Reagents and consumables

Cinnamon powder was purchased from a local distributor. C. cassia was from Carmel Organics, Barukheda, India; C. loureiroi was from Kirkland, D.C, USA; C. zeylanicum was from Sorich Organics, Noida, India. Infliximab was purchased from a local Pharmacy in India. Cell culture reagents were purchased from Corning (NY, USA). All other reagents were purchased from the vendors as listed. Caspase3/7 glo reagent (G8090, Promega Madison, WI, USA), TNF-α (AB155699, Abcam, Waltham, MA, USA), trans-cinnamaldehyde (TCA; C80687, Sigma, Burlington, MA, USA), cinnamic acid (CA; 8.00235, Sigma), eugenol (E51791, Sigma), actinomycin D (A9415, Sigma), trypsin–EDTA (25,200–056, Gibco, Billings, MT, USA), phosphate-buffered saline (PBS), pH 7.2 (20,012–027), and Alamar Blue (DAL1100, Invitrogen, Waltham, MA, USA).

Preparation of aCE

Ground cinnamon (C. zeylanicum, C. cassia, and C. loureiroi) bark powder was dissolved in water (70 °C for 1 h) to obtain 100 mg/mL extracts. All extracts were centrifuged (12,000 rpm for 10 min) to remove the insoluble components. The supernatant was filtered (0.4 µm pore size, HNWP04700, Merck Millipore Burlington, USA), aliquoted, and stored at -80 °C.

Cell lines and cultures

The L929 (ATCC CCL-1) and U937 (ATCC CRL-1593.2) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). The L929 cell line was cultured under aseptic conditions in Eagle’s minimum essential media (EMEM; M4655, Sigma), and the U937 cell line was cultured under aseptic conditions in Roswell Park Memorial Institute-1640 medium (RPMI-1640; M8758, Sigma). Both media were supplemented with 10% (v/v) fetal bovine serum (FBS; 10,082–147, Gibco) and 1% (v/v) penicillin/streptomycin (P4333, Sigma-Aldrich). The cell cultures were maintained in a 37 °C incubator at 95% humidity and in the presence of 5% CO2.

L929 and U937 cell viability assay

The L929 cells were cultured in a 75 cm2 flask in EMEM supplemented with 10% FBS. The assay medium contained 3% FBS in EMEM. The L929 cell suspension was prepared in assay media by trypsinizing the cell monolayer. Fifty microliters of 0.75 × 106 cells/mL (37,500 cells/well) were plated in each well of the 96-well plates. aCE was serially diluted to eight different concentrations ranging from 33.3 to 0.26 mg/mL (final concentrations). 50 µL of aCE and actinomycin D was added to treated and actinomycin control wells. Control wells without aCE or actinomycin D were also established.

The U937 cells were cultured in RPMI-1640 medium supplemented with 10% FBS. The assay medium contained 2% FBS. In total, 25 µL of 1.2 × 106 cells/mL (30,000 cells per/well) was plated in each well of the 96-well plates. aCE was serially diluted to eight different concentrations ranging from 33.3 to 0.26 mg/mL (final concentrations). 25 µL of aCE was added to the treated wells. Control wells without aCE were also established.

The plates were incubated at 37 °C in the presence of 5% CO2 for 18 − 20 h, followed by the addition of 20 µL Alamar Blue to the wells. The plates were further incubated for 6 − 7 h at 37 °C in the presence of 5%. CO2. The color change of alamar blue reagent was measured using a Synergy microplate reader (BioTek, Winooski, VT, U.S.) at 570 nm and 600 nm. The average absorbance of the cell culture medium alone (background) at 600 nm was subtracted from that at 570 nm. The background-subtracted absorbance at 570 nm was plotted against the concentration of the test compound. Viability assays with TCA, cinnamic acid (CA), and eugenol were performed in a manner similar to that described above (the concentration range was 1–0.65 mg/mL).

aCE-infliximab synergy experiments

The L929 cell suspension was prepared by trypsinizing the cell monolayers. In total, 25 μL of the cell suspension (1.5 × 106 cells/mL or 37,500 cells/well) was added to each untreated, treated, TNF-α control and cell control labelled wells in a transparent 96-well microplate plate and incubated for 20 min at 37 °C in an atmosphere of 5% CO2. Twenty-five microliters of aCE or bioactives were added to the cells (labelled treated wells) to final concentrations of 150, 50, 25, and 10 µg/mL or 40, 32, 16, 8, and 4 µg/mL (in duplicates), respectively. Each set was incubated for 15, 30, or 60 min. Twenty-five microliters of the assay medium were added to the control wells (cell control, TNF-α control) and untreated wells in the assay plate. Simultaneously, 100 μL of eight different concentrations of infliximab (30 ng to 0.23 ng/mL, final concentrations) were added to the wells marked as treated and untreated on separate neutralization plate. One hundred microliters of TNF-α (20 IU/mL, final concentration) was added to each treated, untreated, and TNF-α control well and mixed five times (this plate did not contain cells, only infliximab and TNFα in 1:1 volume ratio for neutralization) and incubated for 1 h at 37 °C. One hundred microliters of the assay media were added to the TNF-α control wells and 200 μL to the cell control wells. After 15, 30, or 60 min of incubation of aCE with cells, 50 μL of the neutralization mixture was transferred to the wells in the assay plate marked as treated and untreated. Fifty microliters of 4 μg/mL actinomycin D were added to the treated, untreated, and TNF-α control wells, and 100 μL assay medium was added to the control wells. The cell plates were incubated at 37 °C in the presence of 5% CO2 for 18 − 20 h, followed by the addition of 20 µL alamar blue to the wells and further incubation for 6 − 7 h at 37 °C in an atmosphere of 5%. CO2. The color change was read using a Synergy microplate reader (BioTek) at 570 nm and 600 nm. The average absorbance value of the cell culture medium alone (background) at 600 nm was subtracted from that of the experimental wells at 570 nm. The background-subtracted absorbance at 570 nm was plotted against the infliximab concentration to determine the shift in the half maximal effective concentration (EC50) in the presence of aCE. Similar study was carried out for bioactives (15 min incubation).

The U937 cell suspension was prepared by harvesting the cells, and then centrifuging at 125 rcf for 5 min, resuspending, and counting them in the assay medium. In total, 12.5 μL of the cell suspension (2.4 × 106 cells/mL or 30,000 cells/well) was added to each untreated, treated, TNF-α control and cell control wells in an opaque white 96-well microplate plate (assay plate) and incubated for 10 min at 37 °C in an atmosphere of 5% CO2. aCE (12.5 μL) was added to the cells (labelled treated wells) to final concentrations of 150, 50, 25, and 10 µg/mL, or 12.5 μL bioactives were added to cells to final concentrations of 40, 32, 16, 8, and 4 µg/mL (in duplicates). Each set was incubated for 15 min. The assay medium (12.5 μL) was added to the control wells (cell control, TNF-α control), and untreated wells in the assay plate. Simultaneously, 50 μL of eight different concentrations of infliximab (50 ng to 1.14 ng/mL, final concentration) were added to the wells labelled as treated and untreated on a separate neutralization plate. Fifty microliters of TNF-α (20 IU/mL, final concentration) was added to each treated, untreated, and TNF-α control wells and mixed five times (this plate did not contain cells, only infliximab and TNFα in 1:1 volume ratio for neutralization) and incubated for 1 h at 37 °C. Fifty microliters of the assay media were added to the TNF-α control wells and 100 μL to the cell control wells. After incubation, 25 μL of the mixture was transferred to the wells marked as treated and untreated in the assay plate, and the plate was incubated for 2.5 h at 37 °C in the presence of 5% CO2. Fifty microliters of the caspase 3/7 glo reagent were added to the treated, untreated, TNF-α control and cell control wells, and the plates were further incubated at 30 min at room temperature before taking the reading. Luminescence was measured using a Synergy microplate reader (BioTek).

Gene expression assay

L929 cells were added to 6-well plates (1 × 106 cells per well in 600 µL) in EMEM + 10% FBS (v/v) and incubated for ~ 18 h under sterile conditions. The spent medium was discarded, and the cells were rinsed with EMEM (200 µL/well). In a separate plate, infliximab EC100 (30 ng/mL, final concentration) was mixed with TNF-α (20 IU/mL, final concentration) and incubated for 1 h at 37 °C. aCE samples and concentrations which showed significant synergy (p-value < 0.05) and reproducibility across days in the synergy experiments were selected for gene expression assays. The cells were preincubated with 300 µL of 50 and 25 µg/mL aCE (final concentrations) or 40 and 32 µg/mL (final concentrations) of bioactive for 15 min, followed by the addition of an equal volume of the neutralized TNF-α + infliximab (EC100 mix) and further incubated for 30, 60, or 120 min. Cell control and infliximab control wells (EC100 mix) were also set up. At each time point, the cells were harvested and used for RNA extraction and cDNA preparation. 120 min time point was selected based on data reproducibility. Each sample was analyzed in duplicates.

A U937 cell suspension was prepared in the assay medium and added to 6-well plates (1 × 106 cells per well in 600 µL). In a separate plate, infliximab EC100 (50 ng/mL, final concentration) was mixed with TNF-α (20 IU/mL, final concentration) and incubated for 1 h at 37 °C. aCE samples and concentrations with a significant p-value  in the synergy experiments were selected for gene expression assays. Cells were preincubated with 300 µL of 50 and 25 µg/mL (final concentrations) aCE or 40 and 32 µg/mL (final concentrations) bioactive for 15 min, followed by the addition of 300 µL neutralized TNF-α + infliximab (EC100 mix) and further incubated for 120 min. Cell control and infliximab control (EC100 mix) wells were also set up. At each time point, the cells were harvested and used for RNA extraction and cDNA preparation. Each sample was analyzed in duplicate.

RNA extraction and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

Total RNA from treated, untreated, and control samples was extracted using the RNeasy mini kit (74,101, Qiagen, Hilder, Germany) according to the manufacturer's instructions, and the concentration was measured using Take3 low-volume plates on a Synergy microplate reader (BioTek). cDNA was prepared using Protoscript II (E6560, NEB, Ipswich, MA, USA) per the product datasheet. qRT-PCR was performed using the PowerUp™ SYBR™ Green master mix (A25742, Thermo Fisher Scientific, Waltham, MA, USA) on an ABI 7500 real-time PCR system (Applied Biosystems, Waltham, MA, USA). The conditions used were initial denaturation for 10 min at 95 °C; primer annealing for 15 s at 95 °C, and elongation for 60 s at 60 °C for 40 cycles. A melting curve analysis was also performed where primer specificity was confirmed by the presence of a single peak at melting temperature. Housekeeping genes for data normalization were selected based on Norm Finder. The decrease or increase in TNF-α-induced gene expression was determined in the presence of infliximab, aCE, or bioactive compounds. Changes in gene expression were calculated in terms of fold induction with respect to the untreated cell control using the 2-∆∆CT method. The level of gene expression in TNF-α-treated controls was considered 100%. An increase or decrease in expression after infliximab treatment was the second level of control. The effects of aCE or bioactive treatment in the presence of infliximab + TNFα (EC100) were considered significant when the expression levels of various genes differed significantly from those after the infliximab (EC100) treatment alone. The primers used in this study are listed in Supplementary Table S1.

Statistical analysis

All experiments were performed at least three times unless stated otherwise. All samples were analyzed in duplicates. Means of more than two groups were compared using one-way analysis of variance (ANOVA) with the Kruskal–Wallis test. Statistical significance was set at p < 0.05. Data was analyzed using the GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA).