Cytotoxicity against A549 Human Lung Cancer Cell Line via the Mitochondrial Membrane Potential and Nuclear Condensation Effects of Nepeta paulsenii Briq., a Perennial Herb

Abstract

The genus Nepeta belongs to the largest Lamiaceae family, with 300 species, which are distributed throughout the various regions of Africa, Asia, India, and America. Along with other plant families distinguished by their medicinal and therapeutic values, the Nepeta genus of Lameaceae remains relatively valuable. Hence, the phytochemicals of N. paulsenii Briq. were extracted using different plant parts, i.e., leaves, stem, roots, flowers, and the whole plant by using various solvents (ethanol, water, and ethyl acetate), obtaining 15 fractions. Each extract of dried plant material was analyzed by FT-IR and GC-MS to identify the chemical constituents. The cytotoxicity of each fraction was analyzed by MTT assay and mitochondrial membrane potential and nuclear condensation assays against lung cancer cells. Among the ethyl acetate and ethanolic extracts, the flowers showed the best results, with IC₅₀ values of 51.57 μg/mL and 50.58 μg/mL, respectively. In contrast, among the water extracts of the various plant segments, the stem showed the best results, with an IC₅₀ value of 123.80 μg/mL. 5-flourouracil was used as the standard drug, providing an IC₅₀ value of 83.62 μg/mL. The Hoechst 33342 stain results indicated apoptotic features, i.e., chromatin dissolution and broken down, fragmented, and crescent-shaped nuclei. The ethanolic extracts of the flowers showed more pronounced apoptotic effects on the cells. The mitochondrial membrane potential indicated that rhodamine 123 fluorescence signals suppressed mitochondrial potential due to the treatment with the extracts. Again, the apoptotic index of the ethanolic extract of the flowers remained the highest. Hence it can be concluded that the flower part of N. paulsenii Briq. was found to be the most active against the A459 human lung cancer cell line.

Keywords: A459; Nepeta paulsenii Briq.; adenocarcinomic human alveolar basal epithelial cells; cytotoxicity; in vitro; lung cancer.

Figure 1

A schematic representation of solvent extraction using ethanol (EtOH), water (H₂O), and ethyl acetate (EtOAc) for various plant sections and their mixtures (whole plant). A total of 15 various extracts were obtained.

Figure 2

Chemical species identified by GC-MS in the CH₃COOC₂H₅, aqueous, and ethanolic extracts of the stem, root, leaf, and flower at various retention times.

Figure 3

Dosedependent cytotoxicity effects of stem, leaf, and flower on A549 cell line. (A) Ethyl acetate extract. (B) Ethanolic extract. (C) Aqueous extract. (D) Ethanolic and ethylacetate extracts of roots and mix (leaves, stem, and flower).

Figure 4

A549 cell photos were captured using a digital camera under an inverted contrast phase microscope, following treatment with the extracts.

Figure 5

(A) These photo-micrographs illustrate the pictures of the A549 cells with Hoechst 33342 staining. The untreated cells (negative control) exhibited the cells growing in a lively manner, with clear nuclear and further cellular structures. The cells treated with the extracts showed clear characteristic apoptosis changes. The arrowheads indicate chromatin dissolution, breakdown, and fragmentation. These arrows specify the condensed, fragmented, and crescent-shaped nuclei. Among the extracts tested, EtOH-F (ethanol extract of flower) showed a more pronounced apoptotic effect on the cells. (B) Percentage representation of apoptotic indices for different tested groups. Index apoptotic for untreated (negative control) A549 cells was 7.2 ± 0.8%. In contrast, the apoptotic indices obtained for the treated cells with the ETAC-F, EtOH-F, and EtOH-mix extracts were 49.4 ± 2.7%, 56.2 ± 4.8%, and 36.9 ± 3.6%, respectively. NC = negative control; ETAC-F = ethylacetate extract of flower; EtOH-F = ethanol extract of flower; EtOH-mix = ethanol extract of the mixture. Values are presented as % mean ± SD (n = 6), * represents p < 0.05 and ** represents p < 0.01.

Figure 6

(A) The photo-micrographs call attention to the capability of the extracts to interrupt the potential of the mitochondrial membrane. (B) Graphical illustration of percentage apoptotic indices. The apoptotic index for every test group was expressed as the percentage of the ratio of the unstained cell no./overall cell no. in 10 different fields. NC (7.2 ± 0.8%) = negative control; ETAC-F (49.4 ± 2.7%) = ethyl-acetate extract of flower; EtOH-F (56.2 ± 4.8%) = ethanol extract of flower; EtOH-mix (36.9 ± 3.6%) = ethanol extract of mixture. Values presented as percentage mean ± standard deviation (n = 6), ** represents p < 0.01 and * represents p < 0.05.