To ascertain the embryotoxicity of peritoneal fluid from infertile women with endometriosis (PF-E), on mouse embryos in culture and to examine the effect of pyruvate in the culture medium on PF-E induced embryotoxicity.
Verbena officinalis is widely used by women for maintaining general health and treating various gynaecological disorders during pregnancy. A case report has indicated that the consumption of V. officinalis induced an abortifacient effect. Hence, this study aimed to investigate the prenatal developmental toxicity of this plant according to OECD guideline (no. 414). A total of 50 pregnant female rats (dams) were distributed into five groups (n = 10); 500 mg/kg 1000 mg/2000 mg/kg and 3000 mg/kg of V. offcinalis extracts and the fifth group served as a normal control. All dams received their respective oral single daily treatment from the 6th to the 20th day of gestation. Maternal clinical toxicity signs, body weight and weight gain were recorded. Caesarean sections were performed on day 21 to evaluate embryo-foetal developmental toxicity. For dams, ovaries were harvested and weighed. The number of corpora lutea, implantation sites, and resorptions were recorded. No mortality was observed in dams, but their body weight gain was significantly reduced particularly in dams treated with 2000 and 3000 mg/kg V. officinalis. Asymmetrical distribution of implantation sites and embryos were observed. Embryo-fetotoxicity retardation was observed as evident by the decrease in foetal weight, head cranium, tail length, and higher incidence in the pre-and post-implantation loss. Some foetal skeleton abnormalities such as incomplete ossification of skull, sternebrae, and metatarsal bones were observed in foetuses of the 2000 and 3000 mg/kg V. officinalis-treated dams. LC/MS analysis identified the major constituents including geniposidic acid, tuberonic acid glucoside, luteolin 7, 3'-digalacturonide, iridotrial and apigenin. The glycosylated flavonoids such as apigenin and luteolin could be responsible for the reported prenatal developmental toxicity. In conclusion, the use of V. officinalis during pregnancy is not safe indicating evidence-based toxic effects on the reproductive performance of dams and dose-dependent risk potentials to the foetuses.
Tyrosinase inhibitors have become increasingly important targets for hyperpigmentation disease treatment. Kojic monooleate (KMO), synthesized from the esterification of kojic acid and oleic acid, has shown a better depigmenting effect than kojic acid. In this study, the process parameters include the speed of high shear, the time of high shear and the speed of the stirrer in the production of nanoemulsion containing KMO was optimized using Response Surface Methodology (RSM), as well as evaluated in terms of its physicochemical properties, safety and efficacy. The optimized condition for the formulation of KMO nanoemulsion was 8.04 min (time of high shear), 4905.42 rpm (speed of high shear), and 271.77 rpm (speed of stirrer), which resulted in a droplet size of 103.97 nm. An analysis of variance (ANOVA) showed that the fitness of the quadratic polynomial fit the experimental data with large F-values (148.79) and small p-values (p < 0.0001) and an insignificant lack of fit. The optimized nanoemulsion containing KMO with a pH value of 5.75, showed a high conductivity value (3.98 mS/cm), which indicated that the nanoemulsion containing KMO was identified as an oil-in-water type of nanoemulsion. The nanoemulsion remains stable (no phase separation) under a centrifugation test and displays accelerated stability during storage at 4, 25 and 45 °C over 90 days. The cytotoxicity assay showed that the optimized nanoemulsion was less toxic, with a 50% inhibition of cell viability (IC50) > 500 μg/mL, and that it can inhibit 67.12% of tyrosinase activity. This study reveals that KMO is a promising candidate for the development of a safe cosmetic agent to prevent hyperpigmentation.
In this study, fibroblast cells were stably transfected with mouse POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) to investigate the effect of S-adenosylhomocysteine (SAH), the reversible non-toxic inhibitor of DNA-methyltransferases (DNMTs), at different intervals post-fusion on in vitro development of cloned bovine embryos. Treatment with SAH for 12 hr resulted in 54.6 ± 7.7% blastocyst production, which was significantly greater than in vitro fertilized embryos (IVF: 37.2 ± 2.7%), cloned embryos treated with SAH for 72 hr (31.0 ± 7.6%), and control cloned embryos (34.6 ± 3.6%). The fluorescence intensities of the EGFP-POU5F1 reporter gene at all intervals of SAH treatment, except of 72 hr, were significantly higher than control somatic cell nuclear transfers (SCNT) embryos. The intensity of DNA-methylation in cloned embryos treated with SAH for 48 hr was similar to that of IVF embryos, and was significantly lower than the other SCNT groups. The levels of H3K9 acetylation in all SCNT groups were significantly lower than IVF embryos. Real-time PCR analysis of gene expression revealed significantly higher expression of POU5F1 in cloned versus IVF blastocysts. Neither embryo production method (SCNT vs. IVF) nor the SAH treatment interval affected expression of the BCL2 gene. Cloned embryos at all intervals of SAH treatment, except for 24 hr, had significantly increased VEGF transcript compared to IVF and control SCNT embryos. It was suggested that the time interval of DNMT inhibition may have important consequences on different in vitro features of bovine SCNT, and the improving effects of DNMT inhibition on developmental competency of cloned embryos are restricted to a specific period of time preceding de novo methylation.