A Bufadienolide-Enriched Fraction of Bryophyllum pinnatum Inhibits Human Myometrial Contractility In Vitro

• Abstract
• Introduction
• Results
• Discussion
• Material and Methods
• Chemicals
• Plant material
• Bryophyllum pinnatum leaf press juice
• Flavonoid- and bufadienolide-enriched fractions
• Flavonoid aglycon mix
• Acid hydrolysis of Bryophyllum pinnatum leaf press juice and quantification of flavonoid aglycons
• Measurement of myometrial contractility in vitro
• Myograph data processing
• Cell culture
• Cell viability
• Statistical analysis
• References

Abstract

Bryophyllum pinnatum has been used since the 1970s to prevent premature labour, first in anthroposophic hospitals and, more recently, also in the main Swiss perinatal centres. However, it is not known which compounds in B. pinnatum leaves contribute to the tocolytic effect. Here we studied the effects of a flavonoid-enriched fraction, the corresponding flavonoid aglycon mixture, a bufadienolide-enriched fraction, and B. pinnatum leaf press juice on human myometrial contractility in vitro. The strength (area under the curve and amplitude) and frequency of contractions were recorded using strips of human myometrium mounted in an organ bath system. Cell viability assays were performed with the human myometrium hTERT-C3 and PHM1 – 41 cell lines. Repeated addition of the flavonoid-enriched fraction, flavonoid aglycon mixture, bufadienolide-enriched fraction, or B. pinnatum leaf press juice led to a progressive decrease of contraction strength, without jeopardising the vitality of myometrium strips. The bufadienolide-enriched fraction was the most active, since 1 µg/mL of the bufadienolide-enriched fraction lowered the area under the curve to 40.1 ± 11.8% of the initial value, whereas 150 µg/mL of the flavonoid-enriched fraction, 6.2 µg/mL of the flavonoid aglycon mixture, and 10 mg/mL of the B. pinnatum leaf press juice were required to achieve comparable inhibition. A progressive increase of contraction frequency was observed, except in the case of the flavonoid aglycon mixture, which did not affect frequency. None of the test substances decreased myometrial cell viability, even at concentrations of 500 µg/mL of the flavonoid-enriched fraction, 40 µg/mL of the flavonoid aglycon mixture, 3.8 µg/mL of the bufadienolide-enriched fraction, and 75 mg/mL of the B. pinnatum leaf press juice, i.e., higher than those used in the myometrium experiments. Given the concentrations of flavonoids in the flavonoid-enriched fraction and B. pinnatum leaf press juice, and of bufadienolides in the bufadienolide-enriched fraction and B. pinnatum leaf press juice, it appears that bufadienolides may be mainly responsible for the relaxant effect.

Introduction

Bryophyllum pinnatum (Lam.) Oken [syn. Kalanchoe pinnata (Lam.) Pers.; family Crassulaceae] is a succulent perennial plant native to Madagascar that now grows widely in tropical and subtropical regions around the globe. In ethnomedicine, B. pinnatum has multiple uses including the treatment of wounds, diabetes mellitus, joint pain, headache, hypertension, and kidney stones, and nausea and vomiting in cancer patients. For B. pinnatum extracts, antifungal, antimicrobial, anti-inflammatory, and analgesic properties have been reported [1], [2].

In Europe, the use of remedies prepared from B. pinnatum leaves was limited for a long time to anthroposophic medicine. Initially, they were used for the treatment of various hyperactivity disorders [3], [4], and only in the 1970s was B. pinnatum introduced by the German gynaecologist Werner Hassauer (1928 – 1993) as a routine treatment of preterm labour [5]. In Switzerland, B. pinnatum is used in gynaecology and obstetrics against premature contractions, restlessness, and overactive bladder [6]. A recent assessment of the internal treatment recommendations in the main Swiss obstetrics centres revealed that B. pinnatum preparations are suggested for the treatment of preterm contractions, as well as anxiety, restlessness, and sleep disorders [7]. Several clinical studies documented a very good tolerability of B. pinnatum preparations when used for these indications [3], [4], [6], [8], [9], [10], [11], [12]. Moreover, pharmacovigilance data for the Bryophyllum preparation used in Switzerland (B. pinnatum 50% tablets) and for a comparable preparation in use in Germany and France (B. pinnatum 50% powder) corroborate the very good tolerability (Weleda AG, pharmacovigilance data).

Preterm labour and prematurity (i.e., birth before 37 weeks of pregnancy) are the main determinants of perinatal mortality and long-term morbidity [13], [14]. Since preterm uterine contractions correlate highly with preterm birth, their inhibition by tocolytics (beta-adrenergic receptor agonists, calcium channel blockers, or oxytocin receptor antagonists) constitute a major treatment element. Most studies have shown that administration of tocolytics may delay pregnancy for 48 h with the goal to administer antenatal corticosteroid therapy for foetal lung maturation and to allow intrauterine transfer to a tertiary care perinatal centre [15], [16]. However, tocolysis was not shown to prevent preterm birth from occurring or to reduce neonatal morbidity or mortality [17], [18]. Therefore, additional therapeutic options are needed. As a tocolytic agent, B. pinnatum was shown to be effective and to lead to significantly less maternal adverse effects than beta-adrenergic receptor agonists [10]. In vitro studies showed that B. pinnatum (aqueous extract, 100 mg/mL, and leaf press juice) inhibited spontaneous contractions of human myometrial strips [19], [20]. In human myometrium cells, BPJ lowered the oxytocin-induced increase of intracellular calcium concentrations [21].

Flavonoid glycosides and bufadienolides are the major classes of secondary metabolites in B. pinnatum leaves [22], [23]. The presence of nine different glycosides of kaempferol, quercetin, myricetin, acacetin, and diosmetin was shown, and four bufadienolides, namely, bersaldegenin-1-acetate, bryophyllin A, bersaldegenin-3-acetate, and bersaldegenin-1,3,5-orthoacetate, were identified [22]. Fractions enriched in flavonoid glycosides (FEF) and bufadienolides (BEF) were prepared, and their effects on human myometrial contractility were characterised in vitro. The activity of fractions was compared with that of BPJ, the starting material for the B. pinnatum tablets used in clinical practice, and with a mixture of flavonoid aglycons (A-mix) that corresponded to the composition of aglycons in FEF.

Results

To investigate the contribution of different constituents of B. pinnatum leaves on human myometrial contractility, spontaneously contracting strips of myometrium mounted in an organ bath system were repeatedly exposed to increasing concentrations of FEF, A-Mix, BEF, and BPJ. A schematic representation of the experimental design is given in [Fig. 1]. The concentration ranges for FEF, BEF, and BPJ ([Table 1]) were determined in preliminary experiments to ensure that an effect on the strength of contractions, measured as the AUC and amplitude, would be visible upon two to three additions. The concentration range of A-Mix was such that the concentration of the main flavonoid aglycons in the organ bath were identical to those of the corresponding flavonoid glycosides in FEF.

The AUC of myometrial contractions decreased progressively and in a statistically significant way with the repeated addition of FEF and the corresponding A-Mix (FEF: p < 0.001, A-Mix: p < 0.001; Friedman test) ([Fig. 2 A]). Compared to addition 0, the values of the AUC were significantly lower after the 3rd and 4th additions of FEF (final concentrations of 150 and 200 µg/mL, p = 0.01 and p < 0.001, respectively; Dunnʼs multiple comparisons test) ([Fig. 2 A]). The repeated addition of A-Mix also significantly lowered the AUC after the 3rd and 4th additions (concentrations of 6.2 and 8.3 µg/mL), again when compared to addition 0 (p = 0.008 and p = 0.002, respectively) ([Fig. 2 A]). Comparable inhibitory effects of FEF and A-Mix on the amplitude were observed (FEF: p = 0.01, A-Mix: p < 0.001) ([Fig. 2 B]). Compared to addition 0, the 4th addition of FEF and the 3rd addition of A-Mix led to significantly lower amplitudes (in each case p = 0.01) ([Fig. 2 B]). The repeated addition of FEF promoted a progressive increase in contraction frequency (p = 0.001), with significant differences after the 3rd and 4th additions (p = 0.004 and p = 0.002, respectively). In contrast, increased concentrations of A-Mix did not affect contraction frequency ([Fig. 2 C]).

As shown in [Fig. 3], the effect of BEF on the AUC of myometrial contractility was concentration dependent (p < 0.001). When each addition was compared to addition 0, a significant difference was obtained with the 3rd and 4th additions (concentrations of 1.0 and 1.3 µg/mL, p = 0.02 and p < 0.001, respectively) ([Fig. 3 A]). The amplitude of the contractions also decreased with consecutive additions of BEF (p = 0.001) and was significantly different from addition 0 after the 4th addition (final concentration of BEF in the organ bath was 1.3 µg/mL, p = 0.003) ([Fig. 3 B]). The frequency of the myometrial contractions increased with the repeated additions of BEF (p = 0.001), with significant differences, relative to addition 0, being obtained after the 3rd and 4th additions (p = 0.004 and p = 0.002, respectively) ([Fig. 3 C]).

To compare the effects described above with the active ingredient of commercially available 50% B. pinnatum tablets, BPJ was also tested. The addition of BPJ led to a progressive decrease of the AUC compared to addition 0 (p < 0.001). After the 3rd and 4th additions [final concentrations of 10 and 13 mg/mL (1.0 and 1.3%) of BPJ], the values of the AUC were significantly lower than after addition 0 (p = 0.01 and p < 0.001, respectively) ([Fig. 4 A]). A progressive decrease of the amplitude was also observed (p < 0.003). As shown in [Fig. 4 B], the 4th addition of BPJ resulted in a significant decrease of contraction amplitudes compared to addition 0 (p = 0.01, final concentration of 13 mg/mL). BPJ led to a strong increase in contraction frequency (p < 0.001) to 860.0 ± 219.4% (p = 0.007) at the 3rd addition, and 1120.0 ± 214.2% of the initial value at the 4th addition ([Fig. 4 C]).

Nifedipine, a known tocolytic drug frequently used in clinical practice [7], [24], was used as the PC. The repeated addition of nifedipine led to a progressive decrease of the contraction strength assessed either as the AUC or as amplitude (in each case p < 0.001) ([Fig. 4 A, B]), with statistically significant values after the 3rd and 4th additions (concentrations of 16.2 and 21.6 nM) when compared to addition 0 (in each case p = 0.01 and p = 0.0003, respectively). The frequency of the myometrial contractions was not affected by increasing concentrations of nifedipine (p < 0.5) ([Fig. 4 C]).

The bufadienolide content in BEF and BPJ, and the flavonoid aglycons in FEF, had been previously determined [23], [25]. To be able to compare the effects of FEF with those of BPJ, the main flavonoid aglycons in BPJ were now determined by HPLC-PDA analysis after hydrolysis. BPJ contained 0.034 ± 0.006 mg/mL of flavonoid aglycons in the relative proportions of 78.7% quercetin, 4.8% myricetin, 11.5% diosmetin, and 4.9% kaempferol (Fig. 1S, Supporting Information).

To verify whether the test substances could be toxic to myometrial tissue, the ability of the strips to contract again after the washing step at the end of the myograph experiments was determined. All strips contracted again (Fig. 2S, Supporting Information), either spontaneously or after the addition of oxytocin (final concentration of 1 U/L). This indicated that test substances did not jeopardise the viability of the myometrium strips. In some myograph experiments, however, a decrease of contraction strength was still apparent after the washing step. Therefore, we assessed the cytotoxicity of the test substances during longer periods using two human myometrial cell lines. When hTERT myometrium cells were incubated with test substances for 24 h (data not shown) and 48 h, A-Mix, BEF, and BPJ did not affect cell viability at concentrations up to 40 µg/mL, 15 µg/mL, and 150 mg/mL, respectively. Moreover, FEF was cytotoxic only at a concentration of 1000 µg/mL when compared to the untreated control (p < 0.05) ([Fig. 5]). When PHM1 – 41 cells were incubated with test substances for 24 h (data not shown) and 48 h, FEF and A-Mix did not affect cell viability at concentrations up to 1000 µg/mL and 40 µg/mL, respectively. BEF decreased cell viability when compared to the untreated control only after 48 h, at concentrations of 7.5 and 15 µg/mL (p < 0.05), while BPJ was cytotoxic only at 150 mg/mL (p < 0.05) ([Fig. 5]). DMSO used to dissolve the test samples only decreased cell viability at concentrations higher than 1.5% (Fig. 3S, Supporting Information).

Discussion

Fractions prepared from B. pinnatum leaves and enriched in flavonoid glycosides or in bufadienolides led to a concentration-dependent decrease of myometrial contraction strength in vitro. Therefore, our data suggest that FEF and BEF contain compounds that contribute to the inhibitory effect of BJP ([Fig. 6]), the starting material for the 50% B. pinnatum tablets that have been used in several prospective clinical studies [6], [8], [11], [12]. These B. pinnatum press juice tablets are taken orally, and the intestinal metabolisation of compounds thus needs to be considered. Flavonoid glycosides are known to be hydrolysed, leading to the release of flavonoid aglycons [26], [27], whereas bufadienolides in B. pinnatum are less likely to be metabolised by gut microbiota (own unpublished preliminary observations with bufalin). From a translational point of view, the similar abilities of FEF and A-mix to lower myometrium contractions in vitro suggest that the intestinal metabolisation step is not required for FEF effects on this tissue.

The concentrations in the organ bath of flavonoid aglycons after the addition of FEF and BPJ, and of bufadienolides after the addition of BEF and BPJ was calculated based on previous [23], [25] and present data, and are shown in [Table 1]. It is apparent that the concentration of flavonoid aglycons in BPJ additions was markedly lower than with FEF. In contrast, bufadienolide concentrations after the addition of BEF and BPJ were comparable. The repeated addition of FEF, A-Mix, BEF, and BPJ led to a comparable lowering of the AUC and amplitude ([Fig. 6]), albeit at much higher concentrations for FEF and A-mix. From a translational point of view, this suggests that bufadienolides are mainly responsible for the inhibitory effect of BPJ on myometrium contractility, and that flavonoids only play a minor role. Therefore, special attention should be paid to the amount of bufadienolides present in B. pinnatum preparations administered for the prevention of preterm birth. In general, bufadienolides increasingly appear to be the major pharmacologically active compounds in Bryophyllum spp. [28].

Human myometrium strips are the most relevant model for assessing the effects of substances on uterine contractions. The downside of the model is the highly limited availability of myometrium strips (they have to be freshly taken from a Caesarean section, with prior consent) and the very low throughput that can be achieved with an organ bath model. To study concentration dependency in this model, we have therefore increased the concentration of the various test substances in the myograph chamber with consecutive additions. The inclusion of vehicle (and concomitantly time) controls in the various experiments supported the concentration dependency of the effects. Moreover, the intervals of 20 min after each addition allow the compounds to penetrate into the tissue and thus to exert their effect.

The results obtained in the organ bath seem to represent a true inhibition of contractions since myometrium strips were still vital after the washout, i.e., they could still contract if given enough time and/or if stimulated with oxytocin (Fig. 2S, Supporting Information). Since the contraction strength of the strip treatment with the test substances was still lower than prior to the additions, we investigated the cytotoxic effects of the test substances on two human myometrium cell lines (hTERT-C3 and PHM1 – 41). The test substances were not cytotoxic even at concentrations far higher than those used in the organ bath experiments, and exposure times that were significantly longer ([Figs. 5] and 3S, Supporting Information).

Our results with the myometrium model show that the fractions obtained from B. pinnatum leaves (FEF and BEF) not only reduced contraction strength, but also increased the frequency of spontaneous contractions ([Fig. 6]). This is in line with previous studies [19], [20], but different from what was observed with A-mix. The reason for the frequency increase with BPJ, FEF, and BEF is not clear at this point in time. As for the clinical practice, there are no published reports that B. pinnatum preparations would lead to an increase in frequency of the strong and rhythmical contractions that eventually lead to delivery.

Taken together, our data corroborate the relaxant properties of BPJ and fractions on myometrium strips and, therefore, support the use of B. pinnatum preparations in the management of preterm labour. Considering the qualitative and quantitative composition of the press juice and the fractions tested on one side, and the potency of relaxant effects on the other, bufadienolides appear to be the major active compounds of B. pinnatum.

Material and Methods

Chemicals

Quercetin (purity ≥ 95%), myricetin (purity ≥ 96%), diosmetin (purity ≥ 98%), kaempferol (purity ≥ 97%), NaHCO₃, glucose (ACS grade), KCl (purity ≥ 99%), and EDTA were obtained from Sigma-Aldrich. KH₂PO₄ (purity ≥ 99.5%) and CaCl₂ (purity ≥ 95%) were purchased from Merck. MgSO₄ (purity ≥ 99%) was purchased from Fluka and NaCl (purity ≥ 99.5%) from PanReac AppliChem.

Plant material

Plant material originated from two different harvests. Weleda Brazil provided leaves harvested on 25 March 2014 in S. Roque, Brazil. Identification of this material was done by Moacyr Copani and Paulo Copani, Weleda Brazil. A voucher specimen (ZSS 29717) has been deposited at the Zurich Succulent Plant collection. Leaves were sent by airmail to Weleda AG, Arlesheim, Switzerland, in a refrigerated box. In addition, the Weleda branch located in Schwäbisch Gmünd, Germany, provided leaves harvested in July and August 2010. Identification of this material was done by Michael Straub, Weleda Germany. A voucher specimen (ZSS 29715) was deposited at the Zurich Succulent Plant collection. After harvesting, the leaves were frozen and stored at − 20 °C until processing.

Bryophyllum pinnatum leaf press juice

BPJ was prepared from leaves harvested in S. Roque, Brazil. The press juice was obtained by the mechanical pressing of the leaves in a roller mill. The procedure was identical to the initial steps of the protocol used for the production of the active ingredient of Weleda Bryophyllum 50% chewing tablets (Weleda AG). Unfiltered press juice was kept at − 80 °C until use.

Flavonoid- and bufadienolide-enriched fractions

FEF and BEF used in the present study originated from earlier investigations, and chromatograms of these fractions have been published (see [29] and [25], respectively). Briefly, frozen fresh leaves of B. pinnatum (from Schwäbisch Gmünd, Germany) were lyophilised and powdered in a mortar, and the powder (1065 g) was extracted with MeOH. After evaporation, a portion of the MeOH extract (112 g) was partitioned between CH₂Cl₂ and H₂O, and the aqueous phase (2.2 g) was fractionated by column chromatography on Diaion HP-20 to provide the FEF (610 mg). Evaporation of the CH₂Cl₂ phase yielded a residue (10.8 g) [29] that was purified by solid-phase extraction on RP-18 to afford 268 mg of BEF [23]. For testing, FEF and BEF stock solutions (150 mg/mL and 1.0 mg/mL, respectively) were prepared in DMSO.

Flavonoid aglycon mix

A content of 4.14% of total flavonoid aglycons after hydrolysis of FEF was previously determined, and the relative proportions were 74.6% quercetin, 16.7% myricetin, 4.6% diosmetin, and 4.0% kaempferol [25]. A mixture of the four aglycons in these proportions (A-Mix) was prepared in DMSO at a concentration of 6.21 mg/mL.

Acid hydrolysis of Bryophyllum pinnatum leaf press juice and quantification of flavonoid aglycons

Concentrated HCl (4.05 mL) was added to B. pinnatum juice (20 mL) to a final concentration of 2 N. The mixture was heated under reflux at 90 °C for 1.5 h. The reaction mixture was allowed to cool and was neutralised with NaHCO₃. The solution was extracted with ethyl acetate (3 × 20 mL), and the combined organic layers were evaporated under reduced pressure. The solid residue was redissolved in DMSO (5 mL). Hydrolysis was performed in triplicate.

HPLC analysis was performed on a Shimadzu LCMS 8030 system with a photodiode array detector using an Atlantis dC18 column (4.6 × 150 mm, 3 µm; Waters). The mobile phase consisted of H₂O (A) and MeOH (B), both containing 0.1% formic acid. After isocratic elution with 50% B for 2 min, a gradient of 50 – 75% B in 28 min was applied. The flow rate was 0.6 mL/min, and detection was at 254 nm. The injection volume was 10 µL. The identity of the aglycons was confirmed by electrospray ionisation mass spectrometry and by comparison with reference standards. Quantification of the five aglycons was performed using a calibration curve for quercetin (12.5 – 200 µg/mL) and a previously determined response factor [25]. The HPLC-UV chromatogram of the hydrolysed juice and the calibration curve are shown as Supporting Information (Fig. 1S, Supporting Information).

Measurement of myometrial contractility in vitro

The study was approved by the ethics committee of canton Zurich (KEK-ZH-Nr. 2014 – 0717). Written informed consent of the study participants was obtained on the day before surgery. Inclusion criteria were planned first caesarean section, single pregnancy, negative HIV test, age > 18 years, and no tocolysis within 2 weeks before the caesarean section.

A myometrial biopsy of approximately 5 g was taken from each study participant at the cranial edge of the uterotomy during the elective caesarean section. The myometrial biopsies were immediately stored in Ringer solution and transferred to the laboratory for experiments.

Longitudinal muscle strips of approximately 15 × 2 × 1 mm were prepared and mounted between two clamps of a myograph bath chamber containing 6 mL of Krebs solution (118 mM NaCl, 24.9 mM NaHCO₃, 4.7 mM KCl, 1.24 mM KH₂PO₄, 2.48 mM CaCl₂, 1.21 mM MgSO₄, 10 mM glucose, 0.034 mM EDTA, pH = 7.4), with the temperature set at 37 °C, and aerated with 95% O₂ and 5% CO₂ (PanGas). Contractions were recorded by a DMT800MS myograph (Muscle Strip Myograph system, DMT, ADInstruments) and transferred to a PC via a transducer (ADInstruments PowerLab 4/30).

Regular spontaneous contractions were recorded for 20 min. Then, Krebs solution was added (addition 0), and contractility was recorded for 20 min. Each strip was treated with 1 test substance by adding 4 times, at time intervals of 20 min, the same volume of a stock solution of the test compound, and recording contractility for 20 min after each addition. Test solutions included: control, 20 µL Krebs solution or 2 µL DMSO; FEF, 2 µL of 150.0 mg/mL; A-Mix, 2 µL of 6.21 mg/mL; BEF, 2 µL of 1.0 mg/mL; or BPJ, 20 µL. Contractions as a measure of vitality of the tissue was determined after a 30-min period of washing, where Krebs solution was changed at 5, 10, 20, and 30 min ([Fig. 1]). If the strip did not contract spontaneously after the washing, 1 U of oxytocin was added to stimulate contractions. After observing contractility, the experiment was stopped. Nifedipine was used as a positive control (3 µL of 10.8 µM in Krebs solution). For each substance tested, five different biopsies were used (n = 5).

Myograph data processing

Myometrium contractions were recorded by LabChart Pro 8.0.6 (ADInstruments) and analysed with the peak analysis module. AUC and amplitude of each contraction were calculated. Average AUC and average amplitude were taken as measures for the strength of contractility in each of the 20-min phases. The number of contractions in each phase was also recorded (frequency). Spontaneous contractions (before any addition, i.e., baseline) were set at 100%. The effect after addition was expressed as percentage of the initial value. The values of the various biopsies were used for further statistical analysis (n = 5 per test substance).

Cell culture

A human myometrial telomerase reverse transcriptase cell line (hTERT-C3) [30], [31], provided by M. Grãos (University of Coimbra, Portugal), was cultured in a 1 : 1 mixture of DMEM and F-12 supplemented with antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) and 10% (v/v) heat-inactivated FBS (all from Gibco). Human uterine myometrium smooth muscle cells (PHM1-41), obtained from ATCC (CRL-3046) were maintained in ATCC-formulated DMEM (ATCC No. 30-2002) supplemented with 0.1 mg/mL G-418 (Carl Roth), 2 mM glutamine, and 10% (v/v) heat-inactivated FBS (Gibco).

Cell viability

Cells were seeded into transparent 96-well microplates. hTERT-C3 cells were seeded at a density of 5 × 10⁴ cells/mL (5 × 10³ cells per well) and PHM1 – 41 at a density of 8 × 10⁴ cells/mL (8 × 10³ cells per well). One day after seeding, cells were exposed to FEF (30 – 1000 µg/mL), A-Mix (1.3 – 40 µg/mL), BEF (0.5 – 15 µg/mL), BPJ (5 – 150 mg/mL), and DMSO (0.2 – 6.0%) for 24 and 48 h. After exposure, resazurin was added to the cells (final concentration 1.0 mg/mL), and the plate was incubated at 37 °C for 4 h. The extent of resazurin reduction was measured in a microplate reader (SpectraMax Paradigm, Molecular Devices) at 570 and 600 nm. For each substance tested, four independent experiments were carried out at least in quadruplicate. Ethyl methanesulfonate (30 mM) [32] was used as a positive control. In each experiment, wells with no test substance added to the culture medium served as an untreated control (100% viability). Cell viability was determined according to the following equation:

Statistical analysis

Statistical analyses were performed using GraphPad Prism software. For each test substance, myograph measurements (n = 5) and cell viability data (n = 4) were analysed with the paired, nonparametric Friedman test. In all cases, this test was followed by Dunnʼs multiple comparisons test in which the various additions or concentrations were compared to addition 0 or untreated control, respectively. A significance level of p < 0.05 was considered statistically significant. Myograph measurements data are presented as box plots (median, 25th percentile and 75th percentile, and minimum and maximum values) and as the mean ± standard error of the mean (SEM). Cell viability data is given as the mean ± SEM.

Conflict of Interest

M. M. is an employee of Weleda AG, the company that produces preparations of B. pinnatum. A. P. S. W. received research funding from Weleda AG during the last 5 years.

Acknowledgements

The authors are grateful to Alexandra Dolder for technical support, and to the staff at the Department of Obstetrics, University Hospital Zurich, who facilitated the collection of myometrium samples. We are indebted to the members of the Bryophyllum Study Group for critical discussions, and to Karin Fürer for preparation of FEF and of a bufadienolide-enriched fraction that was eventually purified to obtain BEF. Financial support was provided by Weleda AG and the Johannes Kreyenbühl Foundation.

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• 16 Schleußner E. The prevention, diagnosis and treatment of premature labor. Dtsch Ärztebl Int 2013; 110: 227
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• 17 Hubinont C, Debiève F. Prevention of preterm labour: 2011 update on tocolysis. J Pregnancy 2011; 2011: 941057
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• 18 ACOG: American college of Obstetricians and Gynecologists. Practice Bulletin No. 171: Management of preterm Labor. Obstet Gynecol 2016; 128: e155-e164
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• 19 Gwehenberger B, Rist L, Huch R, von Mandach U. Effect of Bryophyllum pinnatum versus fenoterol on uterine contractility. Eur J Obstet Gynecol Reprod Biol 2004; 113: 164-171
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• 20 Wächter R, Brenneisen R, Hamburger M, Mennet M, Schnelle M, Worel A, Simões-Wüst AP, von Mandach U. Leaf press juice from Bryophyllum pinnatum (Lamarck) Oken induces myometrial relaxation. Phytomedicine 2011; 19: 74-82
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• 21 Simões-Wüst AP, Grãos M, Duarte C, Brenneisen R, Hamburger M, Mennet M, Ramos M, Schnelle M, Wächter R, Worel A. Juice of Bryophyllum pinnatum (Lam.) inhibits oxytocin-induced increase of the intracellular calcium concentration in human myometrial cells. Phytomedicine 2010; 17: 980-986
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• 22 Fürer K, Raith M, Brenneisen R, Mennet M, Simões-Wüst AP, von Mandach U, Hamburger M, Potterat O. Two new flavonol glycosides and a metabolite profile of Bryophyllum pinnatum, a phytotherapeutic used in obstetrics and gynaecology. Planta Med 2013; 79: 1565-1571
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• 23 Oufir M, Seiler C, Gerodetti M, Gerber J, Fürer K, Mennet-von Eiff M, Elsas SM, Brenneisen R, von Mandach U, Hamburger M. Quantification of bufadienolides in Bryophyllum pinnatum leaves and manufactured products by UHPLC-ESIMS/MS. Planta Med 2015; 81: 1190-1197
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• 24 Vogel JP, Nardin JM, Dowswell T, West HM, Oladapo OT. Combination of tocolytic agents for inhibiting preterm labour. Cochrane Database Syst Rev 2014; (07) CD006169
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• 25 Bachmann S, Betschart C, Gerber J, Fürer K, Mennet M, Hamburger M, Potterat O, von Mandach U, Simões-Wüst AP. Potential of Bryophyllum pinnatum as a detrusor relaxant: an in vitro exploratory study. Planta Med 2017; 83: 1274-1280
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• 26 Cassidy A, Minihane AM. The role of metabolism (and the microbiome) in defining the clinical efficacy of dietary flavonoids. Am J Clin Nutr 2017; 105: 10-22
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• 27 Braune A, Blaut M. Bacterial species involved in the conversion of dietary flavonoids in the human gut. Gut Microbes 2016; 7: 216-234
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• 28 Kolodziejczyk-Czepas J, Stochmal A. Bufadienolides of Kalanchoe species: an overview of chemical structure, biological activity and prospects for pharmacological use. Phytochem Rev 2017; 16: 1155-1171
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• 29 Fürer K, Eberli D, Betschart C, Brenneisen R, De Mieri M, Hamburger M, Mennet-von Eiff M, Potterat O, Schnelle M, Simões-Wüst AP, von Mandach U. Inhibition of porcine detrusor contractility by the flavonoid fraction of Bryophyllum pinnatum – a potential phytotherapeutic drug for the treatment of the overactive bladder syndrome. Phytomedicine 2015; 22: 158-164
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• 30 Condon J, Yin S, Mayhew B, Word RA, Wright W, Shay J, Rainey WE. Telomerase immortalization of human myometrial cells. Biol Reprod 2002; 67: 506-514
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• 31 Devost D, Zingg HH. Novel in vitro system for functional assessment of oxytocin action. Am J Physiol Endocrinol Metab 2007; 292: E1-E6
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• 32 Ray SD, Fariss MW. Role of cellular energy status in tocopheryl hemisuccinate cytoprotection against ethyl methanesulfonate-induced toxicity. Arch Biochem Biophys 1994; 311: 180-190
  CrossrefPubMedSearch in Google Scholar

Correspondence

Publication History

Received: 20 August 2018

Accepted after revision: 27 November 2018

Publication Date:
18 December 2018 (online)

© 2018. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

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  PubMed
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  CrossrefPubMedSearch in Google Scholar
• 16 Schleußner E. The prevention, diagnosis and treatment of premature labor. Dtsch Ärztebl Int 2013; 110: 227
  PubMedSearch in Google Scholar
• 17 Hubinont C, Debiève F. Prevention of preterm labour: 2011 update on tocolysis. J Pregnancy 2011; 2011: 941057
  CrossrefPubMedSearch in Google Scholar
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  Search in Google Scholar
• 19 Gwehenberger B, Rist L, Huch R, von Mandach U. Effect of Bryophyllum pinnatum versus fenoterol on uterine contractility. Eur J Obstet Gynecol Reprod Biol 2004; 113: 164-171
  CrossrefPubMedSearch in Google Scholar
• 20 Wächter R, Brenneisen R, Hamburger M, Mennet M, Schnelle M, Worel A, Simões-Wüst AP, von Mandach U. Leaf press juice from Bryophyllum pinnatum (Lamarck) Oken induces myometrial relaxation. Phytomedicine 2011; 19: 74-82
  CrossrefPubMedSearch in Google Scholar
• 21 Simões-Wüst AP, Grãos M, Duarte C, Brenneisen R, Hamburger M, Mennet M, Ramos M, Schnelle M, Wächter R, Worel A. Juice of Bryophyllum pinnatum (Lam.) inhibits oxytocin-induced increase of the intracellular calcium concentration in human myometrial cells. Phytomedicine 2010; 17: 980-986
  CrossrefPubMedSearch in Google Scholar
• 22 Fürer K, Raith M, Brenneisen R, Mennet M, Simões-Wüst AP, von Mandach U, Hamburger M, Potterat O. Two new flavonol glycosides and a metabolite profile of Bryophyllum pinnatum, a phytotherapeutic used in obstetrics and gynaecology. Planta Med 2013; 79: 1565-1571
  Thieme ConnectPubMedSearch in Google Scholar
• 23 Oufir M, Seiler C, Gerodetti M, Gerber J, Fürer K, Mennet-von Eiff M, Elsas SM, Brenneisen R, von Mandach U, Hamburger M. Quantification of bufadienolides in Bryophyllum pinnatum leaves and manufactured products by UHPLC-ESIMS/MS. Planta Med 2015; 81: 1190-1197
  Thieme ConnectPubMedSearch in Google Scholar
• 24 Vogel JP, Nardin JM, Dowswell T, West HM, Oladapo OT. Combination of tocolytic agents for inhibiting preterm labour. Cochrane Database Syst Rev 2014; (07) CD006169
  PubMedSearch in Google Scholar
• 25 Bachmann S, Betschart C, Gerber J, Fürer K, Mennet M, Hamburger M, Potterat O, von Mandach U, Simões-Wüst AP. Potential of Bryophyllum pinnatum as a detrusor relaxant: an in vitro exploratory study. Planta Med 2017; 83: 1274-1280
  Thieme ConnectPubMedSearch in Google Scholar
• 26 Cassidy A, Minihane AM. The role of metabolism (and the microbiome) in defining the clinical efficacy of dietary flavonoids. Am J Clin Nutr 2017; 105: 10-22
  CrossrefPubMedSearch in Google Scholar
• 27 Braune A, Blaut M. Bacterial species involved in the conversion of dietary flavonoids in the human gut. Gut Microbes 2016; 7: 216-234
  CrossrefPubMedSearch in Google Scholar
• 28 Kolodziejczyk-Czepas J, Stochmal A. Bufadienolides of Kalanchoe species: an overview of chemical structure, biological activity and prospects for pharmacological use. Phytochem Rev 2017; 16: 1155-1171
  CrossrefPubMedSearch in Google Scholar
• 29 Fürer K, Eberli D, Betschart C, Brenneisen R, De Mieri M, Hamburger M, Mennet-von Eiff M, Potterat O, Schnelle M, Simões-Wüst AP, von Mandach U. Inhibition of porcine detrusor contractility by the flavonoid fraction of Bryophyllum pinnatum – a potential phytotherapeutic drug for the treatment of the overactive bladder syndrome. Phytomedicine 2015; 22: 158-164
  CrossrefPubMedSearch in Google Scholar
• 30 Condon J, Yin S, Mayhew B, Word RA, Wright W, Shay J, Rainey WE. Telomerase immortalization of human myometrial cells. Biol Reprod 2002; 67: 506-514
  CrossrefPubMedSearch in Google Scholar
• 31 Devost D, Zingg HH. Novel in vitro system for functional assessment of oxytocin action. Am J Physiol Endocrinol Metab 2007; 292: E1-E6
  CrossrefPubMedSearch in Google Scholar
• 32 Ray SD, Fariss MW. Role of cellular energy status in tocopheryl hemisuccinate cytoprotection against ethyl methanesulfonate-induced toxicity. Arch Biochem Biophys 1994; 311: 180-190
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material