last few years and specifically after the pandemic, ingredients that stimulate immunity are playing a growingly important role in the supplements sector due to a big increase in consumer demand.
At the same time, potential buyers have started to be aware of the benefits that propolis provides, expanding a recently existing culture based on natural and beehive products that now showcase on the most popular beauty and wellness blogs.
Even though this is a global market trend, Europe is currently the largest player in terms of propolis market share, with Germany, UK, France, Italy and Turkey taking the lead. Only in Europe, more than 250 products containing propolis extract as its main active ingredient have been launched in the past 5 years.
The growing European demand for propolis can be attributed to its functional benefits, which respond to the rising health awareness and its ability to boost the immune system, digestive health, and to reduce aging effects amongst adults.
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Propolis: a huge opportunity for the pharma and food supplements sector
Propolis is a strategic ingredient for the pharma and food supplements sector, as it responds to the current customer priorities, above mentioned. Healthcare and wellbeing businesses can greatly benefit from the following properties found in propolis, as a way to reinforce their brand and strengthen their product line.
Natural ingredient with antibiotic properties
Propolis is a natural ingredient that creates a long-term connection with the consumer, adopting it not only as a product, but as a way of life. Many studies have verified a synergistic activity between some antibiotics and propolis, with greatest effectiveness shown against the influenza virus has been shown due to its antibiotic properties.
Biodiversity
Propolis collection and consumption contribute to the preservation of the ecosystem, supporting the beekeeping sector. Bees are pollinators with a key role in conserving biodiversity. In fact, close to 75% of the world’s food crops depend to some extent on pollination. With the number of bees plummeting as a result of climate change, reaping the benefits of propolis production is more demanding and urgent than ever.
Preventive health and immune system
Propolis is a powerful compound found in new natural product developments aimed to boost preventive health. Recent scientific research has strongly suggested propolis as one of the most promising immunomodulation agents. The antimicrobial activities of propolis occur through a direct action on microorganisms and indirectly also through the stimulation of the immune system.
Personalization
Propolis extract is becoming more and more available commercially in the form of capsules, mouthwash solutions, creams, throat lozenges, powder and many other formats that can adapt to tailored preferences and needs. Beehive products such as royal jelly, honey, and bee pollen are often combined and blended with this natural ingredient to offer value-added food and pharma products.
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Health benefits of bee propolis extract
Beyond prevention, propolis has many applications in treating various diseases because of its healing substances. As a proofed antiseptic, anti-inflammatory, antioxidant, antibacterial, antimycotic, antifungal, antiulcer, anticancer, and immunomodulatory natural ingredient, propolis can make a less invasive alternative or a complement to existing forms of medicine.
Antibacterial and antifungal qualities
The antifungal activity of propolis extract has been proved to be extremely effective against different fungi. One of the most common antimicrobial applications is its formulation for cold syndromes, such as respiratory and flu-like infections of the upper respiratory tract.
Dermatological and dental care
As a natural biomaterial for dental and oral healthcare, propolis extract is largely used in mouthwashes and toothpastes to prevent supragingival plaque, caries and to treat gingivitis, periodontitis and stomatitis within others.
Although propolis’ majority use is oral, the popularity of propolis as a skin treatment has been increasing. This incredible natural substance is efficacious in helping heal wounds and skin conditions, such as acne, comedones, herpes, burns and eczema.
Antioxidant characteristics
Propolis extract, in all of its different forms, possess antioxidant properties that help fight free radicals in the body. These radicals are compounds that can cause illness and aging if their levels become too high. For these reasons, propolis extract is popularly used in cosmetics, food and pharma supplements as a skin rejuvenator and as a cell regenerator.
Anti-inflammatory mechanisms
Terpenoids extracted from propolis are potent anti-inflammatory agents, making it a non-invasive and effective formula to prevent gastrointestinal disorders and lower high blood pressure, both derived from inflammation. Furthermore, it protects bone health by inhibiting osteoclastogenesis and promoting osteoblastogenesis, promoting health tissue.
Antidiabetic properties
This natural remedy has an antidiabetic effect on human bodies; reducing the expression of glucose and acting as a potential agent for the treatment of insulin-insensitive diabetes.
Foster your natural wellness portfolio with propolis-based supplements
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