Preparation of the chloroform fraction from Piper aduncum

Piper aduncum leaves were collected from the lower Kimiri area, district of La Merced, province of Chanchamayo, department of Junin, at coordinates 11°02'16.5 "S 75°18'54.0 "W. Leaves were collected in the wild, and one sample was taxonomically identified as Piper aduncum L. by the natural history museum of the Universidad Nacional Mayor de San Marcos. The preparation of the Piper aduncum methanolic extract (PAMo) and the Piper aduncum chloroform fraction (PAMoCI) was completed under standardized protocols at the laboratory of Chemistry and Biochemistry of Natural Products of the Universidad Científica del Sur ¹¹.

The leaves were cleaned and dried at 40 °C during two days, then crushed and sieved with a 1 mm mesh. Then 70 g of powdered leaves were weighed, 300 mL of methanol was added and filtered using a Whatman No. 1 filter paper. This procedure was repeated eight times, from which the last three were sonicated for 2 hours. The entire PAMo was filtered using a 0.44 µm membrane. To obtain the PAMoCI, the PAMo was concentrated to 200 mL and extracted by adding 300 mL of chloroform in a 1 L decanting tube. This process was repeated 8 times. The PAMoCl was concentrated at reduced pressure with a rotary evaporator. The PAMo and the PAMoCl were analyzed by thin-layer chromatography (TLC) using silica gel 60 for the stationary phase and benzene-acetone 8:1 as the mobile phase; and it was then developed with iodine and ultraviolet (UV) light ¹². Complementary tests were conducted to identify chemical groups for both PAMo and PAMoCl according to protocols described by Lock ¹³. Finally, the PAMoCl was treated to a concentration of 32 mg/mL in dimethyl sulfoxide (DMSO) and stored at -80 °C for later use. The analytical solvents were purchased from Merck.

Cell line culture

The human gastric cancer cell lines AGS (primary) and KATO III (metastatic) were acquired from the European Authenticated Cell Culture Collection (ECACC 89090402 and 86093004), and cell line 293T (ATCC® CRL-3216TM) from the laboratory of Molecular Genetics of the Universidad Cientifica del Sur.

DMEM-F12 medium (Biowest), supplemented with 10% fetal bovine serum and 1% antibiotic-antifungal solution (full DMEM-F12), was used to culture AGS and 293T cells. RPMI medium (Biowest) supplemented with 20% fetal bovine serum and 1% antibiotic-antifungal solution (Biowest) was used to culture the KATO III cell line. All cells were incubated at 37 °C with 5% CO₂ and subcultured when confluence was 70-80%.

Cellular viability assay

The cell viability assay was performed as previously described ¹⁴. AGS and 293T cells were counted using a Neubauer chamber and 5 × 10³ cells/well were seeded in 96-well plates. KATO III cells were seeded in a quantity of 10⁴ cells/well. All cells were incubated for 12 hours, then treated with 1.25 µg/mL, 2.5 µg/mL, 5 µg/mL, 10 µg/mL, 20 µg/mL, 40 µg/mL, 80 µg/mL, and 160 µg/mL of PAMoCl, and incubated again for 24 and 48 hours. In addition, a control group with the vehicle (DMSO at 0.5%), not the PAMoCl, was included. For cell viability assay, 20 µL of resazurine (0.15 mg/mL) was added to each well and incubated for 3 hours. Finally, the 96-well plates were read with a Synergy LX (Biotek) multimodal plate reader by spectrophotometry at wavelengths of 570 nm and 600 nm. The PAMoCl concentration, that causes 50% cell growth inhibition (GI₅₀) with respect to the control group growth, was calculated.

Cellular morphology observation

Changes in cell morphology after incubation of AGS, KATO III and 293T cells with PAMoCl were observed and photographed on an inverted phase-contrast microscope (Nikon Eclipse TI) after 24 and 48 hours.

Cellular cycle evaluation by flow cytometry

The Darzynkiewicz et al. protocol ¹⁵, with slight modifications, was used to evaluate the effect of PAMoCl on the cell cycle. A total of 350,000 AGS and 500,000 KATO III cells were seeded in 100 × 15 mm petri dishes with DMEM-F12 and RPMI media. After 24 hours, the culture medium was replaced with a new DMEM-F12 medium and a complete RPMI containing PAMoCl at concentrations of 19.62 and 39.23 µg/mL for AGS cells and 87.49 and 160 µg/mL for KATO III cells; these concentrations were obtained from the cell viability assay. All plates were incubated for 24 hours at 37 °C and 5% CO₂. After 24 hours, the plates were washed twice with phosphate buffer saline (PBS) 1X and trypsinized for 5 minutes. The resuspended cells were collected by centrifugation and fixed with cold 70% ethanol; then they were incubated at 4 °C for 30 minutes. Afterwards, the cells were stained with a propidium iodide solution (50 µg/mL) and RNase (100 µg/mL) for an additional 30 minutes and immediately analyzed in the Guava EasyCyte flow cytometer (Merck).

Data analysis

The absorbance data obtained from the cell viability assay were exported to a Microsoft Excel file and expressed as percentages with respect to the control group. For the dose-response relationship and the calculation of GI₅₀, a non-linear regression model was used. The significant differences between groups were compared using the one-way ANOVA test with the Tukey test, as a post hoc test (p < 0.05), using the GraphPad Prism program. The cell cycle results were analyzed using the FCS 7 Express program (DeNovo solutions). The analyzed data are the result of three independent experiments.

Effect of the chloroform fraction of the Piper aduncum methanolic extract on cell viability

In the cell viability assay, all evaluated cell lines showed cytotoxic activity of PAMoCl with the following GI₅₀ results at 24 hours: 39.23 µg/mL, 87.49 µg/mL and 74.10 µg/mL; and at 48 hours 49.47 µg/mL; 64.68 µg/mL and 101.8 µg/mL for AGS, KATO III and 293T cell lines, respectively (Figure 2). At 48 hours, PAMoCl GI₅₀ was significantly lower for AGS and KATO III lines compared to 293T (cytotoxicity control) with values of p < 0.01 and p < 0.05, respectively (Figure 3).

Figure 2 Effect of the chloroform fraction of Piper aduncum methanolic extract (PAMoCl) on cell viability. Cell lines 293T, AGS and KATO III were treated at different concentrations of PAMoCl (1.25; 2.5; 5; 10; 20; 40; 80 and 160 µg/mL) during 24 and 48 hours. GI₅₀ graphs are shown, which are log10 dependent cell viability curves (%) of PAMoCl concentration (µg/mL) for each cell line. In the upper right part of each graph, GI₅₀ values are shown at 24 and 48 hours. 

Figure 3 Comparison of 50% cell growth inhibition (GI₅₀) values for each cell line at (A) 24 hours and (B) 48 hours 

Observation of cell morphology

PAMoCl caused dose-dependent changes in the morphology of AGS, KATO III and 293T cells (Figure 4). After a 24-hour treatment with PAMoCl, starting at 20 µg/mL, AGS cells began to contract and others resuspended. At higher concentrations, such as 80 µg/mL and 160 µg/mL, more cell fragments, few resuspended cells and none attached were observed. After 48 hours the effect was similar. In the KATO III cell line, dead cells (Figure 4) are observed starting from 80 µg/mL, at both 24 and 48 hours. After a 24-hour treatment with PAMoCl, 293T cells began to contract at 80 µg/mL. At 160 µg/mL more contracted and resuspended cells were observed, however, cells could still be found adhering to the culture plate. At 48 hours, the observed effect was similar.

Figure 4 Effect of the chloroform fraction of Piper aduncum methanolic extract at 5 µg/mL, 20 µg/mL and 80 µg/mL concentrations on the morphology of gastric cancer cell lines AGS, KATO III and human kidney 293T. A control group of cells without treatment but with the vehicle (DMSO 0.5%) is also shown for each cell line. T.M. 100X. Red arrow: resuspended cells; blue arrow: attached cells; white arrow: contracted cells; and black arrow: dead cells. 

Effect on the cell cycle

Results showed an effect on the cell cycle from the gastric cancer lines. The percentage of AGS cells in G2/M phase was 31.8%; 44.1% and 52.7% for the concentrations of 0 µg/mL; 19.62 µg/mL and 39.23 µg/mL of PAMoCl, respectively (Figure 5 A-C). The percentage of KATO III cells in G2/M phase was 30.9%; 29.3% and 49.0% for the concentrations of 0 µg/mL; 87.49 µg/mL and 160 µg/mL of PAMoCl, respectively (Figure 5 D-F).

Figure 5 Flow cytometry analysis for the cell cycle of gastric cancer cell lines AGS and KATO III that were treated with the chloroform fraction of Piper aduncum methanolic extract (PAMoCl). Histograms are shown indicating the amount of DNA marked by propidium iodide. Three groups are considered for each cell line: A) control group with AGS cells without PAMoCl, but with the vehicle; B) AGS cells treated with 19.62 μg/mL of PAMoCl; C) AGS cells treated with 39.23 μg/mL of PAMoCl; D) control group with KATO III cells without treatment but with the vehicle; E) KATO III cells treated with 87.49 μg/mL of PAMoCl; F) KATO III cells treated with 160 μg/mL of PAMoCl. Raw data from flow cytometry (black curve), G1: Gap 1 (red curve), S: synthesis (blue curve), and G2/M: Gap 2/mitosis (green curve), cell cycle phases.