Use of quantitative flow cytometry to measure ex vivo immunostimulant activity of echinacea: the case for polysaccharides

Abstract

Introduction: When directly exposed to various echinacea fractions, human leukocytes ex vivo are strongly stimulated to proliferate and to produce immunostimulation and inflammatory cytokines. A comparison of fractions containing lipoidal small molecules and high-molecular-weight water-soluble polysaccharides indicates that the latter are substantially more potent as immunostimulants. Echinacea purpurea (L.) Moench, E. angustifolia DC, and E. pallida (Nutt.), Nutt. extracts, and each plant part contain significantly potent constituents. Flow cytometric techniques were utilized.

Objectives: This study was undertaken to determine whether flow cytometry could measure immunostimulant activity present in echinacea and, if so, which species produced more activity, which plant part was the most active, and whether the organic soluble or the aqueous extractables were more active. Ex vivo human clinical material was employed.

Design: Echinacea extracts were analyzed using flow cytometric techniques. The immunostimulation assays were measured in triplicate.

Methods: Samples dissolved in dimethyl sulfoxide (DMSO) were added to 200 microL of heparinized blood mixed with 50 muL of phosphate buffer, vortexed, and incubated to allow adequate time for immune-cell stimulation. Fifty (50) microL of the stimulated blood samples were added to each of a reagent cocktail consisting of 20 microL of CD4FITC/CD69PE/CD3PerCP expressed on the helper/inducer T-lymphocyte subset; CD8FITC/CD69/PE/ CD3PerCP expressed on the human suppresser/cytotoxic T-lymphocytes and on a subset of natural killer lymphocytes; CD19FITC/CD69PE/CD45PerCP expressed on B-lymphocytes; or CD56FITC/CD69PE/CD45PerCP expressed on NK lymphocytes. Four hundred and fifty (450) microL of 1 X FACS lysing solution was added and incubated in the dark (rt, 30 minutes) and then subjected to flow cytometric analysis. All reported readings are the average of several determinations. Positive controls consisted of phorbol myristyl acetate (PMA) (50 ng/mL), phytohemagglutinin (10 microg/mL), CD2/CD2R (positive activation control)(5 microL/250 muL of reaction), and negative controls consisted of dimethyl sulfoxide (2% in RPMI-1640), RPMI-1640 medium, and cyclosporin A (10 microg/mL).

Results: The main immunostimulatory activity of echinacea resides in the water-soluble materials rather than the lipoidal small molecules. E. purpurea, E. Pallida, and E. angustifolia leaves, stems, flowering tops, and roots all produce substantial immunostimulatory activity.

Conclusions: The use of flow cytometry demonstrates a link between the polysaccharides in echinacea and the biologic immunostimulatory effect that has therapeutic relevance, and strong evidence for this immunostimulant property is presented.