User:Andreas Kontopidis/sandbox



Propolis or bee glue is a resinous mixture that honey bees produce by mixing saliva and beeswax with exudate gathered from tree buds, sap flows, or other botanical sources. It is used as a sealant for unwanted open spaces in the hive. Propolis is used for small gaps (approximately 6 mm or less), while larger spaces are usually filled with beeswax. Its color varies depending on its botanical source, the most common being dark brown. Propolis is sticky at, and above, room temperature (20 C). At lower temperatures, it becomes hard and very brittle.

The term Propolis comes from the greek saying “before the city”. Humans have used propolis for centuries for its pharmacological properties. Recently, an interest in propolis, particularly for its antimicrobial characteristics, has risen in the biomedical community. As a result, a surge of scientific publications revealing the diverse properties and potential beneficial applications of propolis has emerged. Along with antimicrobial properties, propolis has been known to show immune-stimulating and anti-tumoral effects.

Worker bees make up the majority of the matured bee population. They act as foragers and carry out various functions and tasks for the hive. The primary resources desired to bring back to the hive are pollen and nectar. However, they also are responsible for bringing back water and tree resin necessary for the production of propolis. Propolis is not something naturally found in the environment, it is created by bees using tree resin as their primary ingredient. Different conditions globally (as well as resources) change the chemical composition and nature of propolis from region to region. Once bees add saliva and beeswax, which act as catalysts, to collected resin to promote biochemical changes and form propolis. From the tree, to the resin, to the saliva of the bee; the catalytic process is hand picked and modified to benefit specific tasks desired by bees upon creation for the hive and queen. The bees transform the resin to optimize the benefits of propolis.

Purpose
For centuries, beekeepers assumed that bees sealed the beehive with propolis to protect the colony from the elements, such as rain and cold winter drafts. However, 20th-century research has revealed that bees not only survive, but also thrive, with increased ventilation during the winter months throughout most temperate regions of the world. The beehive is considered the most sterile environment found in nature. Despite being full of food, sugar, organisms, resin, pollen, and more; disease and infection are avoided due to its antiseptic properties. Propolis is therefore considered a natural defense and immune system of the hive.

Functions of propolis in the hive:


 * reinforce the structural stability and provide insulation to the hive
 * reduce vibration;
 * make the hive more defensible by sealing alternative entrances: propolis is commonly used to patch holes in the hive. The hive will have a propolis storage for emergency patch jobs, sometimes up to 1 pound of ready to use propolis.
 * prevent diseases and parasites from entering the hive, and to inhibit fungal and bacterial growth; by sealing every crack and coating a thin layer over surfaces of honeycomb cells, inside and outside, the hive prevents the spread of bacteria and fungi from entering the hive.
 * mitigate putrefaction within the hive. Bees usually carry waste out of and away from the hive. However, if a small lizard or mouse, for example, finds its way into the hive and dies there, bees may be unable to carry it out through the hive entrance. In that case, they would attempt instead to seal the carcass in propolis, essentially mummifying it and making it odorless and harmless.
 * Formation of a winding tunnel: propolis is used extensively at the entrance of the hive to form a tight tunnel that bees will crawl through to enter and leave the hive. The bees will be cleansed of microbes as they enter and lever. This maintains a sensitive sterility balance in the hive.

Composition
The composition of propolis varies from hive to hive, from district to district, and from season to season. Normally, it is dark brown in color, but it can be found in green, red, black, and white hues, depending on the sources of resin found in the particular hive area. Honey bees are opportunists, gathering what they need from available sources, and detailed analyses show that the chemical composition of propolis varies considerably from region to region, along with the vegetation. In northern temperate climates, for example, bees collect resins from trees, such as poplars and conifers (the biological role of resin in trees is to seal wounds and defend against bacteria, fungi and insects). "Typical" northern temperate propolis has approximately 50 constituents, primarily resins and vegetable balsams (50%), waxes (30%), essential oils (10%), and pollen (5%). Propolis also contains persistent lipophilic acaricides, a natural pesticide that deters mite infestations.

In neotropical regions, in addition to a large variety of trees, bees may also gather resin from flowers in the genera Clusia and Dalechampia, which are the only known plant genera that produce floral resins to attract pollinators. Clusia resin contains polyprenylated benzophenones. In some areas of Chile, propolis contains viscidone, a terpene from Baccharis shrubs, and in Brazil, naphthoquinone epoxide has recently been isolated from red propolis, and prenylated acids such as 4-hydroxy-3,5-diprenyl cinnamic acid have been documented. An analysis of propolis from Henan, China found sinapinic acid, isoferulic acid, caffeic acid, and chrysin, with the first three compounds demonstrating antibacterial properties. Also, Brazilian red propolis, largely derived from Dalbergia ecastaphyllum plant resin, has high relative percentages of the isoflavonoids 3-hydroxy-8,9-dimethoxypterocarpan and medicarpin. Other flavonoids commonly present include galangin and pinocembrin. Caffeic acid phenethyl ester (CAPE) is also a component of some varieties of propolis from New Zealand.

Occasionally, worker bees will even gather various caulking compounds of human manufacture, when the usual sources are more difficult to obtain. The properties of the propolis depend on the exact sources used by each individual hive; therefore any potential medicinal properties that may be present in one hive's propolis may be absent from another's, or from another sample in the same hive.

Regional differences in composition:

Case study in Greece and Turkey:

A study in Greece and Turkey looked at a severe bacterial disease historically damaging the larvae of honey bees. This disease studied is known as American foulbroud (AFB) and is comprised of a gram positive and spore forming bacterium. Greece and Turkey were both selected for this study because of their rich plant vegetation, high biodiversity/percentage of endemic plants, and high concentrations of diterpenoids found in their mediterranean propolis. The results of these tests showed that propolis inhibitory spectrum is rather broad, however, very strong even at very low concentrations. Antimicrobial activity was confirmed. A key clarification regarding this research is that the results reflected antimicrobial activity in reference to bee health, studies on human health or human usage were not examined. A breakdown of the various components that make up the region specific propolis is provided to the right in two regions found in Greece.

Samples during this study were then collected and stored in below freezing temperatures until chemical analysis. The samples were dissolved in ethanol, incubated, and underwent supernatant filtration. The final supernatant was analysed by GC-MS. The results according to the analysis showed that the largest difference between Greek and Turkish samples concerned the terpene and flavonoid concentrations.

Typically, treatment of honey bees infected with P. larvae was primarily through the use of antibiotics, especially in the US. This use of antibiotics has furthermore been forbidden in some EU countries due to lack of necessary Ministerial registration. A secondary treatment has been extensively looked for and propolis showed to be a potential candidate. Historically, various strains of P. larvae show resistance to antibiotics. The extensive use can lead to an accumulation of residues in hive products decreasing their quality. Due to these legal and biological concerns, bee scientists have been examining the natural antimicrobial products of propolis derived from plant resins and produced by honey bees. In conclusion, both Greek and Turkish propolis had effective antimicrobial activities against P. larvae. Propolis has potential as an alternative natural hive product, perhaps as a disinfectant solution for the prevention of P. larvae infection in honey bee colonies. Results of region specific chemical compositions of propolis are shown in the images to the right.

Flavonoids
Flavonoids are a major component in the diets of many. The compounds are found in tea, apples, tomatoes, onions, and other fruits and vegetables. Flavonoids are known for their broad spectrum of biological activities such as antitumor, and antibacterial.

Antioxidative activity
Some of the biological activity of flavonoids are due to the antioxidative properties. The catechol group in the B ring and 3 hydroxyl group in the structure of flavonoids is responsible for the antioxidative properties. The hydroxyl and catechol groups in the flavonoids are key to the scavenging of reactive oxygen species, reactive nitrogen species, and chelation of metal ions. The catechol group and 3 hydroxyl group are able to donate hydrogen and electron to the reactive species and free radicals, thus stabilizing the free radicals and preventing tissue damage. The flavonoids are helpful in preventing the oxidation of proteins and lipids. Lipid oxidation deletes lipid rafts on the cell’s membrane, which lowers cellular signaling transduction.

Anti-tumor activity
Free radicals cause aging and carcinogenesis. The propolis in moderate climate regions is also anti-tumor as a result of the antioxidative properties of the flavonoids. An example is free radicals caused by sun damage. Hydrogen peroxide (H2O2) damage the genes through peroxidation of DNA and induction of cellular aging. When genes are damaged, there is cell mutation, and formation of the cancer. Propolis was found to inhibit (H2O2-) induced damage to DNA. A study concluded that the flavonoids provided propolis with anti-tumor properties by inhibiting DNA and RNA synthesis of cancerous cells. Specifically, the flavonoids prevented the incorporation of thymidine and uridine into the DNA and RNA molecules.

Anti-inflammatory activity
The anti-inflammatory properties of flavonoids from propolis are also due to anti-oxidative properties. The flavonoids are able to prevent oxidative damage to tissues  caused by the generation of free radicals by macrophages during the inflammatory process. Flavonoids were also found to inhibit the secretions of pro-inflammatory cytokines by the lymphocytes and macrophages.

Antibacterial activity
The flavonoids in the propolis from moderate climate regions have antibacterial activities due to disruption of the bacterial and microbial membranes. The flavonoids require a balance of hydrophilicity and lipophilicity for membrane affinity. The presence of hydroxyl groups in the flavonoid structure are important in membrane disruptive activities and number of hydroxyl groups dictate lipophillicity of the structure. By infiltrating the bacterial membrane, the flavonoids alter membrane fluidity in hydrophilic and lipophilic regions, thereby reducing fluidity of both inner and outer layers of gram negative bacteria. The membrane fluidity was also decreased in gram positive bacteria. Increased rigidity of membranes reduces the motility of bacteria and prevents migration of the bacteria to other regions of the body. Reactive oxygen species are produced bylipopolysaccharides on bacterial membranes. The scavenging activities of flavonoids reduces the reactive oxygen species caused by the bacteria from damaging tissues. The catechol groups of flavonoids are able to cause leakage of small molecules due to bacterial membrane damage. The inner bacterial membrane potential must be maintained for the production of ATP by the ATPase. If there is leakage of protons, then the electrochemical gradient cannot be maintained and ATP synthesis in bacteria is reduced. The B ring of the flavonoids are able to form hydrogen bonds with nucleic acid bases, thus disrupting the process of DNA and RNA synthesis. It was found that DNA synthesis was most inhibited in Proteus vulgaris, while RNA synthesis was most affected in Staphylococcus aureus, both gram positive bacteria. Protein and lipid synthesis activity in bacteria are affected by the disruption of DNA and RNA synthesis.

Anti-viral activity
Flavonoids were found to be anti-viral. The catechol groups of the flavonoids was found to inhibit HIV reverse transcriptase and HIV 1 proteinase. In the same manner of disruption of DNA and RNA synthesis in bacteria, the flavonoids were found to inhibit the replication of the Herpes simplex virus (HSV).

Caffeic acid phenethyl ester (CAPE)
CAPE is a compound responsible for biological activities such as inhibition of cellular proliferation, inducing apoptosis and cellular arrest. CAPE causes the inhibition of DNA synthesis, leading to anti tumor properties. CAPE is also responsible for the interruption of growth signal transduction, inducing apoptosis, and inhibiting the growth of new blood vessels.

Phenolic acids
Phenolic acids are another constituent in propolis, specifically Polish propolis, that have antioxidative properties in the same structure - functional relationship as the flavonoids. The antioxidative properties of phenolic acids are due to the number of hydroxyl groups in the structure and the hydroxyl group position on a ring. On the aromatic ring, the bond energy between hydrogen and oxygen atoms on the hydroxyl groups is smaller than the bond energy on aliphatic portions of the structure. The hydrogen can easily detach from a hydroxyl group on the aromatic ring to stabilize free radical and other reactive species. After the hydrogen detachment, the phenolic acid becomes a phenoxyl radical, which is stable.

Terpenoids
The terpenoids are found in Brazilian and Mediterranean propolis. The presence of terpenoids are useful for ascertaining original propolis from the fake propolis. The terpenoids are volatile compounds that are responsible for the smell of resin in the propolis, and allows for bees to use propolis as air freshener in the hive The terpenoids also exhibit antioxidant and  antimicrobial activities. The anti-bacterial property of the terpenoids is due to penetration is rupture of the bacterial membrane. The hydrogen bond donor group located at the lipophilic decalinic ring system allows terpenoids to puncture cell membranes. The antioxidative properties are also due to the hydrogen bond donor groups, which can stabilize free radicals.

Current uses
A group studied the effects of propolis when added to the lipstick. Two test groups of lipstick were created, one group containing propolis and the other group without propolis. The lipsticks were subjected to rheometer testing and it was found that both samples of lipstick show shear thinning. It was also found that the lipstick with added propolis is able to retain its structure better after application to the skin.

In Korea, there are commercialized products that contain propolis. The concentration of propolis in liquid foundation is 3% and 2% in Propoleo cream. It was reported that Propoleo cream is favorable for oily skin and facial acne. Eye creams contain 1% propolis, and provided anti-bacterial properties. Over 80% of consumers in Korea that purchased products containing propolis were reportedly satisfied with their purchase.

In a study investigating the effects of propolis containing toothpaste on oral and periodontal health, sixteen subjects who underwent oral implants were involved in the study. The subject were divided into two groups: one group was instructed to use propolis containing toothpaste, while one group received toothpaste without the active ingredient. It was found that the subjects that used propolis containing toothpaste had a reduction in microbiota spectrum in the mouth.

Propolis has been experimented in some unique cases. It is used by some string instrument makers (violin, viola, cello and bass) as a varnish ingredient. Some workers use it to seal the surface of newly made bridges. Propolis was purportedly used by Antonio Stradivari in the varnish of his instruments. Additionally, propolis is used to bring about a chemical reaction to convert fats and oils into automobile wax during application.

Studies and Research
Propolis is a natural bioactive compound with anti-microbial, anti-flammatory, anti-oxidant, and anti-septic properties. It contains flavonoids, steroids, polyphenols, aldehydes, amino acids and quinones which contribute to its diverse properties. Propolis has been tested in a number of case studies which apply its unique properties for potential commercial use.

The polyphenolic compounds in propolis are good for reducing inflammation and the wound healing period. A study at Isfahan University of Technology explored the use of propolis in wound bandages made of poly-vinyl alcohol (PVA); PVA is a synthetic biodegradable polymer hydrogel. The material shows high absorption, adhesion, and high flexibility. In the study, Iranian propolis in the form of hydroalcoholic extract of propolis was loaded by electrospinning with PVA solution. A nanofibrous mat, microfibrous mat, and film were developed and later tested with staphylocuccus aureaus virus. It was found that the nanofibrous mat had a better releasing rate and antibacterial maintenance than commercial microfibrous wound dressings, meaning the hydroalcoholic extract of propolis and PVA nanofibrous mat could be used as more efficient controlled delivery system for wound healing.



Propolis has also been tested for use as an eco-friendly fungicide. This study is in attempt to combat phytopathogenic microorganisms which are attacking seedling nurseries, plantations, landscape plants, seed orchards, and native forests. The current control of plant diseases has included highly toxic chemicals which are bad for both the environment and human health. Solution combinations of chitosan oligomers, propolis, and silver nanoparticles were tested against eight fungi and oomycetes in an in vitro study. The results showed that the addition of propolis to chitosan or silver nanoparticles either increased or did not change the inhibitory effect. The results are summarized in the table below with COs being the chitosan oligomers, Ps being propolis, and AgNPs being the silver nanoparticles. There was a lot of success with using propolis, therefore making it a realistic option for fungicide materials.

Propolis was also studied at Islamic Azad University for food health and safety applications. It was proposed to combine ethanolic extract of propolis with a chitosan edible coating to preserve a refrigerated chicken fillet. A preservative made with propolis and a regular chitosan preservative were applied so that the chicken could be evaluated and compared. After 12 days it was found that there was a significant reduction in the growth of bacteria on the propolis coated chicken. Also, the study measured the chemical prevalence, specifically the TBARS, total volatile nitrogen, and peroxide values, and found that the propolis preservative showed a smaller increase than the control preservative. The propolis and chitosan increased the shelf life from three to seven days. There was, however, a minor decrease in sensory qualities such as look, smell, and taste.

Propolis was tested on livestock, such as cattle, fish, poultry, and pigs. Propolis was found to increase growth performance of the livestock. In new fields of propolis application, it is useful replacement for antibiotics in feedings of lifestock. Studies have tested the diffusivity of propolis compounds in polylactic acid (PLA) films for application in active food packaging and anti-microbial packaging films.

A study investigated the effects of propolis embedded into titanium dioxide (TiO2) nanotubes for dental implants. Test rats underwent mandibular implant surgery and were divided into 3 groups. One group received commercially pure TiO2 implants, another group received TiO2 implants with nanotube formations, and last group received TiO2 implants with propolis embedded nanotubes. It was found that the rats with propolis loaded onto the nanotubules in the implants had greater osseointegration compared to the groups without propolis loaded implants.

Propolis has been used in traditional medicine. Propolis is "possibly effective" for treating cold sores, genital herpes, and post-surgery mouth pain, but there is "insufficient evidence" to rate the effectiveness of propolis in treating these or other conditions. Propolis is under preliminary research for the potential development of new drugs associated with control of Candida albicans and immunomodulatory effects.