Chemical Profiling, Antioxidant, Cytotoxic Activities and Molecular Docking Simulation of Carrichtera annua DC. (Cruciferae)

Abstract

Our investigation intended to analyze the chemical composition and the antioxidant activity of Carrichtera annua and to evaluate the antiproliferative effect of C. annua crude and phenolics extracts by MTT assay on a panel of cancerous and non-cancerous breast and liver cell lines. The total flavonoid and phenolic contents of C. annua were 47.3 ± 17.9 mg RE/g and 83.8 ± 5.3 mg respectively. C. annua extract exhibited remarkable antioxidant capacity (50.92 ± 5.64 mg GAE/g) in comparison with BHT (74.86 ± 3.92 mg GAE/g). Moreover, the extract exhibited promising reduction ability (1.17 mMol Fe⁺²/g) in comparison to the positive control (ascorbic acid with 2.75 ± 0.91) and it displayed some definite radical scavenging effect on DPPH (IC₅₀ values of 211.9 ± 3.7 µg/mL). Chemical profiling of C. annua extract was achieved by LC-ESI-TOF-MS/MS analysis. Forty-nine hits mainly polyphenols were detected. Flavonoid fraction of C. annua was more active than the crude extract. It demonstrated selective cytotoxicity against the MCF-7 and HepG2 cells (IC₅₀ = 13.04 and 19.3 µg/mL respectively), induced cell cycle arrest at pre-G1 and G2/M-phases and displayed apoptotic effect. Molecular docking studies supported our findings and revealed that kaempferol-3,7-O-bis-α-L-rhamnoside and kaempferol-3-rutinoside were the most active inhibitors of Bcl-2. Therefore, C. annua herb seems to be a promising candidate to further advance anticancer research. In extrapolation, the intake of C. annua phenolics might be adventitious for alleviating breast and liver malignancies and tumoral proliferation in humans.

Keywords: Carrichtera annua; LC-ESI-TOF-MS/MS; antioxidant; antiproliferative; docking studies.

Figure 1

C. annua total phenolics contents (TPC) expressed as gallic acid equivalent per gram of dry weight GAE/g dw (83.8%) and total flavonoids contents (TFC) expressed as rutin equivalent per gram of dry weight RE/g dw (47.3%).

Figure 2

The scavenging rate (%) of 2,2-diphenyl- 1-picrylhydrazyl (DPPH) by C. annua crude extract. All of the values in the figure are expressed as means (%) and SD of triplicated experiments.

Figure 3

Antioxidant activity of C. annua crude extract (a) The IC₅₀ value of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, (b) ferric-ion reducing antioxidant power (FRAP) assay and (c) total antioxidant capacity (TAC) assay.

Figure 4

Chemical structures of the identified compounds by LC-ESI-TOF-MS/MS.

Figure 4

Chemical structures of the identified compounds by LC-ESI-TOF-MS/MS.

Figure 4

Chemical structures of the identified compounds by LC-ESI-TOF-MS/MS.

Figure 4

Chemical structures of the identified compounds by LC-ESI-TOF-MS/MS.

Figure 5

IC₅₀ nonlinear regression curve fit of percentage of cell viability vs log [con. µg/mL], R square ≈1, using the GraphPad prism software. (A) Cytotoxicity of crude extract against MCF–7, (B) Cytotoxicity of phenolics extract against MCF–7, (C) Cytotoxicity of phenolics extract against HepG2.

Figure 6

FITC/Annexin-V-FITC/PI differential apoptosis/necrosis (A) untreated control, (B) Phenolics extract (IC₅₀ = 13.04 μg/mL, 48 h) and DNA content-flow cytometry aided cell cycle analyses (C) untreated control, (D) Flavonoid extract (IC₅₀ = 13.04 μg/mL, 48 h), (E) bar chart representation) in MCF–7. ** p ≤ 0.05 and *** p ≤ 0.001 are significant different.

Figure 7

FITC/Annexin-V-FITC/PI differential apoptosis/necrosis (A) untreated control, (B) Phenloics extract (IC₅₀ = 19.34 μg/mL, 48 h) and DNA content-flow cytometry aided cell cycle analyses (C) untreated control, (D) Flavonoid extract (IC₅₀ = 13.04 μg/mL, 48 h), (E) bar chart representation in HepG2 cells. ** p ≤ 0.05 and *** p ≤ 0.001 are significant different

Figure 8

RT-PCR analysis of the apoptosis-related genes was performed after the MCF-7 cells were treated with phenolics extract (13.04 μg/mL) for 72 h.

Figure 9

Visualized docked compounds: (A) Kaempferol-3,7-O-bis-α-L-rhamnoside (25) and (B) Kaempferol-3-rutinoside (21) inside the “B-cell lymphoma 2 (Bcl-2) (PDB ID: 4IEH) representing key interactions with bond length (Å) with interactive amino acids (Arg 66 and Tyr 161).