Gründemann et al.

BMC Complementary and Alternative Medicine 2014, 14:283

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RESEARCH ARTICLE Open Access

Equisetum arvense (common horsetail) modulates

the function of inflammatory immunocompetent
cells
Carsten Gründemann1*, Karin Lengen1, Barbara Sauer1, Manuel Garcia-Käufer1, Martin Zehl2 and Roman Huber1

Abstract
Background: In Europe, extracts of Equisetum arvense (common horsetail) have a long tradition in the treatment of
inflammatory disorders. To understand the molecular basis for its use, we investigated the immunomodulatory
capacity of a standardized commercially available common horsetail extract on human primary lymphocyte
function in vitro.
Methods: The standardized extract of Equisetum arvense was phytochemically characterized. Effects on proliferation,
viability and activity of mitogen-activated human lymphocytes were assessed in comparison to cyclosporine A using
annexin V/propidium iodide staining assays and flow cytometry-based surface receptor characterization, respectively.
Intracellular levels of effector molecules (IL-2, IFN-γ and TNF-α) were analyzed with cytokine assays.
Results: T cell proliferation was inhibited dose dependently by the Equisetum extract without induction of apoptosis or
necrosis. This effect was mediated through inhibition of lymphocyte activation, specifically by diminishing CD69 and
IL-2 surface receptor expression and intracellular IL-2 production. Furthermore, treatment with Equisetum arvense
inhibited effector functions, as indicated by reduced production of IFN-γ and TNF-α.
Conclusions: The data indicate that the used extract of Equisetum arvense interferes with the polyfunctionality of
immunocompetent cells thereby providing an anti-inflammatory mode-of-action.
Keywords: Equisetum arvense, Equisetopsida, Horsetail, Inflammation, Lymphocytes, Immunosuppression,
Anthroposophical Medicine

Background The putative medicinal properties are supported by a

Equisetum arvense (common horsetail, field horsetail, or number of studies, which found (I) hepatoprotective [7],
giant horsetail) belongs to the Equisetopsida family and is (II) diuretic [8], (III) anti-bacterial [9,10] or (IV) antioxi-
native to the Arctic and temperate regions of the northern dant effects [11-14]. Furthermore, (V) anti-inflammatory
hemisphere, particularly Europe [1]. Its traditional use as properties for the treatment of wounds or inflammatory
an herbal remedy is documented in several handbooks of diseases such as arthritis have been described [15,16].
phytotherapy [2,3] and continues to be popular in comple- However, to date, there are no studies on the impact of
mentary medicine, especially Anthroposophical Medicine Equisetum arvense extracts on lymphocytes involved in
[4]. Among the species of this genus, only Equisetum inflammatory immune processes. Lymphocytes are the
arvense “Equiseti herba”) is listed in the German commis- body’s second line of defence and T cells are actively re-
sion E Monograph (phytotherapy and herbal substances) cruited to sites of inflammation where they maintain and
of the German Federal Institute for Drugs and Medical activate fibroblasts or bystander dendritic cells and macro-
Devices [5] as well as in the European Pharmacopoeia [6]. phages, transforming them into tissue-destructive effector
cells [17]. T lymphocytes are the dominant cells in inflam-
* Correspondence: [email protected] matory immune diseases [18,19], and their proliferation
1
Center for Complementary Medicine, Department of Environmental Health
Sciences, Medical Center, University of Freiburg, Breisacherstr. 115B, 79106
and mediator release (IFN-γ, TNF-α) are targets for modern
Freiburg, Germany therapies [20]. Immunosuppressants such as glucocorticoids
Full list of author information is available at the end of the article

© 2014 Gründemann et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public
Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this
article, unless otherwise stated.
Gründemann et al. BMC Complementary and Alternative Medicine 2014, 14:283 Page 2 of 10
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[21] or calcineurin inhibitors, e.g. cyclosporine A, which (33 min), followed by a column cleaning and re-
specifically down-regulate the immune system [20], are equilibration step. The eluent flow was split at a 1:4 ratio
the established treatment of choice. Despite the availability before the ESI ion source, which was operated as follows:
of effective conventional medications, approximately 60- capillary voltage: +3.5 kV, nebulizer: 26 psi (N2), dry gas
90% of patients with inflammatory immune disorders re- flow: 9 L/min (N2), and dry temperature: 340°C. MS2, MS3,
sort to alternative or complementary therapies to avoid and MS4 spectra were obtained in an automated data-
side effects [22]. One such therapy involves use of dependent acquisition mode (collision gas: He, isolation
Equisetum arvense. Because we were interested in explor- window: 4 Th, fragmentation amplitude: 1.0 V). 2 μL of
ing the “active principle”, we phytochemically character- the undiluted, centrifuged Equisetum extract were injected.
ized a standardized, commercially available extract of Finally, two of the tentatively identified components were
Equisetum arvense and defined its immunosuppressive confirmed by comparison of the retention times, UV- and
capacity on cell proliferation, activation status and effector MSn-spectra with the reference compounds isoquercitrin
functions using mitogen-activated human lymphocytes. (Carl Roth, Karlsruhe, Germany) and kaempferol-3-O-
glucoside (Extrasynthese, Genay Cedex, France).
Methods Quantification of the main flavonoid isoquercitrin
Plant material (quercetin-3-O-glucoside) in the Equisetum arvense ex-
Equisetum arvense (Equisetopsida), ethanol. Decoctum tract was performed by HPLC-DAD with external stand-
(=Equisetum: D1; Weleda AG, Schwäbisch Gmünd, ard calibration, and the total flavonoid content was
Germany) was used for the experiments. The extract is determined by a spectrophotometric assay adapted from
manufactured according to method no. 19f of the German the European Pharmacopoeia (Ph. Eur.) Monograph on
Homeopathic Pharmacopoeia [23]. The Equisetum arvense Equiseti herba [6] (for details see Additional file 1).
was of European origin and came from wild harvests. The
fresh plant material was kept refrigerated and sent to Ethics statement
Weleda Naturals GmbH, Schwäbisch Gmünd, Germany, Patients gave their written consent for giving blood for
where the identity of the plant material was analyzed by scientific research.
botanic specialists using macroscopic and microscopic All experiments conducted on human material were
methods as well as comparison with depositions of speci- approved by the Ethics committee of the University of
fied botanical reference stocks. The plant material was Freiburg (55/14).
dried, homogenized and a decoct was produced by boiling
one part dried plant material with nine parts ethanol for Preparation and cultivation of human peripheral
4 h. The absolute ethanol concentration of the preparation lymphocytes
used was 36 Vol.-%. We used the same concentration of Human peripheral lymphocytes (PBMC) were isolated from
ethanol for vehicle control experiments and clearly dem- the blood of healthy adult donors obtained from the Blood
onstrated that these amounts have no influence on the Transfusion Centre (Medical Center, University of Freiburg,
behaviour of the cells. After cooling down to room tem- Germany). Venous blood was centrifuged on a Lympho-
perature, the decoct was squeezed out mechanically, fil- Prep™ gradient (density: 1.077 g/cm3, 20 min, 500 × g, 20°C;
tered sterile and filled into 50 mL bottles. Bottles from Progen, Heidelberg, Germany). Cells were washed twice in
sale stocks were sent to our laboratory in Freiburg, medium, and cell viability as well as concentration was de-
Germany, where the experiments were performed. For termined using the trypan blue exclusion test. Cells were
each experiment, a fresh frozen aliquot of the Equisetum cultured in RPMI 1640 medium supplemented with 10%
arvense extract was used, and concentrations of 0.05, 0.1, heat-inactivated fetal calf serum (PAA, Pasching, Austria),
0.2, 0.4 and 0.8 μg/mL were tested. 2 mM L-glutamine, 100 U/mL penicillin and 100 U/mL
streptomycin (all from Life Technologies, Paisley, UK). The
Phytochemical analysis of the Equisetum arvense extract cells were cultured at 37°C in a humidified incubator with
LC-MS analyses were performed on an UltiMate 3000 5% CO2/95% air atmosphere.
RSLC-series system (Dionex, Germering, Germany) coupled
to a 3D quadrupole ion trap mass spectrometer equipped Activation and treatment of lymphocytes and T-cells
with an orthogonal ESI source (HCT, Bruker Daltonics, PBMC (105) were stimulated with phytohemagglutinin-
Bremen, Germany). HPLC separation was carried out on L (PHA-L; 10 μg/mL; Roche Diagnostics, Mannheim,
an Acclaim 120 C18, 2.1 × 150 mm, 3 μm column (Dionex) Germany) or lipopolysaccharide (LPS; 1 μg/mL; Sigma-
at 30°C using 0.1% aqueous formic acid and acetonitrile as Aldrich, Taufkirchen, Germany) in the presence of control
mobile phase A and B, respectively. The flow rate was agents cyclosporine A (CsA; 10 μg/mL obtained by dilu-
0.5 mL/min and the following gradient program was used: tion of Sandimmun® 50 mg/mL, Novartis Pharma GmbH,
5% B (0 min), 5% B (2 min), 11% B (20 min), and 24% B Nürnberg, Germany), camptothecin (CPT; 30 μg/mL;
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Tocris, Eching, Germany) and Triton-X 100 (0.5%; Carl CD69 activation marker and IL-2 surface receptor analysis
Roth, Karlsruhe, Germany), or different concentrations of Cultured cells (24 or 48 h) were washed with PBS
Equisetum arvense extract. After cultivation, the cells were and stained with FITC-labeled anti-human CD69 and
assessed in bioassays as described in the text. PE-labeled anti-human CD25 mAbs (both eBioscience,
Frankfurt, Germany) for 15 min at 4°C. Next, the cells
were washed twice with PBS, resuspended and transferred
Cell division tracking using CFSE into FACS vials. The expression of CD69 and IL-2 surface
PBMC were harvested and washed twice in cold PBS be- receptor α-chain CD25 was measured by FACS analysis
fore they were resuspended in PBS at a concentration of using a FACSCalibur instrument.
5 × 106 cells/mL. CFSE (carboxyfluorescein diacetate suc-
cinimidyl ester, 5 mM; Sigma, Taufkirchen, Germany) was Determination of cytokine production
added in 1/1000 dilution and the PBMC were incubated Cells were treated for 36 h and were restimulated with
for 10 min at 37°C. The staining reaction was stopped by PMA (50 ng/mL) and ionomycin (500 ng/mL) (both from
washing twice with complete medium. Afterwards, the cell Sigma-Aldrich, Taufkirchen, Germany) for additional 6 h.
division progress was analyzed using flow cytometry. Cytokines were measured using intracellular cytokine ana-
lysis. For this purpose, the cells were surface-stained with
anti-human CD8 mAbs (eBioscience), fixed, and perme-
Determination of apoptosis and necrosis using annexin V abilized using 4% paraformaldehyde (Sigma-Aldrich) and
and propidium iodide staining Perm/Wash solution (Becton Dickinson, Franklin Lakes,
PBMC were cultured in the presence of Equisetum arvense NJ), followed by staining with PE-conjugated anti-human
extract for 24 or 48 h, respectively, and the levels of apop- IFN-γ mAb or anti-human TNF-α mAb (both from
tosis and necrosis were determined using Annexin V-FITC eBioscience), respectively. Samples were analyzed with a
apoptosis detection kit (eBioscience, Frankfurt, Germany BD FACSCalibur flow cytometer using BD CellQuest Pro
or BD Bioscience, Heidelberg, Germany) according to the Software.
manufacturer’s instructions. After annexin V staining, pro-
pidium iodide solution (PI; eBioscience or BD Bioscience) Analysis of data
was added and the cells were incubated in the dark, For statistical analysis, data were processed with Microsoft
followed by a flow cytometric analysis to determine the Excel and IBM SPSS software 20.0. Values are presented
amount of apoptosis and necrosis. We used CPT (100 μM) as mean ± SD for the indicated number of independent
and Triton-X 100 (0.5%) as positive controls for apoptosis experiments. Statistical significance was determined by
and necrosis, respectively. one-way ANOVA followed by Dunnett’s post hoc pairwise

15

2
8
1 14
9 13 18
4 6
3 11 12
5 7 10 16 17 19

Figure 1 Secondary plant metabolites identified in the Equisetum arvense extract. The herbal preparation was analysed by LC-MS and the
tentatively identified compounds are labelled in the given HPLC chromatogram showing the DAD response at 340 ± 2 nm: monocaffeoyl-tartaric
acid isomer (1 and 2), monoferuloyl-tartaric acid isomer (3 and 7), kaempferol-3-O-sophoroside-7-O-glucoside (4), quercetin-3,7-di-O-glucoside
(5), caffeoyl-malic acid (6), kaempferol-3,7-di-O-glucoside (8), kaempferol-3-O-rutinoside-7-O-glucoside (9), quercetin-3-O-sophoroside (10),
4-coumaric acid (11), kaempferol-3-O-sophoroside (12), caffeic acid methyl ester (13), protogenkwanin-4’-O-glucoside (14), quercetin-3-O-glucoside
(15), apigenin-O-glucoside (16), kaempferol-3-O-glucoside (17), dicaffeoyl-tartaric acid (18), and genkwanin-O-glucoside (19). The corresponding
MS-data are provided in Additional file 1: Table S1.
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comparisons. For estimation of the inhibitory con- and other polar phenolics. First, an HPLC method was de-
centration (IC50), a probit regression model for the veloped to separate the phenolic compounds in the extract
dose-response curve was used and data were proc- (Figure 1), which were then tentatively identified by LC-
essed by ToxRat® software. The asterisks represent MS with an ESI-ion trap instrument (Additional file 1:
significant differences from controls (*P < 0.05, **P < 0.01, Table S1). Major phenolic constituents of the Equisetum
***P < 0.001). arvense extract were found to be various mono-, di-, and
triglycosides of kaempferol, quercetin, apigenin, genkwa-
Results nin and protogenkwanin, as well as some hydroxycin-
Phytochemical analysis of the Equisetum arvense extract namic acid derivatives, most prominently mono- and
There is no clear correlation between traditional use of dicaffeoyl-tartaric acid. The presence of quercetin-3-O-
this herbal drug and any of its constituent in particular; glucoside (isoquercitrin) and kaempferol-3-O-glucoside
however, the Ph. Eur. prescribes a minimum of 0.3% of was confirmed by comparison of the retention times, UV-
total flavonoids expressed as isoquercitrin in the dried and MSn-spectra with reference compounds. Finally, we
drug [6]. Consequently, we also focused our phytochemical determined the total flavonoid content of the Equisetum
analysis of the Equisetum arvense extract on flavonoids arvense extract by a spectrophotometric assay and the

Figure 2 Effects of Equisetum arvense on proliferation of primary human lymphocytes. CFSE-labeled PHA-L (10 μg/mL)-activated human
PBMC (105) were treated with medium, CsA (10 μg/mL) or increasing concentrations of Equisetum arvense (0.05-0.8 μg/mL). Cell division analysis
was assessed by flow cytometry and depicted as representative dot plots (A) for bulk lymphocytes. Results are summarized in (B) and data are
presented as mean ± SD of five independent experiments and donors. n.d. = no detection. The asterisks (*P < 0.05, **P < 0.01, ***P < 0.001)
represent significant differences from activated untreated controls (CTRL =100%).
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concentration of isoquercitrin by an HPLC-DAD method and necrosis, we used camptothecin and Triton-X 100, re-
(see Additional file 1). spectively. As shown in Figure 3, positive controls signifi-
cantly increased the amount of apoptotic+ (white bars;
Equisetum arvense decreased proliferation of mitogen- 156% ± 22) and necrotic+ (black bars; 3962% ± 725) cells.
activated human lymphocytes Equisetum concentrations had no significant influence on
In order to analyze the immunomodulatory activity of the induction of apoptosis or necrosis compared to stimu-
Equisetum arvense, we evaluated the effects on cell growth lated untreated cells alone (=100%).
and performed proliferation assays with increasing con-
centrations of the Equisetum extract (0.05-0.8 μg/mL) Equisetum arvense influences cell proliferation capacity
using CFSE-labeled mitogen (PHA-L)-activated lympho- through partial inhibition of lymphocyte activation and
cytes (Figure 2). The CFSE dye is inherited from daughter IL-2 biology
cells after cell division and each dividing cell loses fluores- A reduced proliferative capacity could be mediated through
cence intensity without affecting cell viability. The flow cy- inhibition of T cell activation. Figure 4 depicts the respect-
tometry analysis demonstrated that the proliferation of ive experiments and confirms that CsA-treated T-cells ab-
CFSE+ mitogen-activated lymphocytes was strongly inhib- rogate expression of CD69 completely (Figure 4B: 0.5% ±
ited to levels of no detection in the presence of the positive 1.1 and Figure 4C: no detection). Equisetum decreased
control cyclosporine A (CsA) compared to stimulated cells the expression of CD69 (0.05 μg/mL: 95% ± 6.2; 0.1 μg/mL:
alone (=100%). The presence of Equisetum concentration- 91% ± 6; 0.2 μg/mL: 86% ± 7.5; 0.4 μg/mL: 78% ± 8.9;
dependently (0.05 μg/mL: 95% ± 3.2; 0.1 μg/mL: 89% ± 6.6; 0.8 μg/mL: 71% ± 9.7) at early (Figure 4A and B) but not
0.2 μg/mL: 86% ± 5.1; 0.4 μg/mL: 78% ± 9.8; 0.8 μg/mL: at late (Figure 4C) time points compared to controls
54% ± 29.8) decreased the proliferative capacity of human (=100%) in a concentration-dependent manner, reveal-
immunocompetent cells compared to stimulated cells ing that Equisetum exerts its anti-proliferative effect
alone (=100%) with an IC50 value of 1.09 μg/mL. through inhibition of cell activation.
T cell proliferation is also initiated by activation and
Equisetum arvense-mediated inhibition of proliferation is expression of the autocrine growth factor interleukin-2
not mediated through apoptosis or necrosis induction (IL-2). This promotes interaction with the receptor CD25,
In a further step, we aimed to identify the mechanism be- which is up-regulated on the surface of activated T-cells
hind the reduced cell proliferation observed, and quantified [24] and by release of endogenous IL-2. Consequently, the
apoptotic and necrotic effects on activated lymphocytes in influence of the Equisetum arvense extract on IL-2-
the presence of increasing concentrations (0.05-0.8 μg/mL) receptor expression and IL-2 secretion was also analyzed
of the Equisetum extract. As positive controls for apoptosis (Figure 5). The results demonstrated that the Equisetum

400
3963

350 ***
% necrosis

300
(to control)

250

200 ***
% apoptosis

150

100

50

0
Stimulation - + + + + + + + +
CPT - - + - - - - - -
T-x - - - + - - - - -
Equisetum - - - - 0.05 0.1 0.2 0.4 0.8
(µg/mL)

Figure 3 Effects of Equisetum arvense on apoptosis and necrosis induction in primary human lymphocytes. 105 PBMC were cultured for
72 h and left non-stimulated or PHA-L-activated (10 μg/mL) in the presence of medium (CTRL =100%) or in the presence of different Equisetum
arvense concentrations (0.05-0.8 μg/mL). Next, the cells were stained with annexin V and propidium iodide and analyzed using flow cytometry.
CPT (100 μM) and Triton-X100 (0.5%) were used as apoptosis and necrosis control, respectively. Results from analysis of apoptotic (white bars)
and necrotic (black bars) cells are summarized and presented as mean ± SD of four independent experiments and donors. The asterisks represent
significant differences from non-treated stimulated cells alone (***P < 0.001).
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A
CTRL CsA

CD69
61 39 0 0

0.05 0.1 0.2 0.4 0.8

59 31 61 31 63 33 61 31 59 31

CD8
B 120

100 **
*** ***
% CD69 + CD8+ lymphocytes (to control)

80

60

40

20
***
0
C 120

100
**
80

60

40

20 ***
n.d.
0

Stimulation + + + + + + +

CsA - + - - - - -

Equisetum - - 0.05 0.1 0.2 0.4 0.8

(µg/mL)
Figure 4 Effects of Equisetum arvense on activation of primary human lymphocytes. Stimulated CD8+ T cells were cultured in the presence
of medium, CsA (10 μg/mL) or increasing concentrations (0.05-0.8 μg/mL) of the Equisetum extract. Cells were analyzed for CD69 expression using
FITC-labeled anti-human mAbs. (A) Representative dot plots are shown and data were summarized from experiments at early (B; 24 h) or late
(C; 48 h) time points from five independent experiments and donors. n.d. = no detection. The asterisks (**P < 0.01, ***P < 0.001) represent
significant differences from activated untreated controls (CTRL =100%).
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A 140

120

% IL-2-receptor + CD8+ PBMC (to control)

100

80

60

40

20
***
0
B 120

100
*
80

60

40

20
***
0

C 140

120
% IL-2 + CD8+ PBMC

100
* **
(to control)

80

60

40

20 ***
n.d.
0

Stimulation + + + + + + +

CsA - + - - - - -

Equisetum - - 0.05 0.1 0.2 0.4 0.8

(µg/mL)

Figure 5 Effects of Equisetum arvense on IL-2 biology of primary human lymphocytes. Medium-, CsA or Equisetum-treated activated (PHA-L:
10 μg/mL) lymphocytes were surface-stained with anti-human CD25 mAbs and were analyzed at early (A; 24 h) or at late (B; 48 h) time points.
For IL-2 production analysis, cells were treated for 36 h and were restimulated with PMA (50 ng/mL) and ionomycin (500 ng/mL) for additional
6 h followed by surface-staining with anti-human CD8 mAbs and intracellular cytokine assay with PE-conjugated anti-human IFN-γ mAbs or
anti-human TNF-α mAbs (C). Cells were analyzed using flow cytometry. Data were expressed and summarized as mean ± SD of five independent
experiments and donors. The asterisks (*P < 0.05, **P < 0.01, ***P < 0.001) represent significant differences from activated untreated controls
(CTRL =100%).

extract investigated has little impact on the expression of Equisetum arvense slightly affects effector function of
the IL-2 receptor at late time points at high (0.8 μg/mL: activated human lymphocytes
90% ± 14) concentrations only (Figure 5B). It also in- Consequently, it was of interest whether the Equisetum
hibits production of IL-2 (Figure 5C; 0.4 μg/mL: 70% ± extract under investigation only inhibits proliferation of
14; 0.8 μg/mL: 55% ± 35) compared to controls (=100%). lymphocytes or also affects lymphocyte polyfunctionality,
These data indicate that Equisetum arvense reduces which would be directly related to changes in the produc-
proliferation of immunocompetent cells by inhibiting tion of interferon-gamma (IFN-γ) and tumor necrosis
cell activation and IL-2 biology at more than one site. factor-alpha (TNF-α). The production of both mediators
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in the presence of CsA and Equisetum arvense was ana- arvense extract was not mediated by apoptosis and necro-
lyzed using a flow cytometry-based intracellular cytokine sis. Immunosuppressive effects of Equisetum arvense have
approach (Figure 6). The results showed a slight inhibition also been observed by other groups, however, their investi-
of the IFN-γ- (Figure 6A; 0.4 μg/mL: 63% ± 26; 0.8 μg/mL: gations were only done with human cancer cell lines
70% ± 18) as well as TNF-α- (Figure 6B; 0.8 μg/mL: 82% ± [11,25]. T-cell proliferation is a process initiated by ligation
22) production of activated T cells at high Equisetum con- of the T-cell receptor to antigens that triggers a complex
centrations, whereby the presence of CsA reduced secre- T-cell receptor signalling pathway. During this process,
tion of these parameters to undetectable levels. the human transmembrane C-Type lectin protein CD69 is
expressed on the surface of activated T-cells [24]. CD69
Discussion is an early activation marker that is expressed at high
Extracts of Equisetum arvense (common horsetail) have a amounts immediately after activation, and gene as well
long tradition in the treatment of inflammatory disorders, protein expression of this marker rapidly decreases 24 h
for which reason we were interested in clarifying whether after stimulation. We found a decrease in the activation
there is a rational basis for its use. To this end, we charac- status of stimulated T-cells after stimulation, whereby
terized a standardized common horsetail extract in cell- the effects of Equisetum are smaller than that of CsA. T-
based assays using activated immunocompetent cells. lymphocyte proliferation is further initiated by activation
We found a concentration-dependent inhibition of and expression of the autocrine growth factor interleukin-
mitogen-activated lymphocyte proliferation with an IC50 2 (IL-2), which promotes interaction with the receptor
value of 1.09 μg/mL. Furthermore, our data indicate that CD25 that is up-regulated on the surface of activated
the inhibition of proliferation induced by the Equisetum T-cells [24] and by release of endogenous IL-2. The

A 140

120
% IFN-γ + CD8+ PBMC

100 ** *
(to control)

80

60

40

20 ***
n.d.
0
B 140

120 *
% TNF-α + CD8+ PBMC

100
(to control)

80

60

40

20 ***
0
Stimulation + + + + + + +

CsA - + - - - - -

Equisetum - - 0.05 0.1 0.2 0.4 0.8

(µg/mL)

Figure 6 Effects of Equisetum arvense on effector function of primary human lymphocytes. Activated (PHA-L; 10 μg/mL) human PBMC
(105) were treated with medium, CsA (10 μg/mL) or increasing concentrations of Equisetum arvense (0.05-0.8 μg/mL) and were cultured for 36 h
followed by a restimulation period with PMA (50 ng/mL) and ionomycin (500 ng/mL) for additional 6 h. Next, cells were surface-stained with
anti-human CD8 mAbs and intracellular cytokines were detected using PE-conjugated anti-human IFN-γ or anti-human TNF-α mAbs. n.d. = no
detection. The asterisks (*P < 0.05, **P < 0.01, ***P < 0.001) represent significant differences from activated untreated controls (CTRL =100%).
Gründemann et al. BMC Complementary and Alternative Medicine 2014, 14:283 Page 9 of 10
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expression of the IL-2 receptor was indeed slightly affected cells, thereby providing a potential mechanism that ex-
in the presence of Equisetum. Over time, however, inhib- plains its traditional use in the treatment of inflammatory
ition was smaller than that of CsA, which specifically re- disorders. However, further studies should be conducted
duces the early activation state of lymphocytes due to to prove its clinical potency.
suppression of the IL-2 receptor [26,27]. The data further
demonstrate that Equisetum reduces IL-2 cytokine pro- Additional file
duction. Because IL-2 is pivotal for lymphocyte prolifera-
tion, inhibition of its production may at least partially Additional file 1: Quantification of isoquercitrin and total
explain the immunosuppressive effects of Equisetum in flavonoids in the Equisetum arvense extract.

these experiments. In addition, a slight inhibition of IFN-

γ- and TNF-α-production of activated T cells could be Competing interests
The authors declare that they have no competing interests.
shown. Thus, we can conclude that the horsetail extract
interferes with T-cell polyfunctionality, which results in di- Authors’ contribution
minished proliferation of immunocompetent cells. Conceived and designed the experiments: CG, MZ, RH. Performed the
The stems of Equisetum arvense contain high amounts of experiments: CG, KL, BS, MGK, MZ. Analyzed the data: CG, KL, BS, MGK, MZ,
RH. Contributed reagents/materials/analysis tools: CG, MZ, MGK. Wrote the
minerals, in particular silicic acid and silicates (5-8%), po- paper: CG, MZ, RH. All authors read and approved the final manuscript.
tassium and calcium, various flavonoids (0.2-0.9%) and
phenolic acids, the phenolic petrosins onitin and oniti- Acknowledgments
Consumables and fees for writing the manuscript were supported by
9-O-glucoside, equisetumpyrone (only in the very early Weleda AG. The funders had no role in study design, data collection and
developmental stages), triterpenoids and phytosterols. They analysis, decision to publish, or preparation of the manuscript.
also contain low amounts of essential oil, the main cons-
Author details
tituents being hexahydrofarnesyl acetone, cis-geranyl acet- 1
Center for Complementary Medicine, Department of Environmental Health
one, thymol, and trans-phytol as well as a few other Sciences, Medical Center, University of Freiburg, Breisacherstr. 115B, 79106
compounds [28,29]. Especially, the considerable amounts of Freiburg, Germany. 2Department of Pharmacognosy, University of Vienna,
Althanstrasse 14, A-1090 Vienna, Austria.
polyphenols and several compounds of this diverse group
of phytochemicals have attracted interest in the past due to Received: 12 March 2014 Accepted: 22 July 2014
their presumptive physiological activities. However, there is Published: 4 August 2014
no clear correlation between the traditional use of this
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