Figure 1 shows the spread of the compositions of the propolis samples in a PCA model. We have previously characterised most of the major components in propolis from the UK by using accurate mass measurement with LC-MSn20. Although the samples have broadly similar compositions, there are some quite marked variations in individual components. For instance, Fig. 2 shows extracted ion chromatograms for a major component, pinobanksin acetate, across three samples from different positions in the PCA plot. Pinobanksin acetate is most abundant in the Bulgarian samples, which contain ~3.5-fold more of the compound than a sample from Northern Ireland. In contrast, Fig. 3 shows extracted ion traces for a component putatively identified as trimethyl dihydrokaempferol, which is abundant in the Northern Ireland sample but only present at low levels in the Bulgarian sample. Table 1 shows the results obtained in testing the 35 samples of European propolis against Trypanosoma brucei, Trypanosoma congolense and the multidrug resistant strain Trypanosoma brucei B48. Of these, 4 samples displayed high activity, EC 50 values < 5 µg/mL, and 21 displayed intermediate activity between 5 and 10 µg/mL for the standard drug-sensitive strain Lister 427WT. The propolis samples from Norfolk displayed the highest activity, followed by the adjoining county of Suffolk and nearby Northamptonshire. The EC 50 values for the multidrug resistant stain B48 were within ~1.5-fold of the control (Resistance Index (RI) 0.63–1.56; average 0.83 ± 0.04) although the RI for pentamidine was 222 (P < 0.001, Student’s unpaired t-test; Table 1).
Figure 1 PCA plot showing the variation of propolis composition across 35 European propolis samples (Pareto scaled based on 233 components). Full size image
Figure 2 Extracted ion trace showing variation in the levels of pinobanksin acetate across 3 European propolis samples. Full size image
Figure 3 Extracted ion trace showing variation in trimethyl dihydrokaempferol across 3 European propolis samples. Full size image
Table 1 The activity (µg/ml) of 35 European propolis samples against the standard drug-sensitive T. brucei 427WT and multi-drug resistant strain T. brucei B48, and T. congolense. Full size table
OPLS was used to model the activity of the different propolis samples against T. brucei B48 in relation to their composition. It was possible to produce a model for 33 of the samples based on 5 components, including a butyl ester of pinobanksin, which produced a reasonable fit of predicted against observed activity shown in Fig. 4 (the corresponding loadings plot is shown in Fig. S1). The highest activity was associated with a butyl ester of pinobanksin and a propionyl ester of pinobanksin. Table S1 includes MSn data used to further characterise the compounds associated with high activity. It can be seen from the extracted ion trace shown in Fig. 5 that the highest activity sample from Norfolk contains about 4 times the concentration of pinobanksin butyrate present in the lowest activity sample from Leicestershire. The wild type strain of T. brucei 427 gave similar results. Figure S2 shows an OPLS plot of predicted against measured activity with the corresponding loadings plot shown in Fig. S3. The highest activity is again associated with a butyl ester of pinobanksin and two propionyl esters of pinobanksin.
Figure 4 OPLS plot of observed against predicted activity against T. brucei B48 for 33 propolis samples based on five components. Full size image
Figure 5 Extracted ion traces pinobanksin butyrate in samples with high, moderate and low activity against T. brucei. Full size image
The same propolis samples were also tested against the veterinary trypanosome species T. congolense (Table 1) with very similar results, as the average of the ratio of EC 50 (Tbb427WT)/EC 50 (T. congolense) was 1.21 ± 0.11. Interestingly, the two Bulgarian samples were ~3-fold more active against T. congolense than against either of the T. brucei clones, as was one sample from Norfolk, UK. Figure 6 shows the OPLS plot obtained for the activity against T. congolense. The correlation between composition and activity was based on seven components. Figure S4 shows the corresponding loadings plot. There was a stronger fit for this plot than for the activity against T. brucei B48 and all 35 samples could be included in the model. Most active components against T. congolense are different from the most active against T. brucei and thus the OPLS plot highlights, galangin, an isomer of kaempferol, and a methylether of chrysin as the most active components (Table S1).
Figure 6 OPLS plot of observed against predicted activity against T. congolense for 35 propolis samples based on seven components. Full size image
Table 2 shows the data obtained from testing propolis against C. fasciculata which is a closer relative to the trypanosomatids that infect bees than T. brucei is. A wide range of activities were obtained. In many cases the samples were less active against C. fasciculata than against T. brucei. The OPLS model did not give as strong a correlation with the components in the sample as for T. congolense (Fig. 7) although it was possible to reduce the number of variables supporting the plot to thus giving a better indication of which components might be associated with high activity. The corresponding loadings plot is shown in Fig. S5. Galangin methyl ether is associated with high activity and this can be seen in Fig. 8 where one of the most active samples from Essex has about four times the amount of this component in comparison to a sample from Leicestershire.
Table 2 EC 50 values (µg/ml) for European propolis against C. fasciculata (n = 3). Full size table
Figure 7 OPLS model of predicted against observed activity for propolis against C. fasciculata based on 4 components. Full size image
Figure 8 Extracted ion traces for galangin methyl ether in samples with high, moderate and low activity against C. fasciculata. Full size image
Table 3 shows the activity obtained for 25 of the propolis samples against L. mexicana. The activity of the propolis samples against L. mexicana was higher than that obtained against T. brucei, with average EC 50 values below 1 µg/mL for 52% of samples, and all EC 50 values were under 5 µg/mL. The highest activity was obtained for one of the Bulgarian samples, at 0.35 ± 0.03 µg/mL. In most cases activity was equal or superior against the miltefosine APC12-resistant cell line, giving an average Resistance Index of 0.74 ± 0.09, but it was not possible to fit a strong an OPLS model for the data obtained for L. mexicana as for the T. brucei data, probably because the range of activities obtained across the samples is lower than for T. brucei and the number of samples tested was smaller. The activities obtained against Leishmania were an order of magnitude higher than those obtained for C. fasciculata and T. brucei, as shown in Fig. 9.
Table 3 The activity (µg/ml) of propolis against wild type and miltefosine-APC12 resistant L. mexicana (C12Rx). Full size table
Propolis is thought to have several remarkable biological properties, such as antimicrobial, anti-inflammatory, anticarcinogenic, and antioxidant. There has been limited clinical studies by the modern medical community into the effectiveness of propolis, although it has been used for thousands of years by ancient civilizations for its medicinal properties. The ancient Greeks, Romans, and Egyptians were aware of the healing properties of propolis and made extensive use of it as a medicine to treat wounds and abscesses.
There are many products that contain propolis; from toothpaste and skin creams to healing salves, herbal tinctures, syrups and elixirs. Some of the health problems propolis is touted as a natural treatment for include; acne, bacterial infections, burns, canker sores, giardiasis and cold sores amongst many others.
There have also been reported side effects with propolis use particularly in people who are allergic to bees or bee products. Do not use propolis if you have asthma or are allergic to bee by-products (including honey), conifers, poplars, Peru balsam, and salicylates.
The Bee Store can help with all your beekeeping supplies, including propolis traps.
By Laura Tyler, Colorado
There are non-urgent tidbits of beekeeping lore that the experts won’t tell you when you are just getting started with bees. Not because they are secret. But because the amount of information available to new beekeepers is vast, and so much of it is need-to-know, that less pressing but still interesting details—like deciding what to do with that gob of propolis you have been adding to all summer—fall by the wayside. But as you are ready, your willingness to continue learning and trying new things can feel like an initiation drawing you deeper into the world of bees.
WHAT IS PROPOLIS?
Honeybee propolis is a brown or reddish resinous substance made by bees to protect the hive against animal and bacterial invaders. The word “propolis” is a compound of the Greek words “pro” and “polis” and translates to, “Before the city.” Bees use propolis as a building material to fill gaps and crevices, varnish combs and shape entrances, sometimes creating fantastic gobs that supposedly aid ventilation in the hive.
People have observed bees using propolis to corral insect pests like small hive beetles into tiny propolis “jails,” and to embalm dead mice. It has potent antiviral and antibacterial properties that aid in protecting the colony from infection. Made up of plant saps gathered by bees, redolent of beeswax, pollen, and essential oils, propolis has a warm and spicy aroma that suggests comfort and mystery. Its use as a folk medicine dates back thousands of years. Today people use it to treat ailments ranging from oral problems and fungal infections to allergies and sore throat.
The quantity of propolis a bee colony will produce depends on its nature and the conditions in the hive. Some colonies produce big, peanut buttery swaths of propolis that require diligent scraping on your part to move frames around. Others run a drier ship, highlighting the edges and ends of your equipment with a thin, almost delicate, reddish varnish.
Occasionally, when the right trigger is applied, bees will produce a terrific quantity of propolis, the size of a man’s fist or larger, in a single area, typically near the main entrance to the hive. I have seen this happen in my own colonies, usually when something has gone wrong. One time, the bottom edge of a frame came loose, touching the bottom board. The bees took this as an invitation to fill the space between the comb and bottom board with many square inches of potent, immaculate propolis. Another time, a piece of grass that fell into the colony near the entrance inspired a similar behavior. While these feats are exciting to witness, they are difficult to replicate or predict. When I see a colony has a propensity to make propolis, I will insert twigs along the bottom board near the entrance to inspire propolis creation with mixed and often disappointing results.
The simplest and most reliable method of harvesting propolis is to scrape it and save it in a designated bucket each time you work your hive. Look for the bigger, cleaner areas of propolis that collect along the top edges on each frame. Also, there are many fun looking styles and shapes of propolis traps available from beekeeping suppliers.
Beekeeping literature is full of negative information about propolis, how it gums up your equipment and requires continual scraping to maintain frames in moveable condition. Propolis is “unnecessary in modern apiculture, apparently useless to the bees and a disadvantage to the beekeeper,” according to the 34th edition of A.I. Root’s beekeeping classic, The ABC and XYZ of Bee Culture. Curiously, the book goes on to extoll propolis’ importance as, “the base of an important antiseptic preparation used by surgeons…highly recommended as a domestic remedy for wounds and burns.”
That is the nature of propolis. Challenging but important. And highly recommended to beekeepers seeking to expand their role as providers of bee products in their communities.
HOW TO USE PROPOLIS
I swear by propolis as a preventative remedy when I am traveling or feeling run down. I have also found it useful in treating sore throat. I prefer to take propolis raw as opposed extracted in a tincture or blended in a salve. My favorite way to use propolis is the way I learned from a beekeeper friend in my second year of beekeeping:
Collect quality propolis, the rich looking, clean stuff free of bee parts and splinters, as you work your colonies throughout the year.
Store it loose in a sealable container, either a bucket or plastic bags at room temperature. You may also freeze it.
Choose a piece about the size of a pea, roll it into a ball and stick it on the back of a tooth, or the roof of your mouth. Hold it in your mouth for as long as you like, minutes or hours (after awhile it will break down) and then swallow or spit. Do not chew. Propolis has an intense yellow color that will temporarily stain your teeth and mouth. It also has a mild anesthetic quality. A mild tingling or numbness in the mouth is normal when using propolis.
Caution: some people are allergic to honeybee products including propolis. If you experience an allergic reaction, discontinue use and consult a physician.
RECIPE: 20% Propolis Tincture
1 part propolis by weight
4 parts food grade alcohol by weight, 150 proof (75%) or higher. Bacardi 151 or Everclear, depending on your taste.
Clean glass jar with a lid to fit the volume of tincture you are making.
Filter, either a coffee filter or clean piece of tightly woven cotton.
Storage container, jar or bottle with eyedropper
• Place propolis in jar
• Pour alcohol over propolis
• Cover jar with a tight-fitting lid and shake
• Shake jar one or more times per day for two weeks
• Strain solids from your tincture using a coffee filter or woven, cotton cloth
• Decant your finished tincture into storage container
• Label and store away from sunlight
This is a common formula published for centruies. For more information and more detail, we recommend: Bee Propolis: Natural Healing from the Hive by James Fearnley.
Laura Tyler is the director of Sister Bee, a documentary about the life of beekeepers, and lives in Boulder, Colorado, where she raises bees with her husband. If you have questions for her about raising bees, contact her at