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. 2012;2012:429320.
doi: 10.1155/2012/429320. Epub 2012 Mar 27.

Anti-Inflammatory Activities of Cinnamomum cassia Constituents In Vitro and In Vivo

Jung-Chun Liao  1 , Jeng-Shyan Deng, Chuan-Sung Chiu, Wen-Chi Hou, Shyh-Shyun Huang, Pei-Hsin Shie, Guang-Jhong Huang
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Anti-Inflammatory Activities of Cinnamomum cassia Constituents In Vitro and In Vivo

Jung-Chun Liao et al. Evid Based Complement Alternat Med. .
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Abstract

We have investigated the anti-inflammatory effects of Cinnamomum cassia constituents (cinnamic aldehyde, cinnamic alcohol, cinnamic acid, and coumarin) using lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW264.7) and carrageenan (Carr)-induced mouse paw edema model. When RAW264.7 macrophages were treated with cinnamic aldehyde together with LPS, a significant concentration-dependent inhibition of nitric oxide (NO), tumor necrosis factor (TNF-α), and prostaglandin E2 (PGE(2)) levels productions were detected. Western blotting revealed that cinnamic aldehyde blocked protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear transcription factor kappa B (NF-κB), and IκBα, significantly. In the anti-inflammatory test, cinnamic aldehyde decreased the paw edema after Carr administration, and increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the paw tissue. We also demonstrated cinnamic aldehyde attenuated the malondialdehyde (MDA) level and myeloperoxidase (MPO) activity in the edema paw after Carr injection. Cinnamic aldehyde decreased the NO, TNF-α, and PGE(2) levels on the serum level after Carr injection. Western blotting revealed that cinnamic aldehyde decreased Carr-induced iNOS, COX-2, and NF-κB expressions in the edema paw. These findings demonstrated that cinnamic aldehyde has excellent anti-inflammatory activities and thus has great potential to be used as a source for natural health products.

Figures

Figure 1
Chemical structure of Cinnamomum cassia constituents (cinnamic aldehyde, cinnamic alcohol, cinnamic acid, and coumarin) (a) and cytotoxic effects of Cinnamomum cassia constituents in RAW264.7 cells (b). Cells were incubated for 24 h with 100 ng/mL of LPS in the absence or presence of samples (0, 6.25, 12.5, 25, and 50 μM). Samples were added 1 h before incubation with LPS (lipopolysaccharide). Cell viability assay was performed using MTT assay. The data were presented as mean ± S.D. for three different experiments performed in triplicate.
Figure 2
Effects of Cinnamomum cassia constituents (cinnamic aldehyde, cinnamic alcohol, cinnamic acid, and coumarin) on LPS-induced NO production of RAW264.7 macrophages. Cells were incubated for 24 h with 100 ng/mL of LPS in the absence or presence of samples (0, 6.25, 12.5, 25, and 50 μM). Samples were added 1 h before incubation with LPS. Nitrite concentration in the medium was determined using Griess reagent. The data were presented as mean ± S.D. for three different experiments performed in triplicate. ###compared with sample of control group. *P < 0.05, **P < 0.01, and ***P < 0.001 were compared with LPS-alone group.
Figure 3
Inhibition of iNOS and COX-2 protein expression by Cinnamomum cassia constituents (cinnamic aldehyde, cinnamic alcohol, cinnamic acid, and coumarin) in LPS-stimulated RAW264.7 cells. Cells were incubated for 24 h with 100 ng/mL of LPS in the absence or the presence of samples (50 μM). Samples were added 1 h before incubation with LPS. Lysed cells were then prepared and subjected to western blotting using an antibody specific for iNOS and COX-2. β-actin was used as an internal control. (a) A representative western blot from two separate experiments is shown. (b) Relative iNOS and COX-2 protein levels were calculated with reference to an LPS-stimulated culture. ###compared with sample of control group. The data were presented as mean ± S.D. for three different experiments performed in triplicate. *P < 0.05 and ***P < 0.001 were compared with LPS-alone group.
Figure 4
Inhibition of NF-κB and IκBα (a) protein expressions by Cinnamomum cassia constituents (cinnamic aldehyde, cinnamic alcohol, cinnamic acid, and coumarin) in LPS-stimulated RAW264.7 cells. Samples (50 μM) were added into cells 1 h before LPS (100 ng/mL) stimulation and protein samples were prepared for 1 h after LPS stimulation. Activations of signaling molecules were then evaluated by Western blot analysis. Lysed cells were then prepared and subjected to western blotting using an antibody specific for NF-κB (P65) and IκBα in the cytosol. β-actin was used as an internal control. A representative western blot from two separate experiments is shown. Relative NF-κB and IκBα protein levels were calculated with reference to an LPS-stimulated culture (b). ###compared with sample of control group. The data were presented as mean ± S.D. for three different experiments performed in triplicate. *P < 0.05 and ***P < 0.001 were compared with LPS-alone group.
Figure 5
Figure 5
The effects of cinnamic aldehyde on lipopolysaccharide (LPS)-induced TNF-α (a) and PGE2 (b) in LPS-stimulated RAW264.7 cells. Cells were incubated for 24 h with 100 ng/mL of LPS in the absence or in the presence of cinnamic aldehyde (0, 12.5, 25, and 50 μM). Cinnamic aldehyde was added 1 h before the incubation with LPS. TNF-α and PGE2 concentrations in the medium were determined using ELISA kit. The data were presented as mean ± S.D. for three different experiments performed in triplicate. ### P < 0.001 compared with sample of control group.*P < 0.05, **P < 0.01, and ***P < 0.001 were compared with LPS-alone group.
Figure 6
Effects of cinnamic aldehyde and Indo on hind paw edema induced by Carr in mice (a), the tissue MDA (b) and MPO (c) concentrations of foot in mice, Carr-induced NO (d), TNF-α, (e) and PGE2 (f) concentrations of serum at the 5th h in mice. The values are averaged, obtained in individual animals (n = 6). Each value represents as mean ± S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001 as compared with the Carr group.
Figure 7
Inhibition of iNOS, COX-2, and NF-κB protein expressions by cinnamic aldehyde induced by Carr of foot at the 5th h in mice. Tissues suspended were then prepared and subjected to western blotting using an antibody specific for iNOS, COX-2, and NF-κB. β-actin was used as an internal control. (a) A representative western blot from two separate experiments is shown. (b) Relative iNOS, COX-2, and NF-κB protein levels were calculated with reference to a Carr-injected mouse. ###compared with sample of control group. The data were presented as mean ± S.D. for three different experiments performed in triplicate. **P < 0.01 and ***P < 0.001 were compared with Carr-alone group.
Figure 8
Representative light micrographs of mouse hind footpad H&E stained to reveal hemorrhage, edema, and inflammatory cell infiltration in control mice (a), Carr-treated mice demonstrates hemorrhage with moderately extravascular red blood cell and large amount of inflammatory leukocyte mainly neutrophils infiltration in the subdermis interstitial tissue of mice (b), and mice given indomethacin (Indo) (10 mg/kg) before Carr. Cinnamic aldehyde (5 mg/kg) significantly shows morphological alterations (100x) and the numbers of neutrophils in each scope (400 x) compared to subcutaneous injection of Carr only. ### P < 0.001 as compared with the control group. ***P < 0.001 compared with Carr group. Scale bar = 100 μm.
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