Introduction: Propolis is used in Poland as an active ingredient of some drugs administered externally, dietary supplements and cosmetics. According to the literature, propolis is a non-toxic and safe substance, although it may cause allergic contact dermatitis. Aim: The aim of this study was to assess the allergenic properties of propolis and Balsam of Peru. Material and methods: The study was conducted according to the OECD Guideline for testing of chemicals-Skin sensitization with use of Guinea pig maximization test (GPMT). Guinea pigs have similar sensitivity to allergens as human body. Sensitization properties of propolis were tested in comparison with sensitization properties of Balsam of Peru because of the possibility of cross-reaction between those two substances. Results: The skin of guinea pigs in the propolis group showed no visible change compared to the control group during the first (48 h) and second observation (72 h). The skin of guinea pigs from Balsam of Peru group showed discrete erythema in only one case at the first observation (8.33% of the animals). During the second observation, no visible changes were observed compared to the control group. Conclusions: Guinea pig maximization test showed no sensitizing properties of propolis and weak sensitizing properties of Balsam of Peru in concentration of 5%.
Propolis is a substance collected by worker bees from the resin of trees, mainly from poplar buds and conifer. It is used by them as a multifunctional material in constructing and maintaining hives and also as a protection from microbiological contamination. Propolis is widely used in Poland as an active substance in some drugs administered externally for healing wounds, burns, chilblains, and also as dietary supplements and an ingredient of some cosmetics. The most important properties of propolis are an antibacterial, antivirus and antifungal activity, but also anti-inflammatory and regenerative action [
Propolis is known to cause contact allergy, but according to data found in the literature, the hypersensitivity to propolis more often appears in dermatological patients, treated earlier for allergic dermatoses 1.2% - 6.7% (average 3.82%) than in healthy population. In healthy population, allergy to propolis seems to be very rare phenomenon (0.64% - 1.3%) [
The aim of the paper is to assess the sensitizing potential of propolis and Balsam of Peru and to compare those two substances.
The study was submitted to The First Local Ethical Committee on Animal Testing at the Jagiellonian University in Krakow, Poland. The agreement was approved on the 17th October, 2012 (number of agreement 142/2012).
Guinea pigs, all males, weight: 260 - 530 g, were procured from Animal Breeding Laboratory number 0055 according to the List of units eligible for breeding experimental animals approved by the Polish Ministry of Science and Higher Education. Animals were acclimatized to the laboratory conditions for 7 days prior to the test. They were kept at constant temperature of 22˚C and 50% of the humidity, under artificial illumination (12 h of light, 12 h of dark) and were nourished and watered ad libitum. They received 4 drops of 100 mg/ml ascorbic acid solution. Before the study started, guinea pigs were grouped randomly and were weighed. In the propolis group (P) there were 11 animals, in the Balsam of Peru group (B) there were 12 animals and in the control group (C) there were 6 animals. At the beginning the propolis group consisted of 12 animals, but before the study starts one animal was excluded from the study because of its health condition.
Balsam of Peru and Freund’s Adjuvant Complete (FCA) were purchased from Sigma-Aldrich. Ethanol extract of propolis (EEP) was obtained from Przedsiębiorstwo Pszczelarsko-Farmaceutyczne “Apipol-Farma” Sp. z o.o. Its quality was in accordance with the Polish Norm PN-A-77627:1996 The Concetrate of propolis.
The test was carried out according to the OECD Guideline for the Testing of Chemicals-Skin sensitization.
A) Induction: Intradermal injections
On the 0 day three pairs of 0.1 ml injections were given in the shoulder region which was cleared of hair. The propolis group received 3 injections, six in all, on each side as follows: 1) a 1:1 mixture FCA/water for injection, 2) ethanol extract of propolis, 3): ethanol extract of propolis in a 1:1 mixture FCA/water for injection. The Balsam of Peru group received 3 injections, six in all, on each side as follows: 1) a 1:1 mixture FCA/water for injection, 2) Balsam of Peru in ethanol and 3) Balsam of Peru in ethanol in a 1:1 mixture FCA/water for injection. The control group received as follows: 1) a 1:1 mixture FCA/water for injection, 2) ethanol and 3) ethanol in a 1:1 mixture FCA/water for injection.
B) Induction: Topical application
On the 7th day, which means 24 hours before the topical induction application, the test area was shaved. Then on the 8th day a filter paper with appropriate test substance (EEP or Balsam of Peru) or vehicle only (ethanol) was applied and held in contact by an occlusive dressing for 48 hours.
C) Challenge: Topical application
On the 21th day test areas were cleared of hair again. A patch loaded with test substance or vehicle only was applied to one flank. The patches were held in contact by an occlusive dressing for 24 hours.
D) Observations:
21 hours after removing the patch the challenge area was cleared of hair. 3 hours later (approximately 48hours from the start of the challenge application) the skin reaction was observed and recorded according to the grades below:
0―no visible change
1―discrete or patchy erythema
2―moderate and confluent erythema
3―intense erythema and swelling.
At the end (24 hours after the first observation) the second observation (72 hours) was made.
After the test, the animals were weighed again. Their weights ranged from 362 to 594 g.
Animals were humanely euthanized at the end of the study and the skin samples from the challenge site were collected. Tissue samples were fixed in AFA (alcohol, formalin, and acetic acid) fixative, dehydrated and then routinely embedded in paraffin and sliced in 6 μm thick sections. For histopathological evaluation following stains were performed: hematoxylin and eosin, trichromatic Masson Goldner (TGM), May-Grünwald Giemsa. Samples were examined by light microscopy at magnifications of 20×, 40× and 100× to evaluate the various targets.
Peripheral smears were prepared from a freshly drawn blood samples into S- Monovette(R) system with Potassium EDTA as anticoagulant. Blood smears were fixed according to May-Grunwald-Giemsa staining method. Microscopic observations were carried out with magnification lens 100×. Percentage of eosinophils were examined in the blood smears in triplicate and counted with hematologic adder.
Plasma samples were prepared from the blood collected before animals were euthanized. Blood samples were centrifuged for 10 minutes at 1000× g. The supernatant was collected in a tube and stored at −20˚C. IFN gamma concentration was measured by IFN gamma PIG ELISA kit (ABCAM).
The skin of guinea pigs in propolis group shows no visible changes in comparison with the control group during the first and the second observation. The skin of guinea pigs from the Balsam of Peru group show discrete erythema only in one case and in other animals no visible changes in comparison to the control group during the first observation. After the second observation no visible changes were observed in any case. The results of the sensitization experiment are shown in
Histopathologically, macroscopic evaluation of the collected parts of the skin showed no changes. The skin was clear, no swelling, blisters or erosions. In the studied parts of the skin there were no skin lesions typical for inflammation. Neutrophil mobilization was observed mainly in the dermis. In three cases (1 in the propolis group, 2 in the Balsam of Peru group) numerous mast cells in the field of view in the dermis were observed. Most of them were situated below the epidermis and around the capillary (
In other cases, the granulocytes were few or a single in the field of view. Only in one case of the propolis group, numerous neutrophils and monocytes infiltrated epidermis (
Group | Observation 1 | Observation 2 | ||
---|---|---|---|---|
(48 h from the start of the challenge application) | (72 h from the start of the challenge application) | |||
Number. of animals with positive reaction | % of animals with positive reaction | Number of animals with positive reaction | % of animals with positive reaction | |
Propolis | 0/11 | 0% | 0/11 | 0% |
Balsam of Peru | 1/12 | 8.33% | 0/12 | 0% |
Control | 0/6 | 0% | 0/6 | 0% |
In the studied material researchers noticed also:
- the damage of the epidermis with the infiltration of eosinophils in one case of the Balsam of Peru group;
- thin, atypical epidermis; cells of the cuboidal shape with pyknotic nuclei, lack of the proper basal layer in one case of the propolis group;
- a large area of regenerating epidermis and the presence of granulocytes in the dermis under that epidermis in two cases of the Balsam of Peru group.
The observed changes could indicate the early stages of sensitization. In the skin of the control animals no changes within the epidermis were observed and there were only few granulocytes in the field of view in the dermis. The results are shown in
The percentage of animals in which mobilization of granulocytes occurred during the GPMT, which may suggest the early stage of an allergic reaction, is shown in
Although propolis is considered to be a safe and nontoxic substance, there are some reports about sensitizing properties of propolis. Allergic reactions to propolis usually occur as contact dermatitis after topical administration. For assessing the sensitizing potential of a substance it is recommended to apply the Gui-
GROUP | EPIDERMIS | DERMIS | |||
---|---|---|---|---|---|
Changes | Presence | Presence | Vascular Congestions | Oedema | |
in morphology | of leukocytes | of leukocytes | |||
Propolis | 1/11 | 1/11 | 4/11 | 0/11 | 0/11 |
Balsam of Peru | 3/12 | 0/12 | 6/12 | 0/12 | 0/12 |
Control | 0/6 | 0/6 | 0/6 | 0/6 | 0/6 |
Group | No. of animals with granulocytes mobilization | % of animals with granulocytes mobilization |
---|---|---|
Propolis group | 3/11 | 27.3 |
Balsam of Peru group | 5/12 | 41.7 |
Control | 0 | 0 |
nea pig maximization test (GPMT) [
Additionally, guinea pigs were weighed before and after the study. The loss in weight of 10% or more may indicate systemic toxicity of the substance [
The histological examination of animal skin biopsies also showed no sensitization, and only in a few cases, mobilization of leukocytes. Mechanical damage could explain the atypical and regenerating epidermis of animals in one case in the propolis group and in three cases in the Balsam of Peru group. However, in the control group, there were no such cases; therefore, these changes may indicate the early stages of an allergic reaction in animals. The examination of blood smear did not clearly indicate the possible occurrence of allergy, either. Only in 5 cases the elevated level of eosinophils was observed, namely in 3 cases in the propolis group and in 2 cases in the Balsam of Peru group. However, it is worth noting that only in one guinea pig from the Balsam of Peru and only in one guinea pig from the propolis group some changes in morphology of the epidermis during histopathological examination were observed.
The occurrence of allergy was confirmed only in one case of the Balsam of Peru group. During the first observation erythema occurred (severity point 1 according the Magnusson and Kligman scale). It disappeared during the second observation about 72 h after the induction phase. In addition, histopathological examination of the skin revealed the epidermis damage and infiltration of eosinophils. The examination of blood film showed high levels of eosinophils (8%). It can be concluded that Balsam of Peru sensitized one guinea pig of 12 (8.33% of the examined animals), and based on the result of this study it can be qualified as weak contact allergen.
The data from this animal study differ from the results obtained from the guinea pig maximization test conducted by Petersen in 1977 due to the Kligman and Magnusson method (1969). It was shown that 5% propolis in propylene glycol injected intradermally and 50% propolis in ethanol administered topically sensitized 19 out of the 25 animals used in the experiments (76% positive reaction to propolis). However, this experiment was performed using an old version of the maximization test. This method has been criticized, as giving false positive results, by Kligman one of the authors of this method [
This study was conducted according to the OECD Guidelines for the Testing of Chemicals-Skin Sensitization from 1992 included the updated GPMT method. First of all the most potent sensitizer, dinitrochlorobenzene (DNCB) was no longer recommended. For induction exposure, the concentration of test substance should be well-tolerated systematically and should be the highest to cause mild to moderate skin irritation. For the challenge exposure, the concentration of test substance should be the highest nonirritant dose. The modified version of the maximization test has the same sensitivity as the original protocol, but do not lead to positive false results [
The GPMT is used to assess whether the substance has sensitizing properties and more specifically, whether it can cause allergic contact dermatitis (ACD), which is an immune skin reaction to the substance. In humans, ACD can be characterized by itching, erythema, edema, papules, vesicles, filled with transparent, aqueous liquid etc. In other species the reactions may vary and only erythema and edema can be observed according to Magnusson and Kligman grading scale, that is: 0-no visible change, 1-discrete or patchy erythema, 2-moderate and confluent erythema and 3-intense erythema and swelling. The method consists of the induction and the challenge exposure to the test substance. Between the phase of induction and challenge there must be a period of minimum one week during which the hypersensitive state may develop. The concentration of test substance used for each induction exposure should be well-tolerated systemically and should be the highest to cause mild-to-moderate skin irritation. Petersen during the induction exposure for intradermal administration used 5% solution of propolis in propylene glycol and for topical application 50% propolis ethanol solution , which itself may cause skin irritation. As it was shown, irritation threshold for propolis occurs at concentrations higher than 20% [
It should also be noted that in predisposed persons almost any substance may cause contact allergy at high concentrations during the long and frequent exposition [
Differences | GPMT according to Magnussona and Kligmana from 1969 | Updated GPMT according | |
---|---|---|---|
to the OECD Guideline for the Testing | |||
of Chemicals-Skin sensitisation | |||
Use of Dinitrobenzene | Yes | No | |
Propolis concentration | 5%, 50%, 25% | 20%, 20%, 5% | |
Vehicle | Propylene glycol in intradermal injection and ethanol in topical application | Ethanol in intradermal injection and topical application | |
Induction | Intradermal application | 5% propolis solution in propylene glycol | 20% propolis solution in ethanol |
Topical application | 50% propolis solution in ethanol | 20% propolis solution in ethanol | |
Challenge | 25% propolis solution in ethanol | 5% propolis solution in ethanol | |
FCA | NO | YES | |
Observation | I observation (time not mentioned) | 48 h and 72 h after challenge exposure |
Propolis, if used as recommended by the Office for Registration of Medicinal Products, Medical Devices and Biocidal Products in concentrations up to 5% (used in medicinal products registered in Poland) shows no allergenic potential, as confirmed by this research. The guinea pig maximization test showed weak sensitizing properties of Balsam of Peru.
Authors report no conflict of interest.
We thank Mrs. Teresa Dobrut and Mrs. Sylwia Luzar-Piotrowicz for support in this study.
Basista-Sołtys, K., Filipek, B., Zadrożna, M., Nowak, B., Pietrzycka, A. and Kazek, G. (2017) Sensitization Properties of Propolis and Balsam of Peru in Guinea Pig Maximization Test (GPMT. Food and Nutrition Sciences, 8, 714-724. https://doi.org/10.4236/fns.2017.87050