Advanced in Vitro Safety Assessment of Herbal Medicines for the Treatment of Non-Psychotic Mental Disorders in Pregnancy

Abstract

When confronted with non-psychotic mental disorders, pregnant women often refrain from using synthetic drugs and resort to herbal medicines such as St. John's wort, California poppy, valerian, lavender, and hops. Nevertheless, these herbal medicines have not yet been officially approved in pregnancy due to lack of safety data. Using a variety of in vitro methods (determination of cytotoxicity, apoptosis induction, genotoxicity, effects on metabolic properties, and inhibition/induction of differentiation) in a commonly used placental cell line (BeWo b30), we were previously able to show that extracts from these plants are likely to be safe at the usual clinical doses. In the present work, we wanted to extend our safety assessment of these herbal medicines by 1) looking for possible effects on gene expression and 2) using the same in vitro methods to characterize effects of selected phytochemicals that might conceivably lead to safety issues. Proteomics results were promising, as none of the five extracts significantly affected protein expression by up- or down-regulation. Protopine (contained in California poppy), valerenic acid (in valerian), and linalool (in lavender) were inconspicuous in all experiments and showed no adverse effects. Hyperforin and hypericin (two constituents of St. John's wort) and valtrate (typical for valerian) were the most obvious phytochemicals with respect to cytotoxic and apoptotic effects. A decrease in cell viability was evident with hypericin (≥1 µM) and valtrate (≥10 µM), whereas hyperforin (≥3 µM), hypericin (30 µM) and valtrate (≥10 µM) induced cell apoptosis. None of the tested phytochemicals resulted in genotoxic effects at concentrations of 0.1 and 1 µM and thus are not DNA damaging. No decrease in glucose consumption or lactate production was observed under the influence of the phytochemicals, except for valtrate (at all concentrations). No compound affected cell differentiation, except for hyperforin (≥1 µM), which had an inhibitory effect. This study suggests that extracts from St. John's wort, California poppy, valerian, lavender, and hops are likely to be safe during pregnancy. High plasma concentrations of some relevant compounds-hyperforin and hypericin from St. John's wort and valtrate from valerian-deserve special attention, however.

Keywords: Eschscholzia californica; Humulus lupulus; Hypericum perforatum; Lavandula angustifolia; Valeriana officinalis; mental health disorders; pregnancy; safety.

FIGURE 1

Label-free quantification of proteins of treated (herbal extracts) and untreated (0.06% DMSO) BeWo b30 cells for a period of 48 h. Volcano plots representing identified proteins as log₂ fold change (FC) ratio of protein intensity (treated/untreated), plotted against the significance as a function of negative log₁₀ (adjusted p-value). Significantly enriched proteins (adjusted p-value ≤ 0.02) are colored accordingly (• log₂FC > 1, • log₂FC > −1). Insignificantly different proteins are marked in black. Proteins that were only identified in either the treated or untreated sample are displayed in gray (adjusted p-value = 0.001). (A) St. John’s wort, (B) California poppy, (C) valerian, (D) lavender, and (E) hops.

FIGURE 2

Effects of phytochemicals on cell viability of undifferentiated BeWo b30 cells after 72 h of treatment. Among the phytochemicals present in St. John’s wort, such as hyperforin (A) and hypericin (B), only the latter resulted in a significant reduction in cell viability at concentrations of 1, 3, 10, and 30 µM. Protopine (C) (present in California poppy) reduced cell viability by 62.4% at a concentration of 30 µM. Of the phytochemicals present in valerian, such as valerenic acid (D) and valtrate (E), only the latter resulted in a significant reduction in cell viability at concentrations of 10 and 30 µM. Linalool (F) (ingredient of lavender oil) did not show any significant effect in a concentration range from 0.01 up to 30 µM. The effects are shown as fold change compared to the untreated control. Treatments with camptothecin (CPT, 300 µM) and Triton-X-100 (TX, 0.5%) served as toxicity controls. Results were normalized to untreated control signal = 100% (n = 3).

FIGURE 3

Effects of phytochemicals on cell death of undifferentiated BeWo b30 cells after treatment for 72 h: (A) hyperforin, (B) hypericin, (C) protopine, (D) valerenic acid, (E) valtrate, and (F) linalool. Apoptosis only significantly increased for the highest concentrations of hyperforin (≥3 µM), and valtrate (≥10 µM). Hypericin showed a non-significant increase at 1 µM followed by a significant decrease up to the highest concentration due to an overall decrease in detected cells († cell detection was limited due to advanced degradation). Results were normalized to camptothecin (CPT, 300 µM) = 100% (n = 3), which was used as positive control for apoptosis.

FIGURE 4

Effects of phytochemicals (A) hyperforin, (B) hypericin, (C) protopine, (D) valerenic acid, (E) valtrate, and (F) linalool on tail DNA in undifferentiated BeWo b30 cells after exposure for 3 h. No significant genotoxic effects were observed at concentrations of 0.1 and 1 µM. Only the highest concentrations (10 µM) of hyperforin, hypericin, and valtrate led to increased DNA damage of BeWo b30 cells. Results were calculated as fold change compared to the untreated control. Ethyl methanesulfonate (EMS, 3 mM) was used as a positive control (n = 3).

FIGURE 5

Effects of phytochemicals on glucose consumption and lactate production in undifferentiated BeWo b30 cells after treatment for 48 h. Data were normalized per amount of protein (mg). Control consisted of cell culture media containing 0.2% of DMSO. Data were obtained from three independent experiments (n = 3; in triplicate) and are shown as mean ± SD: *p < 0.05. A statistically significant impairment of metabolic activity could not be detected at any of the test concentrations (1, 3, 10, and 30 µM) of the following phytochemicals: protopine (C), valerenic acid (D), and linalool (F). However, valtrate (E) decreased the glycolytic metabolism at concentrations of 1, 3, 10, and 30 µM. Phytochemicals of St. John’s wort led to increased glucose consumption and concomitant lactate production in the case of hyperforin treatment (A) at 1 and 3 µM and increased lactate concentrations in the case of hypericin (B) at 1 µM.

FIGURE 6

Effects of various phytochemicals on the production of β-hCG in BeWo b30 cells. Control consisted of cell culture media containing 0.2% DMSO; 5 µM forskolin (FSK) was used for the FSK control. Data are presented as mean ± SD of at least three independent experiments (n = 3–4; in triplicate): *p < 0.05. (A) Comparison of β-hCG secretion of BeWo b30 cells upon 48-h treatment with increasing concentrations of phytochemicals (1, 3, 10, and 30 µM) vs. FSK control. (B) Effects on inhibition of FSK-induced differentiation of BeWo b30 cells. FSK treatment (5 µM) led to increased β-hCG levels in all phytochemicals after an incubation of 48 h, except for hyperforin, where the FSK-induced differentiation was inhibited at concentrations of 1, 3, and 10 µM. Based on preliminary data (not shown), different concentration gradients (ranging from 0.03 up to 30 µM) of phytochemicals were individually determined in advance (before exposure). Cells were pre-treated with the different phytochemicals for 24 h, before the addition of FSK for another 24 h. (C) Effects of hyperforin on inhibition of FSK-induced differentiation of BeWo b30 cells normalized per amount of protein (μg). FSK treatment (5 µM) led to decreased β-hCG levels after an hyperforin incubation of 48 h at concentrations of 1, 3, and 10 µM.