Focus: Propolis

Propolis is a substance of plant origin, whose use by man dates back to very ancient times (300 BC) and still represents an important natural source used in traditional medicine and modern industry.

The Incas used it as an antipyretic agent, the ancient Egyptians exploited its anti-depressant properties to embalm the corpses of the pharaohs and also well-known figures of ancient Rome and Greece such as Aristotle, Dioscorides, Pliny, Virgil, Varro, Galen knew its healing effects. Its actions antiseptic, disinfectant of the oral cavity and healing wounds have allowed a wide use throughout the Middle Ages. In this era it was applied in the navel dressing of newborns and as a remedy for throat disorders. In the thirteenth century, there were already many propolis-based body care products in Germany and Russia. The London Pharmacopoeia of the eighteenth century classifies it as “officinal drug” (Castaldo, 2002).

In the modern era many countries of the world have rediscovered the interest in the use of natural products to treat various diseases and propolis is a perfect product for this purpose.

The propolis originates from the exudate resinous which is formed on the young leaves, on the buds and in the fissures of the bark of various plants belonging mainly to the genus poplar (“populus”); the characteristic viscous liquid from which it derives has also been found on beech, horse chestnut, birch, pine, alder, willow, although in smaller quantities (Pietta, 2002).

This resin is collected by the bees (Apis Mellifera L.) during the hottest hours of the day, when the consistency of this substance is soft and rubbery, and the digested material is mixed with beeswax and then used in the hives as a material “Sealant” for the construction and protection of their hive, so much so that in jargon is called “glue of bees” (Castaldo, 2002), perhaps because of its etymological analogy with the Greek word “propoliso”, ie substance that paste . The bees use it as a multifunctional substance to fill the cracks in the hives; to cover the inside of the newly built cells, before the queen bee lays there the eggs; to protect the beehive from the invasion of other insects, to avoid water infiltration or drafts (Valcic, 1998). Therefore, as well as a construction material, propolis is also a fundamental source of defense for bees.

It is still used as embalming substance to cover the carcasses of small invaders killed inside the hive: in this way the remains of these dead insects dry up without any putrefactive processes occurring avoiding any danger of infection.

At the base of these observations we can also explain the etymological meaning attributed to the word propoli (from the Greek pros: forward and polis: city), which indicates precisely something that serves to protect, to defend and which is reflected both in the animal world, as we have seen about bees, both in the human world for the innumerable healing properties that this natural substance has shown to possess (Burdock, 1998).

Processing of propolis in the apiary

Regarding the origin of propolis there are two hypotheses: it could derive directly from the resin exudate collected by bees without undergoing any chemical modification, or originated as a result of the action of salivary enzymes, such as β-glucosidase, during mastication ( Marcucci, 1995; Pietta, 2002). Propolis is presented as a lipophilic material, with a pleasant aromatic odor and a variable color (from light yellow to red to dark brown), depending on the place of origin and harvest time. Hard, compact but of fragile consistency when it is cold, it becomes very sticky if heated (Marcucci, 1995; Pietta, 2002). It has a sour taste and gives off a pleasant fragrance when subjected to the flame. The propolis present in the hive has a level of purity that varies between 60 and 80%. The main impurity is the wax that the bees add to the propolis in percentages between 10 and 30% to make it more malleable. Generally in its raw, unprocessed form, it is composed of 50% resin 30% wax, 10% essential and aromatic oils, 5% pollen and 5% various organic substances (Pietta, 2002).

The propolis can not be used as it is, as raw material, nor is it easy to isolate the substances that characterize it from the chemical point of view, due to its complex composition. Usually it undergoes a purification by means of solvent extraction, a process used to remove the inert material without biological activity (waxes) and to isolate the bioactive portion (Pietta, 2002). The usual method was to extract the soluble fraction in ethanol, also called “propolis balsam”. However, extractions were carried out with other solvents, such as methyl alcohol, obtaining the isolation of numerous compounds (Marcucci, 1995). Extraction with ethyl alcohol was particularly suitable for obtaining a propolis extract rich in polyphenolic compounds such as flavonoids and phenolic esters, especially coffee beans and ferulates, known for their antibacterial, antiviral and antioxidant properties (Pietta, 2002).

Chemical composition

Propolis has a very rich and complex chemical composition: more than 180 different compounds have been isolated and identified from this natural product. Numerous chemical studies conducted on different samples of propolis have shown that the main secondary metabolites are phenolic substances, especially flavonoids, (flavanoni, flavones, flavonols, dihydroflavonols) which make up more than 50% of the weight of propolis (Banskota, 1998). These compounds are generally present as aglycones and it is for this reason that they are believed to originate as a result of structural modifications made by the salivary enzymes (glucosidase) of the bees at the time of collection. In fact, this class of compounds, typical of the plant world, is naturally present in the form of glycosidic derivatives. Other phenolic compounds found in propolis are hydroquinones, caffeic acids and related esters and phenolic aldehydes (Pietta, 2002; Kumazawa, 2004). Propolis has also proved to be a rich source of essential elements such as magnesium, nickel, calcium, iron, zinc, cesium, manganese, silver, copper, aluminum, vanadium, vitamins B1, B2, B6, C and E (Marcucci, 1995 ; Castaldo, 2002), as well as amino acids, especially proline and arginine, although in traces (Gabrys, 1986). In addition, some non-phenolic substances have also been found in some propolis, but belonging to different chemical classes such as aliphatic acids, coumarins, aliphatic and aromatic hydrocarbons, terpenoids, steroids and isoprenylated benzophenones.

Chemical variability in relation to the place of origin

The chemical composition of propolis is closely linked to the place of origin, the harvest time and the plant source from which it derives, and it is for this reason that it is difficult to achieve a univocal classification of the chemicals and therapeutic properties contained in the propolis due to of its considerable variability. At the same time it is these remarkable chemical-quantitative differences related to its geographical distribution that diversify and characterize the propolis, making it so unique both from a chemical and biological point of view.

The Tunisian propolis is distinguished by the presence of a characteristic flavonoid, the miricetin 3,7,4 ‘, 5’ tetrametiletere (Martos, 1997), while a study carried out on the propolis of New Zealand origin has shown that the dihydroflavonoids based on pinobanchina and pinocembrin (important especially in the process of vitamin C replacement), make up about 70% of the total flavonoids.

In the Uruguayan and Chinese, however, these dihydroflavonoids are present in less than 10% of the total; while in the Brazilian they make up even 50%. In the Chinese and Uruguayan varieties the predominant flavonoids are essentially flavones and flavonols (Kenneth R. Markham, 1996).

Comparisons on the chemical composition between propolis samples from Europe, South America and Asia have been widely carried out (Marcucci, 1995), and from this it has been possible to establish that European and Chinese propolis abound mainly from various species of flavonoids, phenolic acids and related esters, while the predominant compounds of Brazilian propolis are terpenoids and prenylated derivatives of p-coumaric acid (Marcucci 1995 and Bankova 1987). From this emerges a fundamental fact that there is a considerable difference between the original propolis of the tropical areas (South America), from which completely different compounds were isolated, such as those with a benzophenonic nucleus, and those of the temperate zones (Europe) where the flavonoid composition predominates (Marcucci, 1995).

Biological Activity

Propolis is an ancient natural remedy that is still widely used in modern medicine, thanks to its antiseptic, antiviral, bacteriostatic, antifungal, anti-inflammatory, anesthetic, astringent, spasmolytic, antioxidant and immunomodulatory properties.

Antimicrobial activity

Propolis, given its natural defense role, intrinsically possesses antimicrobial properties; this has been widely demonstrated by scientific studies conducted both on extracts and on pure compounds isolated from different samples. Meresta et al (1985) studied the sensitivity of 75 bacterial species to the extract of propolis, of which 69 were Staphylococci and Streptococci. The antibacterial activity shown against S. aureus was very interesting: MIC (minimum inhibitory concentration) of 10 mg / ml and MBC (minimum bactericidal concentration) of 120 mg / ml. A concentration of 3 mg / ml of propolis ethanol extract (EEP) also inhibits the growth of Escherichia coli and Pseudomonas aeruginosa, but does not affect Klebsiella pneumoniae (Grange and Daney, 1990). Propolis is active against both Gram + (study of 265 strains) and Gram – bacteria, including B. subtilis, S. epidermidis, Streptococcus β – hemoliticus, P. vulgaris, K. Pneumoniae and P. aeruginosa (Fuentes and Hernandez, 1990).

In addition, propolis demonstrated a decrease in tolerance of staphylococci to antibiotics and was noted by in vitro studies (cultures of Staphylococcus aureus), which exists a synergism of action of the propolis associated with antibiotics such as Streptomycin and Cloxacillin (minus Chloramphenicol) (Castaldo, 2002). The mechanism of antimicrobial activity is complicated and could be attributed to a synergism of action among the natural substances that make up the bioactive fraction (Kedzia, 1986).

Most compounds isolated from propolis showed antibacterial activity. It is interesting to note that it is not just a matter of phenolic substances, of which this activity is well known, but also of compounds belonging to other chemical classes. A significant example is propolone A, an isoprenylated benzophenone isolated from a sample of Cuban propolis. In fact, this compound showed an interesting activity against Gram + (especially Streptomyces chartrensis and Streptomyces violochromogenes) and Gram – (Shigella sonnei and Pseudomonas aeruginosa) (Cuesta Rubio, 1999).

As regards antiprotozoal activity, it was detected following in vitro studies on Toxoplasma gondii and on three different strains of Tricomonas vaginalis: the lethal action was obtained at a concentration of 150 mg / ml of EEP (Scheller, 1995) . The 3% EEP association with other antiprotozoal drugs has given remarkable results in experiments on animals infected with Eimeria magna, E. media and E.perforans. Furthermore, an EEP concentration of 11.6 mg / ml promotes a 98% inhibition of the parasitic protozoan Giardia Lamblia (Torres, 1990).

Antiviral activity

Afterwards and in vitro tests it has been shown that the antiviral activity of propolis is directed towards both DNA and RNA viruses, and in particular with regard to herpes simplex type 1 and 2 (HSV-1 and HSV-2) , type 2 adenovirus, vesicular stomatitis virus, poliovirus, HIV, influenza A and B viruses. Reduction of herpetic infection was achieved at a concentration of 30 μg / ml, whereas the stomatitis virus and adenoviruses they showed less susceptibility.

It is believed that the activity shown by propolis against most of these viruses is due to the presence of flavonoids (galangine, crisina, chemferol, acacetin and quercetin), aromatic acids (caffeic acid) and derivatives, such as 3-methylbut-2 -enyl caffeato and 3-methylbutyl ferulatus (Bankova, 1987; Marcucci, 1995), however, however isoprenylated benzophenones, such as isoxanthocol and Guttiferones A and E have shown activity against the HIV retrovirus (Gustafson, 1992).

Also the triterpenoids (moronic, betulonic and anwuweizonic acid), isolated from propolis of Brazilian origin, were tested to evaluate their anti-HIV activity and it was observed that the moronic acid in particular showed a significant antiretroviral action. On the other hand, the activity of betulonic acid was weak, while anwuweizonic acid did not inhibit the proliferation of HIV cells. 3- (3,4-dimethoxyphenyl) -2-propenal, also isolated from Brazilian propolis, was also active in the suppression of T-cells in particular infected with HIV (Junko Ito, 2001).

Antifungal activity

The antifungal activity has been studied in vitro, it is shown in preparations containing 5 or 10% of propolis extract. The ethanolic extract (EEP 10%) was tested on 17 pathogenic fungi, giving significant results especially against Candida albicans, C. parapsilosis, C. tropicalis and C. guilliermondii (Fernandez Junior, 1994). Also for this biological activity the association of 10% EPP with some antifungal drugs showed an interesting synergism against Candida infections. These observations were confirmed by a study conducted on 30 samples of this famous yeast.

Cytotoxic and antitumor activity

The antitumor effects of the samples of propolis studied were ascribed above all to the presence of flavonoids that showed inhibitory activity against DNA synthesis, mitosis and protein synthesis, as they block the incorporation of thymine, uridine and leucine into the mutated cells. (Burdock, 1998). The ethereal propolis fraction (DEEP) has shown an excellent cytotoxic activity especially against nasopharyngeal carcinoma and HeLa cell lines (human carcinoma of the cervix) and it has been experimented that a concentration of 10 mg / ml causes an inhibition 50% of the capacity of these cancer cells to settle in colonies (Ban, 1993; Hladòn, 1980). The effects of EEP instead have been studied also in vivo on male mice with Ehrlich carcinoma. The survival rate after treatment with EEP was compared to that given by Bleomycin (Scheller, 1989; Burdock, 1998).

Hepatoprotective action

Propolis has shown in vivo hepatoprotective action on animals previously treated with CCl4 (carbon tetrachloride). These results were also observed on the hepatotoxic effects resulting from treatment with paracetamol, hexobarbital and in cases of acute hepatic ischemia. This is probably due to the antioxidant activity of propolis and its ability to inhibit microsomal metabolism (Burdock, 1998).

The red Cuban propolis has been studied thanks to the notable hepatoprotective effects demonstrated, as well as for the marked action against free radicals detected. In fact, its effects against hepatitis in rats have been evaluated, induced by administration of galactosamine. This known hepatotoxin produces severe hepatocyte inflammation. The use of the Cuban propolis prevents such cellular alterations, leading to a complete disappearance of the inflammatory state. In fact, it promotes the reduction of the increased activity of alanine aminotransferase and of the concentrations of malondialdehyde in the serum of rats treated with galactosamine, with a decrease in acidophilous degeneration and of phagocytic infiltrations that the same galactosamine had induced (Rodriguez, 1997).

Antioxidant activity

The aqueous extract of propolis has shown, following in vitro studies, an inhibitory activity on lipid peroxidation, in particular, of linoleic acid. At a concentration of 1 to 5 mg / ml the antioxidant activity was higher than that of ascorbic acid (5mM). The scavenger properties of the aqueous extract were also evaluated using the ipoxanthin-xanthine oxidase system: a concentration of 50-100 mg / ml completely inhibits the formation of superoxide anion. The same repressive results were also observed with respect to the DPPH and the hydroxyl radicals. The antioxidant properties shown of the aqueous extract of propolis are due to the presence of phenolic acids, flavonoids, cinnamic acid, caffeic acid, and in particular to some acid-O-caffeoyl-quinic derivatives. Another study conducted by Castaldo et al (2002) found the ability of propolis to preserve the degradation of GSH induced by paracetamol; we recall that GSH is an important endogenous antioxidant that contributes significantly to maintaining the redox balance of cells. Numerous studies have been conducted to compare the antioxidant activity of Japanese, Chinese, Brazilian and US propolis. The Chinese variety was more active than the Brazilian and US (Kumazawa, 2004). In this case it is also important to underline the significant differences between the propoles selected from different geographical areas, differences that, as we have noted, are also noteworthy from the biological point of view. Other properties attributed to propolis are the anti-inflammatory, immunostimulant and anesthetic properties.  The exact anti-inflammatory mechanism has not yet been clarified, but it is likely that it inhibits COX (cyclooxygenase) activity with a dose-dependent mechanism. The immunostimulatory and immunomodulatory function was observed in vitro on macrophages and in vivo (rats) with an increase in the number of CD4 and CD8 cells (Castaldo, 2002).

Commercial applications and preparations

On the market there are numerous pharmaceutical preparations based on propolis (tablets, tablets, lotions, tinctures, syrups, etc.), as well as products for cosmetic use. Among the most common formulations we recall the associations of propolis with other substances of vegetable origin such as effervescent tablets with vitamin C and Aloe vera; tablets with Rosa canina and Echinacea; syrups containing eucalyptus honey and vitamin C or those with myrtle and trace elements; balsamic candies with wild herbs. A spray containing a hydroalcoholic solution, capable of evoking a soothing and calming action in gold-pharyngeal infections, has also proved to be very effective. There is also an ophthalmic cream (GAC 4922) with 1% propolis. Propolis has also been applied in the cosmetic field, above all thanks to the moisturizing, toned and elasticized properties that it can have on the skin; in fact, various cream products are used to act on oily skin.

There are many cosmetic preparations in the form of O / W emulsions, ointments, O / W gels such as gel with isopropylpalmitate, that with emulgina B3 or with Cetiolo HE. The applications of products with pharmacological action in the dermatological field are the most widespread: excellent results have been obtained by applying the propolis in the form of ointment to 50% in the treatment of scalp dermatitis induced by Trichophiton. Other uses are neurodermatitis, eczema, psoriasis, morphea, gangrene, contact dermatitis and treatment of wounds and burns.

In the field of ENT the efficacy was found by studying the benefits obtained following the treatment of patients suffering from otitis externa, mesotympanic otitis and tympanic perforation, as well as in cases of pharyngitis, chronic bronchitis, pharyngo-laryngitis, phlegm and rhinitis.

Compressed products have been active in the treatment of acute and chronic colitis, acute gastric ulcer and acute duodenal ulcer.

In gynecology, tablet and lotion forms are used to treat numerous vaginal infections (Herpes vaginalis, S. pyogenes).

Its use in odontology in the form of mouthwashes (often associated with lemon balm, mauve and / or rosemary), toothpastes, lotions, pastes and powders to be used for gargling or for internal use after melting in water is also well known. In this field, its importance has been observed in the regeneration of the dental pulp, in cases of gingivitis, stomatitis, periodontitis, buccal affections and in the prevention of plaque and caries (Marcucci, 1995).

Toxicity

Numerous studies have been performed both in vivo and in vitro and it has been shown that substantially propolis is not a toxic product, but as most of the substances of natural origin can induce troublesome adverse reactions, although somewhat infrequent. Acute toxicity tests were performed in rats and it was seen that the LD50 is included in a range of 2050 and 7340 mg / kg (Arvouet-Grand, 1993). Instead, other in vivo experiments conducted on the ether extract of propolis have shown toxic effects already at a dose of 350 mg / kg, while the LD50 has been detected, this time, at a concentration of 700 mg / kg, after 19 hours of exposure both with ethereal and alcoholic extracts (Ghisalberti, 1979). This denotes the fact that the toxicity of the substance is not due exclusively to its chemical composition, but also to any external contamination of plant and / or environmental origin: indeed there is a certain correlation between the hypersensitivity reactions induced by propolis and those that occur following contact with the resin of the poplars (Marcucci, 1995). To confirm this, the activity of some flavonoids contained in the propolis was tested and in particular it was found that pinocembrin, one of the main flavonoidic constituents, did not show signs of toxicity when administered orally to mice at a dose of 1000 mg / Kg (Metzner, 1977).

However, the allergic reactions that propolis is able to trigger following the ability of some chemical constituents to induce the immune response are well documented. Contact dermatitis has occurred in the operators of the sector, above all bee growers, maintenance workers of the hives and the collection of the resin, but also in those who have used the preparations based on propolis. The erythema mainly concerns the face, eyelids, neck, hands, arms, vulva and penis as well as irritations to the mucous membranes in the case of oral administration (Hausen, 1987 a). Approximately 200 cases of allergic contact dermatitis were detected, plus one case, much more serious, of dermatitis with granulomatous access and lymphadenopathy. It is believed that the allergen capable of triggering the immune response is the LB-1, which consists of a mixture of caffeic acid esters, especially those of phenylethyl and prenyl. However, the common opinion of many scholars tends to identify LB-1 with the 1,1-dimethylallyl acid of caffeic acid (Hausen, 1987, Marcucci, 1995). The sensitizing properties of LB-1 have been tested in guinea pigs and it has been shown that this compound is the main sensitizing agent contained in propolis (Marcucci, 1995).

The flavonoid tectocrisina, on the other hand, has been evaluated as the second allergen, even though its sensitizing action has been rather moderate (Schmalle, 1986). Observations on the use of oral propolis have suggested that intestinal absorption may play an important role in the sensitization process, especially with regard to mucosal irritations; in fact it has been found that limiting the oral use is limited to allergic reactions (Marcucci, 1995). However, the idea remains that the use of a natural remedy such as this, in addition to the various benefits that it provides thanks to the countless possible applications, produces minimal toxic and far inferior effects compared to the classic medicinal products of synthetic origin.