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The Effect of Echinacea Purpurea, Astragalus Membranaceus and Glycyrrhiza Glabra on CD69 Expression and Immune Cell Activation in Humans

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The Effect of Echinacea Purpurea, Astragalus Membranaceus and Glycyrrhiza Glabra on CD69 Expression and Immune Cell Activation in Humans PHYTOTHERAPY RESEARCH Phytother. Res. 20, 687–695 (2006) Published online 28 June 2006IMMUNE in Wiley InterScienceCELL ACTIVATION BY HERBAL EXTRACTS 687 (www.interscience.wiley.com) DOI: 10.1002/ptr.1938 The Effect of Echinacea purpurea, Astragalus membranaceus and Glycyrrhiza glabra on CD69 Expression and Immune Cell Activation in Humans Julie Brush1, Elissa Mendenhall1, Alena Guggenheim1, Tracy Chan1, Erin Connelly1, Amala Soumyanath2, Randal Buresh2, Richard Barrett1 and Heather Zwickey1* 1Helfgott Research Institute, National College of Naturopathic Medicine, Portland, OR, USA 2Oregon’s Wild Harvest, 43464 SE Phelps Road, Sandy, OR, USA The increasing use of medicinal herbs among the general public has piqued the need for scientific-based research to determine the mechanism of action of herbs administered orally in human subjects. The ability of three herbs, Echinacea purpurea, Astragalus membranaceus and Glycyrrhiza glabra, to activate immune cells in human subjects was assessed in this pilot study. The effect of these herbs when ingested for 7 days was measured both when administered singly, and in combination, using flow cytometry. The primary cell activa- tion marker measured was CD69. The results demonstrate that Echinacea, Astragalus and Glycyrrhiza herbal tinctures stimulated immune cells as quantified by CD69 expression on CD4 and CD8 T cells. This activation took place within 24 h of ingestion, and continued for at least 7 days. In addition, these three herbs had an additive effect on CD69 expression when used in combination. Copyright © 2006 John Wiley & Sons, Ltd. Keywords: CD69; CD8 T cells; CD4 T cells; herbs; botanicals; clinical trial. used in traditional Chinese medicine as a tonic, espe- INTRODUCTION cially for the spleen and lungs, and to prevent colds (McKenna et al., 2002). Glycyrrhiza has been used Medicinal herbs are being used more frequently with medicinally since the beginning of recorded history and the increasing use of complementary medicine among is used widely in Chinese medicine. Glycyrrhiza has the general public in the United States. This has been used historically for respiratory, gastrointestinal, piqued the need for scientific-based research to deter- cardiovascular, genitourinary, eye and skin conditions, mine the specific effects of different herbs within the and for antiviral effects (Fiore et al., 2005). body. While herbal medicine has many years of clinical Most of the research regarding the mechanism of and anecdotal evidence to support it, the mechanisms how these herbs affect the immune system has been of how different herbs elicit clinical effects are not well performed in animal models. In normal, healthy mice described. For example, many herbs are thought to treated with Echinacea, natural killer (NK) cells and ‘boost immunity’. However, the concept of immune monocytes significantly increased in both bone marrow boosting is vague. Immune boosting may involve an and spleen compared with controls (Sun et al., 1999). activation of different cell types of the immune system, Echinacea treated mice with leukemia had a 2.5-fold an increase in cell number, or it may involve another increase in the absolute numbers of NK cells in their process such as a shift from one cytokine pattern to spleens and had a significantly increased survival rate another. compared with untreated mice (Currier and Miller, Three herbs that have known historical and current 2001). In addition to its immune stimulating role, uses for boosting the immune system are Echinacea Echinacea spp. alkamides inhibited LPS-mediated spp. (including E. purpurea Moensch., E. pallida (Nutt.) activation of a murine macrophage line, suggesting Britt. and E. angustifolia (DC) Heller), Astragalus mem- antiinflammatory effects (Chen et al., 2005). branaceus (Fisch.) Bunge and Glycyrrhiza glabra (L.). Much Astragalus research has been focused on its Echinacea has been used on this continent for centur- antitumor effects with its mechanism of action largely ies by Native Americans. It is one of the most common unknown (Nagasawa et al., 2001). Astragalus was able herbs used to treat the symptoms of upper respiratory fully to correct in vitro the deficient T cell function tract infections (Bielory, 2004). Astragalus is primarily found in cells derived from cancer patients (Chu et al., 1988). Triterpene saponins isolated from Astragalus induced interleukin-2, a cytokine produced by activated * Correspondence to: Dr Heather Zwickey, Helfgott Research Institute, T cells, suggesting immunostimulatory and antineo- National College of Naturopathic Medicine, Portland, OR, USA. plastic properties (Yesilada et al., 2005). In contrast, E-mail: [email protected] Contract/grant sponsor: American Medical Association Seed Grant. Astragalus polysaccharides activated mouse B cells Contract/grant sponsor: Helfgott Research Institute Seed Fund; Contract/ and macrophages, but not T cells in another study grant sponsor: Oregon’s Wild Harvest. (Shao et al., 2004). Astragalus root extract significantly Copyright © 2006 John Wiley & Sons, Ltd. Phytother. ReceivedRes. 20, 687–69511 March (2006) 2006 AcceptedDOI: 410.1002/ptr May 2006 Copyright © 2006 John Wiley & Sons, Ltd. 688 J. BRUSH ET AL. increased NK cell activity in patients suffering from This study examines the singular and combined Coxsackie B viral myocarditis with depressed NK cell effects of three potentially immunostimulatory herbs: activity (Yang et al., 1990). Astragalus membranaceus, Echinacea purpurea and The immune effects of Glycyrrhiza are not as well Glycyrrhiza glabra, via the use of immune cell activa- studied. Glycyrrhiza activated peritoneal macrophages tion markers. The main objective of this study was to in a mouse model (Nose et al., 1998). Recent in vitro determine if each herb increased CD69 expression on studies showed its antiviral effects for viruses causing immune cells when administered orally for 7 days. The severe acute respiratory syndrome (SARS) (Cinatl et secondary objective was to determine if the combina- al., 2003), human immunodeficiency virus (HIV) (Sasaki tion of herbs produced a synergistic or additive effect et al., 2002) and Kaposi sarcoma-associated herpes on CD69 expression. virus (KSHV) (Cohen, 2005) through the inhibition of viral replication by an unclear mechanism. In addition, topical Glycyrrhiza gel is effective in treating atopic dermatitis in human subjects, suggesting a potential MATERIALS AND METHODS antiinflammatory role in allergic/inflammatory diseases of the skin (Saeedi et al., 2003). Study design. This study was a placebo-controlled, Several immunological in vitro and in vivo studies double-blind investigation. The study outline is sum- revealed that the total effect of a combination of herbs marized in Fig. 1. with immunostimulating potential can be greater than expected from the sum of effects of the single herbs Subjects. The protocol was approved by the Institu- (Wagner and Jurcic, 1991). Synergistic interactions have tional Review Board at the National College of been documented between different herbs in a formu- Naturopathic Medicine. Subjects were recruited from lation (Williamson, 2001). For example, Revitonil (a the Portland, Oregon, area according to the inclusion combination of extracts of Echinacea and Glycyrrhiza and exclusion criteria listed in Table 1. Of 16 subjects extracts) increased CD69 expression on 30%–50% of completing the study, 3 were male and 13 were female. T-cells in mice, and stimulated phagocytosis by human One additional male subject began the study and was granulocytes in vitro (Wagner and Jurcic, 2002). Much discontinued at 24 h due to stage 1 hypertension meas- knowledge has been gathered from in vitro studies and ured at both 0 and 24 h. Formal informed consent was animal models, however, the true scientific impact of in obtained from each participant before beginning the vitro results depends on their relevance and efficacy in study. Subject compliance was determined to be high humans. by measuring the remaining tinctures in bottles that One way to measure the immune effects of herbs were returned on day 7. In addition, the subjects were is to look at cellular activation markers. Activation interviewed at each timepoint to ensure compliance with markers on immune cells include growth factor receptors instructions. The final number of subjects in each study such as CD69. CD69 is one of the earliest cell surface group were as follows: Echinacea n = 4, Astragalus antigens expressed by T cells following activation. Once n = 4, Glycyrrhiza n = 3, combination n = 3 and placebo expressed, CD69 acts as a co-stimulatory molecule for n = 2. Initially, it was intended to include three subjects T cell activation and proliferation. In addition to ma- in each study group. While the original pilot study ture T cells, CD69 is inducibly expressed by immature design included three subjects in each group, the final thymocytes, B cells, NK cells, monocytes, neutrophils numbers were complicated by a variety of factors. One and eosinophils, and is constitutively expressed by member of the placebo group dropped their bottle. mature thymocytes and platelets (Ziegler et al., 1994). The replacement bottle did not contain placebo. The The presence of CD69 enables us to identify whether additional subject was consented and enrolled with CD4, CD8, NK cells and B-cells have been activated. the expectation of a higher drop-out rate. While the functional significance of CD69 is largely unknown, its mere presence on the surface of a cell Botanical
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