Mentha pulegium L. (Pennyroyal, Lamiaceae) Extracts Impose Abortion or Fetal-Mediated Toxicity in Pregnant Rats; Evidenced by the Modulation of Pregnancy Hormones, MiR-520, MiR-146a, TIMP-1 and MMP-9 Protein Expressions, Inflammatory State, Certain Related Signaling Pathways, and Metabolite Profiling via UPLC-ESI-TOF-MS

Abstract

Pregnant women usually turn to natural products to relieve pregnancy-related ailments which might pose health risks. Mentha pulegium L. (MP, Lamiaceae) is a common insect repellent, and the present work validates its abortifacient capacity, targeting morphological anomalies, biological, and behavioral consequences, compared to misoprostol. The study also includes untargeted metabolite profiling of MP extract and fractions thereof viz. methylene chloride (MecH), ethyl acetate (EtOAc), butanol (But), and the remaining liquor (Rem. Aq.) by UPLC-ESI-MS-TOF, to unravel the constituents provoking abortion. Administration of MP extract/fractions, for three days starting from day 15th of gestation, affected fetal development by disrupting the uterine and placental tissues, or even caused pregnancy termination. These effects also entailed biochemical changes where they decreased progesterone and increased estradiol serum levels, modulated placental gene expressions of both MiR-(146a and 520), decreased uterine MMP-9, and up-regulated TIMP-1 protein expression, and empathized inflammatory responses (TNF-α, IL-1β). In addition, these alterations affected the brain's GFAP, BDNF, and 5-HT content and some of the behavioral parameters escorted by the open field test. All these incidences were also perceived in the misoprostol-treated group. A total of 128 metabolites were identified in the alcoholic extract of MP, including hydroxycinnamates, flavonoid conjugates, quinones, iridoids, and terpenes. MP extract was successful in terminating the pregnancy with minimal behavioral abnormalities and low toxicity margins.

Keywords: IUGR; MMP-9; Mentha pulegium (pennyroyal); UPLC metabolite profiling; abortion; micro-RNA.

Figure 1

Representative images of the female rat uterus dissected on the 18th day of gestation. (C) misoprostol, and (E,F) MP (extract; 250, 500 mg/kg, respectively) uterus with complete abortion but (F) with additional appearance of intensive bleeding and hemorrhage (star) as compared to: (A) normal uterus presentation of unmated female rat, and (B) uterus with normal fetal distribution and normal physiological appearance of pregnancy. However (D) showed no complete abortion but asymmetric distribution of fetuses in the two uterine horns: red brackets; resorption points: green arrows; hematoma: star; and absence of boundaries between fetal balls: yellow arc (Panel I). Photomicrographs images of histopathological uterine tissue alterations after hematoxylin and eosin staining; (A,a,a*) un-mated/control negative, (B,b,b*) pregnant/control positive, (C,c,c*) misoprostol, (D,d,d*), (E,e,e*), and (F,f,f*) MP extract 125, 250, and 500 mg/kg, respectively. (Panel II).

Figure 2

(A) change in weight of pregnant rats in different groups as compared to unmated/unpregnant female through the study (days: 1st, 15th, 16th, 17th, and 18th of gestation), and (B) weight of remaining fetus (Panel I). (A) Two-Way RM ANOVA with one factor repetition (time: day) was utilized to analyze the change in rat weight and (B) the non-parametric data of fetal weight were expressed as the median (min-max) and were analyzed using the non-parametric Kruskal–Wallis followed by Dunn’s as the post hoc test and student t test (Panel I). Common pathological observations and features of uterine tissues. (C) Pregnant rats with normal fetal distribution in the two horns; (D–F) MP (extract/fraction) or misoprostol on the 18th day of gestation. Observations: (D); the uterus with asymmetrical distribution of fetuses in the two horns, complete absence of fetus in one horn, remarkable resorption sites, and reduced number of fetal balls, (E,F); asymmetrical distribution of fetuses in the two horns, absence of boundaries between fetal balls, reduced sized fetal balls, notable resorption sites, and intensive bleeding. The asymmetric distribution of fetuses in the two uterine horns: red brackets; resorption points: green arrows; hematoma: star; and absence of boundaries between fetal balls: yellow arc; reduced fetal balls: black circle (Panel II). MP (extract or fractions) and Misoprostol-mediated structural abnormalities in fetus including height, weight, and physical appearance; (a,a*) pregnant, (b,b*) Misoprostol, and (c,c*) MP; extract or fractions groups respectively (Panel III).

Figure 3

The serum levels of (A) progesterone and (B) estradiol (Panel I). Placental protein expression of (A) MiR-520 and (B) MiR-146a (Panel II). Uterine protein expression of (A) TIMP-1, (B) MMP-9 (Panel III). Values are presented as mean (n = 6) ± SD and statistical analysis was carried out using one-way ANOVA followed by Tukey’s post hoc multiple comparison test. As compared with control (a), misoprostol (b), MP (c); p < 0.05. But.: butanol; EtOAc: ethyl acetate; MecH: methylene chloride; MiR-520: micro ribonucleic acid-520; MiR-146a: micro ribonucleic acid-146a; MMP-9: matrix metalloproteinase-9; MP: Mentha pulegium L.; TIMP-1: tissue inhibitor matrix metalloproteinase-1; Rem. aq.: remaining aqueous.

Figure 4

The serum levels of (A) TNF-α and (B) IL-1β (Panel I). The behavioral changes in an open field test on (A) latency time, (B) rearing frequency, (C) number of squares crossed/3 min, and (D) grooming frequency (Panel II). Cortical contents of (A) GFAP, (B) BDNF, and (C) 5-HT (Panel III). Values are presented as mean (n = 6) ± SD and statistical analysis was carried out using one-way ANOVA followed by Tukey’s post hoc multiple comparison test. As compared with control (a), misoprostol (b), MP (c); p < 0.05. BDNF: brain derived neurotrophic factor; But.: butanol; EtOAc: ethyl acetate; GFAP: Glial fibrillary acidic protein; 5-HT: 5-Hydroxytryptamine; IL-1β: interlukin-1beta; MecH: methylene chloride; MP: Mentha pulegium L.; Rem. Aq.: remaining aqueous, TNF-α: tumor necrosis factor-alfa.

Figure 5

Representative UPLC-ESI-TOF-MS base peak chromatograms of 70% ethanol extract of Mentha pulegium L. (A), (−): negative ESI mode and (B), (+): positive ESI mode. Peak numbers follow metabolites listed in Table 3.

Figure 6

Representative UPLC/TOF-MS base peak chromatograms of fractions of Mentha pulegium L. (MP) in negative mode, (A) Methylene chloride fraction (MecH), (B) Ethyl acetate fraction (EtOAc), (C) Butanol fraction (But.) (D) Remaining aqueous fraction (Rem. Aq.). Peak numbers follow metabolites listed in Table 3.