Bryophyllum pinnatum enhances the inhibitory effect of atosiban and nifedipine on human myometrial contractility: an in vitro study

Abstract

Background: The herbal medicine Bryophyllum pinnatum has been used as a tocolytic agent in anthroposophic medicine and, recently, in conventional settings alone or as an add-on medication with tocolytic agents such as atosiban or nifedipine. We wanted to compare the inhibitory effect of atosiban and nifedipine on human myometrial contractility in vitro in the absence and in the presence of B. pinnatum press juice (BPJ).

Methods: Myometrium biopsies were collected during elective Caesarean sections. Myometrial strips were placed under tension into an organ bath and allowed to contract spontaneously. Test substances alone and at concentrations known to moderately affect contractility in this setup, or in combination, were added to the organ bath, and contractility was recorded throughout the experiments. Changes in the strength (measured as area under the curve (AUC) and amplitude) and frequency of contractions after the addition of all test substances were determined. Cell viability assays were performed with the human myometrium hTERT-C3 and PHM1-41 cell lines.

Results: BPJ (2.5 μg/mL), atosiban (0.27 μg/mL), and nifedipine (3 ng/mL), moderately reduced the strength of spontaneous myometrium contractions. When BPJ was added together with atosiban or nifedipine, inhibition of contraction strength was significantly higher than with the tocolytics alone (p = 0.03 and p < 0.001, respectively). In the case of AUC, BPJ plus atosiban promoted a decrease to 48.8 ± 6.3% of initial, whereas BPJ and atosiban alone lowered it to 70.9 ± 4.7% and to 80.9 ± 4.1% of initial, respectively. Also in the case of AUC, BPJ plus nifedipine promoted a decrease to 39.9 ± 4.6% of initial, at the same time that BPJ and nifedipine alone lowered it to 78.9 ± 3.8% and 71.0 ± 3.4% of initial. Amplitude data supported those AUC data. The inhibitory effects of BPJ plus atosiban and of BPJ plus nifedipine on contractions strength were concentration-dependent. None of the test substances, alone or in combination, decreased myometrial cell viability.

Conclusions: BPJ enhances the inhibitory effect of atosiban and nifedipine on the strength of myometrial contractions, without affecting myometrium tissue or cell viability. The combination treatment of BPJ with atosiban or nifedipine has therapeutic potential.

Keywords: Atosiban; Bryophyllum pinnatum; Contractility; Myometrium; Nifedipine; Preterm.

Conflict of interest statement

MM is an employee of Weleda AG, the company that produces the preparations of Bryophyllum pinnatum. APSW received research funding from Weleda AG during the last 5 years.

Figures

Fig. 1

Experimental design for measurement of myometrial contractions. Test substances were added to the organ bath when myometrium strips had been contracting regularly for 30 min. When the effects of BPJ and/or atosiban were being studied (a), Krebs solution (control), BPJ, atosiban, or the combination of BPJ and atosiban were added, and contractility was recorded for 30 min. When the effects of BPJ and/or nifedipine were being studied (b), Krebs solution (control; two strips) or nifedipine (two strips) was added, contractility was recorded for 30 min, and then BPJ was added to all chambers. Exposure to test substances was followed by a 30 min washout step, with change of Krebs solution at 5, 10, 20 and 30 min

Fig. 2

Effect of BPJ, atosiban, and the combination of BPJ with atosiban on human myometrial contractility in vitro. BPJ (green; 15 μL), atosiban (blue; 4.3 μL of 375 μg/mL) or their combination (red, same concentrations) were added to the myograph chamber. The scatter dot plot shows the AUC (a), the amplitude (b), and the frequency (c) of contractions expressed as percentage of initial. Krebs solution was used as negative control (black, 5 μL). Data were obtained from 11 to 15 different biopsies (n = 11–15) and are presented as mean value ± SEM. *p < 0.05

Fig. 3

Effect of repeated addition of BPJ plus atosiban (15 μL and 4.3 μL of 375 μg/mL, respectively) and of BPJ plus nifedipine (15 μL and 5 μL of 3.7 μg/mL, respectively) on human myometrial contractility in vitro. All test substances were repeatedly added to the myograph chamber. The line chart shows the AUC (a), the amplitude (b), and the frequency (c). Data were obtained with 5 different biopsies (n = 5) and are expressed as percentage of initial. The repeated addition of BPJ was performed for comparison; Krebs solution (5 μL) was used as control. *p < 0.05

Fig. 4

Effect of BPJ, nifedipine, and the combination of BPJ with nifedipine on human myometrial contractility in vitro. BPJ (green; 15 μL), nifedipine (violet; 5 μL of 3.7 μg/mL), or their combination (orange, same concentrations) were added to the myograph chamber. The scatter dot plot shows the AUC (a), the amplitude (b), and the frequency (c) expressed as percentage of initial. Krebs solution was used as negative control (black, 5 μL). Data were obtained from 11 to 13 different biopsies (n = 11–13) and are presented as mean value ± SEM. *p < 0.05

Fig. 5

Effect of BPJ, atosiban, nifedipine, BPJ plus atosiban and BPJ plus nifedipine on myometrium cell viability. (a) Cell viability assays were performed in the presence of BPJ (2.5–10.0 μg/mL), atosiban (0.3–1.1 μg/mL) and nifedipine (3.0–12.0 ng/mL), as well as of BPJ plus atosiban and BPJ plus nifedipine (same concentrations as with single treatments) using hTERT-C3 and PHM1–41 human myometrium cell lines. Cells were incubated with the test substances for 24 h. Triton X-100 (1%) and ethyl methanesulfonate (30 mM) were used as positive controls. Data is presented as mean ± SEM of 4 independent experiments (n = 4), each carried out in quadruplicate; *p < 0.05. (b) Staining of nuclei (blue) and actin (red) from hTERT-C3 cells untreated (i.e. control) or upon treatment with BPJ (10.0 μg/mL), BPJ plus atosiban (10.0 μg/mL and 1.1 μg/mL, respectively), and BPJ plus nifedipine (10.0 μg/mL and 12.0 ng/mL, respectively). The images are representative of four independent cultures