Determination of aristolochic acid I in a thick extract of Aristolochia clematitis herb by HPLC and the establishment of its cytotoxic activity

Abstract

Introduction. Aristolochia clematitis L. is a perennial herbaceous plant with a short rhizome and a characteristic odor. In different countries of the world, including Ukraine, this plant is grown as an ornamental or can be found as a weed. The chemical composition of raw materials of Aristolochia clematitis is represented by different classes of biologically active substances, in particular aristolochic acids.

Aristolochic acid I is the most common aristolochic acid, found in almost all species of the genus Aristolochia, in particular in Aristolochia clematitis. It is known that the raw material of Aristolochia clematitis is toxic, in particular due to the content of aristolochic acids. Studies by various scientists have shown that aristolochic acids can cause nephropathy and inhibit DNA synthesis in hepatoma HepG2 cells in a dose-dependent manner. Therefore, continuing research on the prospects for the use of raw materials of this plant in medicine, we obtained a thick extract of Aristolochia clematitis herb. The aim of the study was to determine aristolochic acid I in a thick extract of Aristolochia clematitis herb and to determine its cytotoxic activity. Materials and methods. For the study, a thick extract obtained from Aristolochia clematitis herb, harvested during the flowering period, was used. The technology of obtaining a thick extract from Aristolochia clematitis herb is as follows: a portion of the crushed raw material into powder was placed in a conical flask and added as an extractant 70% ethanol. First infused for 1 hour at room temperature, then heated under reflux for 2 hours at 60 ° C. The resulting extract was then cooled, filtered and dried to obtain a thick extract. Chromatographic study of a thick extract of Aristolochia clematitis herb was performed on a liquid chromatograph equipped with a diode array detector Shimadzu HPLC-system, ser.20 under the following conditions: Xterra MS C18 speaker, size 150 mm x 4.6 mm, particle size 3.5 μm; column temperature - 400°C; detection wavelength - 390 nm; flow rate of the mobile phase - 0.3 ml / min; volume of the entered sample - 25 μl; mobile phase is reduced¹ Eluent A: 0.1% aqueous solution of trifluoroacetic acid. ² Eluent B: 0.1% solution of acetonitrile in trifluoroacetic acid. Solvent mixture: acetonitrile - water (50:50). Solution of comparison: a portion of a standard sample of aristolochic acid I 1 mg was dissolved in a mixture of solvents and brought to the mark with the same solvent. 1 ml of the resulting solution was transferred into a 10 ml flask and adjusted to the mark with a mixture of solvents. The solution was filtered through a 0.45 μm filter. Determination of cytotoxic activity was performed on an in vitro model at the National University of Pharmacy, on the basis of the problem laboratory of morphofunctional research, under the guidance of Dr. Biol.N., Professor Maloshtan L.M. The study used a line of transplanted animal cell cultures BHK -21 (Baby Hamster Kidney) - a culture of fibroblast-like cells of the kidney of a newborn Syrian hamster. Cytotoxic effects were assessed by reducing cell proliferation in the gentian violet assay. Research results. During the analysis, aristolochic acid I was identified in the obtained extract (retention time - 5,766 ± 0,018 min). The retention time of the standard sample of aristolochic acid I - 5,847 ± 0,022 minutes. The content of aristolochic acid I in the studied extract was 0.39 ± 0.01%. As a result of determining the cytotoxic activity of a thick extract of Aristolochia clematitis herb, it was found that the most pronounced indicators of cytotoxic effects were recorded at a dose of 15-60 mg / ml after incubation for 24 hours. Against the background of doses of 60 mg / ml, almost 98% cytotoxic effect was observed. LC₅₀ was recorded at a dose of 7.5 mg / ml after incubation with BHK-21 cells for 24 hours. Against the background of doses of 3.75 mg / ml and 1.85 mg / ml, approximately the same number of surviving cells was registered, which was 75.0% and 74.88%, respectively. An increase in cytotoxic effects was observed in the 48-hour exposure under the influence of the extract. Conclusions. As a result of the study, aristolochic acid I was identified in a thick extract of Aristolochia clematitis herb, and its quantitative content was determined, which was 0.39%. Using the photocolorimetric method, the cytotoxic effect of the investigated thick extract of Aristolochia clematitis herb on BHK-21 cells was established. Determination of the degree of proliferation of BHK-21 cells in the gentian violet test showed that the ability to proliferate in the presence of the studied extract was dose- and time-dependent. Thus, the obtained data can be used in the standardization of a thick extract of Aristolochia clematitis herb, as well as in the development and production of new drugs based on the obtained extract.

Keywords: Aristolochia clematitis, aristolochic acid I, HPLC, cytotoxic activity.

DOI: 10.5281/zenodo.6350303