Correlation between the major components of Phyllanthus amarus and Phyllanthus urinaria and their inhibitory effects on phagocytic activity of human neutrophils

Background: Recently, we have highlighted the immunomodulatory activity of the standardized extracts of Phyllanthus amarus and P. urinaria. The present study was carried out to correlate between the prevalent constituents of the herbs and their inhibitory effects on phagocytic activity of human neutrophils. Methods: The compounds, gallic acid, ellagic acid, corilagin, geraniin, phyllanthin and hypophyllanthin were identified and quantitatively analyzed in the extracts of Phyllanthus amarus and P. urinaria obtained from Malaysia and Indonesia by using a validated reversed phase high performance liquid chromatography (RP-HPLC) method. The standardized extracts and the pure compounds were evaluated for their effects on chemotaxis, β2 integrin (CD18) expression, phagocytosis and chemiluminescence of human phagocytes. Chemotactic activity was assessed using the Boyden chamber technique, inhibition of CD18 expression and phagocytic ability were tested with the aid of flow cytometry, while effect on the respiratory burst was investigated using a luminol-based chemiluminescence assay. Results: All plant extracts strongly inhibited migration of the phagocytes with the Malaysian P. amarus depicting the highest inhibitory activity. Amongst the compounds tested, geraniin demonstrated the strongest inhibitory activity on chemotaxis of polymorphonuclear leukocytes (PMNs) and monocytes with IC50 values of 1.09 and 1.69 μM, respectively, which were lower than that of ibuprofen. All plant extracts and pure compounds exhibited high inhibitory activity on the oxidative burst of zymosan and PMA stimulated leukocytes. Geraniin and corilagin exhibited exceptionally strong inhibition on the reactive oxygen species (ROS) activity with IC50 values lower than aspirin. The plant extracts exhibited moderate inhibition of E. coli uptake by monocytes but weak effect on PMNs. Of all the compounds, phyllanthin at 50 μg/mL exhibited the highest engulfment inhibitory activity with percentage of phagocytizing cells of 14.2 and 27.1% for PMNs and monocytes, respectively. All plants and compounds tested possessed weak effect on CD18 expression on leukocytes except for hypophyllanthin and phyllanthin which exhibited significant inhibitory effect. Conclusion: The strong inhibition of the extracts on the phagocytic activity of neutrophils was due to the presence of their major constituents especially geraniin, corilagin, phyllanthin and hypophllanthin which were able to modulate the innate response of phagocytes at different steps.