Caryophyllene Oxide, a Bicyclic Terpenoid Isolated from Annona macroprophyllata with Antitumor Activity: In Vivo, In Vitro, and In Silico Studies

Abstract

The Annona genus contains some species used in Mexican traditional medicine for the treatment cancer, including Annona macroprophyllata (A. macroprophyllata). The present study aimed to investigate the anticancer activity of caryophyllene oxide (CO) isolated from A. macroprophyllata using in vivo, in vitro, and in silico approaches. The identification of CO was performed using gas chromatography-mass spectroscopy and NMR methods. Antilymphoma activity was evaluated in male and female Balb/c mice inoculated with U-937 cells. Cytotoxic activity was evaluated using the WST method and flow cytometry was used to determine the type of cell death. Acute oral toxicity was determined, and a molecular docking study was performed using target proteins associated with cancer, including, HMG-CoA, Bcl-2, Mcl-1, and VEGFR-2. Results showed that CO exhibited significant antilymphoma and cytotoxic activities, and its effects were comparable to MTX. In addition, flow cytometry showed that the anticancer activity of CO could be mediated by the induction of late apoptosis and necrosis. The result for the acute oral toxicity of CO was classified in category 4, suggesting it is low risk. Finally, molecular coupling studies showed that CO had more affinity for the enzymes HMG-CoA reductase and Bcl-2. Our study provides evidences that CO is a potential anticancer agent for the treatment of histiocytic lymphoma.

Keywords: Annona macroprophyllata; U-937 cells; acute oral toxicity; antilymphoma activity; caryophyllene oxide; molecular docking.

Figure 1

Pharmacological activities reported for the leaves, seeds and root bark of Annona macroprophyllata.

Figure 2

Structure of caryophyllene oxide obtained from petroleum ether extract from A. macroprophyllata leaves.

Figure 3

Gas chromatography-mass spectrometry analysis of petroleum ether extract from A. macroprophyllata leaves (PEAm) and caryophyllene oxide standard. The x-axis indicates the retention time in minutes, while the y-axis indicates the peak % signal intensity.

Figure 4

Mass spectrum of caryophyllene oxide.

Figure 5

¹H-NMR spectra (A) and ¹³C-NMR spectra (B) of caryophyllene oxide.

Figure 6

Cytotoxic activity of CO in the U-937 cell line. The graphs show the inhibition of cell growth caused by methotrexate (A) and caryophyllene oxide (B) at different concentrations after 24 h of exposure. The assays were performed in triplicate and these data were used to calculate the CC₅₀ by lineal regression analysis.

Figure 7

Lymph node weights (g) of female mice (A) and male mice (B). Healthy control (HC tween 80, 2% v/v in water), untreated control (U-937), methotrexate (MTX) 1.25 mg/kg and caryophyllene oxide (CO) at 1.25, 5 and 10 mg/kg. The graph shows the weight of the axillary and inguinal lymph nodes as follows: left axillary (LA), right axillary (RA), left inguinal (LI), and right inguinal (IR). Results obtained by ANOVA one-way analysis followed by Dunnett’s test for multiple comparison. Data are expressed as mean ± SEM, (n = 6); * p < 0.05 vs. HC, # p < 0.05 vs. U-937, + p < 0.05 vs. MTX.

Figure 8

Apoptosis assay using annexin V/PI staining. The apoptotic and necrotic effect of CO is observed. U-937 cells were exposed for 24 h to, vehicle 0.02% dimethyl sulfoxide (DMSO) (A), methotrexate (CC₅₀ 118.87 µg/mL) (B), and caryophyllene oxide (CC₅₀ 24.25 µg/mL) (C). The histogram zones indicate the following: R1 = necrosis, annexin V-FITC negative/PI positive (− +); R2 = late apoptosis, annexin V-FITC positive/PI positive (+ +); R3 = viable cells, annexin V-FITC negative/PI negative (− −); R4 = early apoptosis, annexin V-FITC positive/PI negative (+ −). The experiments were conducted in triplicate. Data are expressed as means ± SEM, n = 3; *p < 0.05 vs DMSO.

Figure 9

Results of molecular docking. The images show the binding site position and the 3D, and 2D interactions for caryophyllene oxide on each target. Caryophyllene oxide (blue) and interactions with amino acid residues (orange) in HMG-CoA reductase (A); and HMG-CoA substrate (yellow) and interactions with amino acid residues (red) in HMG-CoA reductase (B). For Bcl-2 protein, caryophyllene oxide (blue) (C); and navitoclax (pink) (D), in both cases interactions with amino acid residues (yellow) are highlighted. 2D orange-red interactions are unfavorable interactions, green interactions are polar-type interactions, and lilac interactions are nonpolar interactions.

Figure 10

Results of molecular docking. The images show the binding site position and the 3D, and 2D interactions for caryophyllene oxide on each target. For Mcl-1, caryophyllene oxide (blue) (A); and 9EAs (green)(B), in both cases interactions with amino acid residues (orange) are highlighted. For VEGFR-2, caryophyllene oxide (blue) (C); and axitinib (green) (D), in both cases interactions with amino acid residues (lilac) are highlighted. 2D orange-red interactions are unfavorable interactions, green interactions are polar-type interactions, and lilac interactions are nonpolar interactions.