Research SectionReview of the biological properties and toxicity of bee propolis (propolis)
Introduction
Propolis (CAS No. 9009-62-5) (sometimes also referred to ‘bee glue’) is the generic name for the resinous substance collected by honeybees from various plant sources (CHEMID, 1996). The word propolis is derived from the Greek pro-, for or in defence, and polis-, the city, that is, defence of the city (or the hive) (Ghisalberti, 1979). Propolis is a strongly adhesive, resinous substance collected, transformed and used by bees to seal holes in their honeycombs, smooth out the internal walls and protect the entrance against intruders. Honeybees (Apis mellifera L.) collect the resin from the cracks in the bark of trees and leaf buds. This resin is masticated, salivary enzymes added and the partially digested material is mixed with beeswax and used in the hive (Ghisalberti, 1979; Marcucci, 1995). Although propolis may contain some pollen, it is not pollen nor should it be confused with ‘bee bread’ or ‘royal jelly’, which are wholly different products of the hive.
The United States Department of Agriculture's ‘United States Standards for Grades of Extracted Honey, Effective May 23, 1985’ (adapted from 7 CFR §52.1394) describes propolis as follows (USDA, 1985):
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(l) Propolis means a gum that is gathered by bees from various plants. It may vary in color from light yellow to dark brown. It may cause staining of the comb or frame and may be found in extracted honey.
The precise composition of raw propolis varies with the source. In general, it is composed of 50% resin and vegetable balsam, 30% wax, 10% essential and aromatic oils, 5% pollen and 5% various other substances, including organic debris (Cirasino et al., 1987; Monti et al., 1983). The wax and organic debris are removed during processing, creating propolis tincture.
The substance ‘populus’, a flavour ingredient, has been confused with propolis. This is an understandable misnomer since ‘populus’ is harvested from Populus balsamifera L. (and other Populus species), and it does refer to a resinous material in buds (i.e. before the leaves open in the spring). The buds are protected by a hood which contains a resinous, sticky, varnish-like substance1. This substance is extracted from the buds with a hydrocarbon solvent, producing an oleoresin which may be further steam-distilled or extracted with alcohol. The resulting extractant has a sweet, balsamic odour with a slight cinnamic undertone used in the flavouring of alcoholic beverages (Arctander, 1960; Burdock, 1995). Populus is chemically quite similar to propolis, since this resin is the raw material harvested by bees for manufacture of propolis (see below).
Man's long history of bee domestication has led to a thorough exploitation of bee products, and the many favourable properties of both raw and refined propolis lend to its application in many human pursuits. There is a long history of use of propolis, at least to 300 BC (Ghisalberti, 1979) and its use continues today in home remedies and personal products. Because propolis is reputed to have antiseptic, antimycotic, bacteriostatic, astringent, choleric, spasmolytic, anti-inflammatory, anaesthetic and antioxidant properties, the list of preparations and uses is nearly endless. These applications include over-the-counter dermatological items where it has been claimed useful in wound healing, tissue regeneration, treatment of burns, neurodermatitis, leg ulcers, psoriasis, morphoea, herpes simplex and genitalis, pruritus ani and activity against dermatophytes. It has been marketed as a treatment for rheumatism and sprains; and in dental medicine, it is claimed to be an anaesthetic five times as effective as cocaine. It is used in toothpaste and mouthwash preparations treating gingivitis, cheilitis and stomatitis. It has also found its way into pharmaceutical and cosmetic products such as face creams (vanishing creams and beauty creams), ointments, lotions and solutions. It is marketed in tablets, powder and chewing gum (Ayala et al., 1985; Bankova et al., 1983; Bjorkner, 1994; Dobrowolski et al., 1991; Esser, 1986; Ghisalberti, 1979; Hausen et al., 1987a; Marcucci, 1995). Although Europeans tend to use propolis-containing products more than Americans, it is sold in American health-food stores in capsules (approximately 50 mg/capsule) and is used in mass-marketed dental floss and toothpaste.
Antiquarian non-personal product or medicinal applications include propolis use in Italy in the 17th century, when Stradivari used propolis as an ingredient in the varnish of his stringed instruments (Monti et al., 1983). Today it is still used with musical instruments in rosin for stringed instruments and in the repair of accordions (Monti et al., 1983; Van Ketel and Bruynzeel, 1992). It has been proposed as a chemical preservative in meat products (Han and Park, 1995) and has been tested for bioactivity against larvae of the greater wax moth (Galleria mellonella L.), a common apiary pest, although little effect was noted (Johnson et al., 1994).
Current sales of propolis in the United States are estimated by the primary producer at 40,000 lb/yr (G.A. Burdock, personal communication, 1996). It is not possible to otherwise accurately estimate total sales of propolis in the US because bee-keeping and honey/propolis production is largely a cottage industry.
Propolis is consumed as a constituent of beeswax and honey. The 1975 monograph on beeswax by the Select Committee on GRAS Substances (SCOGS, 1975) notes the presence of 6% of beeswax as unidentified constituents, at least a portion of which is likely to be chrysine (1,3-dioxyflavone), a constituent of propolis. Bisson (1940) notes that the yellow colour of comb wax is due to the presence of chrysine and that propolis is a common constituent of the ‘impurities’ dissolved in beeswax.
Marcucci (1995) has noted that the compounds in propolis resin (raw, unprocessed propolis) originate from three sources: plant exudate collected by bees, secreted substances from bee metabolism, and materials which are introduced during propolis elaboration.
The source of the plant exudate was historically considered to be various indigenous poplar species, but this failed to explain why bees could produce propolis in the area of the equator where no poplars exist. Because the constituents of propolis reflect the source (see below, Chemistry) the advent of more sophisticated chemical analysis identified additional species of trees which could be used as a source of propolis for the foraging bees (Table 1).
As noted above, propolis is a by-product of the beehive. Propolis is collected by the beekeepers who scrape the hive ‘supers’ (superstructure) with a hive tool. This usually takes place in the fall of the year after the honey is extracted. The propolis and wax mixture is shipped to the processor in boxes, barrels or bags.
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Propolis involves little processing:
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The first step in processing is evaluation of the material on its arrival at the plant. If very waxy, it will be put through a cold-water washing process where the extrinsic wax will be removed. The remaining propolis is then air-dried on stainless-steel screens. If very little extrinsic wax is found, it will go immediately to the second step.
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The second step involves dissolving the propolis in 95% ethyl alcohol. Through a proprietary process, the remaining beeswax as well as bee parts and wood chips are removed.
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The final step involves filtration. The propolis tincture is put through a series of filters to remove any remaining small particles of foreign material.
Propolis is a resinous, sticky gum, the colour of which varies from yellow–green to dark brown depending on its source and age. It can be likened to an aromatic glue. It is difficult to remove from the human skin, since it seems to interact strongly with the oils and proteins of the skin. It is hard and brittle when cold, but becomes soft and very sticky when warm (Ghisalberti, 1979; Koltay, 1981).
Some interesting points emerge from the limited work that has been carried out on the constituents of propolis. By far the largest group of compounds isolated are flavonoid pigments, which are ubiquitous in the plant kingdom. It is not surprising, therefore, that the same flavones have been isolated from different samples of propolis and the series of flavonoids isolated from propolis correlate reasonably well with those present in the plants from which honeybees collect propolis. It has been suggested that some of the flavones are modified by an enzyme in the honeybee. If so, it seems likely that any transformation must occur in the presence of enzymes in the saliva of the bees during collection. Also, the simple aromatic compounds found in propolis also occur commonly in plants and their presence in propolis is therefore not unexpected (Ghisalberti, 1979).
Johnson et al. (1994) assayed propolis from three geographical locations and produced the following results (Table 2). The gross composition of North American propolis (percent beeswax and methanol-soluble resin) is variable, but within the range of 16 to 80% reported for European propolis (Cirasino et al., 1987; Ghisalberti, 1979, Monti et al., 1983). The Johnson group note that the proportion of beeswax to plant resin is likely a compromise between availability and use. That is, propolis used to repair honeycomb is often supplemented with large quantities of wax to give it a firmer composition, while propolis applied in a thin coat to the surface of comb usually contains little or no wax (Meyer, 1956). Bees may also incorporate more wax into propolis during periods when resins are scarce or difficult to collect (Meyer, 1956). The low proportion of resin in propolis collected from south Georgia in this study may reflect a low availability of collectable resins in pine forests (Johnson et al., 1994).
Propolis collected from hives in Ohio was more chemically diverse (over 30 compounds detected by paper chromatography) than material from south Georgia (fewer than 10 major compounds) and contained a lower proportion of methanol-insoluble beeswax. Likewise, Rudzki and Grzywa (1983) found at least a slight difference in propolis gathered from the Warsaw region. However, the data of Johnson et al. (1994) revealed little variation in the chemical profile of specific hives over a 6-month period and no differences between propolis samples from adjacent hives.
Simple fractionation of propolis to obtain compounds is difficult due to its complex composition. The usual manner is to extract the fraction soluble in alcohol, called ‘propolis balsam’, leaving the alcohol-insoluble or wax fraction. Although ethanol extract of propolis (EEP) is the most common, extracts with other solvents have been carried out for identification of more than 200 constituents (Marcucci, 1995).
As noted earlier, the largest group of compounds isolated from propolis tincture is flavonoid pigments, which are ubiquitous in the plant kingdom and the series of flavonoids isolated from propolis correlate reasonably well with those present in the plants from which honeybees collect propolis. The substances identified in propolis are familiar constituents of food, food additives and/or generally recognized as safe (GRAS) substances.
Conspicuous among the list of constituents are hydroquinone (0.1%, Greenaway et al., 1987, Greenaway et al., 1991) caffeic acid and its esters (2–20%, Bankova et al., 1995) and quercetin (<0.1–0.7%, Greenaway et al., 1990), each of which have exhibited carcinogenic effects when administered to rodents. However, all three of these substances occur naturally in foods. Hydroquinone is present in beer and coffee (at levels of 1.25 to 40 ppm) and is approved as an indirect additive to food in §175.105; §§176.170, 180 and §177.2420. While quercetin and caffeic acids (and esters of caffeic acid) are not approved for use in food, the contribution of these substances through consumption of propolis is dwarfed when compared with consumption from other natural sources. For example, a single apple (with peel) may contain 5.8 to 26 mg quercetin (IARC, 1983). The estimated average daily intake quercetin by an individual in the US is 25 mg (NTP, 1992). Also, a single serving of lettuce2 may contain 27–56 mg caffeic acid (IARC, 1993). Therefore, propolis contributes an insignificant amount of these substances when compared with the daily diet.
As with any natural product, contaminants from the environment are likely. However, analysis of several different lots confirm an absence of chlorinated hydrocarbons. Analytical data show the levels of lead in raw propolis at or below 9 ppb. Tinctures are generally below 5 ppb, but occasionally rise above this level.
Section snippets
Mechanism of action
As with any natural product, there are a number of constituents in propolis and its extracts that are held in common with other foods, some of which are also known to have biologic activity. Of those substances with biological activity, none contribute more to the observed effects of propolis than the flavonoids.
In his treatise on flavonoids, Havsteen (1983) divides the biochemical effects of flavonoids in animal systems into four categories: (1) binding affinity to biological polymers; (2)
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