Abstract _________________________________________________________________________
Introduction. Echinacea represents a perennial plant indigenous to North America, but also cultured in Europe since a lot of years. Ten species belong to this genus, but only three species show pharmacological properties: E.
angustifolia, E. pallida, E. purpurea.
Up to date, formulations with Echinacea extracts are usually used for their immunomodulatory, antiiflammatory, antiviral and antioxidant activities, even if the mechanism of the action of their component has not yet been investigated.
Our study would understand the mechanism of the action by which Echinacea phytocompounds have an immunoenhancing activity; above all,
we investigated the effects of these extracts on dendritic cell (DC) generation, maturation and biological activity. DCs are the most important antigen-presenting cells: they check both the first phase and the maintenance of adaptative immune responses and the induction of tolerance.
Materials and Methods. DCs are in vitro generated from CD14
+circling monocytes, obtained from mononuclear cells of healthy donors, through an immunomagnetic system. Monocytes are cultured in RPMI 1640 medium, added with fetal bovine serum, L-glutamine, streptomycin and penicillin and supplemented with growth factors (GM-CSF and IL-4), in presence or absence of root extract (total extract, hydrophilic fraction on lipophilic fraction) of the three specie of Echinacea (E. purpurea, E. pallida, E. angustifolia) root extracts. These extracts are tested at a concentration range from 100 to 200 µg/mL. After 6 days of incubation, LPS is added to the culture for further 24 hours, in order to induce maturation.
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Then, morphological and phenotypical analysis are performed respectively by cytospin, May-Grunwald Giemsa staining and cytometric assays. The markers used are CD14 antigen, present on monocytes surface, but not on DC surface, CD40 and CD80 costimulatory molecules, HLA class II (HLA- DR) antigen and CD83 antigen, expressed by mature DCs.
The cytometric analysis of these antigens has also been carried out on dendritic cells cocultured with the cells from the pancreatic cancer line Capan-1, in presence or absence of Echinacea.
Functional studies on DCs generated in presence of Echinacea include cytometric analysis of endocytotic activity mediated by mannose receptor, using the dextran FITC-conjugated as an antigen, and the dosage of culture surnatant cytokines (IL-6, IL-1, IL-10, IL-12, TNF-α, TGF-β) through an enzyme immunoassay. Moreover, we observed the presence of P- glycoprotein on the cell wall of the dendritic cells, in presence or absence of Echinacea total extracts, through an outflow test of rhodamine 123 and through a polymerase chain reaction (PCR).
Results and Discussion. Echinacea phytocompounds don’t compromise DC generation from monocytes (loss of CD14 expression on cells surface).
Neverthless, total extracts interfere with the maturation, as the reduced expression of CD83 antigen shows if compared to the control. Moreover, in presence of Echinacea, a reduced expression (both as percentage and mean) of the CD80 and HLA-DR is observed, if compared to the control; as far the CD40 antigen is concerned, its percentage remains unchanged, while the mean decreases in presence of the extracts, compared to the control. No significant differences of cell phenotype among the different concentrations
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used , nor among the extracts belonging to the three analysed species is noticed.
Otherwise, preliminary results show that treatments with lipophilic and hydrophilic compounds don’t make a blockage of the DC maturation so pronounced than that caused by the total extracts. In fact, after LPS addition CD40, CD80 and CD83 markers are expressed, sometimes higher than on not treated-DCs.
In the cancer microenvironment, we notice that DC hardly generate (the CD14 espression on the 6
thday of culture is higer than the control). The treatment with E. purpurea at a concentration of 100, 150 and 200 µg/mL allows to restore in cocolture the values relevant to CD14 comparable to those of the control. Moreover, it provokes an increase in the percentage and in the expression intensity of CD40 (even if it doesn’t reach the values of the control) and, just in minor, of CD80, of which the intensity had been reduced by the cancer microenvironment.
On the other hand, through rodhamine-123 assay the P-glycoprotein expression isn’t pointed out neither in the control nor in the samples treated with E. pallida, probably because the detection methods we use is not sensitive enough or because the protein has not a pump function on DC. In fact, the glycoprotein is detected by PCR. The P-gp expression is the same in DCs treated with total extract of Echinacea and in the control, both in the immature stage and in the mature state.
When on the 6
thday we perform endocytosis assay, in presence of total root extract of Echinacea we observe no differences compared with the control:
the cells show a marked phagocytic activity. On the contrary, on the 7
thday,
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the cells treated with E. purpurea keep a much more intense activity if compared to the control, even if slightly inferior to that shown at the immature stage.
In presence of lipophilic and hydrophilic fractions, DC endocytic capacity still remains.
Cytokine dosage is carried out on the 7
thday. In the sample treated with Echinacea we remark an increase in the release of IL-1 and a decrease in
the concentration of IL-10, compared with the control. We notice no relevant differences between the three species we used.
Conclusively, our study suggests it is necessary to continue with investigations in this field, studying in deep the activity of lipophilic and hydrophilic fractions of Echinacea extracts, in order to confirm the preliminary results, but also evaluing the effects of separate active principals on DC. It is interesting to identify a possible fraction or species mainly responsible of the observed effects, too.
In fact, as far as the various and reported pharmacological properties is concerned, up to date the immunomodulatory activity is only partially elucidated. Analysing the literature and our data it seems fundamental to understand which are the compounds responsible of the pharmacological activity of the plant and the mechanism of action by which it is realized.
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