The immunomodulatory and anti-inflammatory activities of green propolis extracts from
Green propolis is well known due to the color [
The immunomodulatory effects of natural substances have been considered as alternative adjuvant therapies in the treatment of various diseases [
This study used lyophilized samples of aqueous extracts of propolis produced by Apis Flora, Ribeirão Preto, SP, Brazil. The extracts production follows a patented and standardized process (PI 0405483-0), published in the Revista de Propriedade no. 1778 of 12.01.2005 [
Quantitative analysis of the propolis extracts was carried out in a high-performance liquid chromatography (HPLC-Shimadzu) equipped with a CBM-20A controller, an LC-20AT quaternary pump, an SPD-20A M diode array detector, and a Shimadzu LC software, version 1.21 SP1. A Shim-pack CLC-ODS (M) (4.6 mm × 250 mm, particle diameter of 5 mm, pore size 100 Å) Shimadzu column. The mobile phase consisted of a gradient of methanol (JT Baker) and acidified water with formic acid (0.1% v/v) ranging from 20% to 95%. A run of 77 minutes at a flow rate of 0.8 mL/min, with detection at 275 nm, was performed. The following compounds were used as standards in the HPLC analysis: caffeic acid (Fluka), p-coumaric acid (Fluka) and trans-cinnamic acid (Fluka), artepillin C (Wako), gallic acid (Synth), isosakuranetin (ChromaDex), and 4′O-methyl-ether aromadendrin. These compounds were previously isolated and identified as described by [
Swiss and Balb/c mice (8–12 weeks, 25–30 g) were used. Animals were assigned by the mouse breeding facilities of Federal University of Maranhão. The animals received water and food
The method adopted for granuloma formation was described by Swingle and Shideman [
Treatment protocol for the granuloma model.
To determine the hematological parameters, 100
After the animals were sacrificed, the spleen was removed, weighed, and triturated in 5 mL of phosphate-buffered solution (PBS) using a sieve. To obtain the bone marrow cells, the femur was removed and perfused with 1 mL of PBS. The inguinal lymph nodes were removed, weighed and triturated in 1 mL of PBS. The cell suspensions were kept in an ice bath. All cell counts were performed using crystal violet solution (0.05% in 30% acetic acid) as described by Maciel et al. [
The trachea of the animals was exposed, fitted with a cannula, and 1 mL of cold PBS was injected in the bronchoalveolar space with a syringe. After a short massage on the chest, the solution was aspirated at least three times. The lungs were collected, weighed, and fixed in 10% formalin for subsequent histopathology analysis. To determine the total number of cells in the bronchoalveolar lavage, cell suspensions were stained with crystal violet (0.05%) in 30% acetic acid at a ratio of 9 : 1. Cells were counted in a Neubauer chamber using an optical microscope (×400). For differential counting slides were prepared using a citospin (800 rpm/3 min) and then were fixed and stained using an Instant-Prov Kit (Newprov, Pinhais, Brazil) [
Balb/c mice were divided into three groups (
Treatment protocol for the LPS induced pulmonary inflammation model.
The concentration of the IL-6, IL-10, TNF-
Unpaired Student
The predominant standard in all the tested aqueous extracts of green propolis was p-coumaric acid, but all other standards (caffeic acid, cinnamic, aromadendrin, and isosakuranetin) were also detected in all extracts at a lower level. Artepillin C was not detected in the 1A extract only. The statistical differences in the concentration of these compounds are shown in Table
Chemical characterization of aqueous extracts of green propolis (mg/g).
Markers | Samples | ||||
---|---|---|---|---|---|
E1A | E1B | E10 | E11 | E12 | |
Caffeic acid | 7.33 ± 0.04a | 1.64 ± 0.02b | 3.49 ± 0.11c | 2.83 ± 0.03d | 3.24 ± 0.05e |
p-Coumaric acid | 37.71 ± 0.33a | 10.25 ± 0.04b | 9.43 ± 0.30c | 12.46 ± 0.10d | 19.57 ± 0.18e |
Cinnamic acid | 1.19 ± 0.04a | 0.42 ± 0.02b | 0.53 ± 0.02c | 0.46 ± 0.01bc | 0.80 ± 0.04d |
Aromadendrin | 4.62 ± 0.20a | 0.80 ± 0.08bc | 0.56 ± 0.02b | 0.88 ± 0.11c | 1.44 ± 0.18d |
Isosakuranetin | 16.30 ± 0.26a | 9.51 ± 0.11b | 13.31 ± 0.40c | 6.80 ± 0.05d | 11.24 ± 0.01e |
Artepillin C | 0.00 ± 0.00a | 13.25 ± 0.39b | 41.82 ± 0.42c | 4.03 ± 0.03d | 6.65 ± 0.08e |
The data is presented as the mean ± standard deviation of concentrations (mg/g) of three samples. Samples of green propolis were compared among themselves. The symbols correspond to statistical analysis. For each of the markers different symbols indicate differences among the samples (
Several types of the tested extracts induced different effects in the formation of granuloma, both in relation to total weight (Figure
The effect of treatment with an aqueous propolis extract from
Extracts E1A, E1B and E12 did not induce hematological changes in animals. However, E10 and E11 induced an increase in the number of leukocytes and other white blood cells when compared to the control group, while E11 also induced a reduction in platelet count compared to the control group (Table
The effect of oral treatment with propolis extracts from
Control | E1A | E1B | E10 | E11 | E12 | |
---|---|---|---|---|---|---|
Erythrocytes (×106/ |
|
|
|
|
|
|
Hemoglobin (g/dL) |
|
|
|
|
|
|
Hematocrit (%) |
|
|
|
|
|
|
MCV (fL)a |
|
|
|
|
|
|
MCH (pg)b |
|
|
|
|
|
|
MCHC (g/dL)c |
|
|
|
|
|
|
RDW-CV (%)d |
|
|
|
|
|
|
Leukocytes (×103/ |
|
|
|
|
|
|
Neutrophils (×103/ |
|
|
|
|
|
|
Lymphocytes (×103/ |
|
|
|
|
|
|
Platelets (×103/ |
|
|
|
|
|
|
The results are presented as the mean ± standard deviation, aMCV: mean corpuscular volume, bMCH: mean corpuscular hemoglobin, cMCHC: mean corpuscular hemoglobin concentration, dRDW-CV: red cell distribution width, coefficient of variation. *
Extracts E1B, E10, E11, and E12 induced a decrease in the number of marrow cells while extracts E1A, E1B, and E10 induced a decrease in the number of spleen cells, when compared to the control group. Conversely, extracts E1A, E10, and E11 induced an increase in the number of lymph node cells compared to the control group (Table
The number of cells of the lymphoid organs of mice with granulomatous inflammation orally treated with propolis extracts from
Control | E1A | E1B | E10 | E11 | E12 | |
---|---|---|---|---|---|---|
Bone marrow (×106/mL) | 15.7 ± 0.2 | 14.1 ± 0.9 | 7.9 ± 0.9* | 9.1 ± 0.3* | 8.2 ± 0.3* | 19.0 ± 1.2* |
Spleen (×107/mL) | 2.4 ± 0.2 | 1.6 ± 0.1* | 1.6 ± 0.1* | 1.6 ± 0.2* | 2.4 ± 0.1 | 2.6 ± 0.1 |
Lymph node (×106/mL) | 2.1 ± 0.5 | 7.4 ± 0.7* | 2.2 ± 0.9 | 4.4 ± 0.2* | 5.9 ± 0.5* | 1.6 ± 0.2 |
The data is presented as the mean ± E.P.M. *
To evaluate whether the effect of the treatment with propolis extracts was due to systemic inflammation of the lungs, pulmonary inflammation in animals with granuloma was evaluated. A decrease in the number of inflammatory cells within the total BAL cell count was observed after treatment with the extracts E11 and E12. A significant increase in the number of macrophages was observed in the BAL collected from animals treated with E1B and E10, while the number of neutrophils significantly decreased following treatment with extracts E1B, E10, E11, and E12, when compared to control group. The number of lymphocytes did not change (Figure
The cellular profile of the bronchoalveolar lavage fluid of mice with granulomatous inflammation. Swiss mice that were treated orally for six days with a daily dose of 5 mg/kg were compared to controls, which received apyrogenic water at the same intervals. After treatment, BALs were collected and the different cell-types were counted. (a) The number of cells in the bronchoalveolar lavage fluid of mice with granulomatous inflammation orally treated with extracts of propolis from
Given the effects observed in animals with pulmonary inflammation granuloma, the impact of propolis on acute pulmonary inflammation, induced by LPS, was assessed. For this test, we selected the extracts E10 that induced a proinflammatory effect and E11 that induced an anti-inflammatory effect in the granuloma model. Treatment with E10 and E11 induced a decrease in the number of inflammatory cells, macrophages, neutrophils, and lymphocytes in the BAL (Figure
The cellular profile of bronchoalveolar lavage fluid of mice with lung inflammation induced by LPS. Balb/c mice that were treated orally for 4 days with a daily dose of 5 mg/kg were compared to controls, which received apyrogen water at the same intervals. Induction was done for three consecutive days. One day before the induction of inflammation by LPS, animals were treated with apyrogen water (control), maintained for four days, and then the animals were sacrificed 24 hours after the last LPS treatment, when the bronchoalveolar lavage was performed. (a) The number of cells in the bronchoalveolar lavage fluid of mice with pulmonary inflammation induced by LPS intranasally (in.). (b) The percentage of cells in the bronchoalveolar lavage fluid of mice with pulmonary inflammation induced by LPS intranasally (in.). The data represent the mean ± SD of six animals/group.
There was a decrease in the concentration of TNF-
The effect of treatment with propolis extracts of
This study evaluated the effect of aqueous extract of green propolis in two different models of inflammation. The therapeutic activities of aqueous extracts of propolis are rarely investigated despite of its potential antioxidant and anti-inflammatory activity, and better absorption than ethanolic extract [
This model was used to evaluate the effect of oral treatment with aqueous extracts of propolis. After six days of treatment the E10 extract was observed to induce an increase in edema and inflammatory infiltrate (Figure
The differences observed between the effects of the extracts are probably due to the chemical characteristics of each extract. All extracts contained caffeic acid, p-coumaric and cinnamic, aromadendrin and isosakuranetin. Artepillin C was found in all extracts except for E1A. However, there was a difference in the concentration of these compounds between extracts (Table
The action of acids (p-coumaric, cinnamic and caffeic) in the granuloma model was tested at a dose of 1 mg/kg, but no significant difference in any of the parameters evaluated was observed (data not shown), which makes us think that the anti-inflammatory effect observed in this study is not due to the action of these phenolic acids alone, but by an additive effect between them. However, in other experimental models other actions of these phenolic acids have been demonstrated, such as cinnamic acid isolated from propolis that was shown to act upon both innate and acquired immunity, stimulating the proliferation of lymphocytes, and inducing the production of cytokines [
The complex chemical composition of propolis may be the answer to the existence of numerous activities related to this Beekeeping product. Phenolic compounds are among the most prominent components of propolis because they are considered responsible for most of its properties. This is due to the fact that phenolic compounds exert multiple effects, such as antioxidant, antitumor, anti-inflammatory, and anticancer, among other effects [
Considering the differences observed between the E10 and E11 extracts, such as the biological activity in the granuloma model, as compared to the observed chemical markers, we investigated whether the extracts had any differential action in a systemic way. Firstly hematological data was evaluated, as this provides important indicators of physiological and pathological changes in humans and animals [
To clarify whether the changes observed in the blood would be due to changes in cell production in the bone marrow and to check whether there were changes in recruitment and/or proliferation of leukocytes to the lymph node and spleen, the cells of these organs were also quantified. There was a decrease in the number of cells in the marrow of animals treated with the E1B, E10, E11, and E12 extracts, which may suggest an increase in cell recruitment from the marrow to the blood and explain the increased number of leukocytes in blood. We also observed a decrease in spleen cell in the E1A, E1B, and E10 extracts and increased inguinal lymph node cells by E1A, E10, and E11 extracts (Table
As the E10 and E11 extracts showed systemic effects, we evaluated the effect of the treatment on pulmonary inflammation induced by granuloma. For this, we used the total cell count and differentials in BAL, as this is the standard indicative of inflammatory response in the respiratory tract, where pulmonary macrophages are the predominant cells (>90%) in healthy animals [
We observed a significant decrease in the inflammatory infiltrate in the animals treated with E11 and E12 extracts. In the differential count an increase in the number of macrophages induced by extract E10 and a decrease induced by E11 extract were observed (Figure
From the obtained results that showed that the E10 and E11 extracts induced opposite effects in both the model of granuloma as well as in the macrophage infiltration in the BAL, we investigate whether these opposing effects would also be observed in pulmonary inflammation induced by LPS, a widely used proinflammatory agent. However, in this model, both extracts showed anti-inflammatory effects, as a decrease in the total number of inflammatory cells, macrophages and neutrophils were observed (Figure
Given that all the inflammatory process is conducted with the involvement of cells and their mediators in which the cytokines [
The cytokine IL-6, in addition to being one of the most studied cytokines, has pleiotropic action that influences the antigen-specific immune responses and inflammatory reactions [
However, our results disagree with those obtained by Orsatti et al. [
These effects may be due to synergic effect and/or additive effect of various green propolis compounds thereby decreasing the inflammation observed. The extracts are also capable of modulating the production of proinflammatory and anti-inflammatory cytokines, preventing amplification of the inflammatory process in the pulmonary site. Thus, the tested extracts may become a new therapeutic alternative for use in allergic diseases and inflammation in the respiratory tract.
Further studies will be conducted to characterize the bioactive constituents in other models of inflammation and to evaluate the antioxidant potential of these extracts
Thanks are due to FAPEMA for funding the research (case no. 580342/2008-5). To CNPq (CNPq Call/RHAE n. 67/2008) for funding the research and for the scholarships of masters students Mayara Cristina Pinto da Silva and Aramys Silva dos Reis, and PhD student Graciomar Conceição Costa, and for the research productivity of Prof. Dr. Rosane Nassar Meireles Guerra and Prof. Dr. Flavia Raquel Fernandes do Nascimento and to CAPES for the master’s scholarships of Joleen Lopes Machado, Diêgo de Sousa Arruda Lopes and Anne Karine Martins Assunção.