Preparation of cinnamon water extract

Cinnamon bark (Cinnamomi cassia P RESL ) of Vietnamese origin was purchased from Omni Herb (Daegu, South Korea). The plant was identified by Professor Choi of the Department of Herbology at Kyung Hee University. A voucher specimen sample (CC-2011) was deposited at the Laboratory of Herbology at Kyung Hee University. The plant was pulverized and soaked in one volume of water for 48 hours at room temperature, and further dissolved by sonication for 1 hour. The extract was filtered and evaporated using a freeze dryer (EYELA, Japan) at −70°C. The yield of CWE was about 3.62%. For size fractionation, 1.28 g of CWE was dissolved in 30 ml of distilled water and fractions were collected using 3 kDa and 10kDa Amicon Ultra Centrifugal Filter device (Millipore, Ireland). The yields of a low molecular weight (MW) fraction (below 3 kDa), a middle MW fraction (between 3 kDa and 10 kDa), and a high MW fraction (over 10 kDa) were 60%, 15% and 25% of CWE, respectively. All the final samples were dissolved in PBS and sterilized by passing through a 0.22-μm syringe filter.

Animals

Eight-week-old male BALB/c mice were purchased from the Korean branch of Taconic, SamTaco (Osan, Korea) and fed rodent chow and water ad libitum in a temperature- and humidity-controlled pathogen-free animal facility at the Medical Center of Kyung Hee University Hospital. Mice were maintained in accordance with the Guide for the Care and Use of Laboratory Animals issued by the US National Research Council (1996), and the protocol KHMC-IACUC12-006 was approved by the Kyung Hee University Medical Center Institutional Animal Care and Use committee.

In vivo LPS injection

CWE (20, 100 or 500 mg/kg of body weight) was given to mice via oral gavage for 6 days. Control mice received an equal volume of normal saline during the experimental period. Each group consisted of 12 mice. On day 7, LPS (serotype 055:B5; Sigma, St. Louis, MO, USA) (1.3 mg/kg) was injected intraperitoneally 1 hour before blood sampling. Blood was obtained by cardiac puncture. As a reference drug, dexamethasone (Sigma) (5 mg/kg) was injected intraperitoneally 18 hours before the LPS injection. Blood samples were centrifuged at 800 g for 20 min. The serum samples obtained were stored at −20°C until used.

Isolation and culture of peritoneal macrophages

For the use of in vitro culture of macrophages, normal mice were injected intraperitoneally with 2 ml of sterile thioglycollate medium (BD, France), and macrophages were collected three days later by peritoneal gavage with cold Dulbecco’s modified Eagle’s medium (DMEM). The recovered peritoneal fluid was washed by centrifugation. The cells were resuspended in DMEM with 10% fetal bovine serum and incubated for 3 hours at 37°C with 5% CO 2 . Non-adherent cells were removed.

Viability assay

Cell viability was determined using the MTT method. Macrophages were seeded in 96-well plates and treated with 10, 50, 100, 200, and 400 μg/ml CWE in the presence or absence of LPS for 24 hours. Ten microliters of MTT solution (5 mg/ml) (Sigma) was added to each well and, after 2 hours of incubation, media was aspirated and 100 μl of dimethyl sulfoxide (DMSO) (Sigma) was added. The optical density was read at 560 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

cDNA preparation and real-time PCR

Peritoneal macrophages were seeded in 6-well plates and pre-treated with CWE for 1 hour, then stimulated with 100 ng/ml LPS for 4 hours. Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Germany) and cDNA was reverse-transcribed using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Diluted cDNA was mixed with Power SYBR Green PCR Master mix (Applied Biosystems, Foster City, CA, USA) and 2 pmol of primers for TNF-α or GAPDH and. The following forward and reverse primer sequences were used: TNF-α, forward :5′- ATG ATC GCG GAC GTG GAA-3′ and reverse: 5′-AGG GCC TGG AGT TCT GGA A-3′; GAPDH, forward: 5′-GGC ATG GAC TGT GGT CAT GA-3′ and reverse: 5′-TTC ACC ACC ATG GAG AAG GC-3′. Amplification of cDNA was performed in triplicate using a StepOne realtime PCR system (Applied Biosystems). After an initial heat denaturation at 95°C for 10 min, the PCR conditions were set at 95°C for 15 s and 60°C for 1 min for 40 cycles. For each PCR, a corresponding mRNA sample without RT was included as a negative control. Quantification of each cDNA copy number was determined according to the manufacturer’s protocol. The GAPDH gene was used as an endogenous control.

Cytokine measurement

The levels of cytokines from serum or cell supernatants were measured by enzyme-linked immunosorbent assay (ELISA), according to the manufacturer’s protocol (BD Pharmingen, USA).

Western blot analysis

Peritoneal macrophages were seeded and pretreated with CWE for 1 hour and then stimulated with LPS for 15 min. Cells were rinsed in cold PBS and then lysed on ice in 0.1 ml of RIPA buffer (50 mM Tris–HCl, pH 7.5; 150 mM NaCl; 1 mM EDTA; 20 mM NaF; 0.5% NP-40; and 1% Triton X-100) containing phosphatase inhibitor cocktail (Sigma) and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). After centrifugation at 13,000 g for 10 min, supernatants were collected. Protein concentrations were determined using the Bradford protein assay reagent (Bio-Rad, USA) and the samples were diluted with 6x sodium dodecyl sulfate(SDS) buffer and boiled for 3 min. The samples were separated on a 10% SDS-polyacrylamide gel and were transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 hour. The membranes were incubated with IκBα, IKK, tubulin (Santa Cruz Biotechnology, CA, USA), phospho-IKK, phospho-IκBα, phospho-JNK, JNK, phospho-ERK1/2, ERK1/2, phospho-p38, and p38 diluted in 5% skim milk in TBST overnight at 4°C. The blots were washed with TBST and incubated for 1 hour with anti-rabbit horseradish peroxidase-conjugated antibodies. Immunoreactive bands were visualized by chemiluminescence using ECL (GE Healthcare, Little Chalfont, Buckinghamshire, UK), according to the manufacturer’s instructions.

Quantitation of total polyphenols

Total polyphenols from CWE and the size-based fractions were determined by Folin-Ciocalteau (FC) colorimetry as described previously [14]. Gallic acid solutions were used for a calibration standard curve. Twenty microliters of each fraction or total CWE in 1.58 ml of water was incubated with 100 μl of FC reagent (Sigma) for 5 min at room temperature. Three hundred microliters of 1.88 M sodium carbonate solution was used to quench the FC reagent-mediated reaction to form chromogens. After reading absorbance at 765 nm, the concentration of polyphenols was calculated as gallic acid equivalent per gram of extract.

Statistical analysis

In vivo data are presented as mean ± SEM. Statistical differences among the means of multiple groups were determined by using one-way ANOVA followed by the Scheffe test. In vitro data are presented as mean ± SD. The difference between the two means was assessed using a non-paired Student’s t-test. Calculations were carried out using SPSS version 12. P values of less than 0.05 were considered significant.