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- Title
CYTOTOXIC AND APOPTOSIS-INDUCING POTENTIAL OF POLYMETHOXYLATED FLAVONES ENRICHED FRACTIONS FROM ARTEMISIA ANNUA L. IN HEK293T CELLS.
- Authors
Irma, Durmišević; Irma, Gušić; Selma, Ćurovac; Ilma, Mujković; Džemina, Begović; Sanin, Haverić; Kemal, Durić; Anja, Haverić; Maida, Hadžić Omanović
- Abstract
Artemisia annua L. known as sweet wormwood is a medicinal plant renowned for treatment of different human diseases. Abundant in various bioactive compounds including polymethoxylated flavones such as chrysosplenol D (CHD) and casticin, this plant demonstrates notable antioxidant, antiproliferative and anti-inflammatory activity. These compounds not only augment the bioavailability but also enhance the therapeutic efficacy of well-known artemisinin. The aim of this study was to analyze the effects on cell viability and apoptotic events of three fractions derived from A. annua L. in normal human embryonic kidney cells (HEK293T; ATCC® CRL-3216™). The crude A. annua ethanolic extract was prepared from dried leaves using ultrasound assisted extraction and fractionated by preparative highspeed counter-current chromatography (HSCCC).Monitoring of HSCCC fractions was performed using high-performance liquid chromatography coupled with the diode-array detector. Three fractions were prepared: fraction I enriched in CHD, fraction II enriched in CHD spectrally similar compound and fraction III mixture of two previous. HEK293T cells (105 cells/mL) were cultivated in a standard humidified atmosphere for 48 h. After initial incubation of 24 h, cells were treated with HSCCC fractions purified in polymethoxylated flavones at concentrations of 50, 6.25, 3.125 and 0.3125 µg/mL. Cell viability was assessed through MTT assay and trypan blue exclusion test. Additionally, apoptotic or necrotic stages were identified using the TransDetect® Annexin V-EGFP/PI Cell Apoptosis Detection Kit and analyzed by fluorescent microscopy. Our findings underscore the utility of HSCCC as an effective tool for preparative separation, yielding high purity and excellent recovery of target compounds from the crude extract of A. annua L. The MTT assay revealed a dose-dependent increase in cytotoxicity with fractions II and III, while cultures treated solely with CHD-enriched fractions exhibited high viability. However, the trypan blue results indicated a reduction in cell viability across all fractions, necessitating supplementary assays due to potential interference and limitations of colorimetric tests. A higher frequency of late apoptotic cells was observed in all treated cultures compared to the negative control, indicating potent proapoptotic bioactivity of the tested fractions, and requiring further investigations, particularly in cancer cell lines.
- Subjects
ARTEMISIA annua; FLAVONES; HIGH performance liquid chromatography; COUNTERCURRENT chromatography; TRYPAN blue; COUMARINS
- Publication
Genetics & Applications, 2024, p83
- ISSN
2566-2937
- Publication type
Article