Focused Microwave-Assisted Extraction and LC Determination of Ketoprofen in the Presence of Preservatives in a Pharmaceutical Cream Formulation

A simple and rapid open-vessel focused microwave-assisted extraction (FMAE) method followed by LC analysis was developed for the determination of ketoprofen lysine salt in the presence of methyl p-hydroxybenzoate and propyl p-hydroxybenzoate preservatives in topical cream. Extraction were performed in acetone/potassium dihydrogenphosphate (25 mM, pH 3.0) (70:30 v/v) by reaching a target temperature of 65 °C in a 10 min linear ramp. The chromatographic separation was performed on a Discovery RP-Amide C16 column (250 × 4.6 mm I.D., 5 μm particle size). The optimal mobile phase consisted of acetonitrile/potassium dihydrogen phosphate 25 mM adjusted to pH 3.0 with phosphoric acid (50:50 v/v). The complete analytical procedure was validated with regard to limit of quantification, linearity, precision and accuracy. The method was linear over the concentration range of 0.08–0.12 mg mL⁻¹; the relative standard deviations of intra- and inter-day assays were 1.9–2.3 and 1.8% respectively. The limit of quantification was 0.54 μg mL⁻¹. The proposed method shows many advantages as short extraction time, little solvent consumption without requiring further sample clean-up steps before liquid chromatographic analysis and is proposed for vast scale screening of cream dosage forms aimed to the detection of counterfeit and substandard drugs.

     

Antitumor Effect of Glandora rosmarinifolia (Boraginaceae) Essential Oil through Inhibition of the Activity of the Topo II Enzyme in Acute Myeloid Leukemia

It was previously shown that the antitumor and cytotoxic activity of the essential oil (EO) extracted from the aerial parts of Glandora rosmarinifolia appears to involve a pro-oxidant mechanism in hepatocellular carcinoma (HCC) and in triple-negative breast cancer (TNBC) cell lines. Its most abundant compound is a hydroxy-methyl-naphthoquinone isomer. Important pharmacological activities, such as antitumor, antibacterial, antifungal, antiviral and antiparasitic activities, are attributed to naphthoquinones, probably due to their pro-oxidant or electrophilic potential; for some naphthoquinones, a mechanism of action of topoisomerase inhibition has been reported, in which they appear to act both as catalytic inhibitors and as topoisomerase II poisons. Our aim was to evaluate the cytotoxic activity of the essential oil on an acute myeloid leukemia cell line HL-60 and on its multidrug-resistant (MDR) variant HL-60R and verify its ability to interfere with topoisomerase II activity. MTS assay showed that G. rosmarinifolia EO induced a decrease in tumor cell viability equivalent in the two cell lines; this antitumor effect could depend on the pro-oxidant activity of EO in both cell lines. Furthermore, G. rosmarinifolia EO reduced the activity of Topo II in the nuclear extracts of HL-60 and HL-60R cells, as inferred from the inability to convert the kinetoplast DNA into the decatenated form and then not inducing linear kDNA. Confirming this result, flow cytometric analysis proved that EO induced a G₀-G₁ phase arrest, with cell reduction in the S-phase. In addition, the combination of EO with etoposide showed a good potentiation effect in terms of cytotoxicity in both cell lines. Our results highlight the antitumor activity of EO in the HL-60 cell line and its MDR variant with a peculiar mechanism as a Topo II modulator. Unlike etoposide, EO does not cause stabilization of a covalent Topo II-DNA intermediate but acts as a catalytic inhibitor. These data make G. rosmarinifolia EO a potential anticancer drug candidate due to its cytotoxic action, which is not affected by multidrug resistance.