Phytomedicine 60 (2019) 153013

Contents lists available at ScienceDirect

Phytomedicine
journal homepage: www.elsevier.com/locate/phymed

Synergy quantifications to identify individual contributions of combination T

partners to the overall activity – The example of STW 5
Gudrun Ulrich-Merzenicha, , Lisa Welslaua, Heba Aziz-Kalbhennc, Olaf Kelberc,
⁎

Anastasiia Shcherbakovaa,b
a
Medical Clinic III, UKB, University of Bonn, Venusberg-Campus 1, Building 26, Laboratories UG 65/69, 53127 Bonn, Germany
b
Volga State University of Technology, Lenin sq. 3, 424020 Yoshkar-Ola, Russia
c
Research & Development, Steigerwald Arzneimittelwerk GmbH, Bayer Consumer Health, Havelstr. 5, 64283 Darmstadt, Germany

ARTICLE INFO ABSTRACT

Keywords: Background: The rationale of combinations of plant extracts is often questioned. The common argument for
Multitargeting combinations is a higher efficacy of the combination partners by multitargeting and the elimination of adverse
Synergy events.
Combination index Aim: STW5, a well-known fixed herbal multicomponent preparation, is recommended in the German treatment
IL-8
guidelines for functional gastrointestinal diseases. The study assessed effects of STW5, its single plant compo-
Ca2+
nents and combinations thereof on different targets to identify synergistic, additive or antagonistic effects of the
Reflux-esophagitis
Functional gastrointestinal diseases combination partners.
Study Design/Methods: STW5, its nine components and triple combinations thereof were investigated in two in
vitro models – human esophageal epithelial cells (Het1A) and intestinal smooth muscle cells (HISMC) – in
comparison to Omeprazole (OM) for the release of interleukin 8 (IL-8) as surrogate for inflammation and of Ca2+
as surrogate for motion, under non-inflammatory and inflammatory (Capsaicin 80 µM (CAP)) conditions. The
combination index (CI) of triple combinations was calculated to assess synergistic, antagonistic and additive
effects.
Results: In Het-1A cells, STW5 showed, under non-inflammatory as well as inflammatory conditions, releases of
IL-8 (49.3 ± 4.2 pg/ml, 33.7 ± 2 pg/ml) comparable to the untreated control (46.3 ± 4.8 pg/ml). CAP in-
creased IL-8 releases to 85.8 ± 14 pg/ml (p < 0.005). Among the single plant extracts the Iberis amara extract
(IBE) induced high IL-8 releases under non-inflammatory (441 ± 177 pg/ml) and inflammatory
(625 ± 121 pg/ml) conditions. The Silybum marianum (L.) extract (SM) reduced releases up to 20.1 ± 8 pg/ml
(inflammation). The CI-values of triple combinations with IBE ranged from high synergy (CI<0.03) to antag-
onism (CI:480). Within the triple combinations SM was the most effective combination partner to reduce IL-8.
The combination of Angelica archangelica (L.)/Carum carvi (L.) was also effective. In HISMCs, STW5 induced
concentration dependent higher Ca2+-releases. Only Melissa officinalis (L.) (MO) induced high Ca2+- releases in
HISMCs.
Conclusion: In Het-1A, STW5 inhibited Il-8 releases, although one of its components (IBE) stimulated IL-8
strongly. The combination partners in STW5 assured an overall marked anti-inflammatory action. In the triple
combinations SM was identified as most important combination partner for the IL-8 reduction. CI-measurements
can support the identification of active combination partners in a multicomponent preparation and can give
directions towards the search for multitarget effects.

Abbreviations: A, Angelica; BEGM, Bronchial epithelial cell medium; CAP, Capsaicin; CI, Combination index; CDCA, Chenodeoxycholic acid; C. majus, Chelidonium
majus (L.); C. carvi, Carum carvi (L.); DMEM, Dulbecco's modified Eagle's medium; fa, fractional inhibition; FD, functional dyspepsia; FGID, Functional gastrointestinal
disease; FKS, foetal calf serum; G. glabra, Glycyrrhiza glabr (L.); GTRG, German treatment recommendation guidelines; GERD, gastroesophageal reflux disease; Het-1a,
Human esophageal epithelial cells; HISMC, Human intestinal smooth muscle cells; I, Iberis; IBE, Iberis amara extract; IBS, irritable bowel syndrome; IL-1ß, Interleukin
1ß; IL-8, interleukin 8; M. recutita, Matricaria recutica (L.); M. piperitae, Menthae piperitae (L.); M, Melissa; MO, Mellissa officinalis (L.); OM, Omeprazole; S, Silybum; SM,
Silybum marianum (L.) extract; TNF-α, Tumor necrosis factor alpha
This paper is dedicated to Prof. Dr. hc. mult. Hildebert Wagner at the occasion of his 90th birthday.
⁎
Corresponding author.
E-mail address: gudrun.ulrich-merzenich@ukbonn.de (G. Ulrich-Merzenich).

https://doi.org/10.1016/j.phymed.2019.153013
Received 4 April 2019; Received in revised form 20 June 2019; Accepted 2 July 2019
0944-7113/ © 2019 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
G. Ulrich-Merzenich, et al. Phytomedicine 60 (2019) 153013

Introduction TREM-1 signaling pathway play an important role in the pathogenesis

STW5, a herbal multicomponent preparation for the management of
functional gastrointestinal diseases (FGIDs), is known for its multitarget
effects (Allescher, 2006; Kelber et al. 2006; Allescher and Wagner,
2007; Khayyal et al. 2007; Vinson, 2009; Abdel-Aziz et al., 2010, 2017)
(Fig. 1A and B). The German treatment recommendation guidelines
(GTRG) recommend it for all subtypes of irritable bowel syndrome
(IBS), the most common and best characterized FGID (Abdel-Aziz et al.,
2017), and mention it as treatment option for functional dyspepsia
(FD).
STW5 is a combination of nine different plant extracts (Table 1).
The rationale for this combination has often been discussed and the
common argument is a higher efficacy of the combination partners by
multitargeting. STW5 itself and its single components have been thor-
oughly investigated over the past 30 years demonstrating, that the
combination partners indeed contribute with different pharmacological
activities to the overall mode of action (Fig 1).
We reported earlier that STW5 targets the fatty acid receptor GPR
84 (Abdel-Aziz et al., 2015) and multiple chemokine families on
genome and proteome levels in a murine model of gastroesophageal
reflux disease (GERD). We proposed that interleukin 8 (IL-8) and the
of GERD. This is supported by clinical findings. In FGIDs, IL-8 appears
to be not only associated with the recurrence of GERD (Kahrilas and
Lee, 2005), but is also regarded as one of the central cytokines to
promote inflammation in the upper GI-tract (Richter JE, 2007; de Souza
et al., 2009). Being part of the inflammatory process, IL-8 as a small
heparin binding protein is especially known for mediating the activa-
tion and migration of neutrophils from blood into tissues. We therefore
selected IL-8 as biomarker for inflammation to examine the contribu-
tion of the individual plant extracts and triple combinations of these to
the overall activity of STW5 in Het-1A cells. We chose Het-1A cells since
the prevalence of RE has been directly related to IL-8- concentrations in
the esophageal mucosa (Kahrilas and Lee, 2005; Richter JE, 2007;
de Souza et al., 2009) and STW5 could act directly on these cells in vivo.
Several investigations have been dedicated to the influence of STW5
on motility in the gastrointestinal tract (Wegner and Wagner, 2006;
Schehmann et al., 2006; Abdel-Aziz et al., 2017). Particularly intriguing
was the differential influence of STW5 on the contractility of the sto-
mach. In the corpus STW5 leads to contraction, whereas in the fundus
relaxation is promoted, supporting food transport through the stomach
(Schehmann et al., 2006). We therefore investigated in a second cellular
model the Ca2+-release – an essential component of the coupling of

Fig. 1. Contribution of single constituents of STW 5 to its multitarget activity in different pathomechanisms of (A) functional dyspepsia (epigastric pain syndrome)
and (B) irritable bowel syndrome. The analysis is based on multi-step clustering of published pharmacological data (adapted from Abdel-Aziz et al. 2017). Relative
effect strengths of each extract are presented in the heat-map as color intensity (light yellow for weak effects to dark green for strong effects). (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article.)

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G. Ulrich-Merzenich, et al. Phytomedicine 60 (2019) 153013

Table 1
Composition of STW5.
Extract Single plant Lyophilisate (mg/ml) Percent of STW5

Bot. Name Engl. Name

STW 5 59 100
STW 6 Iberis amara (L.) Bitter candy tuft 18 15
STW 5-KII Menthae piperitae (L.) Peppermint 88 5
STW 5-KIII Matricaria recutita (L.) Chamomille 58 20
STW 5-KIV Glycyrrhiza glabra (L.) Liquorice 100 10
STW 5-KV Angelica archangelica (L.) Angelica 99 10
STW 5-KVI Carum carvi (L.) Caraway 47 10
STW 5-KVII Silybum marianum (L.) Gaertn. Milk thistle 20 10
STW 5-KVIII Melissa officinalis (L.) Mellissa 71 10
STW 5 KIX Chelidonium majus (L.) Greater celandine 75 10

excitation and contraction – as indicator for the influence of STW5 on
motility in human intestinal smooth muscle cells (HISMCs).
In summary, we estimated the IL-8 release in Het-1A cells and the
Ca2+-release in HISMCs under the influence of STW5, its single plant
extracts and triple combinations of the plant extracts under non-in-
flammatory and inflammatory conditions. The combination index (CI)
(Chou and Talalay, 1984; Chou, 2006; Fu et al., 2016; Chou et al.,
2019) was determined to identify potential „lead“-extracts and or ef-
fective combinations.
Materials and methods
Material
Drugs
STW5 (Iberogast®) is a fixed herbal combination preparation con-
sisting of well-characterized hydroethanolic extracts (Kroll and Cordes,
2006; Wegener and Wagner, 2006) of Iberis amara L. (15%), Melissa
officinalis L. (10%), Matricaria recutita L. (20%), Carum carvi L. (10%),
Mentha piperita L. (5%), Angelica archangelica L. (10%), Silybum mar-
ianum L. Gaertner (10%), Chelidonium majus L. (10%) and Glycyrrhiza
glabra L. (10%). STW5 was provided by Steigerwald Arzneimittelwerk
GmbH, Bayer Consumer Health. Capsaicin (CAP) and chenodeoxycholic
acid (CDCA) were procured from Sigma-Aldrich. Other chemicals were
of analytical grade.
STW5 (1–10 µl/ml), its single extracts (Table 1) alone or under the
addition of CAP (80 µM) or CDCA (200 µM). The single plant extracts
were prepared dose-equivalent to STW5 (Table 1). If not stated other-
wise, a minimum of 3 independent experiments was performed in du-
plicate or triplicate each.
Cell viability
Viability was tested by a Resazurin-based assay according to the
instructions of the manufacturer (Tox8, Sigma Aldrich) as described
earlier (Ulrich-Merzenich et al., 2017). Each plant extract was tested in
4 concentrations (equivalent to 1 µl, 3 µl, 5 µl, 10 µl/ml of STW5) under
non-inflammatory and inflammatory conditions (CAP 80 µM, CDCA
200 µM). Omeprazole (OM) was used in concentrations of 10–100 µg/
ml. The experimental conditions did not influence cell viability (Data
not shown).
Cytokine determination
The IL-8 release into the culture medium was measured by ELISA
(Human CXCl8/IL-8 ELISA, R&D systems) according to the instructions
of the manufacturer.
Ca2+ release
The calcium release of HISMC was estimated with the Screen
Quest™ Fluo-8 Calcium Assay Kit (AAT Bioquest ™, Inc) according to the
instructions of the manufacturer.

Cells Synergy quantifications

The normal human esophageal squamous cell line Het-1A was
purchased from ATCC, LGC Standards GmbH, Wesel, Germany.
Originally derived from human normal esophageal autopsy tissue by
transfection with plasmid pRSV-T, these cells retain epithelial mor-
phology, stain positively for cytokeratins and remain non-tumorigenic.
The HIMSC were from SMCN; Science Cell Research Laboratories. They
had been isolated from human intestine.
Cell culture
Cells were cultured in bronchial epithelial cell medium BEGM
(BulletKit; Lonza, Cologne, Germany) in cell culture flasks precoated
with a mixture of 0.01 mg/ml fibronectin, 0.03 mg/ml bovine collagen
type I and 0.01 mg/ml bovine serum albumin (Biochrom, Berlin,
Germany) at 37 °C in a 5% CO2-humidified atmosphere. Cells of passage
numbers 50–67 were used.
HIMSC were cultured in Dulbecco's modified Eagle's medium
(DMEM, Life Science Technologies), 10% foetal calf serum (FKS Gold,
PAA Laboratories) and antibiotics (Penicillin 200 IU/ml, Streptomycin
100 µg/ml). Passages 2–15 after thawing were used.
Prior to the experiments Het-1A and HISMCs were synchronized.
Cells were washed twice with PBS. Culture medium without FKS was
added for 24 h for the synchronization of the cell cycles. Thereafter Het-
1A cells (2 × 103) or HISMCs (2 × 103) were treated (18 hrs) with
For the quantification of synergistic, additive and antagonistic ef-
fects of the different plant extract combinations the Chou–Talalay
combination index (CI) method was applied (Chou-Talalay, 1984, Chou
et al., 2019). The concentrations were chosen based on the composition
of STW5 in a range of 5.9 µl–59 µl/ml for STW5. The single plant ex-
tracts were prepared in dose-equivalents to STW5 (Table 1). Combi-
nations were tested in a minimum of two independent experiments with
two duplicates each. The CI-value was calculated using the CompuSyn
software. The software for the calculation of the CI is based on the
following equations (Chou, 2006; Fu et al., 2016; Chou et al., 2019):
( D )1 (D ) 2 (D )1 (D ) 2
CI = + = +
(D x )1 (D x ) 2 (Dm)1 [fa /(1 fa )]1/ m1 (Dm)2 [fa /(1 fa )]1/ m2
(Dx)1 is the dose of the drug (extract) D1 alone that inhibits a system by
x% (Here: IL-8 release of CAP-stimulated cells). (Dx)2 is the dose of the
drug (extract) D2 alone that inhibits a system by x%. (D)1 and (D)2 are
the doses of the drugs D1 and D2 in the combination which inhibit a
system by x%. Dm is the median-effect dose, fa is the fractional in-
hibition at x% inhibition, m is the slope of the median-effect plot which
depicts the shape of the dose-effect plot (Chou, 2006). A rearrangement
of the median-effect equation allows the calculation of the dose:
D = Dm [fa /(1 fa )]1/m

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G. Ulrich-Merzenich, et al.
The CI for ternary combinations can be calculated from the general
equation for n-drug combinations at x% inhibitions:
n (D ) j
n (CI)
x =
j=1 (Dx ) j
The definition of synergism is CI <1, antagonism is CI >1, and
additive effect is CI = 1. Since our aim was to identify active combi-
nation partners in the fixed combination of STW5, we did not report
data on dose reductions.
Substraction assay
A substraction assay of the plant extract combinations was performed
to support the identification of the plant extract(s) responsible for the
tested effect. Cells were stimulated as described under “Cell culture”
with STW5 alone as well as with STW5 without the Iberis amara extract
and in each subsequent step one additional extract was “removed” up to
the last two extracts (Table 3). The volume of the “removed” plant
extract was substituted by medium. The IC50 – values were determined
for the combinations. A minimum of 2 independent assays with dupli-
cates each was performed.
Statistics
Data were obtained from a minimum of three independent experi-
ments performed in triplicate for each data point if not mentioned
otherwise. The differences between the groups were analysed using the
Student's T-test in case of normally distributed data. Otherwise the
Wilcoxon signed rank test was used.
Results
IL-8 release in Het1A cells
The spontaneous release of IL-8 was 46.3 ± 4.8 pg/ml (Fig. 2A).
The highest release of IL-8 was induced by the Iberis amara extract (IBE)
(441 ± 177 pg/ml), whereas STW5 lead to releases up to
49.3 ± 4.2 pg/ml. Other extracts like Silybum marianum (L.) reduced
releases up to 20.1 ± 8 pg/ml (Fig. 2A). The IL-8 releases induced by
all plant extracts constituting STW5 are shown in Fig. 2A.
The stimulation of Het-1A cells with Capsaicin increased the IL-8
release to 85.8 ± 14 pg/ml (p < 0.005). Under this condition IBE
stimulated an IL-8 release up to 625 ± 121 pg/ml. STW5 reduced IL-8
releases in the presence of Capsaicin up to 33.7 ± 2 pg/ml (Fig. 2B).
In another set of experiments Het-1A cells were stimulated with
CDCA to induce inflammation (Fig. 3A, B). CDCA increased IL-8 re-
leases and STW5 reduced the same (Fig. 3A). The IL-8 releases induced
by the different plant extracts constituting STW5 are shown in Fig. 3B.
Also with CDCA as inflammatory stimulator IBE increased the IL-8 re-
lease whereas STW5 inhibited the same (Fig. 3B).
Combination Index assessments of triple combinations
STW5 is a combination of nine different plant extracts. We initially
intended to identify the three most active plant extracts to reduce in-
flammation and to determine their synergistic activity. When we dis-
covered that IBE alone increased substantially the IL-8 release, but
STW5 did the opposite, we changed our strategy and investigated which
plant combinations would efficiently reduce the IL-8 release induced by
IBE. The CI-values of the tested triple combinations ranged from very
strong synergy (CI<0.03) to very strong antagonism (CI:480) (Table 2).
Silybum marianum (L.) was the most effective combination partner in
the inhibition of IL-8 release within the triple combinations. The
combination of Angelica archangelica (L.)/Carum carvi (L.) was also ef-
fective in reducing the IL-8 release induced by IBE. The combinations of
Silybum marianum (L.) and Glycyrrhiza glabra (L.); of Angelica arch-
angelica (L.) and Carum carvi (L.); and of Silybum marianum (L.) and
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Phytomedicine 60 (2019) 153013
Carum carvi (L.) showed strong synergies in the triple combinations also
at high effect levels (Table 3). The (r)-values of the curves for the in-
dividual drugs and the ones of the combinations acting synergistically
were ≥ 0.9, indicating an acceptable conformity with the mass-action
law. This was not the case for all of the tested combinations.
Substraction assay
In a second approach, the substraction assay, we also aimed at
identifying the plant extracts that are responsible for the overall IL-8
reduction seen for STW5. Interestingly, when removing Silybum mar-
ianum (L.) from the combination, the IC50 for the reduction of IL-8
dramatically increased (Table 3).
Ca2+ -release in HISMC
We investigated the capability of STW5 and its single components to
modulate the Ca2+-release in HISMCs in vitro under non-inflammatory
and inflammatory conditions. Results are shown in Fig. 4. STW5 itself
stimulated a Ca2+-release in HISMCs. The only single plant extract
clearly stimulating a Ca2+ release was Melissa officinalis (L.). (Fig. 4),
however to a lower extent than STW5 and only at the highest con-
centration under non-inflammatory as well as inflammatory conditions
(Fig. 4). All other concentrations did not show significant differences.
Moreover, it seems that for concentrations of 1–5 ul/ml the effect of M.
officinalis (L.) is even lower than the effects of Mentha piperita (L.) and
Matricaria recutita (L.). However, a very small but significant increase in
the Ca2+ release was also observed for Mentha. piperita (L.) and Ma-
tricaria recutita. The opposite was observed for Glycyrrhiza glabra, An-
gelica archangelica und Silybum marianum (Fig. 4). Therefore we tested
the triple combination of Mentha piperita, Matricaria recutita and Melissa
officinalis (Combination 1) as well as the triple combination of Glycyr-
rhiza glabra, Angelica. archangelica und Silybum marianum (Combination
2) (Fig. 5A, B). Combination 1 did increase the Ca2+ release, but not
comparable to STW5. However, compared to Melissa officinalis alone;
the Ca2+ release was slightly higher and concentration dependent
under inflammatory conditions. The response to combination 2 was less
stable (Fig. 5A and B).
Discussion
Our initial plan to first identify the three most active plant extracts
that reduce IL-8 releases and subsequently to quantify their combina-
tory mode of action by the CI-method needed to be modified since in
Het-1A-cells the Iberis amara (L.) extract substantially increased the IL-8
release instead of inhibiting it.
This shows primarily that the downregulation of IL-8 by STW5 in
Het-1A cells is not a simple addition of the dose reductions induced by
the contained individual plant extracts. It is predominantly another
example of current knowledge that cytokines are organized in networks
with feedback control mechanisms, a significant measure of re-
dundancy (Ulrich-Merzenich et al., 2012, 2017) and their combined
effect is therefore difficult to predict. In the intestinal immune system
cytokines are important signal transducing molecules that support the
maintenance of physiological functions. A reduction of IL-8 in epithelial
cells of the GI-tract will be protective against inflammation. The finding
that the IB-extract dramatically increased IL-8 release was not expected.
Therefore we searched for other plant extracts contained in STW5 that
could be responsible for the inhibition of IL-8 releases. We examined all
possible triple combinations with the IBE. Although the combination of
Iberis amara (L.) with Angelica archangelica (L.) and Carum carvi (L.) did
show a synergism in reducing IL-8, the plant extract involved in all
triple combinations that indicated synergism was Silybum marianum
(L.). When comparing our findings regarding Silybum marianum (L.)
with the most recent histogram (Fig. 1) on the modes of action of the
single components of STW5 for FD and the epigastric pain syndrome, it
G. Ulrich-Merzenich, et al. Phytomedicine 60 (2019) 153013

Fig. 2. (A) IL-8 release from Het-1A cells. Cells were incubated for 18 h with STW5, one of its constituents or omeprazole (10–100 µg/ml) as reference drug. The
control represents the IL-8 release of unstimulated cells. *: p < 0.05, **:p < 0.01, ***p < 0.001. Data were calculated vs.control. (B) IL-8 release from Het-1A cells
under Capsaicin-induced inflammation. The cells were incubated with STW5, its different constituents or Omeprazole (10–100 µg/ml) as reference drug for 18 h in
the presence of Capsaicin (80 µM). *: p < 0.05, **:p < 0.01, ***p < 0.001. Data are calculated vs. Capsaicin-stimulation. ++: p < 0.01 vs. control.

was intriguing to note that Silybum marianum is characterized by strong
anti-inflammatory and mucosa-protective properties. These pharmaco-
logical activities coincide highly with our findings.
Also Carum carvi (L.) and Matricaria recutita (L.) possess such
properties, but combined with other pharmacological activities. The
combination of Silybum marianum (L.), Carum carvi (L.) and Iberis amara
(L.) indicated synergism with low IC–values, supporting the importance
of Silybum marianum (L.) for the reduction of IL-8. The anti-in-
flammatory mode of action of Silybum marianum (L.) (Abenavoll et al.,
2018), Matricaria recutita (L.) (Ortiz et al., 2017) or Carum Carvi (L.)
was confirmed earlier in different in vitro and in vivo models (Abdel-
Aziz et al., 2017), in agreement with our findings. Anti-inflammatory
modes of action of the single plants Mentha piperita, Glycyrrhiza glabra
(L.), Melissa officinalis (L.), Chelidonium majus (L.), and also Iberis amara
(L.) have been demonstrated earlier and rather recently
(Okpanyi, 1994; Michael et al., 2009; Sun et al., 2014; Wei et al., 2018;
Liao et al., 2018).
Earlier experiments with Iberis amara (L.) demonstrated an inhibi-
tion of inflammation in rat small intestines (Michael et al., al.2006,
2009). This could be an indication for differences in species or more
likely for different effects of Iberis amara (L.) in the different regions of
the GI-tract. Therefore the existing evidence for an anti-inflammatory
effect of Iberis amara (Okpanyi, 1994; Reichling and Saller, 2006) may
not contradict our result. To our best of knowledge this is the first in
vitro study examining Iberis amara (L.) in human epithelial esophageal
cells. The overall anti-inflammatory activity of STW5 obviously persists
and it will be exciting to investigate further the regulation of the IL-8
release in epithelial cells in this model as well as the IL-8 regulation and
its role in inflammation of the upper GI-tract.
In HISMCs Melissa officinalis (L.) was the plant extract which, like
STW5, clearly increased the Ca2+-release. In comparison with the most
recent histogram (Fig. 1) summarizing the studies on STW5 and its
components, Melissa officinalis (L.) is assigned a strong ability to induce
(antrum) contractility. This corresponds with our findings. The only
plant extract which also possesses the same property is Chelidonium
majus (L.), however in combination with “acid regulation”, whereas
Melissa officinalis (L.) possesses an additional anti-inflammatory mode
of action. Interestingly, Melissa officinalis (L.) is also shown in the his-
togram (Fig. 1b) summarizing the properties of the components of
STW5 in relation to intestinal motility (Fig. 1b). Here Chelidonium majus

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G. Ulrich-Merzenich, et al. Phytomedicine 60 (2019) 153013

Fig. 3. (A) Effect of STW 5 on CDCA induced

IL-8 release from Het1A cells. STW5 reduces
concentration dependently the IL-8 release in
Het1A cells stimulated by CDCA. Het-1A cells
were incubated for 18 h with STW5 in the
presence of the inflammatory stimulant CDCA
(200 µM); each data point represents a
minimum of 5 independent experiments. *:
p < 0.05, **:p < 0.01, ***p < 0.001. Data are
calculated vs. CDCA-stimulation. +++:
p < 0.01 vs. control. (B) induced IL-8 release
from Het-1A cells. Het1A cells were incubated
for 18 h with STW5 or its single components in
the presence of the inflammatory stimulant
CDCA (200 µM). Each data point represents a
minimum of 3 independent experiments. *:
p < 0.05, **:p < 0.01, ***p < 0.001. Data are
calculated vs. CDCA-stimulation. ++:
p < 0.01 vs. control.

Table 2 Table 3
IC50 and IC75 – values of triple Extract-combinations and their combination Substraction assay.
indices (CI).
Plant extract EC50 (µl/ml)

STW5 10.99
STW5 –Iberis amara (C1) 15.33
C1 – Mentha piperitae (C2) 9.57
C2 – Matricaria recutita (C3) 22.63
C3 – Glycyrrhiza glabra (C4) 5.33
C4 – Angelica archangelica (C5) 6.32
C5 – Carum carvi (C6) 2.26
C6- – Silybum marianum (C7) 1091.25

The IC-values are reported as extract concentration equivalent to µl/ml of
STW5. The CI-values correspond with the IC55 and IC75-values. Grey shading
marks synergistic combinations.
(L) plays no role, again supporting our findings of a unique role of
Melissa officinalis (L.) with respect to contractility. The spasmolytic and
tonic effects of STW5 and its components, but also its regional effects,
are summarized in several reviews (Ammon et al., 2006;
Hohenester et al., 2004; Heinle et al., 2006). The finding that Melissa
officinalis in combination with Mentha piperita (L.) and Matricaria.
The table shows the IC-50 values for the IL-8 release of Het-1A cells
stimulated with STW5 and extract combinations thereof as indicated.
recutita (L.) increases the Ca2+ release of HISMCs, especially under
inflammatory conditions, indicates a supporting role of Mentha piperita
(L.) and Matricaria recutita (L.). The summarized findings in the histo-
gram (Fig. 1A) support this property for Matricaria recutita (L.), but not
for Mentha piperita (L.). However, Mentha piperita (L.) as well as Gly-
cyrrhiza glabra (L.) and Angelica archangelica (L.) have been related to
spasmolytic modes of action and thus target the “contractility” of a cell/
tissue.

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G. Ulrich-Merzenich, et al. Phytomedicine 60 (2019) 153013

Fig. 4. (A) Ca2+ release from HISMC in fold changes compared to the untreated control. HIMSC cells were stimulated with STW5, the single components of STW5 or
Omeprazole. Each data point represents 3 independent experiments. *p < 0.05; **p < 0.01, ***p < 0.001 vs. control. (B) Ca2+ release from HISMC in fold changes
of untreated control. Cells were simultaneously stimulated with Capsaicin (80 µM) and with the different plant extracts. Adding the inflammatory stimulator
capsaicin showed that STW5 and Melissa officinalis stimulated a higher Calcium release than Capsaicin alone. Each data point represents 3 independent experiments.
*p < 0.05; **p < 0.01, ***p < 0.001 vs. control.

Limitations
We did not investigate all possible combinations of the nine plant
extracts (dual, tetra, penta, hexa, hepta, or octo-combinations) for the
IL-8 release. The possibility that a tetra-, pental- or higher combination
would lead to different results is imaginable. However, the substractive
approach supports that SM, within the mixture and within the combi-
nation of Carum carvi (L.) and Angelica archangelica (L.), reduces IL-8
releases under non-inflammatory and inflammatory conditions. We also
tested only one sequence in the “substractive approach”. A different
sequence leading to different combinations than those which we
examined might yield another result. But the fact that two approaches
(CI measurements of the triple combinations and the substractive assay)
confirm the same plant (combinations) and coincide with the cluster-
analysis of the published data is a promising finding.
Investigating the combinations of nine plant extracts is challenging.
Some of the plant extracts showed opposing effects with respect to the
IL-8 release. The application of the CI–method like certain other
methods to assess synergy, however, is not appropriate for combination
partners with opposing effects. We examined the Iberis amara extract on
a “non-IL-8-reducing effect level” to calculate the CIs. The curves of the
combinations for CIs indicating synergism had r-values ≥0.9 indicating

Fig. 5. Ca2+-release from HISMC. Cells were

stimulated with (A) combination 1 (Mentha
piperita, Matricaria recutita, Melissa officinalis)
and combination 2 (Glycyrrhiza glabra,
Angelica archangelica, Silybum marianum) alone
or (B) in the presence of Capsaicin (80 µM).
Combination 1 increased the Ca2+ -release
under both conditions.

7
G. Ulrich-Merzenich, et al.
an acceptable conformity with the Mass-action law.
We did not perform sequential dilution assays to calculate con-
fidence intervals at specific effect levels. We did not test different dose
ratios since STW5 is a fixed combination. In the case of the Ca2+ release
of HISMC we did not test all possible combinations. Again it cannot be
ruled out that certain combinations might be effective.
Conclusion
The IL-8 releases (inhibition) induced by the different plant extracts
of STW5 were not simply additive, reflecting that cytokines are orga-
nized in networks with a significant measure of redundancy. The
multicomponent mixture STW 5 showed marked and concentration
dependent anti-inflammatory action as indicated by the IL-8 reduction
in Het-1A cells. Based on the CI measurements Silybum marianum (L.)
was identified as the most effective combination partner for the in-
hibition of IL-8 releases within the triple combinations. The substrac-
tion assay supported this finding. In HISMCs STW 5 and the single plant
extract Mellissa officinalis (L.) induced increased Ca2+-releases.
CI-measurements can be used, besides their known application in
drug development, for the identification of leads in complex mixtures.
However, the prerequisite for applying the CI-method is a known dose-
response relationship of the investigated drugs/plant extracts and a
dose range that does not exceed a 2fold or 3fold dilution since other-
wise the dose range would become too wide. CI-measurements cannot
explain mechanisms of action but can give valuable orientation towards
the identification of multi-target effects using different cellular models.
Conflict of interest
GUM received research grants from Steigerwald Arzneimittelwerk
GmbH, Bayer Consumer Health. HA and OK are employees of
Steigerwald Arzneimittelwerk GmbH, Bayer Consumer Health. AS and
LW declare no conflict of interest.
CRediT authorship contribution statement
Gudrun Ulrich-Merzenich: Conceptualization, Methodology,
Writing - original draft, Investigation, Writing - review & editing. Lisa
Welslau: Investigation, Writing - review & editing. Heba Aziz-
Kalbhenn: Investigation, Writing - review & editing. Olaf Kelber:
Writing - review & editing. Anastasiia Shcherbakova:
Conceptualization, Methodology, Writing - original draft, Investigation,
Writing - review & editing.
Acknowledgments
The study was partially supported by Steigerwald Arzneimittelwerk
GmbH, Bayer Consumer Health. Authors thank Bernd Merzenich for
language editing and support in the preparation of the graphics.
References
Abenavoli, L., Izzo, A.A., Milić, N., Cicala, C., Santini, A., Capasso, R., 2018. Milk thistle
(Silybum marianum): a concise overview on its chemistry, pharmacological, and
nutraceutical uses in liver diseases. Phytother. Res. 32 (11), 2202–2213. https://doi.
org/10.1002/ptr.6171.
Abdel-Aziz, H., Kelber, O., Lorkowski, G., et al., 2017. Evaluating the multitarget effects
of combinations through multistep clustering of pharmacological data: the example
of the commercial preparation iberogast. Planta Med. 83 (14/15), 1130–1140.
https://doi.org/10.1055/s-0043-116852.
Abdel-Aziz, H., Zaki, H.F., Neuhuber, W., Kelber, O., Weiser, D., Khayyal, M.T., 2010.
Effect of an herbal preparation, STW5, in an acute model of reflux oesophagitis in
rats. J. Pharmacol. Sci. 113 (2), 134–142. https://doi.org/10.1254/jphs.09355FP.
Abdel-Aziz, H., Schneider, M., Neuhuber, W., Meguid Kassem, A., Khailah, S., Müller, J.,
Gamaleldeen, H., Khairy, A., Khayyal, M., Efferth, T., Ulrich-Merzenich, G., 2016.
GPR84 and TREM-1 signaling contribute to the pathogenesis of reflux esophagitis.
Mol. Med. 21 (1), 1011–1024. https://doi.org/10.2119/molmed.2015.00098.
Allescher, H.D., Wagner, H., 2007. STW5/Iberogast: multi-target-wirkung bei
8
Phytomedicine 60 (2019) 153013
funktioneller dyspepsie und reizdarmsyndrom. Wiener Medizinische Wochenschrift
157 (13–14), 301–307. https://doi.org/10.1007/s10354-007-0429-3.
Allescher, H.D., 2006. Functional dyspepsia – a multicausal disease and its therapy.
Phytomedicine 13 (SV), 2–11. https://doi.org/10.1016/j.phymed.2006.05.001.
Ammon, H.P.T., Kelber, O., Okpanyi, S.N., 2006. Spasmolytic and tonic effect of Iberogast
(STW 5) in intstinal smooth muscle. Phytomedicine 13 (Suppl. 5), 67–74. https://doi.
org/10.1016/j.phymed.2006.08.004.
Chou, T.C., Talalay, P., 1984. Quantitative analysis of dose—effect relationships: the
combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 22,
27–55. https://doi.org/10.1016/0065-2571(84)90007-4.
Chou, T.C., 2006. Theoretical basis, experimental design, and computerized simulation of
synergism and antagonism in drug combination studies. Pharmacol. Rev. 58,
621–681. http://pharmrev.aspetjournals.org/cgi/reprint/58/3/621.
Chou, T.C., Shapiro, T.A., Fu, J., Chou, J.H., Ulrich-Merzenich, G.S., 2019. Computerized
quantification of drugs synergism in animal studies or in clinical trials using only ten
data points. Synergy 9, 100049. https://doi.org/10.1016/j.synres.2019.100049.
(Accepted).
Fu, J.N., Zhang, N., Chou, J.H., Dong, H.J., Dong, S.F., Lin, S.F., Ulrich-Merzenich, G.S.,
Chou, T.C., 2016. Drug combination in vivo using combination index method: tax-
otere and T-607 against colon carcinoma HCT-116 xenograft tumor in nude mice.
Synergy 3, 15–30. https://doi.org/10.1016/j.synres.2016.06.001.
Heinle, H., Hagelaue, R.D., Pascht, U., Kelber, O., Weise, R.D., 2006. Intestinal spasmo-
lytic effects of STW 5 (Ibrogast) and ist components. Phytomedicine 13 (SV), 75–79.
https://doi.org/10.1016/j.phymed.2006.03.013.
Hohenester, B., Rühl, A., Kelber, O., Schemann, M., 2004. The herbal preparation STW 5
(lberogast) has potent and region-specific effects on gastric motility.
Neurogastroenterol. Motil. 16, 765–773. https://doi.org/10.1111/j.1365-2982.2004.
00548.x.
Kahrilas, P.J., Lee, T.J., 2005. Pathophysiology of gastroesophageal reflux disease.
Thoracis Surgery Clinics 15 (3), 323–333. https://doi.org/10.1007/s00268-017-
3952-4.
Kelber, O., Wittwer, A., Lapke, C., Kroll, U., Weiser, D., Okpanyi, S.N., Heilmann, J.,
2006. Exvivo/in vitro absorptionof STW5(Iberogast)and its extract components.
Phytomedicine 5, 107–113. https://doi.org/10.1016/j.phymed.2006.07.002.
Khayyal, M.T., Abdel-Aziz, H., Wadie, W., Vinson, B., Okpany, S.N., Kelber, O., 2007.
Effect of STW 5 in an experimental model of esophagitis. Planta Med. 73, 842.
https://doi.org/10.1055/s-2007-988356.
Kroll, U., Cordes, C, 2006. Pharmaceutical prerequisites for a multi-target therapy.
Phytomedicine 13 (Suppl 5), 12–19. https://doi.org/10.1016/j.phymed.2006.03.
016.
Liao, W., He, X., Yi, Z, Xiang, W., Ding, Y., 2018. Chelidonine suppresses LPS-Induced
production of inflammatory mediators through the inhibitory of the TLR4/NF-κB
signaling pathway in RAW264.7 macrophages. Biomed. Pharmacother. 107,
1151–1159. https://doi.org/10.1016/j.biopha.2018.08.094.
Michael, S., Kelber, O., Hauschildt, S., Spanel-Borowski, K., Nieber, K., 2009. Inhibition of
inflammation-induced alterations in rat small intestine by the herbal preparations
STW 5 and STW 6. Phytomedicine 16 (2–3), 161–171. https://doi.org/10.1016/j.
phymed.2008.10.011.
Michael, S., Kelber, O., Vinson, B., Nieber, K., 2006. Herbal preparations STW 5 and
STW6 inhibit inflammation-mediated motility disorders in the ileum. Gut 55 (SV),
A206. https://doi.org/10.1016/j.phymed.2008.10.011.
Okpanyi, S.N., 1994. The antiinflammatory activity of the plant extract of Iberis amara.
Planta Med. 59, A 665. https://doi.org/10.1055/s-0032-1320467.
Ortiz, M.I., Cariño-Cortés, R., Ponce-Monter, H.A., González-García, M.P., Castañeda-
Hernández, G., Salinas-Caballero, M., 2017. Synergistic interaction of matricaria
chamomilla extract with diclofenac and indomethacin on carrageenan-induced paw
inflammation in rats. Drug Dev. Res. 78 (7), 360–367. https://doi.org/10.1002/ddr.
21401.
Reichling, J., Saller, R, 2006. Iberis. In: Balsche, W., Ebel, S., Fricke, U., Hackenthal, E.,
Holgrabe, U., Keller, K., Reichling, J., Schulz, V., (Hrsg) (Eds.), Hager ROM 2006.
Hagers Handbuch De Drogen Und Arzneistoff. Springer, Berlin.
Richter, J.E., 2007. Gastroesophageal reflux disease. Best Pract. Res. Clin. Gastroenterol.
21 (4), 609–631.
Schemann, M., Michel, K., Zeller, F., Hohenester, B., Rühl, A., 2006. Region specific ef-
fects of STW5 (Iberogast) and its components in gastric fundus, corpus and antrum.
Phytomedicine 13, 90–99. https://doi.org/10.1016/j.phymed.2006.03.020.
Souza, R.F., Huo, X., Mittal, V., Schuler, C.M., Carmack, S.W., Zhang, H.Y., Zhang, X., Yu,
C., Hormi-Carver, K., Genta, R.M., Spechler, S.J., 2009. Gastroesophageal reflux
might cause esophagitis through a cytokine-mediated mechanism rather than caustic
acid injury. Gastroenterology 137, 1776–1784. https://doi.org/10.1053/j.gastro.
2009.07.055.
Sun, Z., Wang, H., Wang, J., Zhou, L., Yang, P., 2014. Chemical composition and anti-
inflammatory, cytotoxic and antioxidant activities of essential oil from leaves of
mentha piperita grown in China. PLoS One 9 (12), e114767. https://doi.org/10.
1371/journal.pone.0114767.
Ulrich-Merzenich, G., Hartbrod, F., Koptina, A., Müller, J., Kelber, O., Zeitler, H., 2017.
Salicylate based phytopharmaceuticals induce adaptive cytokine and chemokine
network responses in human fibroblast cultures. Phytomedicine 34, 202–211.
https://doi.org/10.1016/j.phymed.2017.08.002.
Ulrich-Merzenich, G., Koptina, A., Kelber, O., Freischmidt, A., Heilmann, J., Mülle, R.J.,
Sadeghlar, F., Zeitler, H., Wagner, H., 2012. Gene expression profiles obtained in vivo
for the prediction of adverse events exemplified for salicylate containing phyto-
pharmaceuticals and the tricyclic antidepressant Imipramine. Phytomedicine 19
(3–4), 322–329. https://doi.org/10.1016/j.phymed.2011.09.078. Epub 2011Nov25.
Vinson, B., 2009. Development of Iberogast: clinical evidence for multicomponent herbal
mixtures. In: Cooper, R., Kronenberg, F. (Eds.), Botanical Medicine, from Bench to
G. Ulrich-Merzenich, et al.
Bedside. Mary Ann Liebert, Inc, NewRochelle, NY, USA, pp. 167–189. https://doi.
org/10.1007/s10354-012-0169-x. (Chapter).
Wegener, T., Wagner, H., 2006. active components and the pharmacological multi target
principle of STW5 (Iberogast). Phytomedicine 13 (Suppl.5), 20–35. https://doi.org/
10.1016/j.phymed.2006.07.001.
Phytomedicine 60 (2019) 153013
Wei, M., Ma, Y., Liu, Y., Zhou, Y., Men, L., Yue, K., Pi, Z., Liu, Z., Liu, Z., 2018. Urinary
metabolomics study on the anti-inflammation effects of flavonoids obtained from
Glycyrrhiza. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 1086, 1–10. https://
doi.org/10.1016/j.jchromb.2018.04.007.