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Immunomodulatory effects of Echinacea laevigata ethanol tinctures produced from different organs

Immunomodulatory effects of Echinacea laevigata ethanol tinctures produced from different organs BioscienceHorizons Volume 9 2016 10.1093/biohorizons/hzw001 Research article Immunomodulatory effects of Echinacea laevigata ethanol tinctures produced from different organs 1, 2 1 Ekta N. Haria *, M. Ann D. N. Perera and David S. Senchina Biology Department, Drake University, 2507 University Ave., Des Moines, IA 50311, USA W. M. Keck Metabolomics Laboratory, Iowa State University, 0124 Molecular Biology Building, Ames, IA 50011, USA *Corresponding author: 2507 University Ave, Des Moines, IA 50311 (c/o David Senchina, Biology Department). Email: hariaekta@gmail.com Echinacea supplements may prevent or reduce symptoms of upper respiratory infections by immunomodulation, possibly by altering the cytokine profile. Compared with other species in the genus, the immunomodulatory properties of Echinacea laeviagata are poorly characterized. The purposes of this study were to compare the diversity and quantity of known bioactive compounds from aboveground organs of E. laevigata, and to characterize the in vitro immumodulatory properties of ethanol tinctures produced from those structures. High-performance liquid chromatography (HPLC) was used to determine the levels of alkamides and caffeic acid derivatives. Peripheral blood mononuclear cells (PBMCs) were obtained from 16 adults and challenged in vitro with extracts. PBMC proliferation and production of the cytokines interleukin-2 (IL-2), IL-10 and tumour necrosis factor-α (TNF-α) were measured. Fresh flower, leaf and root extracts were able to augment TNF and IL-10 and prolif - eration, whereas fresh stem extract was only able to augment TNF. Extracts produced from flowers contained the greatest bioactive compound quantities and diversity. Caftaric acid was the most abundant compound and correlated with some (but not all) observed immune effects. These results suggest that of all aboveground parts, flowers have the greatest abundance and diversity of known bioactive compounds, and both flower and leaf extracts were immunomodulators. Key words: coneflower, cytokine, interleukin, peripheral blood mononuclear cell, proliferation, tumour necrosis factor Submitted on 18 September 2015; accepted on 25 January 2016 Introduction Each of the nine species of Echinacea differs in its diversity and abundance of purported bioactive compounds (Wu et al., Echinacea (Asteraceae) is a genus of nine plants native to the 2004; Pellati et al., 2005; Kraus et al., 2006). Within a single spe- United States (McGregor, 1968; Wu et al., 2009). These spe- cies, phytochemical profiles differ between different organs, such cies have been used for medicinal purposes (Moerman, 1998; as aboveground (aerial) and belowground parts (Qu et al., 2005; Barnes et al., 2005), with current interest in their use as immu- Senchina et al., 2009a). Four classes of bioactive compounds nomodulatory therapies for upper respiratory infections have been identified from Echinacea extracts (alkamides, caffeic (URIs) such as colds and influenza. Clinical reports conflict acid derivatives, ketones and polysaccharides). Only alkamides regarding their utility, with some reporting efficacy and others and caffeic acid derivatives are likely of physiological relevance not (Karsch-Völk, Barrett and Linde, 2015; Schapowal, Klein (Matthias et al., 2005; Ye et al., 2011; Goey et al., 2012), because and Johnston, 2015). Some of the discrepancies may be ketones readily oxidize (Qiang et al., 2013) and polysaccharides explained by the rampant adulteration and mislabeling com- are likely modified in the gut ( Woelkart et al., 2008). mon to many commercial preparations, or the lack of control for such variables (Gilroy et al., 2003; Krochmal et al., 2004; Much research on the therapeutic potential of Echinacea Wolsko et al., 2005). extracts has concentrated on inflammatory pathways. © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Research article Bioscience Horizons • Volume 9 2016 Echinacea bioactive compounds modulate transcription fac- cross-pollinating. A voucher specimen was deposited in the tors (Gertsch et al., 2004; Matthias et al., 2008) that lead to Ada Hayden Herbarium at Iowa State University the modulation of cytokines associated with inflammation, (ISC#447184). Specimens were extracted immediately post- such as tumour necrosis factor (TNF) (Lalone et  al., 2010; harvest. Plants were divided by organ (flower, leaf, stem, Hou, Huang and Shyur, 2011; Senchina et al., 2011; Dapas root), and each organ was processed separately. After separa- et al., 2014) and interleukin-1β (IL-1β) (Senchina et al., 2009a, tion, organs were diced manually using a surgical scalpel. The c; Zhang et al., 2012). Non-inflammatory cytokines, such as diced material was extracted at a ratio of 1:9 plant material– IL-10, may also be modulated (Zhai et al., 2007; Kapai et al., solvent in 50% ethanol–50% cell culture water using meth- 2011; Ritchie et al., 2011; Senchina et al., 2011). Thus, contin- ods described elsewhere (which simulate lay herbalist gent on a multitude of agricultural and experimental factors, a preparations) (Senchina et al., 2006b). Extracts were allowed given Echinacea extract can influence Th1 responses, Th2 to steep for 1 h at room temperature on a horizontal shaker responses or both simultaneously. Consistent with their vary- before being passed through sterilized tulle and stored at ing phytochemical profiles, different Echinacea species have −80°C and tested within 1 week of production. Extracts were different cytokine-modulating effects (Senchina et al., 2006a). vortexed prior to use in any phytochemical or immunomodu- latory assays. Far less is known about Echinacea laevigata compared with other species in the genus (E. augustifolia, E. pallida, Phytochemical profiling E. purpurea), for which the biochemical and immunomodula- Phytochemical composition of the extracts was determined tory properties are relatively well-characterized. Compared via high-performance liquid chromatography (HPLC) with with other Echinacea species, E. laevigata extracts had high UV detector for alkamides, ketones and caffeic acid deriva- caffeic acid derivative content (Pellati et al., 2004, 2005), and tives using previous methods (Senchina et al., 2011). Briefly, contained alkamides 2a, 4–6, 8a, 10a, and 11 (Wu et al., 2004; HPLC analysis was performed with YMC-Pack ODS-AM RP Senchina et al., 2011). Therefore, E. laevigata may harbour C18 (250 × 4.6 mm, 5 μm) analytical column (Waters; relevant phytomedicinal capacity and deserves consideration. Bedford, MA, USA). The solvent system was acetonitrile/H O To our knowledge there is only one report of the immuno- 2 with 0.01% formic acid for lipophilic constituents as well as modulatory properties of E. laevigata (Senchina et al., 2011). hydrophilic constituents but with varying gradients. The flow It demonstrated that E. laevigata tinctures exhibit immuno- rate was maintained at 1.0 ml/min. Alkamides were separated modulatory capacities, but only roots were examined. It is at a linear gradient of 40–80% acetonitrile over 45 min. unknown whether aerial parts of E. laevigata (flowers, leaves, Hydrophilic constituents, e.g. caffeic acid, were determined on stems) harbour similar activities. a linear gradient of 10–35% acetonitrile over 25 min. The col- The purpose of this study was to compare the diversity and umn temperature was 30°C. UV spectra recorded were in the quantity of alkamides and caffeic acid derivatives from differ- range of 200–400 nm, while 330 nm was used for quantifica - ent aboveground organs of E. laevigata, and to characterize tion of caffeic acid derivatives and 254 nm for alkamides. the in vitro immumodulatory properties of ethanol tinctures Bystander endotoxin levels were quantified from all extracts produced from these structures. The research question was: using a colorimetric method. The cell culture model employed do ethanol tinctures produced from E. laevigata aerial organs here is insensitive to endotoxin levels of 10 EU/mL or less demonstrate immunomodulatory activity in an in vitro pri- (Senchina et al., 2006b). mary cell culture model? It was hypothesized that (a) of the aboveground organs, flower extracts would exhibit greater Human subjects and cell isolation abundance and diversity of alkamides and caffeic acid deriva- Approval to work with human subjects was granted by the tives than leaf or stem extracts, and consequently greater Drake University Institutional Review Board (ID 2007- immunomodulatory activity; (b) of all plant organs, the 08015). Sixteen young adults (7 females and 9 males; belowground roots would have the greater abundance and 23.5 ± 3.8 years) gave written informed consent prior to par- diversity of alkamides and caffeic acid derivatives compared ticipation and donated blood. Blood was drawn from the with any of the aboveground organs. antecubital vein following Universal Precautions and peripheral blood mononuclear cells (PBMCs) were separated as described Methods elsewhere (Senchina et al., 2009a, c). Blood was collected in heparinized tubes, and then diluted 1:1 with phosphate-buff- Plant harvesting and extraction ered saline before being layered on top of Ficoll-Paque and Echinacea laevigata (Ames 25 161) plants were harvested as being centrifuged at 1800 rpm and 4°C for 15 min; the cen- whole plants with root bundles intact from a common garden trifuge was allowed to stop without any braking. Leucocytes in September 2007 at the United States Department of Agr- were then extracted from the Ficoll layer using pipettes and iculture (USDA) North Central Regional Plant Introduction washed twice with Hank’s buffered saline solution (HBSS) Station in Ames, Iowa. Plants were identified and provided before being manually counted via hemocytometer and stan- courtesy of Dr. Joe-Ann McCoy, were 3 years old at har- dardized to 1.0 × 10 cells/ml in AIM-V media. Extracts were vest and had been enclosed in pollination cages to prevent diluted 1:12.5 in AIM-V media before being added to cell 2 Bioscience Horizons • Volume 9 2016 Research article culture wells by using 50 μl per 1 ml of cell culture fluids; plant organs (0.05/4 = 0.0125). Significance was defined as more details may be found elsewhere (Perera et al., 2014). A p ≤ α and trends towards significance were defined as negative control (solvent vehicle, just AIM-V media) was run α < p < 2α. Pearson correlations were run to examine whether with all subjects to serve as a baseline. A positive control phytochemical composition or bystander endotoxin levels (phytohemagluttin, PHA; stock 100 μg/ml) was run with a correlated with immune outcomes. subset of subjects (n = 6) to ensure the experimental tech- niques worked. For proliferation, cells were cultured for 72 h Results and then tested via a 3-hour tetrazolium salt assay (Cell Titer, Catalogue #G3580, Promega). For cytokine assays, culture Phytochemistry and correlations time varied by assay, being 24 h (TNF), 48 h (IL-2), or 72 h Several different alkamides and caffeic acid derivatives were (IL-10); culture supernatants were collected at their respec- detected in the extracts (Table  1). Caftaric acid was the most tive time points, stored at −80°C, and assayed by ELISA (BD widely-distributed compound; some alkamides, caftaric acid and Biosciences, Catalogue #555212, #555190, #555157). other caffeic acid derivatives were observed only in individual extracts. Statistics Correlations between alkamide/caffeic acid derivative Paired samples t-tests were used to determine whether prolif- content and immune outcomes are provided in Table 2. All erative or cytokine production differences were seen between detectable compounds except alkamide 8a correlated posi- extract- or PHA-treated cells, with a corrected α-level of tively and significantly with IL-10 production. Only caftaric 0.0125 owing to multiple comparisons for each of the four acid correlated positively with TNF production. None of the compounds correlated with either IL-2 or PBMC prolifera- Table 1. HPLC analysis of the extracts tion. Endotoxin levels from the extracts were (in EU/ml): flower = 2.2, leaf = 0.1, stem < 0.1, root = 1.1. Endotoxin Flower Leaf Stem Root levels never correlated with immune outcomes (all Alkamide 2a ND ND ND 0.001 p ≥ 0.148). Alkamide 8a 0.029 0.001 0.023 ND Immunomodulatory properties Alkamide 14 0.012 ND ND ND Flower, leaf and root extracts significantly increased PBMC proliferation (all p ≤ 0.01) compared with solvent control Caftaric acid 0.004 0.007 0.004 0.007 (Fig. 1A). Leaf, stem and root extracts significantly increased TNF production (all p ≤ 0.007), whereas flower extracts dem - Chlorogenic acid 0.014 ND ND 0.003 onstrated a trend (p = 0.016) in that respect (Fig. 1B). Flower Cichoric acid 0.007 ND ND ND and root extracts significantly increased IL-10 production (both p ≤ 0.007), whereas leaf extract exhibited a trend Echinacoside ND ND ND 0.002 (p = 0.018) in that direction (Fig. 1C). None of the extracts Values are expressed as mg/mL. Although tested for, none of the following produced from fresh material significantly influenced IL-2 compounds were detected: alkamide 2b, alkamide 8b. alkamide 10a, production (control 8.4 ± 1.6, flower 8.7 ± 1.4, leaf 6.3 ± 0.9, alkamide 10b, alkamide 11, alkamide 12, alkamide 13, and ketone 23. ND, not detected. stem 5.6 ± 0.7, root 7.4 ± 1.3). Positive controls behaved as Table 2. Correlations between bioactive compounds and immunomodulatory activity IL-2 IL-10 TNF Proliferation Alkamide 2a 0.014 (0.901) 0.293 (0.018)* 0.179 (0.111) 0.134 (0.238) Alkamide 8a 0.006 (0.958) 0.135 (0.283) 0.009 (0.94) 0.018 (0.872) Alkamide 14 0.145 (0.200) 0.211 (0.091) −0.075 (0.507) 0.320 (0.777) Caftaric Acid −0.111 (0.326) 0.263 (0.034)* 0.290 (0.009)* 0.164 (0.145) Chlorogenic Acid 0.153 (0.176) 0.283 (0.022)* −0.038 (0.738) 0.063 (0.580) Cichoric Acid 0.145 (0.200) 0.211 (0.091) −0.075 (0.507) 0.320 (0.777) Echinacoside 0.014 (0.901) 0.293 (0.018)* 0.179 (0.111) 0.134 (0.238) Values are Pearson correlation coefficients and their associated p-values in parentheses. Asterisks indicate significant relationships whereas daggers indicate trends towards significant relationships. 3 Research article Bioscience Horizons • Volume 9 2016 Discussion Phytochemical differences by plant organ The first part of the hypothesis was that, of the aboveground organs, flower extracts would exhibit greater abundance and diversity of alkamides and caffeic acid derivatives than leaf or stem extracts. Flower extract had greater abundance and diversity of bioactive compounds compared with leaf and stem extracts (Table  1), supporting the hypothesis. The present work is the first report of alkamide and caffeic acid derivative composition of E. laevigata leaves and stems. In a study of E. laevigata inflorescences, it was reported that the most abun - dant compounds in were (in order from greatest to least detected quantities): alkamides 8/9; cichoric acid, caftaric acid and alkamide 16 (Binns et al., 2002). Findings from the present study (Table 1) are largely consistent (Binns et al., 2002), with the exception of alkamide 16 which was not analysed. A study of fresh E. tennesseensis showed both flower and stem extracts had greater (though differing) alkamide levels compared with leaf extracts (Senchina et al., 2009a), again similar to the pres- ent study (Table  1). Compositional differences between Echinacea species are well-documented (Pellati et  al., 2004; Wu et al., 2004; Kraus et al., 2006). Other reports on the phy- tochemistry of Echinacea aboveground parts are difficult to compare with this study because they assayed different com- pounds (Mazza and Cottrell, 1999) or dried material from a different species (Mølgaard et al., 2003) or because they anal- ysed all aboveground parts together (Brown, Chan and Betz, 2010; Brown et al., 2011; Ramasahayam et al., 2011). Though not the primary focus of this investigation, root extracts were also produced from fresh material as a com- parison point with previous research. Alkamides previously reported from Echinacea laevigata root extracts include 2a, 4, 5, 6, 8a, 10a, and 11 (Wu et al., 2004; Senchina et al., 2011). In the present study, only alkamide 2a was found (Table 1), but alkamides 4–6 were not assayed due to a lack of available standards. Caffeic acid derivatives previously reported from E. laevigata root extracts include caftaric acid, cichoric acid and echinacoside (Pellati et al., 2005; Senchina et al., 2011). In the present study, caftaric acid, chlorogenic acid and echi- nacoside (but not cichoric acid) were detected (Table 1). The data on caffeic acid derivatives obtained in this study confirm and extend previous work, whereas levels of alkamides were lower in the present study than that seen in previous reports. Immunomodulatory differences Figure 1. In vitro immunomodulatory effects of Echinacae laevigata by plant organ extracts on human PBMCs: (A) proliferation, (B) TNF, (C) IL-10. Values are means ± standard errors. Asterisks (*) indicate statistically The second part of the hypothesis was that, of the significant differences ( p < 0.017) between treatment and control, aboveground organs, flower extracts would exhibit greater whereas daggers ( ) indicate a trends (0.017 < p < 0.034) towards a immunomodulatory activity. When the immunomodulatory statistically significant difference between treatment and control. capacities of all three extracts produced from aboveground fresh material were compared (Fig. 1), flower extracts influ - enced three of the four immune parameters, whereas leaf expected by increasing cytokine production or PBMC prolif- extracts influenced two and stem extracts influenced only one; eration compared with solvent control in all instances (all thus, the immunomodulatory data also support the first p ≤ 0.037; data not shown). 4 Bioscience Horizons • Volume 9 2016 Research article hypothesis. Though not the primary focus of this investiga- significant relationships. Given the idiosyncratic nature of the tion, root extracts had the strongest immunomodulatory significant relationships, the correlations may not be physio - activity of all extracts significantly modulating three of the logically relevant. It may be more likely that other compounds four immune parameters. are responsible for the effects shown in Fig. 1. A similar lack of correlation between known bioactive compounds and Modulation of cytokine production or cell proliferation may extract immumodulatory activity has been reported previ- relate to the potential utility of Echinacea in the context of URIs. ously for Echinacea (Vimalanathan, Arnason and Hudson, For example, in the context of pathogen-induced inflammation, 2009), and has also been observed in studies of other herbal some inflammation is beneficial as it makes the environment less supplements including Pueraria (Cherdshewasart and Sutjit, hospitable for the pathogen; however, inflammation also causes 2008) and Sanguinaria (Perera et al., 2014). The lack of any cell and tissue damage, and is responsible for many of the symp- robust, consistent correlations is unsurprising given the com- toms associated with infection. As reviewed previously else- plex phytochemical milieu of plants and possible unaccounted where (Senchina, Hallam and Cheney, 2013), several studies of pre-experimental factors, yet it may suggest that the alka- Echinacea extracts and leucocytes have shown a general up- mides and caffeic acid derivatives do not account for all regulation of cytokine production, regardless of whether those immunomodulatory activity from Echinacea extracts. cytokines are pro- or anti-inflammatory. However, other studies (LaLone et  al., 2009; Cech et  al., 2010; Lalone et  al., 2010; Limitations and future directions Ritchie et  al., 2011; Dapas et  al., 2014) have shown that Echinacea phytochemicals may selectively inhibit pro-inflam - Some limitations may be identified from the research con - matory cytokines like TNF while promoting cytokines such as ducted here. All work was performed in vitro, so it is unclear IL-10; this may lead to a reduction of symptom severity in the whether these results would translate directly to clinical sce- context of upper respiratory tract infection. Discrepancies narios. Although all plants grew in the same common garden between the two groups of studies may be explained by phyto- and were the same age, they were grown under outdoor condi- chemical differences in the compounds or supplements tested, tions that are not precisely replicable, and it is unknown what specifically the alkamides, but composition did not always cor - role environmental variables may have played in these results. relate with observed effects (Cech et al., 2010). Findings from Data obtained in this study point naturally to several this study echoed the former pattern of a generalized up-regula- potential future directions. Additional data on the effects of tion of cytokines (Fig. 1). Since the extracts tested in this study pre-harvest conditions (sunlight, hydration, soil conditions, contained alkamides alongside other compounds such as caffeic etc.) or post-harvest conditions (such as extraction methods acid derivatives (Table 1), the most parsimonious explanation is or drying) are needed to better understand heterogeneous that TNF and IL-10 are being modulated by different compound findings between studies that use similar species or extraction classes (discussed in the Limitations and future directions sec- techniques, as these variables would quite likely explain dis- tion). Thus, present results do not resolve the current conflicts parities between studies. More cross-genus comparisons (con- among different reports. trolling for the pre-experimental conditions highlighted The present work is the first report of the immunomodula - previously) would illumine the interplay between species tory properties of E. laeviagata flowers, leaves and stems. In the selection and environmental conditions, helping parse out lone previous report of E. laevigata immuomodulatory activities which immunomodulatory effects are consistent across the (Senchina et al., 2011), root extracts augmented IL-10 but did genus vs. peculiar to individual species or species groups. The not have a signic fi ant effect on IL-2, TNF or proliferation; immuomodulatory properties of one species, E. atrorubens, flower, leaf and stem were not tested. Given all the pre-clinical have not yet been reported. and laboratory factors that can influence extract immunomodu - latory activity (Senchina et al., 2009b) and differences in meth- Conclusions ods and subjects between the two studies, it is likely premature to make any comparisons between the immunomodulatory Echinacea laevigata appears to have phytochemical and capacities of aboveground vs. belowground parts of E. laevigata. immunomodulatory properties similar to other members of its The finding that E. laevigata extracts broadly influenced prolif - genus. Extracts generated from fresh aboveground material eration, TNF and IL-10 (but not IL-2) is consistent with the demonstrated immunomodulatory effects. Similar to other studies regarding the in vitro immunomodulatory properties of Echinacea species, E. laevigata extracts appear able to stimu- other Echinacea species (Rininger et al., 2000; Randolph et al., late both Th1 and Th2 aspects of immunity. 2003; Gertsch et al., 2004; Hwang, Dasgupta and Actor, 2004; Mishima et al., 2004; Senchina et al., 2006a, 2009a). Author biography Phytochemical composition and Ekta Haria obtained her bachelor’s degree from Drake immunomodulatory activity University, Des Moines, Iowa in 2014. She is currently a Physician Assistant student at the University of Nebraska Statistical correlations between extract composition and in Medical Center, Omaha, Nebraska. vitro immunomodulatory activity (Table  2) yielded a few 5 Research article Bioscience Horizons • Volume 9 2016 Gilroy, C. M., Steiner, J. F., Byers, T. et al. (2003) Echinacea and truth in Acknowledgements labeling. Archives of Internal Medicine, 163 (6), 699–704. Ekta Haria wrote this paper with the mentorship of David Goey, A. K. L., Rosing, H., Meijerman, I. et al. (2012) The bioanalysis Senchina. Plants were provided courtesy of Dr. Joe-Ann of the major Echinacea purpurea constituents dodeca-2E,4E,8Z,10E/Z- McCoy. Several undergraduates at Drake University (Jennifer tetraenoic acid isobutylamides in human plasma using LC-MS/MS. H. Strauch, Breanna R. Dumke, Brad L. Laflen, Nisarg B. Journal of Chromatography. B, 902, 151–156. Shah, Griffin B. Hoffmann) helped with plant extraction and Hou, C.-C., Huang, C.-C. and Shyur, L.-F. (2011) Echinacea alkamides pre- immune assays. At Iowa State University, Isaac Miller assisted vent lipopolysaccharide/D-galactosamine-induced acute hepatic with plant harvesting and Amila Dias assisted with phyto- injury through JNK pathway-mediated HO-1 expression. Journal of chemical profiling. David Senchina contributed to all parts Agricultural and Food Chemistry, 59 (22), 11966–11974. of the experimental process. 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Immunomodulatory effects of Echinacea laevigata ethanol tinctures produced from different organs

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BioscienceHorizons Volume 9 2016 10.1093/biohorizons/hzw001 Research article Immunomodulatory effects of Echinacea laevigata ethanol tinctures produced from different organs 1, 2 1 Ekta N. Haria *, M. Ann D. N. Perera and David S. Senchina Biology Department, Drake University, 2507 University Ave., Des Moines, IA 50311, USA W. M. Keck Metabolomics Laboratory, Iowa State University, 0124 Molecular Biology Building, Ames, IA 50011, USA *Corresponding author: 2507 University Ave, Des Moines, IA 50311 (c/o David Senchina, Biology Department). Email: hariaekta@gmail.com Echinacea supplements may prevent or reduce symptoms of upper respiratory infections by immunomodulation, possibly by altering the cytokine profile. Compared with other species in the genus, the immunomodulatory properties of Echinacea laeviagata are poorly characterized. The purposes of this study were to compare the diversity and quantity of known bioactive compounds from aboveground organs of E. laevigata, and to characterize the in vitro immumodulatory properties of ethanol tinctures produced from those structures. High-performance liquid chromatography (HPLC) was used to determine the levels of alkamides and caffeic acid derivatives. Peripheral blood mononuclear cells (PBMCs) were obtained from 16 adults and challenged in vitro with extracts. PBMC proliferation and production of the cytokines interleukin-2 (IL-2), IL-10 and tumour necrosis factor-α (TNF-α) were measured. Fresh flower, leaf and root extracts were able to augment TNF and IL-10 and prolif - eration, whereas fresh stem extract was only able to augment TNF. Extracts produced from flowers contained the greatest bioactive compound quantities and diversity. Caftaric acid was the most abundant compound and correlated with some (but not all) observed immune effects. These results suggest that of all aboveground parts, flowers have the greatest abundance and diversity of known bioactive compounds, and both flower and leaf extracts were immunomodulators. Key words: coneflower, cytokine, interleukin, peripheral blood mononuclear cell, proliferation, tumour necrosis factor Submitted on 18 September 2015; accepted on 25 January 2016 Introduction Each of the nine species of Echinacea differs in its diversity and abundance of purported bioactive compounds (Wu et al., Echinacea (Asteraceae) is a genus of nine plants native to the 2004; Pellati et al., 2005; Kraus et al., 2006). Within a single spe- United States (McGregor, 1968; Wu et al., 2009). These spe- cies, phytochemical profiles differ between different organs, such cies have been used for medicinal purposes (Moerman, 1998; as aboveground (aerial) and belowground parts (Qu et al., 2005; Barnes et al., 2005), with current interest in their use as immu- Senchina et al., 2009a). Four classes of bioactive compounds nomodulatory therapies for upper respiratory infections have been identified from Echinacea extracts (alkamides, caffeic (URIs) such as colds and influenza. Clinical reports conflict acid derivatives, ketones and polysaccharides). Only alkamides regarding their utility, with some reporting efficacy and others and caffeic acid derivatives are likely of physiological relevance not (Karsch-Völk, Barrett and Linde, 2015; Schapowal, Klein (Matthias et al., 2005; Ye et al., 2011; Goey et al., 2012), because and Johnston, 2015). Some of the discrepancies may be ketones readily oxidize (Qiang et al., 2013) and polysaccharides explained by the rampant adulteration and mislabeling com- are likely modified in the gut ( Woelkart et al., 2008). mon to many commercial preparations, or the lack of control for such variables (Gilroy et al., 2003; Krochmal et al., 2004; Much research on the therapeutic potential of Echinacea Wolsko et al., 2005). extracts has concentrated on inflammatory pathways. © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Research article Bioscience Horizons • Volume 9 2016 Echinacea bioactive compounds modulate transcription fac- cross-pollinating. A voucher specimen was deposited in the tors (Gertsch et al., 2004; Matthias et al., 2008) that lead to Ada Hayden Herbarium at Iowa State University the modulation of cytokines associated with inflammation, (ISC#447184). Specimens were extracted immediately post- such as tumour necrosis factor (TNF) (Lalone et  al., 2010; harvest. Plants were divided by organ (flower, leaf, stem, Hou, Huang and Shyur, 2011; Senchina et al., 2011; Dapas root), and each organ was processed separately. After separa- et al., 2014) and interleukin-1β (IL-1β) (Senchina et al., 2009a, tion, organs were diced manually using a surgical scalpel. The c; Zhang et al., 2012). Non-inflammatory cytokines, such as diced material was extracted at a ratio of 1:9 plant material– IL-10, may also be modulated (Zhai et al., 2007; Kapai et al., solvent in 50% ethanol–50% cell culture water using meth- 2011; Ritchie et al., 2011; Senchina et al., 2011). Thus, contin- ods described elsewhere (which simulate lay herbalist gent on a multitude of agricultural and experimental factors, a preparations) (Senchina et al., 2006b). Extracts were allowed given Echinacea extract can influence Th1 responses, Th2 to steep for 1 h at room temperature on a horizontal shaker responses or both simultaneously. Consistent with their vary- before being passed through sterilized tulle and stored at ing phytochemical profiles, different Echinacea species have −80°C and tested within 1 week of production. Extracts were different cytokine-modulating effects (Senchina et al., 2006a). vortexed prior to use in any phytochemical or immunomodu- latory assays. Far less is known about Echinacea laevigata compared with other species in the genus (E. augustifolia, E. pallida, Phytochemical profiling E. purpurea), for which the biochemical and immunomodula- Phytochemical composition of the extracts was determined tory properties are relatively well-characterized. Compared via high-performance liquid chromatography (HPLC) with with other Echinacea species, E. laevigata extracts had high UV detector for alkamides, ketones and caffeic acid deriva- caffeic acid derivative content (Pellati et al., 2004, 2005), and tives using previous methods (Senchina et al., 2011). Briefly, contained alkamides 2a, 4–6, 8a, 10a, and 11 (Wu et al., 2004; HPLC analysis was performed with YMC-Pack ODS-AM RP Senchina et al., 2011). Therefore, E. laevigata may harbour C18 (250 × 4.6 mm, 5 μm) analytical column (Waters; relevant phytomedicinal capacity and deserves consideration. Bedford, MA, USA). The solvent system was acetonitrile/H O To our knowledge there is only one report of the immuno- 2 with 0.01% formic acid for lipophilic constituents as well as modulatory properties of E. laevigata (Senchina et al., 2011). hydrophilic constituents but with varying gradients. The flow It demonstrated that E. laevigata tinctures exhibit immuno- rate was maintained at 1.0 ml/min. Alkamides were separated modulatory capacities, but only roots were examined. It is at a linear gradient of 40–80% acetonitrile over 45 min. unknown whether aerial parts of E. laevigata (flowers, leaves, Hydrophilic constituents, e.g. caffeic acid, were determined on stems) harbour similar activities. a linear gradient of 10–35% acetonitrile over 25 min. The col- The purpose of this study was to compare the diversity and umn temperature was 30°C. UV spectra recorded were in the quantity of alkamides and caffeic acid derivatives from differ- range of 200–400 nm, while 330 nm was used for quantifica - ent aboveground organs of E. laevigata, and to characterize tion of caffeic acid derivatives and 254 nm for alkamides. the in vitro immumodulatory properties of ethanol tinctures Bystander endotoxin levels were quantified from all extracts produced from these structures. The research question was: using a colorimetric method. The cell culture model employed do ethanol tinctures produced from E. laevigata aerial organs here is insensitive to endotoxin levels of 10 EU/mL or less demonstrate immunomodulatory activity in an in vitro pri- (Senchina et al., 2006b). mary cell culture model? It was hypothesized that (a) of the aboveground organs, flower extracts would exhibit greater Human subjects and cell isolation abundance and diversity of alkamides and caffeic acid deriva- Approval to work with human subjects was granted by the tives than leaf or stem extracts, and consequently greater Drake University Institutional Review Board (ID 2007- immunomodulatory activity; (b) of all plant organs, the 08015). Sixteen young adults (7 females and 9 males; belowground roots would have the greater abundance and 23.5 ± 3.8 years) gave written informed consent prior to par- diversity of alkamides and caffeic acid derivatives compared ticipation and donated blood. Blood was drawn from the with any of the aboveground organs. antecubital vein following Universal Precautions and peripheral blood mononuclear cells (PBMCs) were separated as described Methods elsewhere (Senchina et al., 2009a, c). Blood was collected in heparinized tubes, and then diluted 1:1 with phosphate-buff- Plant harvesting and extraction ered saline before being layered on top of Ficoll-Paque and Echinacea laevigata (Ames 25 161) plants were harvested as being centrifuged at 1800 rpm and 4°C for 15 min; the cen- whole plants with root bundles intact from a common garden trifuge was allowed to stop without any braking. Leucocytes in September 2007 at the United States Department of Agr- were then extracted from the Ficoll layer using pipettes and iculture (USDA) North Central Regional Plant Introduction washed twice with Hank’s buffered saline solution (HBSS) Station in Ames, Iowa. Plants were identified and provided before being manually counted via hemocytometer and stan- courtesy of Dr. Joe-Ann McCoy, were 3 years old at har- dardized to 1.0 × 10 cells/ml in AIM-V media. Extracts were vest and had been enclosed in pollination cages to prevent diluted 1:12.5 in AIM-V media before being added to cell 2 Bioscience Horizons • Volume 9 2016 Research article culture wells by using 50 μl per 1 ml of cell culture fluids; plant organs (0.05/4 = 0.0125). Significance was defined as more details may be found elsewhere (Perera et al., 2014). A p ≤ α and trends towards significance were defined as negative control (solvent vehicle, just AIM-V media) was run α < p < 2α. Pearson correlations were run to examine whether with all subjects to serve as a baseline. A positive control phytochemical composition or bystander endotoxin levels (phytohemagluttin, PHA; stock 100 μg/ml) was run with a correlated with immune outcomes. subset of subjects (n = 6) to ensure the experimental tech- niques worked. For proliferation, cells were cultured for 72 h Results and then tested via a 3-hour tetrazolium salt assay (Cell Titer, Catalogue #G3580, Promega). For cytokine assays, culture Phytochemistry and correlations time varied by assay, being 24 h (TNF), 48 h (IL-2), or 72 h Several different alkamides and caffeic acid derivatives were (IL-10); culture supernatants were collected at their respec- detected in the extracts (Table  1). Caftaric acid was the most tive time points, stored at −80°C, and assayed by ELISA (BD widely-distributed compound; some alkamides, caftaric acid and Biosciences, Catalogue #555212, #555190, #555157). other caffeic acid derivatives were observed only in individual extracts. Statistics Correlations between alkamide/caffeic acid derivative Paired samples t-tests were used to determine whether prolif- content and immune outcomes are provided in Table 2. All erative or cytokine production differences were seen between detectable compounds except alkamide 8a correlated posi- extract- or PHA-treated cells, with a corrected α-level of tively and significantly with IL-10 production. Only caftaric 0.0125 owing to multiple comparisons for each of the four acid correlated positively with TNF production. None of the compounds correlated with either IL-2 or PBMC prolifera- Table 1. HPLC analysis of the extracts tion. Endotoxin levels from the extracts were (in EU/ml): flower = 2.2, leaf = 0.1, stem < 0.1, root = 1.1. Endotoxin Flower Leaf Stem Root levels never correlated with immune outcomes (all Alkamide 2a ND ND ND 0.001 p ≥ 0.148). Alkamide 8a 0.029 0.001 0.023 ND Immunomodulatory properties Alkamide 14 0.012 ND ND ND Flower, leaf and root extracts significantly increased PBMC proliferation (all p ≤ 0.01) compared with solvent control Caftaric acid 0.004 0.007 0.004 0.007 (Fig. 1A). Leaf, stem and root extracts significantly increased TNF production (all p ≤ 0.007), whereas flower extracts dem - Chlorogenic acid 0.014 ND ND 0.003 onstrated a trend (p = 0.016) in that respect (Fig. 1B). Flower Cichoric acid 0.007 ND ND ND and root extracts significantly increased IL-10 production (both p ≤ 0.007), whereas leaf extract exhibited a trend Echinacoside ND ND ND 0.002 (p = 0.018) in that direction (Fig. 1C). None of the extracts Values are expressed as mg/mL. Although tested for, none of the following produced from fresh material significantly influenced IL-2 compounds were detected: alkamide 2b, alkamide 8b. alkamide 10a, production (control 8.4 ± 1.6, flower 8.7 ± 1.4, leaf 6.3 ± 0.9, alkamide 10b, alkamide 11, alkamide 12, alkamide 13, and ketone 23. ND, not detected. stem 5.6 ± 0.7, root 7.4 ± 1.3). Positive controls behaved as Table 2. Correlations between bioactive compounds and immunomodulatory activity IL-2 IL-10 TNF Proliferation Alkamide 2a 0.014 (0.901) 0.293 (0.018)* 0.179 (0.111) 0.134 (0.238) Alkamide 8a 0.006 (0.958) 0.135 (0.283) 0.009 (0.94) 0.018 (0.872) Alkamide 14 0.145 (0.200) 0.211 (0.091) −0.075 (0.507) 0.320 (0.777) Caftaric Acid −0.111 (0.326) 0.263 (0.034)* 0.290 (0.009)* 0.164 (0.145) Chlorogenic Acid 0.153 (0.176) 0.283 (0.022)* −0.038 (0.738) 0.063 (0.580) Cichoric Acid 0.145 (0.200) 0.211 (0.091) −0.075 (0.507) 0.320 (0.777) Echinacoside 0.014 (0.901) 0.293 (0.018)* 0.179 (0.111) 0.134 (0.238) Values are Pearson correlation coefficients and their associated p-values in parentheses. Asterisks indicate significant relationships whereas daggers indicate trends towards significant relationships. 3 Research article Bioscience Horizons • Volume 9 2016 Discussion Phytochemical differences by plant organ The first part of the hypothesis was that, of the aboveground organs, flower extracts would exhibit greater abundance and diversity of alkamides and caffeic acid derivatives than leaf or stem extracts. Flower extract had greater abundance and diversity of bioactive compounds compared with leaf and stem extracts (Table  1), supporting the hypothesis. The present work is the first report of alkamide and caffeic acid derivative composition of E. laevigata leaves and stems. In a study of E. laevigata inflorescences, it was reported that the most abun - dant compounds in were (in order from greatest to least detected quantities): alkamides 8/9; cichoric acid, caftaric acid and alkamide 16 (Binns et al., 2002). Findings from the present study (Table 1) are largely consistent (Binns et al., 2002), with the exception of alkamide 16 which was not analysed. A study of fresh E. tennesseensis showed both flower and stem extracts had greater (though differing) alkamide levels compared with leaf extracts (Senchina et al., 2009a), again similar to the pres- ent study (Table  1). Compositional differences between Echinacea species are well-documented (Pellati et  al., 2004; Wu et al., 2004; Kraus et al., 2006). Other reports on the phy- tochemistry of Echinacea aboveground parts are difficult to compare with this study because they assayed different com- pounds (Mazza and Cottrell, 1999) or dried material from a different species (Mølgaard et al., 2003) or because they anal- ysed all aboveground parts together (Brown, Chan and Betz, 2010; Brown et al., 2011; Ramasahayam et al., 2011). Though not the primary focus of this investigation, root extracts were also produced from fresh material as a com- parison point with previous research. Alkamides previously reported from Echinacea laevigata root extracts include 2a, 4, 5, 6, 8a, 10a, and 11 (Wu et al., 2004; Senchina et al., 2011). In the present study, only alkamide 2a was found (Table 1), but alkamides 4–6 were not assayed due to a lack of available standards. Caffeic acid derivatives previously reported from E. laevigata root extracts include caftaric acid, cichoric acid and echinacoside (Pellati et al., 2005; Senchina et al., 2011). In the present study, caftaric acid, chlorogenic acid and echi- nacoside (but not cichoric acid) were detected (Table 1). The data on caffeic acid derivatives obtained in this study confirm and extend previous work, whereas levels of alkamides were lower in the present study than that seen in previous reports. Immunomodulatory differences Figure 1. In vitro immunomodulatory effects of Echinacae laevigata by plant organ extracts on human PBMCs: (A) proliferation, (B) TNF, (C) IL-10. Values are means ± standard errors. Asterisks (*) indicate statistically The second part of the hypothesis was that, of the significant differences ( p < 0.017) between treatment and control, aboveground organs, flower extracts would exhibit greater whereas daggers ( ) indicate a trends (0.017 < p < 0.034) towards a immunomodulatory activity. When the immunomodulatory statistically significant difference between treatment and control. capacities of all three extracts produced from aboveground fresh material were compared (Fig. 1), flower extracts influ - enced three of the four immune parameters, whereas leaf expected by increasing cytokine production or PBMC prolif- extracts influenced two and stem extracts influenced only one; eration compared with solvent control in all instances (all thus, the immunomodulatory data also support the first p ≤ 0.037; data not shown). 4 Bioscience Horizons • Volume 9 2016 Research article hypothesis. Though not the primary focus of this investiga- significant relationships. Given the idiosyncratic nature of the tion, root extracts had the strongest immunomodulatory significant relationships, the correlations may not be physio - activity of all extracts significantly modulating three of the logically relevant. It may be more likely that other compounds four immune parameters. are responsible for the effects shown in Fig. 1. A similar lack of correlation between known bioactive compounds and Modulation of cytokine production or cell proliferation may extract immumodulatory activity has been reported previ- relate to the potential utility of Echinacea in the context of URIs. ously for Echinacea (Vimalanathan, Arnason and Hudson, For example, in the context of pathogen-induced inflammation, 2009), and has also been observed in studies of other herbal some inflammation is beneficial as it makes the environment less supplements including Pueraria (Cherdshewasart and Sutjit, hospitable for the pathogen; however, inflammation also causes 2008) and Sanguinaria (Perera et al., 2014). The lack of any cell and tissue damage, and is responsible for many of the symp- robust, consistent correlations is unsurprising given the com- toms associated with infection. As reviewed previously else- plex phytochemical milieu of plants and possible unaccounted where (Senchina, Hallam and Cheney, 2013), several studies of pre-experimental factors, yet it may suggest that the alka- Echinacea extracts and leucocytes have shown a general up- mides and caffeic acid derivatives do not account for all regulation of cytokine production, regardless of whether those immunomodulatory activity from Echinacea extracts. cytokines are pro- or anti-inflammatory. However, other studies (LaLone et  al., 2009; Cech et  al., 2010; Lalone et  al., 2010; Limitations and future directions Ritchie et  al., 2011; Dapas et  al., 2014) have shown that Echinacea phytochemicals may selectively inhibit pro-inflam - Some limitations may be identified from the research con - matory cytokines like TNF while promoting cytokines such as ducted here. All work was performed in vitro, so it is unclear IL-10; this may lead to a reduction of symptom severity in the whether these results would translate directly to clinical sce- context of upper respiratory tract infection. Discrepancies narios. Although all plants grew in the same common garden between the two groups of studies may be explained by phyto- and were the same age, they were grown under outdoor condi- chemical differences in the compounds or supplements tested, tions that are not precisely replicable, and it is unknown what specifically the alkamides, but composition did not always cor - role environmental variables may have played in these results. relate with observed effects (Cech et al., 2010). Findings from Data obtained in this study point naturally to several this study echoed the former pattern of a generalized up-regula- potential future directions. Additional data on the effects of tion of cytokines (Fig. 1). Since the extracts tested in this study pre-harvest conditions (sunlight, hydration, soil conditions, contained alkamides alongside other compounds such as caffeic etc.) or post-harvest conditions (such as extraction methods acid derivatives (Table 1), the most parsimonious explanation is or drying) are needed to better understand heterogeneous that TNF and IL-10 are being modulated by different compound findings between studies that use similar species or extraction classes (discussed in the Limitations and future directions sec- techniques, as these variables would quite likely explain dis- tion). Thus, present results do not resolve the current conflicts parities between studies. More cross-genus comparisons (con- among different reports. trolling for the pre-experimental conditions highlighted The present work is the first report of the immunomodula - previously) would illumine the interplay between species tory properties of E. laeviagata flowers, leaves and stems. In the selection and environmental conditions, helping parse out lone previous report of E. laevigata immuomodulatory activities which immunomodulatory effects are consistent across the (Senchina et al., 2011), root extracts augmented IL-10 but did genus vs. peculiar to individual species or species groups. The not have a signic fi ant effect on IL-2, TNF or proliferation; immuomodulatory properties of one species, E. atrorubens, flower, leaf and stem were not tested. Given all the pre-clinical have not yet been reported. and laboratory factors that can influence extract immunomodu - latory activity (Senchina et al., 2009b) and differences in meth- Conclusions ods and subjects between the two studies, it is likely premature to make any comparisons between the immunomodulatory Echinacea laevigata appears to have phytochemical and capacities of aboveground vs. belowground parts of E. laevigata. immunomodulatory properties similar to other members of its The finding that E. laevigata extracts broadly influenced prolif - genus. Extracts generated from fresh aboveground material eration, TNF and IL-10 (but not IL-2) is consistent with the demonstrated immunomodulatory effects. Similar to other studies regarding the in vitro immunomodulatory properties of Echinacea species, E. laevigata extracts appear able to stimu- other Echinacea species (Rininger et al., 2000; Randolph et al., late both Th1 and Th2 aspects of immunity. 2003; Gertsch et al., 2004; Hwang, Dasgupta and Actor, 2004; Mishima et al., 2004; Senchina et al., 2006a, 2009a). Author biography Phytochemical composition and Ekta Haria obtained her bachelor’s degree from Drake immunomodulatory activity University, Des Moines, Iowa in 2014. She is currently a Physician Assistant student at the University of Nebraska Statistical correlations between extract composition and in Medical Center, Omaha, Nebraska. vitro immunomodulatory activity (Table  2) yielded a few 5 Research article Bioscience Horizons • Volume 9 2016 Gilroy, C. M., Steiner, J. F., Byers, T. et al. (2003) Echinacea and truth in Acknowledgements labeling. Archives of Internal Medicine, 163 (6), 699–704. Ekta Haria wrote this paper with the mentorship of David Goey, A. K. L., Rosing, H., Meijerman, I. et al. (2012) The bioanalysis Senchina. Plants were provided courtesy of Dr. Joe-Ann of the major Echinacea purpurea constituents dodeca-2E,4E,8Z,10E/Z- McCoy. Several undergraduates at Drake University (Jennifer tetraenoic acid isobutylamides in human plasma using LC-MS/MS. H. Strauch, Breanna R. Dumke, Brad L. Laflen, Nisarg B. Journal of Chromatography. B, 902, 151–156. Shah, Griffin B. Hoffmann) helped with plant extraction and Hou, C.-C., Huang, C.-C. and Shyur, L.-F. (2011) Echinacea alkamides pre- immune assays. At Iowa State University, Isaac Miller assisted vent lipopolysaccharide/D-galactosamine-induced acute hepatic with plant harvesting and Amila Dias assisted with phyto- injury through JNK pathway-mediated HO-1 expression. Journal of chemical profiling. David Senchina contributed to all parts Agricultural and Food Chemistry, 59 (22), 11966–11974. of the experimental process. 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